Background:The molecular mechanism underlying the involvement of the Transferrin receptor(TFRC)in cervical cancer remains poorly understood.This study aims to elucidate the role of TFRC in cervical cancer by analyzing...Background:The molecular mechanism underlying the involvement of the Transferrin receptor(TFRC)in cervical cancer remains poorly understood.This study aims to elucidate the role of TFRC in cervical cancer by analyzing data from The Cancer Genome Atlas(TCGA)and Genotype-Tissue Expression(GTEx)databases.Methods:TFRC protein expression was obtained from Human Protein Altas(HPA).All datas were collected from TCGA and GTEx.In this study,we analyzed the expression of TFRC in cervical cancer and its clinical significance.Through Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene set enrichment analyses(GSEA),investigated the related molecular pathways of TFRC.The relationship between TFRC and immune infiltration was then examined.The prognosis of different immune cell subsets was then analyzed after dividing cervical cancer patients into high and low expression of TFRC groups.Results:TFRC is highly expressed in various tumor tissues compared to control normal tissues,including cervical cancer.An increased expression of TFRC was associated with higher Tumor(T)and Node(N)stage,as well as a higher clinical stage.Kaplan–Meier(KM)survival analysis investigated that higher TFRC expression patients have a poor overall survival(OS),disease specific survival(DSS)and progress free interval(PFI).Both KEGG and GSEA enriched signaling pathway by high TFRC and low TFRC groups.There was a significant negative linear correlation between TFRC expression and immune infiltration.TFRC affects the prognosis of cervical cancer patients through immune pathway.Conclusions:Cervical cancer patients with TFRC expression may have a worse prognosis.展开更多
Iron is an essential trophic element that is required for cell viability and differentiation, especially in oligodendrocytes, which consume relatively high rates of energy to produce myelin. Multiple iron metabolism p...Iron is an essential trophic element that is required for cell viability and differentiation, especially in oligodendrocytes, which consume relatively high rates of energy to produce myelin. Multiple iron metabolism proteins are expressed in the brain including transferrin receptor and ferritin-H. However, it is still unknown whether they are developmentally regulated in oligodendrocyte lineage cells for myelination. Here, using an in vitro cultured differentiation model of oligodendrocytes, we found that both transferrin receptor and ferritin-H are significantly upregulated during oligodendrocyte maturation, implying the essential role of iron in the development of oligodendrocytes. Additional different doses of Fe3＋ in the cultured medium did not affect oligodendrocyte precursor cell maturation or ferritin-H expression but decreased the expression of the transferrin receptor. These results indicate that upregulation of both transferrin receptor and ferritin-H contributes to maturation and myelination of oligodendrocyte precursor cells.展开更多
AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL ...AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.展开更多
Objective To establish and evaluate a protein serum ferritin （SF） and soluble transferrin receptor microarray method for combined measurement of （sTfR）. Methods Microarrayer was used to print both anti-SF antibodi...Objective To establish and evaluate a protein serum ferritin （SF） and soluble transferrin receptor microarray method for combined measurement of （sTfR）. Methods Microarrayer was used to print both anti-SF antibodies I and anti-sTfR antibodies I on each protein microarray. Anti-SF antibodies II and anti-sTfR antibodies II were used as detection antibodies and goat antibodies coupled to Cy3 were used as antibodies Ill. The detection conditions of the quantitative analysis method for simultaneous measurement of SF and sTfR with protein microarray were optimized and evaluated. The protein microarray was compared with commercially available traditional tests with 26 serum samples. Results By comparison experiment, mouse monoclonal antibodies were chosen as the probes and contact printing was chosen as the printing method. The concentrations of SF and sTfR probes were 0.5 mg/mL and 0.5 mg/mL respectively, while those of SF and sTfR detection antibodies were 5 μg/mL and 0.36 μg/mL respectively. Intra- and inter-assay variability was between 3.26% and 18.38% for all tests. The regression coefficients comparing protein microarray with traditional test assays were better than 0.81 for SF and sTfR. Conclusion The present study has established a protein microarray method for combined measurement of SF and sTfR.展开更多
To obtain single chain variable fragment （scFv） and bivalent single chain variable fragment （bsFv） against transferrin receptor, up-stream and down-stream primers were designed according to the complementary seque...To obtain single chain variable fragment （scFv） and bivalent single chain variable fragment （bsFv） against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy （VH） and FR4 of variable light （VL）, respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.展开更多
Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies fo...Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies for clinical application,we constructed TfR bispecific T-cell engager(BiTE)in two extensively applied formats,including single-chain tandem singlechain variable fragments(scFvs)and double-chain diabodies,and evaluated their functional characterizations in vitro.Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+HepG2 cells.However,compared to two・chain diabodies,scFvs were more efficient in antigen binding and TfR target killing.Furthermore,different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies.Thus,the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.展开更多
Objective: To investigate the effect of high throughput hemodialysis on soluble transferrin receptor in hemodialysis patients and the improvement of renal anemia. Methods: 132 patients receiving maintenance hemodialys...Objective: To investigate the effect of high throughput hemodialysis on soluble transferrin receptor in hemodialysis patients and the improvement of renal anemia. Methods: 132 patients receiving maintenance hemodialysis in our hospital from July 2017 to July 2019 were selected and divided into control group and observation group according to the random number table method, with 66 cases each. The observation group was treated with high-flux hemodialysis, while the control group was treated with low-flux hemodialysis for 6 months. Compare two groups before and after treatment serum beta 2 microglobulin (beta 2 - MG), serum creatinine (Scr), blood urea nitrogen (BUN) level, anemia related index [red blood cells deposited (HCT), hemoglobin (Hb), reticulocyte percentage (Ret%)], iron metabolism index [serum ferritin (SF), transferrin saturation (TSAT)、Hepcidin(Hepc)], soluble transferrin receptor (sTfR) levels and adverse reactions. Results: the levels of 2-MG, Scr and BUN in the two groups before treatment were compared (P>0.05). After treatment, Scr and BUN levels in the two groups were significantly decreased (P<0.05), but were compared between the two groups (P>0.05). The level of 2-MG in the observation group was lower than that in the control group (P<0.05). Before treatment, sTfR, Hb, HCT level and Ret% of the two groups were compared(P>0.05). After treatment, Hb and HCT levels in the observation group were higher than those in the control group, while Ret% were lower than those in the control group, (P<0.05). Before treatment, the levels of ST、TAST、sTfR and Hepc in the two groups were compared (P>0.05). After treatment, the level of ST and TAST in the observation group was higher than that in the control group, The levels of sTfR and Hepc were lower than the control group (P<0.05). The overall incidence of adverse reactions in the observation group (8.93%) was lower than that in the control group (10.14%), with no significant difference (P>0.05). Conclusion: The high-throughput hemodialysis department significantly improved renal anemia in hemodialysis patients, reduced serum sTfR level, and had fewer adverse reactions and higher safety.展开更多
Hypochromic microcytic anaemia includes iron deficiency, anaemia of chronic disorders, beta thalassemia trait and sideroblastic anaemia. To rule out the cause of hypochromic microcytic anaemia is a diagnostic difficul...Hypochromic microcytic anaemia includes iron deficiency, anaemia of chronic disorders, beta thalassemia trait and sideroblastic anaemia. To rule out the cause of hypochromic microcytic anaemia is a diagnostic difficulty. The conventional laboratory tests used for diagnosis have few disadvantages. Serum transferrin receptor (sTfR) is the most reliable method for assessment of body iron. Eighty four children were included in this study. They were further divided into four groups: iron deficiency anaemia (IDA), anaemia of chronic disorders (ACD), beta thalassemia trait (β TT) and controls. Children withIDAand ACD were diagnosed on the basis of history and serum iron profile. Subjects with β TT had HbA2 > 3.5%. sTfR were performed on all subjects. Level of sTfR in patients withIDAwas 5.79 μg/ml ± 1.3 μg/ml. In patients with anaemia of chronic disorders (ACD), β thalassemia trait and controls mean sTfR were 2.18 μg/ml ± 0.6 μg/ml, 2.1μg/ml ± 0.5 μg/ml and 2.0 μg/ml ± 0.5 μg/ml respectively. These results show level of sTfR was raised in IDA when compared with controls or ACD and β TT (p展开更多
Hepatitis C virus(HCV) is the main pathogen causing chronic hepatitis and primary liver cancer. Various viral proteins and host cell molecules are involved in the HCV cell entry, but the mechanism of infection has not...Hepatitis C virus(HCV) is the main pathogen causing chronic hepatitis and primary liver cancer. Various viral proteins and host cell molecules are involved in the HCV cell entry, but the mechanism of infection has not been completely elucidated. The transferrin receptor can act as a receptor for many viruses during cell entry. The transferrin receptor is not only closely related to HCV-induced iron metabolism disorders but also mediates the fusion of HCV with the host cell membrane as a specific receptor for CD81-dependent viral adhesion.展开更多
The immunosuppressive phenotype of tumor cells extensively attenuates the immune activation effects of traditional treatments.In this work,a transferrin receptor(TfR)targeted immunostimulant(PTI)is fabricated for phot...The immunosuppressive phenotype of tumor cells extensively attenuates the immune activation effects of traditional treatments.In this work,a transferrin receptor(TfR)targeted immunostimulant(PTI)is fabricated for photodynamic immunotherapy against metastatic tumors by interrupting β-catenin signal pathway.To synthesize PTI,the photosensitizer conjugated TfR targeting peptide moiety(Palmitic-K(PpIX)-HAIYPRH)is unitized to encapsulate the transcription interrupter of ICG-001.On the one hand,the recognition of PTI and TfR can promote drug delivery into tumor cells to destruct primary tumors through photodynamic therapy and initiate an immunogenic cell death with the release of tumorassociated antigens.On the other hand,PTI will interrupt the binding between b-catenin andcAMP response element-binding protein(CREB),regulating the gene transcription to downregulate programmed death ligand 1(PD-L1)while upregulating CeC motif chemokine ligand 4(CCL4).Furthermore,the elevated CCL4 can recruit the dendritic cells to present tumor-specific antigens and promote T cells activation and infiltration,and the downregulated PD-L1 can avoid the immune evasion of tumor cells and activate systemic anti-tumor immunity to eradicate lung metastasis.This work may inspire the development of antibody antibody-free strategy to activate systemic immune response in consideration of immunosuppressive conditions.展开更多
The membrane transferrin receptor-mediated endocytosis has been exploited for developing novel targeted drug delivery systems, which could have a variety of applications in the site-specific delivery of anticancer dru...The membrane transferrin receptor-mediated endocytosis has been exploited for developing novel targeted drug delivery systems, which could have a variety of applications in the site-specific delivery of anticancer drugs, proteins and therapeutic genes into proliferating malignant cells that overexpress the transferrin receptors. This is achieved by coupling transferrin or monoclonal antibody to transferrin receptor with therapeutic drugs or drug delivery vesicles. The transferrin conjugates can be obtained by use of either bifunctional chemical linkers or by genetic infusion of therapeutic peptides/proteins into the structure of transferrin / and monoclonal antibody to transferrin receptor. A variety of drug carriers such as liposomes, nanoparticles and DNApolymer complexes （i.e. polyplex and lipoplex） were used to efficiently deliver the Wansferrin conjugates. Use of transferrin conjugates results in improvement in drug efficacy, selectivity and drug release as well as reduction in drug toxicity. This paper reviews the basic biochemistry of transferrin and the transferrin receptor as well as the strategy for developing targeted drug delivery system.展开更多
IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis. 1 The histopathology of IgAN is characterized by abundance of mesangial matrix and proliferation of mesangial cells. IgA_1 deposition in t...IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis. 1 The histopathology of IgAN is characterized by abundance of mesangial matrix and proliferation of mesangial cells. IgA_1 deposition in the mesangium plays an important role in the inflammatory process in this disease.展开更多
Recently, considerable attention in the field of cancer therapy has been focused on the mammalian rapamycin target(m TOR), inhibition of which could result in autophagic cell death(ACD). Though novel combination chemo...Recently, considerable attention in the field of cancer therapy has been focused on the mammalian rapamycin target(m TOR), inhibition of which could result in autophagic cell death(ACD). Though novel combination chemotherapy of autophagy inducers with chemotherapeutic agents is extensively investigated, nanomedicine-based combination therapy for ACD remains in infancy. In attempt to actively trigger ACD for synergistic chemotherapy, here we incorporated autophagy inducer rapamycin(RAP) into 7 pep-modified PEG-DSPE polymer micelles(7 pep-M-RAP) to specifically target and efficiently priming ACD of MCF-7 human breast cancer cells with high expression of transferrin receptor(Tf R). Cytotoxic paclitaxel(PTX)-loaded micelle(7 pep-M-PTX) was regarded as chemotherapeutic drug model. We discovered that with superior intracellular uptake in vitro and more tumor accumulation of micelles in vivo, 7 pep-M-RAP exhibited excellent autophagy induction and synergistic antitumor efficacy with 7 pep-M-PTX. Mechanism study further revealed that 7 pep-M-RAP and 7 pep-MPTX used in combination provided enhanced efficacy through induction of both apoptosis-and mitochondria-associated autophagic cell death. Together, our findings suggested that the targeted excess autophagy may provide a rational strategy to improve therapeutic outcome of breast cancer, and simultaneous induction of ACD and apoptosis may be a promising anticancer modality.展开更多
The aim of this research was to explore the effects and signaling pathway of ultraviolet-B(UVB)irradiation on the expression of hypoxia-inducible factor 1a(HIF-1α)and transferrin receptor(TfR).HIF-1α protein was mea...The aim of this research was to explore the effects and signaling pathway of ultraviolet-B(UVB)irradiation on the expression of hypoxia-inducible factor 1a(HIF-1α)and transferrin receptor(TfR).HIF-1α protein was measured by Western blot method.Expressions of epidermal growth factor receptor(EGFR),phosphor-EGF-R and TfR after UVB irradiation were determined with flow cytometry.After UVB irradiation,mRNA levels of HIF-1α and TfR were detected by real time-PCR.Results showed that compared with control groups,UVB was able to induce HIF1α and TfR protein expression in a dose-and time-dependent manner in HaCat cells(P<0.05).TfR mRNA was expressed in a dose-dependent manner and reached a peak at the 8th hour in HaCat cells(P<0.05)whereas HIF-1α mRNA expression was not affected by UVB treatment(P>0.05).The EGFR/PI3K/AKT signaling pathway was required for the induction of HIF-1α and TfR expression induced by UVB.UVB induced activation of EGFR in HaCat cells and EGFR regulated expression of TfR and HIF-1α.EGFR(−/−)MEF did not increase the HIF1 expression following UVB irradiation(P>0.05).In contrast,EGFR(+/+)MEF strongly enhanced HIF1a expression after UVB irradiation(P<0.05).PD153035,a selective inhibitor of EGFR tyrosine kinase,inhibited the TfR protein expression in UVB-treated cells in a dose-dependent manner(P<0.05).PI3K inhibitors,LY294002 and wortmannin,inhibited HIF-1a and TfR expressions induced by UVB(P<0.05).The DEC1(−/−)Ha-Cat cells did not increase their TfR and HIF-1α expressions following UVB irradiation(P>0.05).In contrast,DEC1(+/+)HaCat cells strongly enhanced TfR and HIF-1α protein expression after UVB irradiation(P<0.05).We conclude that UVB induces TfR and HIF-1α expressions via EGFR/PI3K/AKT/DEC1 signaling pathway.展开更多
Previous studies have shown that the receptor tyrosine kinase Eph receptor A4(EphA4) is abundantly expressed in the nervous system. The EphA4 signaling pathway plays an important role in regulating motor neuron ferrop...Previous studies have shown that the receptor tyrosine kinase Eph receptor A4(EphA4) is abundantly expressed in the nervous system. The EphA4 signaling pathway plays an important role in regulating motor neuron ferroptosis in motor neuron disease. To investigate whether EphA4 signaling is involved in ferroptosis in spinal cord ischemia/reperfusion injury, in this study we established a rat model of spinal cord ischemia/reperfusion injury by clamping the left carotid artery and the left subclavian artery. We found that spinal cord ischemia/reperfusion injury increased EphA4 expression in the neurons of anterior horn, markedly worsened ferroptosis-related indicators, substantially increased the number of mitochondria exhibiting features consistent with ferroptosis, promoted deterioration of motor nerve function, increased the permeability of the blood-spinal cord barrier, and increased the rate of motor neuron death. Inhibition of EphA4 largely rescued these effects. However, intrathecal administration of the ferroptosis inducer Erastin counteracted the beneficial effects conferred by treatment with the EphA4 inhibitor. Mass spectrometry and a PubMed search were performed to identify proteins that interact with EphA4, with the most notable being Beclin1 and Erk1/2. Our results showed that inhibition of EphA4 expression reduced binding to Beclin1, markedly reduced p-Beclin1, and reduced Beclin1-XCT complex formation. Inhibition of EphA4 also reduced binding to p-Erk1/2 and markedly decreased the expression of c-Myc, transferrin receptor 1, and p-Erk1/2. Additionally, we observed co-localization of EphA4 and p-Beclin1 and of EphA4 and p-ERK1/2 in neurons in the anterior horn. In conclusion, EphA4 participates in regulating ferroptosis of spinal motor neurons in the anterior horn in spinal cord ischemia/reperfusion injury by promoting formation of the Beclin1-XCT complex and activating the Erk1/2/c-Myc/transferrin receptor 1 axis.展开更多
Objective: To explore the expression of transferrin(Tf) and transferrin receptor(Tf R) in hematoma brain tissue at different stage after intracerebral hemorrhage(ICH) in rats. Methods: ICH rats model were established ...Objective: To explore the expression of transferrin(Tf) and transferrin receptor(Tf R) in hematoma brain tissue at different stage after intracerebral hemorrhage(ICH) in rats. Methods: ICH rats model were established by collagenase method, and rats were sacrificed at 24 h, 72 h, 7 d and 14 d after operation. The levels of Tf and Tf R in different periods of rats were detected by immunohistochemical method, and correlation between two groups was analyzed. Results: Tf, Tf R-positive cells at each time after operation in observation group were significantly higher than that in control group(P<0.05). Tf, Tf R-positive cells began to increase from 24 h after the operation and reached the peak 72 h-7 d after surgery, but then gradually decreased. Tf was mainly expressed in nucleus and cytoplasm of neurons and glial cells around the hematoma, but Tf R was mainly expressed in nucleus and cytoplasm of neurons and choroid plexus endothelial cells. Correlation analysis showed that the Tf-positive cell was significantly positively correlated with Tf R-positive cell expression(r=0.447, P=0.022). Conclusions: Tf and Tf R were important transporters in brain tissue excessive load iron transport after ICH, and detecting the expression levels of the two indicators can provide a reference for prognosis treatmentin ICH.展开更多
基金supported by the National Natural Science Foundation of China[No.81602020]the Tianjin Medical University Cancer Institute&Hospital Research Project[No.1805].
文摘Background:The molecular mechanism underlying the involvement of the Transferrin receptor(TFRC)in cervical cancer remains poorly understood.This study aims to elucidate the role of TFRC in cervical cancer by analyzing data from The Cancer Genome Atlas(TCGA)and Genotype-Tissue Expression(GTEx)databases.Methods:TFRC protein expression was obtained from Human Protein Altas(HPA).All datas were collected from TCGA and GTEx.In this study,we analyzed the expression of TFRC in cervical cancer and its clinical significance.Through Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene set enrichment analyses(GSEA),investigated the related molecular pathways of TFRC.The relationship between TFRC and immune infiltration was then examined.The prognosis of different immune cell subsets was then analyzed after dividing cervical cancer patients into high and low expression of TFRC groups.Results:TFRC is highly expressed in various tumor tissues compared to control normal tissues,including cervical cancer.An increased expression of TFRC was associated with higher Tumor(T)and Node(N)stage,as well as a higher clinical stage.Kaplan–Meier(KM)survival analysis investigated that higher TFRC expression patients have a poor overall survival(OS),disease specific survival(DSS)and progress free interval(PFI).Both KEGG and GSEA enriched signaling pathway by high TFRC and low TFRC groups.There was a significant negative linear correlation between TFRC expression and immune infiltration.TFRC affects the prognosis of cervical cancer patients through immune pathway.Conclusions:Cervical cancer patients with TFRC expression may have a worse prognosis.
基金funded by Shanghai Municipal Health Bureau,No.KPB-WSJ1004the National Natural Science Foundation of China,No.81200971
文摘Iron is an essential trophic element that is required for cell viability and differentiation, especially in oligodendrocytes, which consume relatively high rates of energy to produce myelin. Multiple iron metabolism proteins are expressed in the brain including transferrin receptor and ferritin-H. However, it is still unknown whether they are developmentally regulated in oligodendrocyte lineage cells for myelination. Here, using an in vitro cultured differentiation model of oligodendrocytes, we found that both transferrin receptor and ferritin-H are significantly upregulated during oligodendrocyte maturation, implying the essential role of iron in the development of oligodendrocytes. Additional different doses of Fe3＋ in the cultured medium did not affect oligodendrocyte precursor cell maturation or ferritin-H expression but decreased the expression of the transferrin receptor. These results indicate that upregulation of both transferrin receptor and ferritin-H contributes to maturation and myelination of oligodendrocyte precursor cells.
基金Supported by Natural Key and Basic Research Development Program,No.2002CB513109
文摘AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.
基金funded by the 863 Program entitled as"The research and exploration of nutrition fortified food for improving growth and development(2010AA023004)"performed by the Trace Elements Nutrition Key Laboratory of the Ministry of Health
文摘Objective To establish and evaluate a protein serum ferritin （SF） and soluble transferrin receptor microarray method for combined measurement of （sTfR）. Methods Microarrayer was used to print both anti-SF antibodies I and anti-sTfR antibodies I on each protein microarray. Anti-SF antibodies II and anti-sTfR antibodies II were used as detection antibodies and goat antibodies coupled to Cy3 were used as antibodies Ill. The detection conditions of the quantitative analysis method for simultaneous measurement of SF and sTfR with protein microarray were optimized and evaluated. The protein microarray was compared with commercially available traditional tests with 26 serum samples. Results By comparison experiment, mouse monoclonal antibodies were chosen as the probes and contact printing was chosen as the printing method. The concentrations of SF and sTfR probes were 0.5 mg/mL and 0.5 mg/mL respectively, while those of SF and sTfR detection antibodies were 5 μg/mL and 0.36 μg/mL respectively. Intra- and inter-assay variability was between 3.26% and 18.38% for all tests. The regression coefficients comparing protein microarray with traditional test assays were better than 0.81 for SF and sTfR. Conclusion The present study has established a protein microarray method for combined measurement of SF and sTfR.
基金supported by a grant from "863" program of China (No. 2006AA02Z158)the Ministry of Education Science Foundation of China (No. 20060487024)Science and Technology project of Jiangxi Province Education Department (No. 2006-86).
文摘To obtain single chain variable fragment （scFv） and bivalent single chain variable fragment （bsFv） against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy （VH） and FR4 of variable light （VL）, respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.
基金National Natural Science Foundation of China(No.31570937 and No.81871391)Natural Science Foundation of Hubei Province of China(No.2017CFB707)+1 种基金the Fundamental Research Funds for the Central Universities of China(No.HUST:2018KFYYXJJ086)Graduates'Innovation Foundation of Huazhong University of Science and Technology(No.5003510001).
文摘Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies for clinical application,we constructed TfR bispecific T-cell engager(BiTE)in two extensively applied formats,including single-chain tandem singlechain variable fragments(scFvs)and double-chain diabodies,and evaluated their functional characterizations in vitro.Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+HepG2 cells.However,compared to two・chain diabodies,scFvs were more efficient in antigen binding and TfR target killing.Furthermore,different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies.Thus,the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.
基金Zhongshan Municipal Science and Technology Plan Project(No.2017B1061)
文摘Objective: To investigate the effect of high throughput hemodialysis on soluble transferrin receptor in hemodialysis patients and the improvement of renal anemia. Methods: 132 patients receiving maintenance hemodialysis in our hospital from July 2017 to July 2019 were selected and divided into control group and observation group according to the random number table method, with 66 cases each. The observation group was treated with high-flux hemodialysis, while the control group was treated with low-flux hemodialysis for 6 months. Compare two groups before and after treatment serum beta 2 microglobulin (beta 2 - MG), serum creatinine (Scr), blood urea nitrogen (BUN) level, anemia related index [red blood cells deposited (HCT), hemoglobin (Hb), reticulocyte percentage (Ret%)], iron metabolism index [serum ferritin (SF), transferrin saturation (TSAT)、Hepcidin(Hepc)], soluble transferrin receptor (sTfR) levels and adverse reactions. Results: the levels of 2-MG, Scr and BUN in the two groups before treatment were compared (P>0.05). After treatment, Scr and BUN levels in the two groups were significantly decreased (P<0.05), but were compared between the two groups (P>0.05). The level of 2-MG in the observation group was lower than that in the control group (P<0.05). Before treatment, sTfR, Hb, HCT level and Ret% of the two groups were compared(P>0.05). After treatment, Hb and HCT levels in the observation group were higher than those in the control group, while Ret% were lower than those in the control group, (P<0.05). Before treatment, the levels of ST、TAST、sTfR and Hepc in the two groups were compared (P>0.05). After treatment, the level of ST and TAST in the observation group was higher than that in the control group, The levels of sTfR and Hepc were lower than the control group (P<0.05). The overall incidence of adverse reactions in the observation group (8.93%) was lower than that in the control group (10.14%), with no significant difference (P>0.05). Conclusion: The high-throughput hemodialysis department significantly improved renal anemia in hemodialysis patients, reduced serum sTfR level, and had fewer adverse reactions and higher safety.
文摘Hypochromic microcytic anaemia includes iron deficiency, anaemia of chronic disorders, beta thalassemia trait and sideroblastic anaemia. To rule out the cause of hypochromic microcytic anaemia is a diagnostic difficulty. The conventional laboratory tests used for diagnosis have few disadvantages. Serum transferrin receptor (sTfR) is the most reliable method for assessment of body iron. Eighty four children were included in this study. They were further divided into four groups: iron deficiency anaemia (IDA), anaemia of chronic disorders (ACD), beta thalassemia trait (β TT) and controls. Children withIDAand ACD were diagnosed on the basis of history and serum iron profile. Subjects with β TT had HbA2 > 3.5%. sTfR were performed on all subjects. Level of sTfR in patients withIDAwas 5.79 μg/ml ± 1.3 μg/ml. In patients with anaemia of chronic disorders (ACD), β thalassemia trait and controls mean sTfR were 2.18 μg/ml ± 0.6 μg/ml, 2.1μg/ml ± 0.5 μg/ml and 2.0 μg/ml ± 0.5 μg/ml respectively. These results show level of sTfR was raised in IDA when compared with controls or ACD and β TT (p
文摘Hepatitis C virus(HCV) is the main pathogen causing chronic hepatitis and primary liver cancer. Various viral proteins and host cell molecules are involved in the HCV cell entry, but the mechanism of infection has not been completely elucidated. The transferrin receptor can act as a receptor for many viruses during cell entry. The transferrin receptor is not only closely related to HCV-induced iron metabolism disorders but also mediates the fusion of HCV with the host cell membrane as a specific receptor for CD81-dependent viral adhesion.
基金the financial support of National Key R&D Program of China(No.2021YFD1800600)the National Natural Science Foundation of China(No.32371394)+2 种基金the Guangdong Basic and Applied Basic Research Foundation(No.2021B1515020043,China)the Special Projects in Key Areas of Colleges and Universities in Guangdong Province(No.2022ZDZX2046,China)Open Research Foundation of State Key Laboratory of Respiratory Diseases(No.SKLRD-OP-202204,China).
文摘The immunosuppressive phenotype of tumor cells extensively attenuates the immune activation effects of traditional treatments.In this work,a transferrin receptor(TfR)targeted immunostimulant(PTI)is fabricated for photodynamic immunotherapy against metastatic tumors by interrupting β-catenin signal pathway.To synthesize PTI,the photosensitizer conjugated TfR targeting peptide moiety(Palmitic-K(PpIX)-HAIYPRH)is unitized to encapsulate the transcription interrupter of ICG-001.On the one hand,the recognition of PTI and TfR can promote drug delivery into tumor cells to destruct primary tumors through photodynamic therapy and initiate an immunogenic cell death with the release of tumorassociated antigens.On the other hand,PTI will interrupt the binding between b-catenin andcAMP response element-binding protein(CREB),regulating the gene transcription to downregulate programmed death ligand 1(PD-L1)while upregulating CeC motif chemokine ligand 4(CCL4).Furthermore,the elevated CCL4 can recruit the dendritic cells to present tumor-specific antigens and promote T cells activation and infiltration,and the downregulated PD-L1 can avoid the immune evasion of tumor cells and activate systemic anti-tumor immunity to eradicate lung metastasis.This work may inspire the development of antibody antibody-free strategy to activate systemic immune response in consideration of immunosuppressive conditions.
基金supported by National High Technology Research and Development Program of China (2004AA2Z3783)Beijing Municipal Special-purpose Science Foundation of China (Z0004105040311)
文摘The membrane transferrin receptor-mediated endocytosis has been exploited for developing novel targeted drug delivery systems, which could have a variety of applications in the site-specific delivery of anticancer drugs, proteins and therapeutic genes into proliferating malignant cells that overexpress the transferrin receptors. This is achieved by coupling transferrin or monoclonal antibody to transferrin receptor with therapeutic drugs or drug delivery vesicles. The transferrin conjugates can be obtained by use of either bifunctional chemical linkers or by genetic infusion of therapeutic peptides/proteins into the structure of transferrin / and monoclonal antibody to transferrin receptor. A variety of drug carriers such as liposomes, nanoparticles and DNApolymer complexes （i.e. polyplex and lipoplex） were used to efficiently deliver the Wansferrin conjugates. Use of transferrin conjugates results in improvement in drug efficacy, selectivity and drug release as well as reduction in drug toxicity. This paper reviews the basic biochemistry of transferrin and the transferrin receptor as well as the strategy for developing targeted drug delivery system.
基金ThestudywassupportedbyNational"211 Project"inPekingUniversityandtheSpecialFundforPromotionofEducationMinistryofEducation China
文摘IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis. 1 The histopathology of IgAN is characterized by abundance of mesangial matrix and proliferation of mesangial cells. IgA_1 deposition in the mesangium plays an important role in the inflammatory process in this disease.
基金supported by the National Natural Science Foundation of China(81690264)Key Project from the Ministry of Science and Technology(Grant No.2018ZX09721003)Scientific Research Incubation Fund of Beijing Children’s Hospital,Capital Medical University(Grant No.GPY201711,China)
文摘Recently, considerable attention in the field of cancer therapy has been focused on the mammalian rapamycin target(m TOR), inhibition of which could result in autophagic cell death(ACD). Though novel combination chemotherapy of autophagy inducers with chemotherapeutic agents is extensively investigated, nanomedicine-based combination therapy for ACD remains in infancy. In attempt to actively trigger ACD for synergistic chemotherapy, here we incorporated autophagy inducer rapamycin(RAP) into 7 pep-modified PEG-DSPE polymer micelles(7 pep-M-RAP) to specifically target and efficiently priming ACD of MCF-7 human breast cancer cells with high expression of transferrin receptor(Tf R). Cytotoxic paclitaxel(PTX)-loaded micelle(7 pep-M-PTX) was regarded as chemotherapeutic drug model. We discovered that with superior intracellular uptake in vitro and more tumor accumulation of micelles in vivo, 7 pep-M-RAP exhibited excellent autophagy induction and synergistic antitumor efficacy with 7 pep-M-PTX. Mechanism study further revealed that 7 pep-M-RAP and 7 pep-MPTX used in combination provided enhanced efficacy through induction of both apoptosis-and mitochondria-associated autophagic cell death. Together, our findings suggested that the targeted excess autophagy may provide a rational strategy to improve therapeutic outcome of breast cancer, and simultaneous induction of ACD and apoptosis may be a promising anticancer modality.
基金This research was supported in part by a grant from the National Natural Science Foundation of China(Grant No.30271195)a grant from NIH(P20 RR016457 from the BRIN Program of the National Center for Research Resources)+1 种基金a grant for biomedical research from Rhode Island Foundation,a grant from the Committee on Aid to Faculty Research from Providence Collegea grant from Slater Center for Environmental Biotechnology.
文摘The aim of this research was to explore the effects and signaling pathway of ultraviolet-B(UVB)irradiation on the expression of hypoxia-inducible factor 1a(HIF-1α)and transferrin receptor(TfR).HIF-1α protein was measured by Western blot method.Expressions of epidermal growth factor receptor(EGFR),phosphor-EGF-R and TfR after UVB irradiation were determined with flow cytometry.After UVB irradiation,mRNA levels of HIF-1α and TfR were detected by real time-PCR.Results showed that compared with control groups,UVB was able to induce HIF1α and TfR protein expression in a dose-and time-dependent manner in HaCat cells(P<0.05).TfR mRNA was expressed in a dose-dependent manner and reached a peak at the 8th hour in HaCat cells(P<0.05)whereas HIF-1α mRNA expression was not affected by UVB treatment(P>0.05).The EGFR/PI3K/AKT signaling pathway was required for the induction of HIF-1α and TfR expression induced by UVB.UVB induced activation of EGFR in HaCat cells and EGFR regulated expression of TfR and HIF-1α.EGFR(−/−)MEF did not increase the HIF1 expression following UVB irradiation(P>0.05).In contrast,EGFR(+/+)MEF strongly enhanced HIF1a expression after UVB irradiation(P<0.05).PD153035,a selective inhibitor of EGFR tyrosine kinase,inhibited the TfR protein expression in UVB-treated cells in a dose-dependent manner(P<0.05).PI3K inhibitors,LY294002 and wortmannin,inhibited HIF-1a and TfR expressions induced by UVB(P<0.05).The DEC1(−/−)Ha-Cat cells did not increase their TfR and HIF-1α expressions following UVB irradiation(P>0.05).In contrast,DEC1(+/+)HaCat cells strongly enhanced TfR and HIF-1α protein expression after UVB irradiation(P<0.05).We conclude that UVB induces TfR and HIF-1α expressions via EGFR/PI3K/AKT/DEC1 signaling pathway.
基金supported by the National Natural Science Foundation of China,No.81771342 (to HM)。
文摘Previous studies have shown that the receptor tyrosine kinase Eph receptor A4(EphA4) is abundantly expressed in the nervous system. The EphA4 signaling pathway plays an important role in regulating motor neuron ferroptosis in motor neuron disease. To investigate whether EphA4 signaling is involved in ferroptosis in spinal cord ischemia/reperfusion injury, in this study we established a rat model of spinal cord ischemia/reperfusion injury by clamping the left carotid artery and the left subclavian artery. We found that spinal cord ischemia/reperfusion injury increased EphA4 expression in the neurons of anterior horn, markedly worsened ferroptosis-related indicators, substantially increased the number of mitochondria exhibiting features consistent with ferroptosis, promoted deterioration of motor nerve function, increased the permeability of the blood-spinal cord barrier, and increased the rate of motor neuron death. Inhibition of EphA4 largely rescued these effects. However, intrathecal administration of the ferroptosis inducer Erastin counteracted the beneficial effects conferred by treatment with the EphA4 inhibitor. Mass spectrometry and a PubMed search were performed to identify proteins that interact with EphA4, with the most notable being Beclin1 and Erk1/2. Our results showed that inhibition of EphA4 expression reduced binding to Beclin1, markedly reduced p-Beclin1, and reduced Beclin1-XCT complex formation. Inhibition of EphA4 also reduced binding to p-Erk1/2 and markedly decreased the expression of c-Myc, transferrin receptor 1, and p-Erk1/2. Additionally, we observed co-localization of EphA4 and p-Beclin1 and of EphA4 and p-ERK1/2 in neurons in the anterior horn. In conclusion, EphA4 participates in regulating ferroptosis of spinal motor neurons in the anterior horn in spinal cord ischemia/reperfusion injury by promoting formation of the Beclin1-XCT complex and activating the Erk1/2/c-Myc/transferrin receptor 1 axis.
基金supported by 2012 Kunshan Instruction Subject(KS1254)
文摘Objective: To explore the expression of transferrin(Tf) and transferrin receptor(Tf R) in hematoma brain tissue at different stage after intracerebral hemorrhage(ICH) in rats. Methods: ICH rats model were established by collagenase method, and rats were sacrificed at 24 h, 72 h, 7 d and 14 d after operation. The levels of Tf and Tf R in different periods of rats were detected by immunohistochemical method, and correlation between two groups was analyzed. Results: Tf, Tf R-positive cells at each time after operation in observation group were significantly higher than that in control group(P<0.05). Tf, Tf R-positive cells began to increase from 24 h after the operation and reached the peak 72 h-7 d after surgery, but then gradually decreased. Tf was mainly expressed in nucleus and cytoplasm of neurons and glial cells around the hematoma, but Tf R was mainly expressed in nucleus and cytoplasm of neurons and choroid plexus endothelial cells. Correlation analysis showed that the Tf-positive cell was significantly positively correlated with Tf R-positive cell expression(r=0.447, P=0.022). Conclusions: Tf and Tf R were important transporters in brain tissue excessive load iron transport after ICH, and detecting the expression levels of the two indicators can provide a reference for prognosis treatmentin ICH.
