Objective: Application of magnetic nanoparticles as gene carrier in gene therapy has developed quickly. This study was designed to investigate the preparation of superparamagnetic dextran-coated iron oxide nanoparticl...Objective: Application of magnetic nanoparticles as gene carrier in gene therapy has developed quickly. This study was designed to investigate the preparation of superparamagnetic dextran-coated iron oxide nanoparticles (SDION) and the feasibility of SDION used as a novel gene carrier for plasmid DNA in vitro. Methods: SDION were prepared by chemical coprecipitation and separated by gel filtration on Sephacryl S-300HR, characterized by TEM, laser scattering system and Vibrating Sample Magnetometer Signal Processor. The green fluorescent protein (pGFP-C2) plasmid DNA was used as target gene. SDION-pGFP-C2 conjugate compounds were produced by means of oxidoreduction reaction. The connection ratio of SDION and pGFP-C2 DNA was analyzed and evaluated by agarose electrophoresis and the concentration of pGFP-C2 in supernatant was measured. Using liposome as control, the transfection efficiency of SDION and liposome was respectively evaluated under fluorescence microscope in vitro. Results: The diameter of SDION ranges from 3 nm to 8 nm, the effective diameter was 59.2 nm and the saturation magnetization was 0.23 emu/g. After SDION were reasonably oxidized, SDION could connect with pGFP-C2 to a high degree. The transfection efficiency of SDION as gene carrier was higher than that of liposome. Conclusion: The successes in connecting SDION with pGFP-C2 plasmid by means of oxidoreduction reaction and in transferring pGFP-C2 gene into human bladder cancer BIU-87 cells in vitro provided the experimental evidence for the feasibility of SDION used as a novel gene carrier.展开更多
The development of nanotechnology provides a new method for genetic engineering.However,the nanoparticles as gene carriers have been mainly used in the mammalian cells so far.We observed that ZnS nanoparticles modifie...The development of nanotechnology provides a new method for genetic engineering.However,the nanoparticles as gene carriers have been mainly used in the mammalian cells so far.We observed that ZnS nanoparticles modified with positively charged poly-L-lysine（PLL） successfully delivered GUS-encoding plasmid DNA into tobacco cells by means of ultrasound-assisted method.Polymerase chain reaction（PCR） detection,Southern blot analysis and GUS histochemical staining were carried out for the regenerated plants.The stable genetic modified plants mediated by ZnS nanoparticles can be obtained.This article demonstrates the great potential of nanoparticles as gene carrier in plant transformation and proves a novel approach for plant genetic decoration.展开更多
To investigate the enhancement of immunological activity of CpG ODN by chitosan gene carrier in mice, the effect of lymphocyte proliferation was detected in mice by using MTT, the levels of IgG and cytokines （IL-2 an...To investigate the enhancement of immunological activity of CpG ODN by chitosan gene carrier in mice, the effect of lymphocyte proliferation was detected in mice by using MTT, the levels of IgG and cytokines （IL-2 and IL-12） in serum were measured by ELISA and peripheral blood T lymphocyte subsets CD4^＋, CD8^＋ were analyzed by flow cytometry. Our results showed that spleen lymphocytes isolated from the CS-CpG ODN group of mice showed the strongest proliferation （SI =1.551）, and the levels of IgG, IL-2 and IL-12 in serum were higher than those of other groups. Compared with the immunization with CpG ODN, the immunization with CS-CpG ODN gene carrier was more efficient in up-regulating the percentage of CD4^＋T cells and the ratio of CD4^＋/CD8^＋ of mice, It was concluded that CS gene carrier of CpG ODN was much more effective in improving immunity of CpG ODN in mice.展开更多
In recent years,various carriers for gene delivery nave been developed for biomedical applications.Among all kinds of gene carriers,cationic polymeric carriers for delivery therapeutic gene as non-viral carriers have ...In recent years,various carriers for gene delivery nave been developed for biomedical applications.Among all kinds of gene carriers,cationic polymeric carriers for delivery therapeutic gene as non-viral carriers have received growing interests due to their improved high transfection efficiency with the relative safety.In particular,the advancement of novel polymeric gene carriers has gained much progress in the development of effective anticancer therapy.Herein,this review focused on the development of cationic polymeric carriers for cancer therapy,including polyethylenimine(PEI),polyamidoamine(PAMAM) dendrimers,polylysine(PLL),chitosan and modified cationic polymers.And recent progresses in the development of novel polymeric carriers for gene delivery,such as targeted gene carriers,responsive gene carriers and multifunctional gene carriers,were summarized.Finally,the future perspectives in the development of novel polymeric carriers for delivery gene were presented.展开更多
Hydroxyapatite(HA)is a representative substance that induces bone regeneration.Our research team extracted nanohydroxyapatite(EH)from natural resources,especially equine bones,and developed it as a molecular biologica...Hydroxyapatite(HA)is a representative substance that induces bone regeneration.Our research team extracted nanohydroxyapatite(EH)from natural resources,especially equine bones,and developed it as a molecular biological tool.Polyethylenimine(PEI)was used to coat the EH to develop a gene carrier.To verify that PEI is well coated in the EH,we first observed the morphology and dispersity of PEI-coated EH(pEH)by electron microscopy.The pEH particles were well distributed,while only the EH particles were not distributed and aggregated.Then,the existence of nitrogen elements of PEI on the surface of the pEH was confirmed by EDS,calcium concentration measurement and fourier transform infrared spectroscopy(FT-IR).Additionally,the pEH was confirmed to have a more positive charge than the 25 kD PEI by comparing the zeta potentials.As a result of pGL3 transfection,pEH was better able to transport genes to cells than 25 kD PEI.After verification as a gene carrier for pEH,we induced osteogenic differentiation of DPSCs by loading the BMP-2 gene in pEH(BMP-2/pEH)and delivering it to the cells.As a result,it was confirmed that osteogenic differentiation was promoted by showing that the expression of osteopontin(OPN),osteocalcin(OCN),and runt-related transcription factor 2(RUNX2)was significantly increased in the group treated with BMP-2/pEH.In conclusion,we have not only developed a novel nonviral gene carrier that is better performing and less toxic than 25 kD PEI by modifying natural HA(the agricultural byproduct)but also proved that bone differentiation can be effectively promoted by delivering BMP-2 with pEH to stem cells.展开更多
Cationic polysaccharides have been receiving more attentions and used as nonviral gene delivery vectors. In this paper, quaternized hydroxyethylcellulose （QHEC） derivatives were studied as gene carriers for their ef...Cationic polysaccharides have been receiving more attentions and used as nonviral gene delivery vectors. In this paper, quaternized hydroxyethylcellulose （QHEC） derivatives were studied as gene carriers for their efficient DNA binding abilities. All QHECs could form stable QHEC/DNA complexes and resist the degradation of DNase I. And the dynamic light scatter （DLS） results showed that all QHEC/DNA complexes could form compact particles. These QHEC/DNA complexes exhibited effective transfeetion abilities in comparison to the naked DNA. The cytotox- icities of QHEC and QHEC/DNA complexes were also evaluated in four cell lines which were relatively low com- pared with 25 kDa bPEI. All results indicated that these quaternized hydroxyethylcelluloses could be used as poten- tial gene delivery vectors.展开更多
Anion starch nanoparticle (StNP) with a diameter of 50 nm was prepared in wa- ter-in-oil microemulsion, with soluble starch as raw materials and POCl3 as crosslinking agent. PLL-StNP was prepared by linking poly-L-lys...Anion starch nanoparticle (StNP) with a diameter of 50 nm was prepared in wa- ter-in-oil microemulsion, with soluble starch as raw materials and POCl3 as crosslinking agent. PLL-StNP was prepared by linking poly-L-lysine (PLL) on the surface of StNP. At the same time, the size of PLL-StNP and its stability in aqueous solution were checked by AFM. The analysis of plasmid DNA binding, DNase I enzymatic degradation, toxicity and transfection were done. We discovered that PLL-StNP may be used as non-virus nanoparticle gene carrier. And we devel- oped the method of preparing PLL-StNP gene carrier and used it in cell transfection. As non-virus gene carrier, PLL-StNP has some advantages, such as large load of DNA, high transfection effi- ciency, low cell toxicity and biodegradability.展开更多
Uniform-sized amino-modified silica nanopar-ticles have been prepared by the controlled synchronous hydrolysis of tetraethoxysilane and N-(?amimoethyl)-- aminopropyltriethoxysilane in water nanodroplet of the wa-ter-i...Uniform-sized amino-modified silica nanopar-ticles have been prepared by the controlled synchronous hydrolysis of tetraethoxysilane and N-(?amimoethyl)-- aminopropyltriethoxysilane in water nanodroplet of the wa-ter-in-oil microemulsion. These nanoparticles display posi-tive charge potential at definited pH. This is due to the pres-ence of amino groups on the surface of the nanoparticles. Nanoparticles-plasmid DNA complexes can easily form through electrostatical binding between the positive charges of the amino-modified silica nanoparticles and the negative charges of the plasmid DNA. The complexes can be also dis-sociated under alkaline pH or high ionic strength conditions. And enzymatic digestion of the plasmid DNA is almost in-hibited by these nanoparticles complexes. A novel non-viral gene carrier based on the amino-modified silica nanoparti-cles is proposed under the combination of nanotechnology, biotechnology and gene engineering technology. The plasmid DNA can successfully cross various systemic barriers to COS-7 cells as well as mediate high expression of Green Fluorescence Protein (GFP) gene in cells by use of this novel gene carrier.展开更多
Low molecular weight polyethylenimine-poly(gamma-benzyl L-glutamate) (PEI-PBLG) was crosslinked by N,N'-cystaminebisacrylamide (CBA) to get the polymer named as CBA-PEI-PBLG (CPP).CPP not only inherits PEI-PBLG...Low molecular weight polyethylenimine-poly(gamma-benzyl L-glutamate) (PEI-PBLG) was crosslinked by N,N'-cystaminebisacrylamide (CBA) to get the polymer named as CBA-PEI-PBLG (CPP).CPP not only inherits PEI-PBLG's amphiphilic advantages,but also possesses reducible properties.CPP can complex with DNA to form nanoparticles.CPP/DNA complex particles were characterized by particle size and zeta potential analysis.The result showed that the complex particles have suitable size and surface charges for gene delivery.And gel retardation assay also prove CBA-PEI-PBLG has proper condensing ability for DNA.CPP has good reducible property,and also has good biocompatibility because of introducing PBLG segment.The cytotoxicity of CPP was evaluated using MTT assay,and the results showed CPP has lower cytotoxicity compared with PEI 25 K.The transfection properties were characterized in different cells by using plasmid DNA as a reporter.CPP showed higher transfection efficiencies and lower cytotoxicity in HeLa cells.This was attributed to bioreducible and biocompatibility properties of the CPP.These results suggested that CPP is a promising low-toxic,highly effective non-viral gene carrier.展开更多
This study is performed to investigate the mitochondrial carrier gene family in silkworm genome. In total, 30 genes are identified and claded into eight well-conserved groups. Gene duplication contributes to the expan...This study is performed to investigate the mitochondrial carrier gene family in silkworm genome. In total, 30 genes are identified and claded into eight well-conserved groups. Gene duplication contributes to the expansion and complexity of this family. Diverse expression patterns suggest their functional differentiation. Analyses of the sitespecific profiles reveal critical amino acid residues for functional divergence. This study highlights the molecular evolution of the mitochondrial carrier gene family in silkworm and may provide a starting point for further experimental verification.展开更多
Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liv...Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.展开更多
Objective The study is to identify the carrier rate of common deafness mutation in Chinese pregnant women via detecting deafness gene mutations with gene chip. Methods The pregnant women in obstetric clinic without he...Objective The study is to identify the carrier rate of common deafness mutation in Chinese pregnant women via detecting deafness gene mutations with gene chip. Methods The pregnant women in obstetric clinic without hearing impairment and hearing disorders family history were selected. The informed consent was signed. Peripheral blood was taken to extract genom- ic DNA. Application of genetic deafness gene chip for detecting 9 mutational hot spot of the most common 4 Chinese deafness genes, namely GJB2 （35delG, 176del16bp, 235delC, 299delAT）, GJB3 （C538T） ,SLC26A4 （ IVS72A〉G, A2168G） and mito- chondrial DNA 12S rRNA （A1555G, C1494T） . Further genetic testing were provided to the spouses and newborns of the screened carriers. Results Peripheral blood of 430 pregnant women were detected, detection of deafness gene mutation carri- ers in 24 cases（4.2%）, including 13 cases of the GJB2 heterozygous mutation, 3 cases of SLC26A4 heterozygous mutation, 1 cases of GJB3 heterozygous mutation, and 1 case of mitochondrial 12S rRNA mutation. 18 spouses and 17 newborns took further genetic tests, and 6 newborns inherited the mutation from their mother. Conclusion The common deafness genes muta- tion has a high carrier rate in pregnant women group, 235delC and IVS7-2A〉G heterozygous mutations are common.展开更多
Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase （SAD） is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of ...Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase （SAD） is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame （ORF）. Analysis in the BLAST on NCBI shows that Jatropha curcas SAD （JSAD） gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD （RSAD） is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.展开更多
Polyethyleneimine(PEI),as a widely used polymer material in the field of gene delivery,has been extensively studied for modification and shielding to reduce its cytotoxicity.However,research aimed at preparing degrada...Polyethyleneimine(PEI),as a widely used polymer material in the field of gene delivery,has been extensively studied for modification and shielding to reduce its cytotoxicity.However,research aimed at preparing degradable PEI is scarce.In this work,the hydrogen peroxide(H_(2)O_(2))oxidation method was used to introduce degradable amide groups in the PEI and a series of oxidized PEI22k(oxPEI22k)with different degrees of oxidation were synthesized by regulating the dosage of H_(2)O_(2).The relationship between the oxidation degree of oxPEI22k and the gene transfection efficiency of oxPEI22k was studied in detail,confirming that the oxPEI22k with oxidation degrees of 16.7%and 28.6%achieved improved transfection efficiency compared to unmodified PEI.These oxPEI22k also proved reduced cytotoxicity and improved degradability.Further,this strategy was extended to the synthesis of low-molecular-weight oxPEI1.8k.The oxPEI1.8k with suitable oxidation degree also achieved improved transfection efficiency and reduced cytotoxicity.In brief,this work provided high-efficiency and low-cytotoxicity degradable gene delivery carriers by regulating the oxidation degree of PEI,which was of great significance for promoting clinical applications of PEI.展开更多
基金This project was supported by a grant from the National Natural Science Foundation of China (No. 30271300).
文摘Objective: Application of magnetic nanoparticles as gene carrier in gene therapy has developed quickly. This study was designed to investigate the preparation of superparamagnetic dextran-coated iron oxide nanoparticles (SDION) and the feasibility of SDION used as a novel gene carrier for plasmid DNA in vitro. Methods: SDION were prepared by chemical coprecipitation and separated by gel filtration on Sephacryl S-300HR, characterized by TEM, laser scattering system and Vibrating Sample Magnetometer Signal Processor. The green fluorescent protein (pGFP-C2) plasmid DNA was used as target gene. SDION-pGFP-C2 conjugate compounds were produced by means of oxidoreduction reaction. The connection ratio of SDION and pGFP-C2 DNA was analyzed and evaluated by agarose electrophoresis and the concentration of pGFP-C2 in supernatant was measured. Using liposome as control, the transfection efficiency of SDION and liposome was respectively evaluated under fluorescence microscope in vitro. Results: The diameter of SDION ranges from 3 nm to 8 nm, the effective diameter was 59.2 nm and the saturation magnetization was 0.23 emu/g. After SDION were reasonably oxidized, SDION could connect with pGFP-C2 to a high degree. The transfection efficiency of SDION as gene carrier was higher than that of liposome. Conclusion: The successes in connecting SDION with pGFP-C2 plasmid by means of oxidoreduction reaction and in transferring pGFP-C2 gene into human bladder cancer BIU-87 cells in vitro provided the experimental evidence for the feasibility of SDION used as a novel gene carrier.
基金Supported by the National Natural Science Foundation of China(No.21074019)the China Postdoctoral Science Foundation Funded Project(No.200904501024)+1 种基金the Natural Science Foundation of Jilin Province,China(No.20101539)the Jilin Province Science and Technology Development Project,China(No.20090155)
文摘The development of nanotechnology provides a new method for genetic engineering.However,the nanoparticles as gene carriers have been mainly used in the mammalian cells so far.We observed that ZnS nanoparticles modified with positively charged poly-L-lysine（PLL） successfully delivered GUS-encoding plasmid DNA into tobacco cells by means of ultrasound-assisted method.Polymerase chain reaction（PCR） detection,Southern blot analysis and GUS histochemical staining were carried out for the regenerated plants.The stable genetic modified plants mediated by ZnS nanoparticles can be obtained.This article demonstrates the great potential of nanoparticles as gene carrier in plant transformation and proves a novel approach for plant genetic decoration.
基金This work was supported by a grant from Natural Science Foundation of Hubei province (No.2006ABA134)National Natural Sciences Foundation (No.30170051)
文摘To investigate the enhancement of immunological activity of CpG ODN by chitosan gene carrier in mice, the effect of lymphocyte proliferation was detected in mice by using MTT, the levels of IgG and cytokines （IL-2 and IL-12） in serum were measured by ELISA and peripheral blood T lymphocyte subsets CD4^＋, CD8^＋ were analyzed by flow cytometry. Our results showed that spleen lymphocytes isolated from the CS-CpG ODN group of mice showed the strongest proliferation （SI =1.551）, and the levels of IgG, IL-2 and IL-12 in serum were higher than those of other groups. Compared with the immunization with CpG ODN, the immunization with CS-CpG ODN gene carrier was more efficient in up-regulating the percentage of CD4^＋T cells and the ratio of CD4^＋/CD8^＋ of mice, It was concluded that CS gene carrier of CpG ODN was much more effective in improving immunity of CpG ODN in mice.
基金supported by the National Natural Science Foundation of China(51503200,21474104,51233004,51520105004, 51390484)Jilin Province Science and Technology Development Program (20160204032GX)the National Program for Support of Top-notch Young Professionals
文摘In recent years,various carriers for gene delivery nave been developed for biomedical applications.Among all kinds of gene carriers,cationic polymeric carriers for delivery therapeutic gene as non-viral carriers have received growing interests due to their improved high transfection efficiency with the relative safety.In particular,the advancement of novel polymeric gene carriers has gained much progress in the development of effective anticancer therapy.Herein,this review focused on the development of cationic polymeric carriers for cancer therapy,including polyethylenimine(PEI),polyamidoamine(PAMAM) dendrimers,polylysine(PLL),chitosan and modified cationic polymers.And recent progresses in the development of novel polymeric carriers for gene delivery,such as targeted gene carriers,responsive gene carriers and multifunctional gene carriers,were summarized.Finally,the future perspectives in the development of novel polymeric carriers for delivery gene were presented.
基金This study was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science,ICT&Future Planning(NRF-2020R1F1A1067439,NRF-2020R1I1A1A01068262)the Korea Institute of Energy Technology Evaluation and Planning(KETEP)and the Ministry of Trade,Industry&Energy(MOTIE)of the Republic of Korea(20194210100230).
文摘Hydroxyapatite(HA)is a representative substance that induces bone regeneration.Our research team extracted nanohydroxyapatite(EH)from natural resources,especially equine bones,and developed it as a molecular biological tool.Polyethylenimine(PEI)was used to coat the EH to develop a gene carrier.To verify that PEI is well coated in the EH,we first observed the morphology and dispersity of PEI-coated EH(pEH)by electron microscopy.The pEH particles were well distributed,while only the EH particles were not distributed and aggregated.Then,the existence of nitrogen elements of PEI on the surface of the pEH was confirmed by EDS,calcium concentration measurement and fourier transform infrared spectroscopy(FT-IR).Additionally,the pEH was confirmed to have a more positive charge than the 25 kD PEI by comparing the zeta potentials.As a result of pGL3 transfection,pEH was better able to transport genes to cells than 25 kD PEI.After verification as a gene carrier for pEH,we induced osteogenic differentiation of DPSCs by loading the BMP-2 gene in pEH(BMP-2/pEH)and delivering it to the cells.As a result,it was confirmed that osteogenic differentiation was promoted by showing that the expression of osteopontin(OPN),osteocalcin(OCN),and runt-related transcription factor 2(RUNX2)was significantly increased in the group treated with BMP-2/pEH.In conclusion,we have not only developed a novel nonviral gene carrier that is better performing and less toxic than 25 kD PEI by modifying natural HA(the agricultural byproduct)but also proved that bone differentiation can be effectively promoted by delivering BMP-2 with pEH to stem cells.
文摘Cationic polysaccharides have been receiving more attentions and used as nonviral gene delivery vectors. In this paper, quaternized hydroxyethylcellulose （QHEC） derivatives were studied as gene carriers for their efficient DNA binding abilities. All QHECs could form stable QHEC/DNA complexes and resist the degradation of DNase I. And the dynamic light scatter （DLS） results showed that all QHEC/DNA complexes could form compact particles. These QHEC/DNA complexes exhibited effective transfeetion abilities in comparison to the naked DNA. The cytotox- icities of QHEC and QHEC/DNA complexes were also evaluated in four cell lines which were relatively low com- pared with 25 kDa bPEI. All results indicated that these quaternized hydroxyethylcelluloses could be used as poten- tial gene delivery vectors.
文摘Anion starch nanoparticle (StNP) with a diameter of 50 nm was prepared in wa- ter-in-oil microemulsion, with soluble starch as raw materials and POCl3 as crosslinking agent. PLL-StNP was prepared by linking poly-L-lysine (PLL) on the surface of StNP. At the same time, the size of PLL-StNP and its stability in aqueous solution were checked by AFM. The analysis of plasmid DNA binding, DNase I enzymatic degradation, toxicity and transfection were done. We discovered that PLL-StNP may be used as non-virus nanoparticle gene carrier. And we devel- oped the method of preparing PLL-StNP gene carrier and used it in cell transfection. As non-virus gene carrier, PLL-StNP has some advantages, such as large load of DNA, high transfection effi- ciency, low cell toxicity and biodegradability.
基金supported by the Pre-Key Project of Basic Research of Ministry of Science and Technology of the People’s Republic of China(Grant No.2001-51)the Key Project of the National Natural Science Foundation of China(Grant No.20135010)+4 种基金the National Outstanding Youth Foundation of China(Grant No.29825110)the Key Project Foundation of the Education Ministry of China(Grant No.2000-156)the Leading Teacher Foundation of the Education Ministry of China(Grant No,2000-65)the Oversea Youth Scholar Co-research Foundation of China(Grant No.20028506)the Natural Science Foundation of Hunan Province(Grant Nos.00GKY1011 and 01JJY2012)
文摘Uniform-sized amino-modified silica nanopar-ticles have been prepared by the controlled synchronous hydrolysis of tetraethoxysilane and N-(?amimoethyl)-- aminopropyltriethoxysilane in water nanodroplet of the wa-ter-in-oil microemulsion. These nanoparticles display posi-tive charge potential at definited pH. This is due to the pres-ence of amino groups on the surface of the nanoparticles. Nanoparticles-plasmid DNA complexes can easily form through electrostatical binding between the positive charges of the amino-modified silica nanoparticles and the negative charges of the plasmid DNA. The complexes can be also dis-sociated under alkaline pH or high ionic strength conditions. And enzymatic digestion of the plasmid DNA is almost in-hibited by these nanoparticles complexes. A novel non-viral gene carrier based on the amino-modified silica nanoparti-cles is proposed under the combination of nanotechnology, biotechnology and gene engineering technology. The plasmid DNA can successfully cross various systemic barriers to COS-7 cells as well as mediate high expression of Green Fluorescence Protein (GFP) gene in cells by use of this novel gene carrier.
基金the National Natural Science Foundation of China(50873102,50903009 and Key Program:50733003)the National Natural Science Foundation of China - A3 Foresight Program(20621140369)the Program of Scientific Development of Jilin Province(20090128,20096018)for financial support to this work
文摘Low molecular weight polyethylenimine-poly(gamma-benzyl L-glutamate) (PEI-PBLG) was crosslinked by N,N'-cystaminebisacrylamide (CBA) to get the polymer named as CBA-PEI-PBLG (CPP).CPP not only inherits PEI-PBLG's amphiphilic advantages,but also possesses reducible properties.CPP can complex with DNA to form nanoparticles.CPP/DNA complex particles were characterized by particle size and zeta potential analysis.The result showed that the complex particles have suitable size and surface charges for gene delivery.And gel retardation assay also prove CBA-PEI-PBLG has proper condensing ability for DNA.CPP has good reducible property,and also has good biocompatibility because of introducing PBLG segment.The cytotoxicity of CPP was evaluated using MTT assay,and the results showed CPP has lower cytotoxicity compared with PEI 25 K.The transfection properties were characterized in different cells by using plasmid DNA as a reporter.CPP showed higher transfection efficiencies and lower cytotoxicity in HeLa cells.This was attributed to bioreducible and biocompatibility properties of the CPP.These results suggested that CPP is a promising low-toxic,highly effective non-viral gene carrier.
基金supported by the Jiangsu University Senior Personnel Research Grants, China (10JDG027)
文摘This study is performed to investigate the mitochondrial carrier gene family in silkworm genome. In total, 30 genes are identified and claded into eight well-conserved groups. Gene duplication contributes to the expansion and complexity of this family. Diverse expression patterns suggest their functional differentiation. Analyses of the sitespecific profiles reveal critical amino acid residues for functional divergence. This study highlights the molecular evolution of the mitochondrial carrier gene family in silkworm and may provide a starting point for further experimental verification.
基金supported by Wuhan Municipal Science and Technology Bureau of applied basic research project(No.2013062301010823)Wuhan City health planning medieal research project(No.WX14A11)
文摘Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.
文摘Objective The study is to identify the carrier rate of common deafness mutation in Chinese pregnant women via detecting deafness gene mutations with gene chip. Methods The pregnant women in obstetric clinic without hearing impairment and hearing disorders family history were selected. The informed consent was signed. Peripheral blood was taken to extract genom- ic DNA. Application of genetic deafness gene chip for detecting 9 mutational hot spot of the most common 4 Chinese deafness genes, namely GJB2 （35delG, 176del16bp, 235delC, 299delAT）, GJB3 （C538T） ,SLC26A4 （ IVS72A〉G, A2168G） and mito- chondrial DNA 12S rRNA （A1555G, C1494T） . Further genetic testing were provided to the spouses and newborns of the screened carriers. Results Peripheral blood of 430 pregnant women were detected, detection of deafness gene mutation carri- ers in 24 cases（4.2%）, including 13 cases of the GJB2 heterozygous mutation, 3 cases of SLC26A4 heterozygous mutation, 1 cases of GJB3 heterozygous mutation, and 1 case of mitochondrial 12S rRNA mutation. 18 spouses and 17 newborns took further genetic tests, and 6 newborns inherited the mutation from their mother. Conclusion The common deafness genes muta- tion has a high carrier rate in pregnant women group, 235delC and IVS7-2A〉G heterozygous mutations are common.
基金Project supported by"Tenth Five Years"Key Program of the State Science and Technology Commission in China(Grant Nos.2002BA901A15,2004BA411B01)
文摘Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase （SAD） is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame （ORF）. Analysis in the BLAST on NCBI shows that Jatropha curcas SAD （JSAD） gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD （RSAD） is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.
基金financially supported by National Key Research and Development Program of China(No.2021YFB3800900)the National Natural Science Foundation of China(Nos.51925305,51833010 and 52203183)+2 种基金Natural Science Foundation of Xiamen,China(No.3502Z202371004)Fundamental Research Funds for the Central Universities(No.20720230004)the talent cultivation project Funds for the Innovation Laboratory for Sciences and Technologies of Energy Materials of Fujian Province(No.HRTP-[2022]52)。
文摘Polyethyleneimine(PEI),as a widely used polymer material in the field of gene delivery,has been extensively studied for modification and shielding to reduce its cytotoxicity.However,research aimed at preparing degradable PEI is scarce.In this work,the hydrogen peroxide(H_(2)O_(2))oxidation method was used to introduce degradable amide groups in the PEI and a series of oxidized PEI22k(oxPEI22k)with different degrees of oxidation were synthesized by regulating the dosage of H_(2)O_(2).The relationship between the oxidation degree of oxPEI22k and the gene transfection efficiency of oxPEI22k was studied in detail,confirming that the oxPEI22k with oxidation degrees of 16.7%and 28.6%achieved improved transfection efficiency compared to unmodified PEI.These oxPEI22k also proved reduced cytotoxicity and improved degradability.Further,this strategy was extended to the synthesis of low-molecular-weight oxPEI1.8k.The oxPEI1.8k with suitable oxidation degree also achieved improved transfection efficiency and reduced cytotoxicity.In brief,this work provided high-efficiency and low-cytotoxicity degradable gene delivery carriers by regulating the oxidation degree of PEI,which was of great significance for promoting clinical applications of PEI.
