AIM: To investigate the cytotoxic activity of extracts of trichosanthes root tubers (EOT) on HepA-H cells and HeLa cells compared with trichosanthin (TCS), and to explore the possible mechanism of growth inhibitory ef...AIM: To investigate the cytotoxic activity of extracts of trichosanthes root tubers (EOT) on HepA-H cells and HeLa cells compared with trichosanthin (TCS), and to explore the possible mechanism of growth inhibitory effect of EOT on HeLa cells.METHODS: Tumor cells were cultured in vitro, and then microculture tetrzoalium assay (MTT) was used to investigate drugs' cytotoxic activity. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe ultrastructural changes of cells, and electrophoresis was performed to detect changes of biochemical characteristics of intercellular DNA.RESULTS: TCS and EOT had no obvious effects on HepA-H cells (P＞0.05), but had remarkable effects on HeLa cells in a time and dose dependent manner (r＞0.864,P＜0.05 or P＜0.01). The inhibitory rate of EOT was much higher than that of TCS (P＜0.01). Median inhibitory rates (IC50) of TCS and EOT on HeLa cells were 610.9 mg/L and 115.6 mg/L for 36 h, and 130.7 mg/L and 33.4 mg/L for 48 h respectively. Marked morphologic changes were observed including microvillus disappearance or reduction, cell membrane bledding, cell shrinkage, condensation of chromosomes and apoptotic bodies with complete membranes. Meanwhile, apoptosis of HeLa cells was confirmed by DNA ladder formation on gel electrophoresis. CONCLUSION: TCS and EOT have no obvious effects on HepA-H cells, but have significant inhibitory effects on HeLa cells, indicating that EOT is superior to TCS in anti-tumor activity.展开更多
Recent studies indicate that cell-cycle checkpoints are tightly correlated with the regulation of apoptosis, in which p53 plays an important role. Our present works show that the expression of E6/E7 oncogenes of human...Recent studies indicate that cell-cycle checkpoints are tightly correlated with the regulation of apoptosis, in which p53 plays an important role. Our present works show that the expression of E6/E7 oncogenes of human papillomavirus in HeLa cells is inhibited in the presence of anti-tumor reagent tripchlorolide (TC), which results in the up-regulation of p53 in HeLa cells. Interestingly, under the same TC-treatment, the cells at the early S-phase are more susceptible to apoptosis than those at the middle S-phase although p53 protein is stabilized to the same level in both situations. Significant difference is exhibited between the two specified expression profiles. Further analysis demonstrates that anti-apoptotic gene survivin is up-regulated by p53 in the TC-treated middle-S cells, whereas it is down-regulated by p53 in the TC-treated early-S cells. Taken together, the present study indicates that the differential p53-regulated expres- sion of survivin at different stages of the cell cycle results in different cellular outputs under the same apoptosis-inducer.展开更多
AIM: To investigate the inhibitory effect of Chinese herbal medicine on the transcription of hepatitis C virus (HCV) structural gene in Hela D cells.METHODS: Hela cell line was transfected with recombinant pBK-CMV-HCV...AIM: To investigate the inhibitory effect of Chinese herbal medicine on the transcription of hepatitis C virus (HCV) structural gene in Hela D cells.METHODS: Hela cell line was transfected with recombinant pBK-CMV-HCV containing HCV structural gene by Lipofectamine. RT-nested-PCR and Western blot assay were used to testify the HCV gene expression in Hela cells. The Hela cells expressing HCV structural protein were named Hela D cells. Prescriptions of Xiao chaihu Decoction (XCHD),Fufang Huangqi (FFHQ) and Bingganling (BGL) wererespectively added to Hela D cells in various concentrations. Semi-quantitative RT-nested-PCR product analysis was performed according to the fluorescent density between HCV DNA band and GAPDH DNA band in gel electrophoresisafter screened. RESULTS: Recombinant pBK-CMV-HCV could correctly express the HCV structural gene in Hela D cells. After coculture of Hela D cells with three prescriptional different concentrations for 48 h respectively, the transcription of HCVgene decreased with increasing of the concentration of each prescription. The lightness ratio of HCV product bands to GAPDH product bands was 0.24, 0.10 and 0.12 in Hela D cells incubated with 0.1 g/mL of XCHD, FFHQand BGL respectively and the lightness ratio HCV product bands to GAPDH product bands was 0.75, 0.67 and 0.61respectively in the control cells. CONCLUSION: The prescriptions of XCHD, FFHQ and BGL partly inhibit the transcription of HCV structural gene inHela D cells.展开更多
Objective： To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosomajaponicum,（SjAslp） and transfer it into mammalian cells to express the objecti...Objective： To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosomajaponicum,（SjAslp） and transfer it into mammalian cells to express the objective protein. Methods： By polymerase chain reaction （PCR） technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK＋/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1（＋）. After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Results： The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion： SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.展开更多
OBJECTIVE To study the mechanism of apoptosis induced by cisplatin in vitro in HeLa cells cervical cancer cell line. METHODS The inhibitory effect of cisplatin on HeLa cell growth was analyzed by the MTT assay. Cell a...OBJECTIVE To study the mechanism of apoptosis induced by cisplatin in vitro in HeLa cells cervical cancer cell line. METHODS The inhibitory effect of cisplatin on HeLa cell growth was analyzed by the MTT assay. Cell apoptosis was measured with flow cytometry and Hoechst 33258 staining following cisplatin treatment. The effect of cisplatin on transcription of HPV E6 was analyzed by RT-PCR and protein expression of E6, P53, p21, Bax and Bcl-2 was studied by Western blots. RESULTS Cisplatin inhibited cellular proliferation in a time and dose- dependant manner. The sub-G1 peak by flow cytometry showed a higher apoptotic rate in the experimental group compared to the controls and Hoechst 33258 staining indicated that apoptosis was induced by cisplatin. Results of RT-PCR demonstrated that cisplatin decreased tran- scription of E6. Western Blots showed that cisplatin decreased protein expression of E6 and increased protein expression of P53, p21 and Bax but had no effect on protein expression of Bcl-2. CONCLUSION Cisplatin induces apoptosis and death of HeLa cells through the suppression of HPV E6 and restoration of p53 function.展开更多
基金Supported by the Science and Technoiogy Department of Zhejiang Province,No.2003C30057
文摘AIM: To investigate the cytotoxic activity of extracts of trichosanthes root tubers (EOT) on HepA-H cells and HeLa cells compared with trichosanthin (TCS), and to explore the possible mechanism of growth inhibitory effect of EOT on HeLa cells.METHODS: Tumor cells were cultured in vitro, and then microculture tetrzoalium assay (MTT) was used to investigate drugs' cytotoxic activity. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe ultrastructural changes of cells, and electrophoresis was performed to detect changes of biochemical characteristics of intercellular DNA.RESULTS: TCS and EOT had no obvious effects on HepA-H cells (P＞0.05), but had remarkable effects on HeLa cells in a time and dose dependent manner (r＞0.864,P＜0.05 or P＜0.01). The inhibitory rate of EOT was much higher than that of TCS (P＜0.01). Median inhibitory rates (IC50) of TCS and EOT on HeLa cells were 610.9 mg/L and 115.6 mg/L for 36 h, and 130.7 mg/L and 33.4 mg/L for 48 h respectively. Marked morphologic changes were observed including microvillus disappearance or reduction, cell membrane bledding, cell shrinkage, condensation of chromosomes and apoptotic bodies with complete membranes. Meanwhile, apoptosis of HeLa cells was confirmed by DNA ladder formation on gel electrophoresis. CONCLUSION: TCS and EOT have no obvious effects on HepA-H cells, but have significant inhibitory effects on HeLa cells, indicating that EOT is superior to TCS in anti-tumor activity.
基金This work was supported by a grant of National Natu-ral Science Foundation of China(No.30230110)a grant of Science and Technology Committee of Shanghai Mu-nicipality(No.04DZ14901)a grant of Chinese Acad-emy of Sciences#KSCX2-SW-203 to Jia Rui WU.
文摘Recent studies indicate that cell-cycle checkpoints are tightly correlated with the regulation of apoptosis, in which p53 plays an important role. Our present works show that the expression of E6/E7 oncogenes of human papillomavirus in HeLa cells is inhibited in the presence of anti-tumor reagent tripchlorolide (TC), which results in the up-regulation of p53 in HeLa cells. Interestingly, under the same TC-treatment, the cells at the early S-phase are more susceptible to apoptosis than those at the middle S-phase although p53 protein is stabilized to the same level in both situations. Significant difference is exhibited between the two specified expression profiles. Further analysis demonstrates that anti-apoptotic gene survivin is up-regulated by p53 in the TC-treated middle-S cells, whereas it is down-regulated by p53 in the TC-treated early-S cells. Taken together, the present study indicates that the differential p53-regulated expres- sion of survivin at different stages of the cell cycle results in different cellular outputs under the same apoptosis-inducer.
基金Supported by the Chinese medicine and pharmacology bureau of Jiangsu Province in China
文摘AIM: To investigate the inhibitory effect of Chinese herbal medicine on the transcription of hepatitis C virus (HCV) structural gene in Hela D cells.METHODS: Hela cell line was transfected with recombinant pBK-CMV-HCV containing HCV structural gene by Lipofectamine. RT-nested-PCR and Western blot assay were used to testify the HCV gene expression in Hela cells. The Hela cells expressing HCV structural protein were named Hela D cells. Prescriptions of Xiao chaihu Decoction (XCHD),Fufang Huangqi (FFHQ) and Bingganling (BGL) wererespectively added to Hela D cells in various concentrations. Semi-quantitative RT-nested-PCR product analysis was performed according to the fluorescent density between HCV DNA band and GAPDH DNA band in gel electrophoresisafter screened. RESULTS: Recombinant pBK-CMV-HCV could correctly express the HCV structural gene in Hela D cells. After coculture of Hela D cells with three prescriptional different concentrations for 48 h respectively, the transcription of HCVgene decreased with increasing of the concentration of each prescription. The lightness ratio of HCV product bands to GAPDH product bands was 0.24, 0.10 and 0.12 in Hela D cells incubated with 0.1 g/mL of XCHD, FFHQand BGL respectively and the lightness ratio HCV product bands to GAPDH product bands was 0.75, 0.67 and 0.61respectively in the control cells. CONCLUSION: The prescriptions of XCHD, FFHQ and BGL partly inhibit the transcription of HCV structural gene inHela D cells.
基金the Scientific Research Foundation of Education Committee of Hunan (07C713 and 07C708)
文摘Objective： To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosomajaponicum,（SjAslp） and transfer it into mammalian cells to express the objective protein. Methods： By polymerase chain reaction （PCR） technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK＋/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1（＋）. After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Results： The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion： SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.
文摘OBJECTIVE To study the mechanism of apoptosis induced by cisplatin in vitro in HeLa cells cervical cancer cell line. METHODS The inhibitory effect of cisplatin on HeLa cell growth was analyzed by the MTT assay. Cell apoptosis was measured with flow cytometry and Hoechst 33258 staining following cisplatin treatment. The effect of cisplatin on transcription of HPV E6 was analyzed by RT-PCR and protein expression of E6, P53, p21, Bax and Bcl-2 was studied by Western blots. RESULTS Cisplatin inhibited cellular proliferation in a time and dose- dependant manner. The sub-G1 peak by flow cytometry showed a higher apoptotic rate in the experimental group compared to the controls and Hoechst 33258 staining indicated that apoptosis was induced by cisplatin. Results of RT-PCR demonstrated that cisplatin decreased tran- scription of E6. Western Blots showed that cisplatin decreased protein expression of E6 and increased protein expression of P53, p21 and Bax but had no effect on protein expression of Bcl-2. CONCLUSION Cisplatin induces apoptosis and death of HeLa cells through the suppression of HPV E6 and restoration of p53 function.
