Objective.To develop monoclonal antibodies again st the catalytic subunit of human telomerase reverse transcriptasefor i ts expression detection of human tumors.Methods.A dominant epitope in hTERT was automatically sy...Objective.To develop monoclonal antibodies again st the catalytic subunit of human telomerase reverse transcriptasefor i ts expression detection of human tumors.Methods.A dominant epitope in hTERT was automatically synthesized based on Fmoc method,and was used to immuni ze Balb/c mice.Hybridomas were generated and screened by ELISA for specific mo noclonal antibodies,and the characterization was performed by Western blotting and immunohistochemical staining.The heavy chain variable region of antibody wa s cloned by RT-PCR and sequenced.Results.Antigenic peptide hTERT 7 was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis.One hybr idoma cell line secreting anti-hTERT 7 antibodies designated as M2was established after primary screening and cons equent 3rounds of limited dilution.M2was IgG1in isotyping.The competi-tive assay showed that the M2antibody was hTERT 7-specific,and the affinity constant was about 1×10 6 mol-1 .The antibody reacted with cell extracts from HeLa cancer cells but not wi th those from normal2BS cells in ELISA assay.For in situ staining of immunohis tochemistry,the positive staining presented in the nuclear compartment of HeLa ,while2BS was negative.The heavy chain variable region from M2re-vealed tha t the monoclonal antibody was mouse origin.Conclusions.The developed mouse mon oclonal antibody is hTERT-specific and able to recognize native cellular hTERT in ELISA and immunohistochemistry,which makes the immuno-detection of telom-e rase hTERT expression in cancer cells or tissues possible.展开更多
Objective: To investigate the influence of anti-keratin autoantibodies (AK auto Abs) on telom-erase activity of squamous cell carcinoma cultured in vitro and the mechanisms of the inhibitory effects of AK auto Abs on ...Objective: To investigate the influence of anti-keratin autoantibodies (AK auto Abs) on telom-erase activity of squamous cell carcinoma cultured in vitro and the mechanisms of the inhibitory effects of AK auto Abs on squamous cell carcinoma. Methods: Influence of AK auto Abs on the proliferation of Tca cells was observed by MTT colorimetry. Telomerase activity of cultured Tca cells and human keratinocytes was determined by telomeric repeat amplication protocol-ELISA (TRAP-ELISA) and polyacrylamide gel elec-trophoresis (PAGE). After being treated with AK auto Abs for 36 h at a concentration of 4, 8, 16 mg/L respectively, the changes of telomerase activity of Tea cells were also detected by TRAP-ELISA and PAGE. Results: MTT colorimetric determination showed that the capacity of proliferation of Tca cells correlated negatively with the concentration of AK auto Abs (r= -0. 74, P<0. 01). TRAP-ELISA and PAGE showed that telomerase activity of Tca cells increased significantly compared to that of cultured human keratinocytes (t = 3. 5396, P<0. 01). AK auto Abs at a concentrations of 4, 8, 16 mg/L had significant dose-dependent inhibitory effects on telomerase activity of Tca cells (r= - 0. 8358, P<0. 01). Conclusion: AK auto Abs have a significant dose-dependent inhibitory effect on the proliferation of cultured Tea cells. AK auto Abs inhibit telomerase activity of cultured Tca cells with dose-dependent pattern. It suggests that decrease of telomerase activity may play an important role in the inhibitory effects of AK auto Abs on squamous cell carcinoma.展开更多
Objective To study the safety and effect of the umbilical cord blood （UCB）-derived mesenchymal stem cells （MSCs） on apoptosis of human cardiomyocytes （HCM）. Methods UCB was collected at the time of delivery with...Objective To study the safety and effect of the umbilical cord blood （UCB）-derived mesenchymal stem cells （MSCs） on apoptosis of human cardiomyocytes （HCM）. Methods UCB was collected at the time of delivery with informed consent obtained from 10 donors. The UCB-derived MSCs were treated with 5-azaserube （5-AZA） and were further induced to differentiate into cardiomyocytes. Telomerase activity, G-banding patterns of chromosomal karyotypes, tumor formation in nude mice, RT-PCR, and the effect of inhibiting apoptosis of HCM were investigated. Results MSCs derived from UCB were differentiated into cardiomyocytes in vitro, which possessed telomerase activity after 5-AZA induction, and no abnormal chromosomal karyotypes were observed. Expression of p53, cyclin A, cdk2, ~3 -actin, C-fos, h-TERT and c-myc were similar in MSCs before and after 5-AZA treatment. There was no tumor formation in nude mice after injection of UCB-derived MSCs. UCB-derived MSCs significantly inhibited apoptosis of HCM. Conclusion UCB-derived MSCs are a valuable, safe and effective source of cell-transplantation treatment .展开更多
文摘Objective.To develop monoclonal antibodies again st the catalytic subunit of human telomerase reverse transcriptasefor i ts expression detection of human tumors.Methods.A dominant epitope in hTERT was automatically synthesized based on Fmoc method,and was used to immuni ze Balb/c mice.Hybridomas were generated and screened by ELISA for specific mo noclonal antibodies,and the characterization was performed by Western blotting and immunohistochemical staining.The heavy chain variable region of antibody wa s cloned by RT-PCR and sequenced.Results.Antigenic peptide hTERT 7 was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis.One hybr idoma cell line secreting anti-hTERT 7 antibodies designated as M2was established after primary screening and cons equent 3rounds of limited dilution.M2was IgG1in isotyping.The competi-tive assay showed that the M2antibody was hTERT 7-specific,and the affinity constant was about 1×10 6 mol-1 .The antibody reacted with cell extracts from HeLa cancer cells but not wi th those from normal2BS cells in ELISA assay.For in situ staining of immunohis tochemistry,the positive staining presented in the nuclear compartment of HeLa ,while2BS was negative.The heavy chain variable region from M2re-vealed tha t the monoclonal antibody was mouse origin.Conclusions.The developed mouse mon oclonal antibody is hTERT-specific and able to recognize native cellular hTERT in ELISA and immunohistochemistry,which makes the immuno-detection of telom-e rase hTERT expression in cancer cells or tissues possible.
文摘Objective: To investigate the influence of anti-keratin autoantibodies (AK auto Abs) on telom-erase activity of squamous cell carcinoma cultured in vitro and the mechanisms of the inhibitory effects of AK auto Abs on squamous cell carcinoma. Methods: Influence of AK auto Abs on the proliferation of Tca cells was observed by MTT colorimetry. Telomerase activity of cultured Tca cells and human keratinocytes was determined by telomeric repeat amplication protocol-ELISA (TRAP-ELISA) and polyacrylamide gel elec-trophoresis (PAGE). After being treated with AK auto Abs for 36 h at a concentration of 4, 8, 16 mg/L respectively, the changes of telomerase activity of Tea cells were also detected by TRAP-ELISA and PAGE. Results: MTT colorimetric determination showed that the capacity of proliferation of Tca cells correlated negatively with the concentration of AK auto Abs (r= -0. 74, P<0. 01). TRAP-ELISA and PAGE showed that telomerase activity of Tca cells increased significantly compared to that of cultured human keratinocytes (t = 3. 5396, P<0. 01). AK auto Abs at a concentrations of 4, 8, 16 mg/L had significant dose-dependent inhibitory effects on telomerase activity of Tca cells (r= - 0. 8358, P<0. 01). Conclusion: AK auto Abs have a significant dose-dependent inhibitory effect on the proliferation of cultured Tea cells. AK auto Abs inhibit telomerase activity of cultured Tca cells with dose-dependent pattern. It suggests that decrease of telomerase activity may play an important role in the inhibitory effects of AK auto Abs on squamous cell carcinoma.
文摘Objective To study the safety and effect of the umbilical cord blood （UCB）-derived mesenchymal stem cells （MSCs） on apoptosis of human cardiomyocytes （HCM）. Methods UCB was collected at the time of delivery with informed consent obtained from 10 donors. The UCB-derived MSCs were treated with 5-azaserube （5-AZA） and were further induced to differentiate into cardiomyocytes. Telomerase activity, G-banding patterns of chromosomal karyotypes, tumor formation in nude mice, RT-PCR, and the effect of inhibiting apoptosis of HCM were investigated. Results MSCs derived from UCB were differentiated into cardiomyocytes in vitro, which possessed telomerase activity after 5-AZA induction, and no abnormal chromosomal karyotypes were observed. Expression of p53, cyclin A, cdk2, ~3 -actin, C-fos, h-TERT and c-myc were similar in MSCs before and after 5-AZA treatment. There was no tumor formation in nude mice after injection of UCB-derived MSCs. UCB-derived MSCs significantly inhibited apoptosis of HCM. Conclusion UCB-derived MSCs are a valuable, safe and effective source of cell-transplantation treatment .
