OBJECTIVE: To analysis the proliferation properties and telomerase activity of human embryonic tendon cells transformed by ptsA58H plasmid cultured in vitro continuously. METHODS: The 40th, 70th, and 75th passages of transformed human embryonic tendon cells (THETC) were adopted. The collagen secretion of THETC was detected by immunohistochemical methods, the growth curve of different passages of THETC was compared, and chromosome karyotype was analyzed. Total RNA of THETC were extracted to detect human telomerase reverse transcriptase (hTERT) mRNA expression by RT-PCR technique. RESULTS: When THETC were subcultured to 70 passages, the morphological characteristics of cells changed and began replicative senescence. THETC still could secret type I collagen normally. The chromosome of THETC was heteroploid (2n = 94). There were no hTERT mRNA expression. CONCLUSION: SV40 transfection can not make human embryonic tendon cells immortalization, on the other hand, human embryonic tendon cells transformed by ptsA58H plasmid has no tendency of malignant transformation.
Objective To investigate the effect of the 8-bromum-cyclic adenosine monophosphate (8-Br-cAMP) on the telomerase activity and changes of cell cycle in retinoblastoma （RB） cells. Methods The cultured RB cells were divided into the experimental group (8-Br-cAMP) and control group. After cultured for 24, 48 and 72 hours in vitro, the telomerase activity of RB cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and the changes of cell cycle were detected by flow-cytometry. Results The difference of telomerase activity was significant between the experimental groups and control group (Plt;0.01). There was a negative correlation between the A value of absorbance and the time in the experimental groups (r=-0.778 9, F=33.936, Plt;0.01). The changes of the cell cycle were that the percentages increased in G1 phase and decreased in S phases. Conclusion 8-Br-cAMP may weaken telomerase activity, affect the cell cycle, and inhibit the proliferation of RB cells. (Chin J Ocul Fundus Dis，2004，20：358-360)
OBJECTIVE: To elongate the proliferation life-span of human umbilicus vein endothelial cell (HUVEC). METHODS: We synthesized the human telomerase reverse transcriptase mRNA (hTERT mRNA) by in vitro transcription, then transferred the hTERT mRNA into HUVEC in quicent stage by lipofect introduction. RESULTS: Telomerase expressed transiently in HUVEC, and the cell life-span was elongated for 7 population doublings. CONCLUSION: Telomerase can be reconstructed controllably and transiently in HUVEC by hTERT mRNA introduction, this method has the potential to be used to elongate the lifespan of cells cultured in vitro.
ObjectiveTo comprehensively analyze the recent advancements in the field of mesenchymal stem cells (MSCs) aging,and summary its achievements and its difficulty at the present. MethodsThe literature about MSCs aging was reviewed and analyzed. ResultsInducible telomerase reactivation of MSCs is successful to extend the life span of senescent cells,but it also has potential safety hazard.The age range presented in the research of age-related cell senescence is inconsistent,resulting in different outcomes.Many ways to improve cell in vitro culture conditions will help delay aging.Recent research indicates that oxidative stress theory is seemed to not completely explain cell aging. ConclusionFurther research of MSCs aging mechanism will help the tissue engineering transform to clinical application.
Objective To investigate the variety of telomerase activity in the course of liver cancer development, and the possibility of using telomerase as a marker of HCC. Methods Human liver specimens, comprising 22 HCC and adjacent peritumoral tissues, 12 liver cirrhosistissues, 6 nodulat regenerative hyperplasia (NRH) tissues and 10 normal liver tissues, were examined for telomerase activity by TRAP assay based on PCR. Results Twenty of 22 HCC and 14 of 22 adjacent tissue specimens were positive for telomerase activity with a positive rate of 90.9% and 63.6% respectively. Ten of 12 liver cirrhosis tissues were positive with a positive rate of 83.3%. 5 of 6 NRH were positive with a positive rate of 83.3%. Telomerase activity was negative in 10 normal liver tissues. Conclusion Telomerase may occur in the progress of hepatocarcinogenesis. Telomerase can be used as a tumor marker of HCC.
Objective To investigate the expressions of CD90, IGF1R, and hTERT protein in hepatocellular carcinoma, and the correlations of each other in the development of carcinoma. Methods The expressions of CD90, IGF1R, and hTERT protein in hepatocellular carcinoma were detected by S-P immunohistochemical staining, 20 cases of normal liver tissues were collected as contrast, and to compare the relations between expression and prognosis or survival rate. Results The positive rate of CD90, IGF1R, and hTERT protein in hepatocellular carcinoma group were obviously higher than that in contrast group（P＜0.05）, which was 63.9% vs. 0, 52.8% vs.5.0%, and 47.2% vs.0, respectively. The positive rate of CD90, IGF1R, and hTERT protein were higher in UICC Ⅲ-Ⅳ stage group than that in UICC stage Ⅰ-Ⅱ group（P＜0.05）, which was 79.2% vs.33.3%, 70.8% vs.16.7%, and 62.5% vs.16.7%, respectively. There was a statistically significant positive correlation observed between the expressions of CD90 and IGF1R protein （Kendall’s tau-b=0.563 1, P＜0.05）, so it was with CD90 and hTERT protein （Kendall’s tau-b=0.363 6, P＜0.05）. The survival rates of positive expressions of CD90, IGF1R, and hTERT protein were lower than negative expressions of CD90, IGF1R, and hTERT（P＜0.05）, which was 21.7% vs.50.0%, 17.6% vs.43.8%, and 20.0% vs.38.9%, respectively. Conclusions The expressions of CD90, IGF1R, and hTERT may have correlations with the progress of HCC, and may serve as a marker for HCC prognosis potentially.
Objective To introduce telomeres, telomerase and their expression in gastric carcinoma.MethodsThe related literatures were collected and reviewed.Rsults In summary, telomerase activity could be detected in 85%-90% of gastric cancer. Moreover, the patient with telomerase-positive tumors showed poorer prognosis than those with telomerase-negative tumours, indicating that telomerase-positive gastric cancer might have more malignant potential. ConclusionKnowledge of telomerase activity in gastric cancer may be useful in cancer diagnosis, as well as a prognostic indicator of clinical outcome. Future development of drugs aimed at telomerase inhibition may potentially provide a therapy with relatively less side effects.
ObjectiveTo summarize the research situation of mesenchymal stem cells (MSCs) senescence, including the characteristics and mechanisms of senescence. MethodsThe original articles in recent years about MSCs senescence were extensively reviewed, and comprehensively analyzed. ResultsThe senescence of MSCs which manifests as morphological senescence, reduced proliferation and differentiation potential, altered immunoregulation are found during the cultivation in experiment, which profoundly affects clinical application of MSCs. The research about the mechanisms of MSCs senescence includes telomere and telomerase, and stress-mediated injury etc, involving regulation of telomerase, and regulation of signal pathways of p53/p21, P13K/Akt, and Wnt/β-catenin etc. ConclusionThe further study of senescence mechanisms will help to accelerate the clinical application of MSCs in the future.
【摘要】 目的 观察晚期糖基化终产物（advanced glycosylation end prodrcts，AGE）对人结肠癌细胞株SW-480增殖的影响，并探讨其可能机制。 方法 不同浓度AGE干预SW-480细胞，噻唑蓝（MTT）法比较各组细胞活力，流式细胞术观察AGE对SW-480细胞周期的影响，蛋白质印迹法观察AGE对SW-480细胞CyclinD1表达的影响，端粒重复序列扩增法（telomeric repeat amplification protocol，TRAP）银染法观察AGE对SW-480细胞端粒酶活性的影响。MTT测细胞活力的检测设置空白对照组、100 μg/mL小牛血清白蛋白（bovine serum albumin，BSA）组及50、100、500 μg/mL AGE组，其余检测只设置100 μg/mL BSA组和100 μg/mL AGE组。 结果 MTT结果示AGE促进SW-480细胞的增殖，且呈浓度依赖性。100 μg/mL BSA组与100 μg/mL AGE组72 h后的细胞G0/G1期所占百分比分别为56.02%±0.58%、51.93%±1.01%，差异有统计学意义（Plt;0.05）。蛋白质印迹法示100 μg/mL AGE组72 h后CyclinD1的表达较100 μg/mL BSA组增加，差异有统计学意义（Plt;0.05）。TRAP银染法检测示100 μg/mL AGE干预SW-480细胞72 h后可以增加端粒酶活性（Plt;0.05）。 结论 AGE可促进人结肠癌细胞SW-480生长，呈剂量依赖性。其作用机制可能与AGE上调CyclinD1的表达加速G1/S期转换及增加端粒酶活性有关。【Abstract】 Objective To observe the effects of advanced glycosylation end products (AGE) on proliferation of SW-480 cells and study the possible mechanism. Methods Various concentrations of AGE were designed to have impact on SW-480 cells. Proliferation of SW-480 cells was assessed by thiazolyl blue tetrazolium bromide (MTT) assay; The impact of AGE on the cell cycle of SW-480 cells was analyzed by flow cytometry (FCM); the influence of AGE on expression of CyclinD1 was checked by Western blotting; and the impact of AGE on telomerase activity was examined by telomeric repeat amplification proctol (TRAP) sliver staining. For the MTT assay, blank control group, 100 μg/mL bovine serum albumin (BSA) group, 50, 100 and 500 μg/mL AGE groups were designed, while for other examinations, there were only 100 μg/mL BSA group and 100 μg/mL AGE group. Results MTT result showed that AGE increased the proliferation of SW-480 cells in a dose-dependent mode. The proportion of the cells at G0/G1 stage of the 100 μg/mL BSA group and the 100 μg/mL AGE experimental group were (56.02±0.58)% and (51.93±1.01)% respectively after 72 hours, with a significant difference (Plt;0.05); western blotting showed that the expression of CyclinD1 in the 100 μg/mL AGE group was significantly higher than that in the 100 μg/mL BSA group after 72 hours; TRAP silver staining demonstrated that telomerase activity increased significantly after treated with 100 μg/mL AGE for 72 hours. Conclusions AGE can promote the growth of SW-480 cells in a dose-dependent mode. Its mechanism is mainly by up-regulating the expression of CyclinD1 to shorten G0/G1 and increasing the telomerase activity significantly.
OBJECTIVE: To construct a plasmid which has a reporter gene for exploring the role of human telomerase reverse transcriptase(hTRT) in in-vitro cell cultivation. METHODS: hTRT was cut by restricted enzyme from plasmid pGRN145 and inserted to plasmid pEGFP-C1 (enhanced green fluorescent protein). RESULTS: Restricted enzyme analysis and DNA sequencing showed that the sequence of the pEGFP -hTRT transgenic plasmid was correct. CONCLUSION: The recombinant vector pEGFP-hTRT has been successfully constructed, and it can be used as a transgenic plasmid in generating immortalized cell lines.