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 analysis the biological characteristics of human fibroblasts transfected by human telomerase reverse transcriptase (hTERT) eucaryotic expression plasmid pGRN145. METHODS: Fibroblasts from children’s foreskin were isolated and cultured in vitro, and the fibroblasts were transfected by pGRN145 with Lipofec-tAMINE PLUS Reagent. After strict screening of hygromycin B, the positive clones were subcultured. The telomerase activity was detected by RT-PCR and TRAP-PCR technique. The cell generation cycle and apoptosis rate were detected by flow cytometry to investigate the proliferative characteristics after transfection, and the chromosome karyotype of transformed cells was analyzed. The collagen secreted by transformed cells was detected by immunohistochemical staining. RESULTS: The morphological properties of fibroblasts did not change obviously after transfection. There were telomerase activity in transfected fibroblasts, while it could not be detected in pre-transfection fibroblasts. The cell generation cycle had no obvious changes between pre-transfection and post-transfection. However, the apoptosis rate of transfected fibroblasts were decreased compared with that of pre-transfection. The fibroblasts transfected by pGRN145 maintained the normal diploid karyotype, as well as the cells could normally secret type I and III collagen. CONCLUSION: The human fibroblasts transfected by pGRN145 has telomerase activity with prolonged life span of culture, which preliminarily proves the availability of establishing standard seeding cell lines of tissue engineering by hTERT plasmid transfection techniques.
Objective To review the research process of telomerase reverse transcriptase (TERT) in the restoration of neurological diseases. Methods The related l iterature on TERT in the restoration of neurological diseases was extensively reviewed and comprehensively analyzed. Results TERT was the significant component of telomerase and the critical regulator of telomerase activity. It played an important role in the pathomechanism of neurological diseases including tumors,neurodevelopmental deficits, and nerve injury. TERT was becoming a research focus in the reparative therapy of neurological diseases. Conclusion TERT has manifested its great academic significance and appl ication prospects in the reparative therapy of neurological diseases, which deserves a further investigation.
【Abstract】Objective To explore the relation between the expression of telomerase and DNA ploidy with biliarypancreatic system cancer, so as to find a better way to diagnose and distinguish jaundice between malignance and benign disease.Methods Endoscopic retrograde cholangiopancreatography (ERCP) were performed before operation in patients with obstructive jaundice. The bile and pancreatice juice were collected before ERCP. Biopsy specimens from part of patients were obtained during ERCP. All cancer specimens were possessed once again during operation and were assessed by the activity of telomerase and DNA ploidy. Results ① Telomerase positive rate 〔87.50%(56/64)〕 of tissue specimens in malignant obstructive jaundice were higher than that in benign obstructive jaundice 〔3.33%(2/60)〕，P=0.000. ② Telomerase positive rate〔71.88%(46/64)〕of Bile and pancreatice juice in malignant obstructive jaundice were higher than that in benign obstructive jaundice 〔3.33%(2/60)〕， P=0.000, tissue specimens obtained by endoscopy with malignant obstructive jaundice had detectable telomerase activity, positive rate was 83.33%（20/24）. ③ The rate of DNA heteroploid with malignant obstructive jaundice was 62.50%（40/64）, that of diploid can be seen in all patients with benign obstructive jaundice, the difference was statistically significant (P=0.000). ④ The rate of telomerase positive and DNA heteroploid in high differentiation tumor were significantly lower than in middlelow differentiation tumor （P=0.028，P=0.001）.Conclusion Applying the duodenoscope we collected the bile and pancreatic fluid before operation and obtain biopsy specimens whose telomerase activity and DNA ploid were detected. This is simple, safe, quick method which can identify the malignant and benign obstructive jaundice.
Objective To investigate the expression of telomerase reverse transcriptase (TERT) and cell apoptosis in neonatal rats with hypoxia ischemia brain damage (HIBD). Methods A total of 42 7-day-old SD rats (12-18 g, male or female) were randomly allocated into sham-operation group (n=6) and hypoxia-ischemia (HI) group (n=36). In HI group, the rats were anesthetized with ethylether. The right common carotid artery (CCA) was exposed and permanently l igated with a 7-0silk suture through a midl ine cervical incision. A duration of 2.5 hours of hypoxia (8%O2 / 92%N2) was used to produce HIBD model. For sham-operation group, the CCA was exposed without l igation or hypoxia. The brain tissues were harvested at 4, 8, 12, 24, 48, and 72 hours after completion of an HI insult. The expressions of TERT and CC3 were detected by immunohistochemical staining. The apoptosis cells were detected with TUNEL staining method. Results The expression of TERT was increased at 4 hours after HI injury, significantly increased at 24-48 hours and then decreased at 72 hours. The expression of CC3 was increased at 4 hours after HI injury, significantly increased at 24 hours and still maintained high expression at 48 hours and 72 hours. However, in the sham-operation group, both the expressions of TERT and CC3 were extremely low. The expression of TERT and CC3 were higher in the HI group than in the sham-operation group at different time points, and the differences were significant (P lt; 0.05). The TUNEL staining showed that the positive cells in hippocampus and cortical areas were increased at 4 hours after HI injury, significantly increased at 24-48 hours and maintained a high level at 72 hours. However, there was few positive cells in the sham-operation group. There were significant differences between the HI group and the sham-operation group at different time points (P lt; 0.05). Conclusion TERT could be induced by HI in neonatal rats, and might have a protective role in regulating the cell apoptosis in the neonatal HIBD.
【Abstract】Objective To design the hammerhead ribozyme gene according to the hTR sequence in the gallbladder cancer cell, and build it into the eukaryon expression vector pTriEx-4. Methods According to the hTR cDNA sequence, the authors designed the primers and take the hTR template area gene from the gallbladder cancer cells by RT-PCR.The hammerhead ribozyme gene was synthesize according to the result of sequencing, and combine them with eukaryon expressing vector. Identified the exactitude of recombine vector by digestion.Results The 68 bp sequence extracted from the cell through the RT-PCR had the same template sequence comparing with the hTR cDNA. The recombinant plasmid with the hammerhead ribozyme gene was correct by digestion identification. Conclusion The RT-PCR method can extract the gallbladder cancer cell’s hTR gene. We construct the eukaryon expression vector containing the hammerhead ribozyme gene successfully which is the foundation for gene therapy of gallbladder cancer.
【Abstract】Objective To investigate the expression of PCNA in gastric cancer and its relationship with telomerase activity of peritoneal washings and peritoneal dissemination, and to compare the efficacy of telomerase activity and cytology of peritoneal washings for prediction of peritoneal metastasis of gastric cancer. MethodsTelomeric repeated amplification protocol (TRAP)enzymelinked immunosorbent assay (ELISA) was performed to measure the telomerase activity of peritoneal washings collected from 60 patients with gastric cancer. Exfoliate cytologic analysis of the corresponding samples was used for comparison.Expression of PCNA was measured with immunohistochemical staining.Their relationship with clinicopathologic features were evaluated. ResultsThe positive rate of telomerase activity in peritoneal washing collected from patients with gastric cancer was 41.7%,which well related to serosal invasion, histology types, depth of infiltration and peritoneal metastasis of gastric cancer. The positive rate of telomerase activity increased with the increased depth of infiltration and serosal involvement areas (P＜0.05).The positive rate of exfoliative cytology was 25.0%, which was obviously high in the group with macroscopic peritoneal metastasis (the group of P1-3). The positive rate of exfoliative cytology also increased with the increased depth of infiltration and serosal involvement areas (P＜0.05). Although the positive rate of telomerase activity in peritoneal washing collected from patients with gastric cancer was not significantly higher than that of exfoliative cytology in general, it was significantly higher than that of exfoliative cytology in the group of pT4, P1-3 and undifferentiated type.The PCNA proliferation index (PI) of positive telomerase activity group was significantly higher than that of negative. The PCNA PI was significantly higher in the group of P1-3 and serosal invasion thanthat of P0 and without serosal invasion. ConclusionTo detect telomerase activity in peritoneal washings and to detect tumor cells by cytologic method are useful to predict subclinical metastasis to the peritoneum in patients with gastric cancer,but telomerase activity is more sensitive than the other one.Telomerase activity is well related to proliferating activity of gastric cancer,which was the very important reason of peritoneal metastasis and serosal invasion.
ObjectiveTo construct tumor specific tubercle bacillus antigen Ag85A gene lentiviral vector driven by murine telomerase catalytic subunit promoter (PmTERT), paving the way for further research in tumor targeting immuno-gene therapy. MethodsPmTERT was amplified by PCR method, with murine genomic DNA as template. Then, transcriptional activities of PmTERT in various murine and human cell strains were studied by luciferase assay. Ag85A expression lentiviral vectors driven by cytomegalo virus (CMV) promoter and PmTERT respectively (pLVX-Ag85ACMV and pLVX-Ag85A-PmTERT) were constructed with nucleic acid cloning approach. And above recombinants were verified with DNA sequencing and Western blot. ResultsLucifease assay revealed that 331 bp PmTERT cloned in present research had transcriptional activity in murine Lewis lung cancer cells, human lung adenocarcinoma cells A549, and human esophageal cancer cells EC-109, while no transcriptional activity in murine fibroblasts NIH3T3 and human embryo fibroblasts MRC-5. Western blot revealed expression of Ag85A in pLVX-Ag85A-CMV transfected Lewis and NIH3T3 cells, pLVX-Ag85A-PmTERT transfected Lewis cells, no expression in pLVX-Ag85A-PmTERT transfected NIH3T3 cells. ConclusionPmTERT has tumor specific transcriptional activity. Ag85A gene can express selectively in tumor cells, driven by PmTERT.
Objective Telomerase reverse transcriptase (TERT) is the key factor to determine cell growth and l ifespan. Meanwhile, it is tightly related to resistance of cell to stress and apoptosis. However, up till now l ittle is known about the role TERT plays in nervous system. To investigate the effect of conditioned medium from astrocytes (AS) transfected with TERT on neurons subjected to hypoxia-ischemia-reperfusion (HI-RP) through construction of in vitro HI-RP model of neurons. Methods An eukaryote expression plasmids containing rat full length TERT gene was constructed as pcDNA3-TERT. Twenty newborn rats at age of 3 days were sacrificed and their cerebral cortex were collected for isolation and cultivationof AS. Then AS were transfected with pcDNA3-TERT through l iposomes mediation, and positive clones were selected by G418 and expanded for continuous culture to establ ish the plamid pcDNA3-TERT transfection group. Meanwhile, the empty plasmid pcDNA3 transfection group and the non-transfection group were establ ished as control. The expression of gl ial fibrillary acidic protein (GFAP), which was the specific marker of the AS, was detected by immunocytochemistry, as well as the expression of TERT. Astrocyte conditioned medium (ACM) of the plamid pcDNA3-TERT transfection group was collected as TERT-ACM, while the ACM of the empty plasmid pcDNA3 transfection group and the non-transfection group were collected respectively as p-ACM and ACM. Next, 60 rats at age of 1 day were sacrificed and their cerebral cortex were collected for isolation and cultivation of neurons. The neurons were randomly divided into experimental group and normal group, the experimental group were further divided into 4 groups including control group, ACM group, p-ACM group, and TERT-ACM group. The neurons of control group were subjected to HI damage in serum-free DMEM, and the neurons of ACM group, p-ACM group, and TERTACM group were subjected to HI damage in different medium which contained ACM, p-ACM, and TERT-ACM, respectively. After duration of HI for 3 hours under the environment with 5%CO2, 1%O2, and 94%N2; the neurons of experimental groups were placed in CO2 incubator to imitate RP for 3, 6, 18, 24, and 36 hours in vitro. The neurons of normal group were not subjected to HI and RP treatment. During the treatment of HI-RP, the survival ratio of neurons was detected by means of MTT, the lactate dehydrogenase (LDH) activity of neuron medium with LDH detection kit, and the neuronal apoptosis by means of TUNEL. Results The percentages of GFAP positive cells were 98%, 99%, and 98% in non-transfection group, plasmid pcDNA3-TERT transfection group, and plasmid pcDNA3 transfection group, respectively. There was no expression of TERT in no-transfection group and plasmid pcDNA3 transfection group, and the percentage of TERT positive cells in plasmid pcDNA3- TERT transfection group was 98%. Compared with normal group, the survival ratio of ......（余見正文）
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.