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 prevent the senescence of ’seed cells’ for tissue engineering, the life span of human fibroblasts is extended by reconstitution of telomerase activity, and the osteogenic potential of these fibroblasts are tested. METHODS: The pGRN145 plasmids encoding human telomerase reverse transcriptase (hTERT) were introduced into the normal human primary fibroblasts by electroporation. Telomerase activity was analyzed by TRAP-PCR assay. The beta-galactosidase stain was used to indicate the signs of cell senescence. The hTERT positive fibroblasts were then induced to form bone nodules. The bone nodules were stained by tetracycline and Alizarin Red S. RESULTS: Stable telomerase activity could be detected in the transfected fibroblasts and no signs of cell senescence were found in the fibroblasts cultured for more than 50 doublings. The hTERT positive fibroblasts could form bone nodules when they were cultured in vitro induced by bone morphogentic protein 2 and tumor necrosis factor-alpha. CONCLUSION: The fibroblasts with reconstituted telomerase activity reserve their osteogenic potential.
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.
【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.
【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 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 ......（余见正文）
ObjectiveTo explore the effects of exogenous estrogen receptor β1 (ERβ1) gene on the expression of human telomerase reverse transcriptase (hTERT) as well as the changes of proliferation ability in MDA-MB-231 cell line by transfecting recombinant eukaryotic expressing vector containing ERβ1 cDNA into human breast cancer MDA-MB-231 cell. MethodsRecombinant eukaryotic expressing vector containing ERβ1 cDNA was transfected into human breast cancer MDA-MB-231 cell by using cationic liposome as transfecting agent (acted as pcDNA3.1ERβ1 transfection group), empty vector group and non-transfection group acted as controls. The expression levels in both the mRNA and protein of both the ERβ1 and hTERT were tested by real-time PCR and Western blot, respectively. The change of proliferation ability in MDA-MB-231 cell was displayed by cell growth curve, and the change of cell apoptosis was detected by flow cytometry. ResultsThe expression level of ERβ1 mRNA in the pcDNA3.1-ERβ1 transfection group (0.449±0.077) significantly increased as compared with the nontransfection group (0.153±0.035) or the empty vector group (0.160±0.020), P=0.001 or P=0.000. The expression level of ERβ1 protein in the pcDNA3.1-ERβ1 transfection group (0.847±0.065) significantly increased as compared with the non-transfection group (0.356±0.050) or the empty vector group (0.390±0.030), P=0.001 or P=0.000. The expression level of hTERT mRNA in the pcDNA3.1-ERβ1 transfection group (0.127±0.020) significantly decreased as compared with the non-transfection group (0.283±0.025) or the empty vector group (0.283±0.049), P=0.001 or P=0.002. The expression level of hTERT protein in the pcDNA3.1-ERβ1 transfection group (0.147±0.023) significantly decreased as compared with the non-transfection group (0.783±0.025) or the empty vector group (0.802±0.019), P=0.001 or P=0.002. The rate of cell apoptosis in the pcDNA3.1-ERβ1 transfection group 〔(6.15±0.94)%〕 was higher than that in the non-transfection group 〔(1.41±0.42)%〕， P=0.001. Cell proliferation curve showed that proliferation ability significantly decreased in the pcDNA3.1-ERβ1 transfected groups as compared with the non-transfection group (Plt;0.05). ConclusionERβ1 could inhibit cell growth of human breast cancer MDA-MB-231 cell by down-regulating the expression of hTERT.
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.
ObjectiveTo build a lentiviral expression vector regulated by two targets 5 copies of HREs and hTERTp, express the target gene CDX2, and to test the activity of hTERT promoter by using LoVo cells for transfection. MethodsAfter the primer sets were designed, the hTERT promoter was cloned by PCR amplification from the genome of colon cancer. The CEA promoter was removed from the original vector pLEGFP-5HRE-CEAp by double digestion and PCR method, and then the hTERTp was introduced into the vector to construct the recombinant plasmid pLEGFP-5HRE-hTERTp. 5HRE-hTERTp was obtained by PCR, while the CMV promoter was removed from the original vector pLVX-EGFP-3FLAG by double digestion and PCR method, and then the 5HRE-hTERTp was introduced into the vector to construct the recombinant plasmid pLVX-5HRE-hTERTp-EGFP-3FLAG. The CDX2 was cloned by PCR amplification from GV230-CDX2-EGFP, and the EGFP was removed from the vector pLVX-5HRE-hTERTp-EGFP-3FLAG by double digestion, and then the CDX2 was introduced into the vector to construct the recombinant plasmid pLVX-5HRE-hTERTp-CDX2-3FLAG. LoVo cells ex vivo was transiently transfected by pLVX-5HRE-hTERTp-EGFP-3FLAG to evaluate the activity of hTERTp by detecting the expression of green fluorescence protein EGFP. ResultsPCR and sequencing analyzing showed that pLEGFP-5HRE-hTERTp, pLVX-5HRE-hTERTp-EGFP-3FLAG, and pLVX-5HRE-hTERTp-CDX2-3FLAG were sequenced correctly and the same as our designed. pLVX-5HRE-hTERTp-EGFP-3FLAG was successfully transfected into LoVo cells ex vivo and expressed green fluorescence protein EGFP, which showed that hTERTp was activated and promoted the expression of downstream gene. ConclusionThe lentiviral expression vector, pLVX-5HREhTERTp-EGFP-3FLAG and pLVX-5HRE-hTERTp-CDX2-3FLAG are successfully constructed, which lays the foundation of further research. But the function of dual-target regulation needs further proof.