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 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.
Objective To evaluated the role of wt-P53 protein in telomerase regulation in keloid fibroblasts(KFBs). Methods The fibroblasts were derived from humankeloid tissue which was proved by pathological diagnosis. KFBs were divided into 2 groups, the transfection group and the untransfection group. wt-p53 gene was transfected into the fibroblasts by adenovirus vectors in the transfection group. The KFBs untransfected with wt-p53 gene served as control (untransfection group). After 48 hours of transfection, the expression of wt-P53 protein was analyzed by both Western blotting and immunofluorescence method, respectively. The telomerase activity was evaluated by TRAP-ELISA after 1-7 days of transfection. Results All the KFBs from 2 groups expressed wt-P53 protein. But the expression level of wt-P53 protein in the transfection group was significantly higher than that in the untransfection group.At the same time of high expression of wt-P53 protein, the telomeraseactivity of KFBs in transfection group was significantly lower than that in theuntransfection group(P＜0.05). Conclusion High level expression of wt-P53 protein can transiently inhibit the telomerase activity of KFBs.
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.
ObjectiveTo investigate the effects of telomerase reverse transcriptase (TERT) promoter mutation and TERT mRNA expression on the prognosis of patients with hepatocellular carcinoma (HCC) after radical resection, and the clinicopathological factors affecting the prognosis of patients with HCC after radical resection were explored.MethodsClinical data of 212 HCC patients underwent radical resection from Jan. 2009 to Jan. 2016 in The Affiliated Cancer Hospital of University of Electronic Science and Technology of China were selected and analyzed. The mutations of TERT, TP53, and catenin beta 1 (CTNNB1) were detected by Sanger sequencing, and the expression of TERT mRNA was detected by SYBR. Patients were followed up routinely and their overall survival (OS) and disease-free survival (DFS) were recorded.ResultsThe mutation rates of the TERT promoter, TP53, and CTNNB1 gene were 17.9% (38/212), 40.1% (85/212), and 13.7% (29/212), respectively. The TERT promoter mutation had significant correlation with Child-Pugh classification and preoperative albumin value (P<0.05). Expression level of TERT mRNA had significant correlation with HBV infection, Child-Pugh classification, preoperative AST value and ALT value (P<0.05). Cox proportional hazards regression result showed that anatomical hepatectomy, tumor diameter>5 cm, and high expression of TERT mRNA were significant prognostic factors of OS (P<0.05); preoperative platelets count≤100×109/L, tumor diameter>5 cm, and high expression of TERT mRNA were significant prognostic factors of DFS (P<0.05).ConclusionFor patients after HCC surgery, high expression of TERT mRNA may be a key factor affecting the prognosis of HCC patients.
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 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.
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.
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.