【Abstract】ObjectiveTo investigate the expressions of hTERT mRNA and BRCA1 protein and to analyze the correlation between these two factors in breast cancer. MethodsThe expression of hTERT mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). The expression of BRCA1 protein was examined by immunohistochemistry. ResultsThe positive rates of hTERT mRNA and BRCA1 protein were 72.1%（31/43） and 34.9%（15/43） in breast cancer tissue, were 5.0%(2/40) and 77.5%(31/40) in paracancerous breast tissue respectively. Significant difference existed between breast cancer tissue and paracancerous breast tissue (P＜0.05). Significant negative correlation existed between the expression of BRCA1 protein and expression of hTERT mRNA （r=-0.995, P＜0.01）. ConclusionThe expression of hTERT mRNA is upregulated in breast cancer, and expression of BRCA1 protein is downregulated in breast cancer. BRCA1 protein expression may be associated with expression of hTERT mRNA in breast cancer, which may be involved in the carcinogenesis of breast cancer.
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 explore the SV40-mediated immortalization, the related factors and their roles in cell immortalization. METHODS: The original articles about cell immortalization and replicative senescence in recent decade were reviewed. RESULTS: Cell immortalization was a multifaceted phenomenon, it was involved in viral DNA integration, activation of telomerase, inactivation of growth suppressors, and so on, and their roles were closely related. CONCLUSION: The research on cell immortalization may be expected to provide important insights into a broad range of cellular biological phenomenon, and the immortalized cells can play important roles in the research of cell engineering and tissue engineering as standard cells.
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.
ObjectiveTo investigate the expressions and clinical significance of human telomerase reverse transcriptase (hTERT) mRNA and γglutamyl transpeptidase mRNA-H (GGT mRNA-H) in the peripheral blood of small hepatocellular carcinoma (HCC) patients. MethodsThe expressions of hTERT mRNA and GGT mRNA-H were detected in the peripheral blood of thirty patients with small HCC by RT-PCR, eighteen patients with benign liver diseases, and twelve normal volunteers. ResultsThe positive rate of hTERT mRNA and GGT mRNA-H expression in patients with small HCC were 80.0% (24/30) and 46.7%(14/30), respectively. In patients with hepatitic cirrhosis the positive rate of hTERT mRNA expression was 33.3% (6/18), while the expression of GGT mRNA was not detected. Both the expressions of hTERT mRNA and GGT mRNA-H were negative in all normal volunteers. The combination analysis of hTERT mRNA and GGT mRNA-H expression achieved positive rate of 86.7% in the diagnosis of small HCC, which was significantly higher than the positive rate of AFP (26.7%)， Plt;0.05. ConclusionThe hTERT mRNA and GGT mRNA-H are significantly expressed in small HCC patients, the combination analysis of hTERT mRNA and GGT mRNA-H seems to be useful in the early diagnosis of small HCC.
ObjectiveTo investigate the expression change characteristic of stemness-related markers for recurrent hepatocellular carcinoma (HCC), and to discuss the relationship between stemness-related markers and clinicopathologic characteristics of HCC.MethodsWe collected 25 recurrent HCC patients who also had the first liver resection for HCC in Sichuan Cancer Hospital from Jan. 2010 to Oct. 2018. Immunohistochemistry was used to compare expressions of CD133, CD90, CD117, and epithelial cell adhesion molecule (EpCAM) in HCC tissues. Fluorescence in situ hybridization was used to detect telomere length.ResultsThe primary HCC had higher platelet count, larger tumor, less microvascular invasion (MVI), and less multiple HCC than the recurrent HCC (P<0.05), but the expressions of CD90, CD133, CD117, and EpCAM were not significantly differed after recurrence (P>0.05). The expressions of CD90, CD133, CD117, and EpCAM were not associated with tumor size, tumor number, Barcelona Clinic Liver Cancer Staging (BCLC staging), satellite nodules, and differentiation (P>0.05). The MVI-positive group had a significantly higher expression level of EpCAM (P=0.016) and longer telomere length (P=0.001). The telomere length was longer for tumors diameter less than 5 cm (P=0.038) and poor differentiation (P=0.046). Correlation analysis found that there was no relationship between telomere length and expression levels of EpCAM (r=–0.092, P=0.513), CD90 (r=–0.235, P=0.100), CD133 (r=0.024, P=0.867), and CD117 (r=–0.277, P=0.052), but an apparent positive correlation between expression levels of EpCAM and CD133 was found (r=0.358, P=0.011). Survival analysis found that poor differentiation (P=0.003) and BCLC B–C staging (P=0.040) were the risk factors of disease-free survival for patients after first HCC resection, and BCLC B–C staging (P=0.017) and tumor diameter more than 5 cm (P=0.035) were the risk factors for recurrent HCC.ConclusionsRecurrent HCC had similar stemness-related markers expression and longer telomere length. Expression level of EpCAM and telomere length were associated with MVI.
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.
【摘要】 目的 觀察晚期糖基化終產物（advanced glycosylation end prodrcts，AGE）對人結腸癌細胞株SW-480增殖的影響，并探討其可能機制。 方法 不同濃度AGE干預SW-480細胞，噻唑藍（MTT）法比較各組細胞活力，流式細胞術觀察AGE對SW-480細胞周期的影響，蛋白質印跡法觀察AGE對SW-480細胞CyclinD1表達的影響，端粒重復序列擴增法（telomeric repeat amplification protocol，TRAP）銀染法觀察AGE對SW-480細胞端粒酶活性的影響。MTT測細胞活力的檢測設置空白對照組、100 μg/mL小牛血清白蛋白（bovine serum albumin，BSA）組及50、100、500 μg/mL AGE組，其余檢測只設置100 μg/mL BSA組和100 μg/mL AGE組。 結果 MTT結果示AGE促進SW-480細胞的增殖，且呈濃度依賴性。100 μg/mL BSA組與100 μg/mL AGE組72 h后的細胞G0/G1期所占百分比分別為56.02%±0.58%、51.93%±1.01%，差異有統計學意義（Plt;0.05）。蛋白質印跡法示100 μg/mL AGE組72 h后CyclinD1的表達較100 μg/mL BSA組增加，差異有統計學意義（Plt;0.05）。TRAP銀染法檢測示100 μg/mL AGE干預SW-480細胞72 h后可以增加端粒酶活性（Plt;0.05）。 結論 AGE可促進人結腸癌細胞SW-480生長，呈劑量依賴性。其作用機制可能與AGE上調CyclinD1的表達加速G1/S期轉換及增加端粒酶活性有關。【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 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.