【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 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.
【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.