论文部分内容阅读
目的 :探讨雷帕霉素(rapamycin,RAPA)对骨肉瘤MG63细胞中巢蛋白(nestin)表达的影响及可能的作用机制。方法 :MTT法检测RAPA对MG63细胞增殖的影响,并采用反转录聚合酶链式反应(reverse transcription-polymerase chine reaction,RT-PCR)和蛋白质印迹法检测RAPA对nestin m RNA和蛋白表达的影响;分别采用细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)抑制剂U0126、蛋白激酶B(protein kinase B,PKB,又称Akt)抑制剂wortmannin、糖原合成激酶-3β(glycogen synthase kinase-3β,GSK-3β)抑制剂氯化锂(lithium chloride,Li Cl)或瞬时转染特异性针对GSK-3β的小干扰RNA(small interfering RNA,si RNA)以及蛋白激酶A(protein kinase A,PKA)抑制剂H89预处理MG63细胞,再用RAPA进行处理,蛋白质印迹法检测抑制ERK、Akt、GSK-3β以及PKA的活性对RAPA导致的nestin蛋白上调的影响;蛋白质印迹法检测RAPA处理后对Akt和GSK-3β活性[磷酸化Akt(phospho-Akt,p-Akt)/Akt和p-GSK-3β/GSK-3β的比值]的影响。结果 :RAPA对细胞的相对增殖率无明显影响;RAPA(100 nmol/L)处理MG63细胞48 h后,nestin蛋白的表达水平明显提高,为对照组的1.93倍(P<0.05);24和48 h时,nestin m RNA的表达水平分别为对照组的1.89倍和1.84倍(P值均<0.05)。Wortmannin增强了RAPA导致的nestin表达升高,Li Cl或GSK-3β-si RNA干扰GSK-3β表达抑制了RAPA导致的nestin表达升高,而U0126和H89则对nestin的表达无明显影响。p-Akt/Akt比值在RAPA处理24和48 h后分别下调21%和63%,而p-GSK-3β/GSK-3β比值则分别下调了26%和42%。结论 :RAPA通过抑制Akt的活性和增强GSK-3β的活性上调了MG63细胞中nestin的表达。
Objective: To investigate the effect of rapamycin (RAPA) on the expression of nestin in osteosarcoma MG63 cells and its possible mechanism. METHODS: The effect of RAPA on the proliferation of MG63 cells was detected by MTT assay. The effect of RAPA on the expression of nestin mRNA and m RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting U0126, wortmannin, an inhibitor of extracellular signal-regulated kinase (ERK), inhibitor of protein kinase B (PKB), glycogen synthase kinase- 3β, GSK-3β) inhibitor lithium chloride (LiCl) or transiently transfected small interfering RNA (si RNA) specific to GSK-3β and protein kinase A ) Inhibitor H89 pretreatment MG63 cells, and then treated with RAPA, Western blotting to detect the inhibition of ERK, Akt, GSK-3β and PKA activity of RAPA-induced nestin protein upregulation; Western blot detection of RAPA treatment of Akt And the ratio of GSK-3β activity [phospho-Akt (p-Akt) / Akt and p-GSK-3β / GSK-3β]. RESULTS: After treated with RAPA (100 nmol / L) for 48 h, the expression of nestin protein increased significantly by 1.93 folds (P <0.05) in control group (P <0.05) h, the expression levels of nestin m RNA were 1.89 and 1.84 times higher than that of the control group (all P <0.05). Wortmannin enhanced the expression of nestin induced by RAPA. The interference of LiCl or GSK-3β-si RNA with GSK-3βinhibited the increase of nestin expression induced by RAPA, while U0126 and H89 had no effect on the expression of nestin. The p-Akt / Akt ratio was down-regulated by 21% and 63%, respectively, at 24 and 48 h after RAPA treatment, whereas the p-GSK-3β / GSK-3β ratio was down-regulated by 26 and 42%, respectively. Conclusion: RAPA up-regulated the expression of nestin in MG63 cells by inhibiting the activity of Akt and enhancing the activity of GSK-3β.