雷帕霉素抑制人神经母细胞瘤细胞株生长及诱导凋亡的机制研究

目的研究雷帕霉素对人神经母细胞瘤细胞株SH-SY5Y的生长抑制和诱导凋亡作用，以及对靶向信号通路磷脂酰肌醇3-激酶／蛋白激酶B／哺乳动物雷帕霉素靶蛋白（P13K／AkCmTOR）的影响。方敞用不同浓度的雷帕霉素（10、15、20μmol／L）处理SH-SY5Y细胞（雷帕霉素干预组），以加入相同体积二甲基亚砜（DMSO）的SH-SY5Y细胞作为阴性对照组，设立空白对照组。CCK．8法测定细胞生长率；采用流式细胞仪检测细胞周期，通过AnnexinV-FITC／PI双染法检测细胞凋亡率；Westernblotting检测easpase-3剪切体及P13K／Akt/mTOR信号通路上下游分子PDKp85、Akt、mTOR、4E-BPl表达及其磷酸化水平的变化。结果与阴性对照组和空白对照组比较，雷帕霉素干预组细胞生长率显著降低（P〈0．05或P〈0．01）；G。／G，期细胞百分比显著增高（P〈0．05）；细胞凋亡率显著升高（P〈0．01）。Westernblotting检测结果显示：与阴性对照组和空白对照组比较，雷帕霉素干预组easpase-3剪切体表达水平较高；P13K／Akt/mTOR信号通路上下游分子的活性均被抑制，P13Kp85、Akt、mTOR、4E-BP1的磷酸化水平均明显降低。结论雷帕霉素对人神经母细胞瘤细胞株SH-SY5Y具有抑制生长和诱导凋亡的作用，其机制可能与抑制P13K／Akt/mTOR信号通路有关。

Mechanism of effects of rapamycin on growth inhibition and inducing apoptosis of human neuroblastoma cell line

Objective To investigate the effects of rapamycin on growth inhibition and inducing apoptosis of human neuroblastoma cell line SH-SYSY and the target signaling pathway of phosphatidylinositol-3-kinase/ protein kinase B/the mammalian target of rapamycin （PIBK/Akt/mTOR）. Methods The rapamycin intervention group contained SH-SYSY cells treated by the rapamycin of different concentrations （10, 15, 20 pmol/L）. The negative control group contained SH-SYSY cells by adding the same volumes of DMSO. The blank control group was also established. The CCK-8 assay, flow cytometry assay, and annexin V-FITG/PI double staining were used to determine cell proliferation rate, cell cycle, and apoptosis rate of SH-SYSY cells, respectively. Western blotting was performed to determine expressions and changes of phosphorylation level of cleaved caspase-3 and upstream and downstream molecules of signaling pathway of PI3K/Akt/roTOR, including PI3Kp85, Akt, roTOR, and 4E-BP1. Results Compared to the negative control group and blank control group, the cell growth rate of rapamycin intervention group decreased significantly （P〈0.05 or P〈0. 01）. Cell cycle analysis further showed that rapamycin could arrest the cell cycle at G0/G1 phase （P〈0.05）. Early and late apoptosis in SH-SYSY cells treated by rapamycin were evident through annexin V-FITC/PI staining assay （P〈0. 01）. The results of Western blotting showed that the expression level of cleaved caspase-3 of rapamycinintervention group was higher than that of the negative control group and blank control group; the activities of upstream and downstream molecules of signaling pathway of PI3K/Akt/mTOR were inhibited; and the phosphorylation levels of PI3K, Akt, roTOR, and 4E-BP1 were significantly decreased. Conclusion The rapamycin can inhibit the cell growth and induce the apoptosis of human neuroblastoma cell line SH-SY5Y. The mechanism may be relevant to the suppression of signaling pathway of PI3K/Akt/roTOR.