论著(基础研究)

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

  • 陈 盛 ,
  • 顾 硕 ,
  • 徐 敏 ,
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  • 上海交通大学 医学院附属上海儿童医学中心外科, 上海 200127
陈 盛(1982—), 男, 硕士, 主治医师; 电子信箱: chensheng_2007@hotmail.com。

网络出版日期: 2014-05-13

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

  • CHEN Sheng ,
  • GU Shuo ,
  • XU Min ,
  • et al
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  • Department of Pediatric Surgery, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

Online published: 2014-05-13

摘要

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(PI3K/Akt/mTOR)的影响。方法 用不同浓度的雷帕霉素(10、15、20 μmol/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;Western blotting检测caspase-3剪切体及PI3K/Akt/mTOR信号通路上下游分子PI3Kp85、Akt、mTOR、4E-BP1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);G0/G1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)。Western blotting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caspase-3剪切体表达水平较高;PI3K/Akt/mTOR信号通路上下游分子的活性均被抑制,PI3Kp85、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PI3K/Akt/mTOR信号通路有关。

本文引用格式

陈 盛 , 顾 硕 , 徐 敏 , . 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[J]. 上海交通大学学报(医学版), 2014 , 34(4) : 481 . DOI: 10.3969/j.issn.1674-8115.2014.04.016

Abstract

Objective To investigate the effects of rapamycin on growth inhibition and inducing apoptosis of human neuroblastoma cell line SH-SY5Y and the target signaling pathway of phosphatidylinositol-3-kinase/protein kinase B/the mammalian target of rapamycin (PI3K/Akt/mTOR). Methods The rapamycin intervention group contained SH-SY5Y cells treated by the rapamycin of different concentrations (10, 15, 20 μmol/L). The negative control group contained SH-SY5Y 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-FITC/PI double staining were used to determine cell proliferation rate, cell cycle, and apoptosis rate of SH-SY5Y 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/mTOR, including PI3Kp85, Akt, mTOR, 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-SY5Y 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 rapamycin intervention 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, mTOR, 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/mTOR.
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