减毒沙门菌携带survivin特异性siRNA对肝细胞癌的生长抑制作用及其相关机制

J4 ›› 2011, Vol. 37 ›› Issue (5): 855-860.

减毒沙门菌携带survivin特异性siRNA对肝细胞癌的生长抑制作用及其相关机制

陈丽华^1,2,沈维高²,芦丽莉²,陈丽松²,刘艳波²

1. 北华大学学报编辑部|吉林吉林 132033；2. 北华大学基础医学院病理生理学教研室|吉林吉林 132013

收稿日期:2011-07-01 出版日期:2011-09-28 发布日期:2011-09-27
通讯作者: 刘艳波（Tel：0432-64608106，E-mail: liuyanbobeihua@163.com） E-mail:liuyanbobeihua@163.com
作者简介:陈丽华（1968-）|女|吉林省吉林市人|副编审|医学硕士|主要从事肝癌的发病机制研究。
基金资助:
吉林省教育厅科研基金资助课题（2011146）

Growth inhibition of survivin specific siRNA carried by attenuated Salmonella typhimurium on hepatocellular
carcinoma and its mechanism

CHEN Li-hua^1,2,SHEN Wei-gao²,LU Li-li²,CHEN Li-song²,LIU Yan-bo²

1. Department of Edition of Journal of Beihua University,Jilin 132033,China;2. Department of Pathophysiology,School of Basic Medical Sciences,Beihua University,Jilin 132013,China

Received:2011-07-01 Online:2011-09-28 Published:2011-09-27

摘要/Abstract

摘要：

目的：探讨survivin特异性siRNA对肝细胞癌HepG2细胞的生长抑制作用及减毒沙门菌携带survivin siRNA对裸鼠肝癌皮下移植瘤生长抑制作用及其相关机制。方法：针对survivin的siRNA表达载体，脂质体和空质粒2个对照组分别转染肝细胞癌HepG2细胞后，应用RT-PCR和Western blotting分别检测细胞survivin mRNA及蛋白表达水平，应用MTT法检测细胞增殖能力，流式细胞术检测细胞周期和凋亡水平。构建裸鼠肝癌皮下移植瘤模型，将携带survivin特异的 siRNA载体的减毒沙门菌腹腔注射，观察其对移植瘤的生长抑制作用，应用免疫组织化学染色观察肿瘤细胞内survivin表达，应用TUNEL法观察肿瘤细胞凋亡情况。结果：转染48 h后，实验组细胞survivin mRNA分别为脂质体组和空质粒组的（32.0±6.2)%和（27.8%±4.6)%，蛋白表达水平分别为（27.3±4.0)%和（20.8±2.8)%；实验组细胞的生长速度是脂质体组的（29.2±4.8)%；流式细胞术检测，实验组G1期细胞比例明显高于2个对照组，S期细胞比例减少(P<0.05)。成功构建裸鼠肝癌皮下移植瘤模型，将携带survivin-siRNA质粒减毒沙门菌腹腔注射后，实验组动物移植瘤生长速度相对缓慢，与对照组比较差异有统计学意义（P<0.05）；免疫组织化学检测，实验组动物肿瘤细胞内survivin表达明显降低；TUNEL实验，肿瘤细胞凋亡率明显增高，与对照组比较差异有统计学意义(P<0.05）。结论： survivin　特异性siRNA体外可明显抑制肝癌HepG2细胞增殖，并促进其凋亡，将携带survivin siRNA质粒减毒沙门菌腹腔注射后可明显抑制肝癌皮下移植瘤的生长，其机制可能与细胞凋亡有关。

关键词: RNA干扰；survivin；肝肿瘤；HepG2；细胞凋亡

Abstract:

Objective To explore the growth inhibition of survivin specific siRNA on hepatocellula carcinoma HepG2 cells in vitro and to investigate the growth inhibition of survivin specific siRNA carried by attenuated Salmonella typhimurium on subcutaneous hepatocellular carcinoma in nude mice and its mechanism.Methods According to the expression of vector siRNA, the lipofectin control group and plasmid control group were transfected to HepG2 cells.The expressions of survivin mRNA and protein were detected by RT-PCR and Western blotting respectively.The proliferation abilities of cells were measured by MTT,and the cell cycle and apoptosis level were detected by flow cytometry (FCM).The subcutaneous hepatocellular carcinoma model was set up in nude mice and the survivin specific siRNA carrid by attenuated Salmonella typhimurium was injected into abdominal of mice and the growth inhibition of cells was observed.The expression of survivin was detected by immunohistochemistry and the apoptosis was detected by TUNEL staining.Results After 48 h transfection,the level of survivin mRNA in the cells in siRNA group was (32.0±6.2)% and (27.8±4.6)% of those in lipofectin control group and plasmid control group,respectively;and expression of survivin protein was (27.3±4.0)% and (20.8±2.8)% of inlipofecton control group and plasmid control group, respectively.The proliferation rate of the cells in survivn siRNA group was (29.2±4.8)% of that in lipofectin group.The cell proportion in G1 phase in survivin siRNA group was significantly higher than that in two control groups,while the cell proportion in S phase was lower(P<0.05).Subcutaneous hepatocellular carcinoma model was successfully set up in nude mice, and after injected with survivin siRNA carried by specific attenuated Salmonella typhimurium the growth rate was lower in siRNA group than the others (P<0.05).The expression of survivin in survivin siRNA group was lower than that in controls detected by immunohistochemical method. The apoptotic rate of the cells was increased obviously than those in controls detected by TUNEL (P<0.05).Conclusion Survivin specific siRNA could inhibit the proliferation of liver cancer HepG2 cells and promote the apoptosis in vitro and survivin specific siRNA carried by attenuated Salmonella typhimurium could inhibit the growth of liver subcutaneous tumor and the mechanism may be related to the apoptosis.

Key words: RNA interference;survivin;liver neoplasms;HepG2;cell apoptosis

中图分类号:

陈丽华,沈维高,芦丽莉,陈丽松,刘艳波. 减毒沙门菌携带survivin特异性siRNA对肝细胞癌的生长抑制作用及其相关机制[J]. J4, 2011, 37(5): 855-860.

CHEN Li-hua,SHEN Wei-gao,LU Li-li,CHEN Li-song,LIU Yan-bo. Growth inhibition of survivin specific siRNA carried by attenuated Salmonella typhimurium on hepatocellular
carcinoma and its mechanism[J]. J4, 2011, 37(5): 855-860.

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