热休克蛋白70反义寡核苷酸对肝癌细胞增殖及凋亡的影响

Effect of heat shock protein 70 antisense oligonucleotide on proliferation and apoptosis of human hepatocellular carcinoma cell

目的探讨热休克蛋白70反义寡核苷酸(HSP70 ASODN)对肝癌细胞增殖、凋亡的影响及其可能机制。方法合成特异性靶向HSP70 ASODN,并将其转染SMMC-7721细胞;四甲基噻唑蓝(MTT)法检测HSP70 ASODN对SMMC-7721细胞增殖的影响,计算生长抑制率;荧光显微镜观察细胞凋亡形态;流式细胞仪分析细胞周期分布,计算细胞增殖指数;免疫细胞化学观察细胞内HSP70、Bcl-2、Bax的表达。结果 ASODN组细胞生长抑制率明显高于正义寡核苷酸(NSODN)组、转染试剂对照组及空白对照组(P<0.05);形态学观察到典型瘤细胞凋亡特征;流式细胞仪分析证实HSP70 ASODN主要阻止瘤细胞进入S期,细胞增殖指数下降,出现明显的凋亡峰;免疫细胞化学显示可降低细胞内HSP70、Bcl-2水平(P<0.05),提升Bax水平(P<0.05)。结论HSP70 ASODN可抑制人肝癌SMMC-7721细胞的增殖,诱导细胞的凋亡,可能机制为中和了HSP70和细胞周期调节蛋白间的相互作用,导致细胞周期阻滞,打破了肿瘤细胞逃逸凋亡平衡,促使细胞发生凋亡。

Objective To investigate the effect of heat shock protein 70 antisense oligonucleotide on proliferation and apoptosis of human hepatocellular carcinoma cell line SMMC-7721. Methods Human HCC cell line SMMC-7721 was transfected in vitro with heat shock protein antisense oligonucleotide mediated by SofastTM. Cell growth inhibition was determined as percentage by the colorimetric MTr cell viability/proliferation assay. Cell morphological change of apoptosis was observed with fluorescence microscope. Distribution of cell cycle was analyzed by flow cytometry. Immunocytochemistry staining was used to observe the intra-cellular expression of HSP70,Bcl-2,Bax. Results The inhibiting proliferation rate of SMMC-7721 cells increased with the treatment dose and incubation time of HSP70 ASODN. These effects were not observed in the cells treated with normal sense oligonucleotide or SofastTM Transfection Reagent. Apoptotic bodies were observed in SMMC-7721 cells treated with HSPT0 ASODN at a concentration of 400nmol/L for 48h. Cytometric analysis showed a significant decrease in the percentage of G2/M and S-phases while a significant increase in the percentage of G0/G1 among the total cells treated with HSP70 ASODN The results showed that treatment of SMMC-7721 cells with HSP70 ASODN for 48h resulted in a inhibition of HSP70 and Bcl-2 expression, and an increased expression of Bax. While treatment with HSP70 normal sense oligonucleotide for 48h had no effect on expression of these proteins. Conclusion The abrogation of HSP70 by HSP70 ASODN exclusively can inhibit cell growth and induce cell apoptosis.