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目的:探讨腺病毒介导的shRNA下调第10号染色体缺失的磷酸酶和张力蛋白同源物基因(PTEN)表达对体外培养的大鼠活化肝星状细胞(HSCs)增殖和凋亡的影响及其信号转导机制。方法:体外培养活化HSCs,以腺病毒为载体将靶向PTEN的shRNA干扰重组体转染至体外活化的大鼠HSCs;四甲基偶氮唑盐(MTT)法检测HSCs增殖;末端转移酶标记技术(TUNEL)及流式细胞术测定HSCs凋亡;Western blotting方法检测PTEN、Bax、Bcl-2、Akt、p-Akt、ERK1/2及p-ERK1/2蛋白表达情况;实时荧光定量PCR方法检测PTEN、Akt及ERK1 mRNA表达情况。结果:(1)靶向PTEN的RNA干扰重组腺病毒成功感染体外活化HSCs,并在一定范围内呈时间依赖性地促进HSCs增殖,腺病毒感染HSCs后72 h,HSCs凋亡率显著下降(P<0.05);(2)Bax表达降低,Bcl-2表达增加(P<0.05);(3)p-Akt和p-ERK1/2蛋白表达显著增加(P<0.05);而Akt蛋白及其mRNA、ERK1蛋白及其mRNA表达均无显著改变(P>0.05)。结论:RNA干扰下调PTEN基因表达可能通过Bcl-2/Bax途径促进体外活化HSCs增殖并抑制其凋亡,此外,RNA干扰下调PTEN基因表达促进p-Akt和p-ERK1/2表达增多,提示PTEN可能通过影响PI3K/Akt和ERK1/2信号通路而在调控HSCs增殖和凋亡中发挥重要作用。
AIM: To investigate the effects of adenovirus mediated shRNA knockdown of PTEN expression on the proliferation and apoptosis of rat activated hepatic stellate cells (HSCs) in vitro. Its signal transduction mechanism. Methods: The activated HSCs were cultured in vitro. The recombinant adenovirus vector targeting PTEN was transfected into HSCs in vitro. The proliferation of HSCs was detected by MTT assay. The expression of terminal transferase The expression of PTEN, Bax, Bcl-2, Akt, p-Akt, ERK1 / 2 and p-ERK1 / 2 protein were detected by Western blotting and real-time fluorescence quantitative PCR Detection of PTEN, Akt and ERK1 mRNA expression. Results: (1) The recombinant adenovirus targeting PTEN successfully infected activated HSCs in vitro and promoted the proliferation of HSCs in a certain range in a time-dependent manner. The apoptosis rate of HSCs was significantly decreased at 72 h after adenovirus infection (P (P <0.05). (2) The expression of Bax decreased and the expression of Bcl-2 increased (P <0.05). (3) The protein expressions of p-Akt and p-ERK1 / 2 increased significantly , ERK1 protein and its mRNA expression had no significant change (P> 0.05). Conclusion: The down-regulation of PTEN gene expression by RNA interference may promote the proliferation and inhibit the apoptosis of HSCs through Bcl-2 / Bax pathway. In addition, RNA interference down-regulates the expression of PTEN gene to promote the expression of p-Akt and p-ERK1 / 2, It may play an important role in the regulation of HSCs proliferation and apoptosis by affecting the PI3K / Akt and ERK1 / 2 signaling pathways.