野生型PTEN 过表达对体外活化肝星状细胞内钙离子浓度的影响

Effect of wild-type PTEN over-expression on intracellular calcium ion concentration in activated hepatic stellate cells in vitro

摘要:

目的 探讨野生型第10 号染色体缺失的磷酸酶张力蛋白同源物基因（PTEN）过表达对体外活化肝星状细胞（HSC）内钙离子浓度的影响。方法 体外培养活化大鼠肝星状细胞系（HSC-T6），利用腺病毒载体，将野生型PTEN 基因转染活化HSC ；Western blot 及实时荧光定量聚合酶链反应（qrt-PCR）检测HSC 的PTEN 蛋白及mRNA 表达；采用钙离子荧光探针Rhod-2/AM，于激光扫描共聚焦显微镜下检测HSC 内钙离子浓度变化。实验分组：① Control 组：腺病毒转染时以DMEM 代替病毒液；② Ad-GFP 组：转染表达绿色荧光蛋白（GFP）的对照空病毒Ad-GFP ；③ Ad-PTEN 组：转染携带野生型PTEN 基因并表达GFP 的重组腺病毒Ad-PTEN。结果 野生型PTEN 在活化大鼠HSC 大量表达，3 组HSC 内钙离子浓度比较，差异有统计学意义（P <0.05），Ad-PTEN 组HSC 内钙离子浓度（251.60±90.88）低于Control 组（1 953.95±132.99）及Ad-GFP 组（1 937.57±115.17），而Control 组与Ad-GFP 组之间HSC 内钙离子浓度比较，差异无统计学意义（P >0.05）。结论 野生型PTEN 过表达可降低体外活化大鼠HSC 内钙离子浓度。

Abstract:

Objective To investigate the effect of over-expression of wild-type phosphatase and tensin homology deleted on chromosome ten (PTEN) on the intracellular calcium ion concentration in activated hepatic stellate cells (HSCs) in vitro. Methods Using adenoviral vector, wild-type PTEN gene was transduced into activated rat HSC (HSC-T6) in vitro, and the expressions of PTEN mRNA and protein were detected by qRT-PCR and Western blot. Then with the help of laser scanning confocal microscope (LSCM), the changes of intracellular calcium ion concentration in HSCs were detected using calcium ion fluorescence probe Rhod-2/AM. The cells were grouped as follows: control group, in which the viral medium was replaced by DMEM when HSCs were transfected with adenovirus; Ad-GFP group, in which HSCs were transfected with adenovirus expressing green fluorescent protein (GFP) alone; and Ad-PTEN group, in which HSCs were transfected with adenovirus harboring both wildtype PTEN and GFP genes. Results The wild-type PTEN was successfully transduced into activated HSCs by adenoviral vector. The intracellular calcium ion concentration of the HSCs in the Ad-PTEN group significantly decreased compared with the control group and the Ad-GFP group (P < 0.05). However, no significant difference was observed in the intracellular calcium ion concentration of HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusions The over-expression of wild-type PTEN can significantly reduce the intracellular calcium ion concentration in activated hepatic stellate cells in vitro.