Objective: To investigate the effect of overexpression of wild-type phosphatase and tensin homolog (PTEN) deleted on chromosome 10 and its mutant G129E (exhibiting the activity of protein phosphatase and losing the activity of lipid phosphatase) on F-actin in activated hepatic stellate cells (HSCs) cultured in vitro. Methods: The activated hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and activated HSCs were transfected with adenovirus that carried wild-type PTEN gene and G129E gene using transient transfection. The HSCs were divided into the following groups: control group, which was transfected with DMEM medium instead of virus solution; Ad-GFP group, which was transfected with the empty adenovirus vector with the expression of green fluorescent protein (GFP); Ad-PTEN group, which was transfected with the recombinant adenovirus with wild-type PTEN gene and GFP expression; Ad-G129E group, which was transfected with the recombinant adenovirus with G129E gene and GFP expression. Western blot and quantitative real-time PCR were used to measure the protein and mRNA expression of PTEN in activated HSCs; under a laser scanning confocal microscope (LSCM), phalloidine labeled with the fluorescein tetramethylrhodamine isothiocyanate (TRITC) was used to observe the morphology of HSCs, distribution and fluorescence intensity of F-actin, and changes in pseudopodia and stress fibers, and a calcium fluorescence probe (Rhod-2/AM) was used to measure the changes in Ca(2+) concentration in HSCs. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference test was used for comparison between two groups. Results: Wild-type PTEN and G129E genes were highly expressed in activated HSCs. In the control group and the Ad-GFP group, HSCs had a starlike or polygonal shape, F-actin was reconfigured and formed a large number of stress fibers which stretched across the whole cell, and layered pseudopodia were seen around the cell. In the Ad-PTEN group and the Ad-G129E group, the HSCs had a fusiform shape, F-actin was mainly seen around the cell, a small number of stress fibers were seen inside the cell, and layered pseudopodia around the cell disappeared. The Ad-PTEN group and the Ad-G129E group had significant reductions in the fluorescence intensity of F-actin compared with the control group and the Ad-GFP group (357.67±13.39/377.25±14.55 vs 961.87±27.33/954.68±20.71, F = 1783.486, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05). The Ad-PTEN group and the Ad-G129E group had significant reductions in the relative concentration of Ca(2+) compared with the control group and the Ad-GFP group (251.60±90.88/352.18±146.01 vs 1953.95±132.99/1937.57±115.17, F = 834.988, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05). Conclusion: The overexpressed wild-type PTEN and its mutant G129E can significantly inhibit the formation and reconfiguration of cytoskeletal protein F-actin and reduce the concentration of Ca2+ in activated HSCs in vitro. In addition, there are no significant differences in the above effects between wild-type PTEN and G129E.
目的： 探讨过表达野生型第10号染色体缺失的磷酸酶张力蛋白同源物基因（PTEN）及其突变体G129E（仅保留蛋白磷酸酶活性而丧失脂质磷酸酶活性）对体外培养的活化肝星状细胞（HSC）纤维状肌动蛋白（F-actin）的影响。 方法： 体外培养活化大鼠肝星状细胞（HSC-T6），用瞬时转染技术，将携带野生型PTEN基因及G129E基因的腺病毒转染活化HSC。实验分组如下：对照组：腺病毒转染时以DMEM培养基代替病毒液；Ad-GFP组：转染表达绿色荧光蛋白（GFP）的载体空病毒Ad-GFP；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；Ad-G129E组：转染携带G129E基因并表达GFP的重组腺病毒Ad-G129E。应用Western blot及实时荧光定量PCR技术检测活化HSC的PTEN蛋白及mRNA表达；借助激光扫描共聚焦显微镜（LSCM），应用荧光素四甲基异硫氰酸罗丹明（TRITC）标记的鬼笔环肽检测HSC形态、F-actin的分布及荧光强度、伪足以及应力纤维的变化，并采用钙荧光探针Rhod-2/AM负载检测HSC内钙离子（Ca(2+)）浓度的变化。多组间比较采用单因素方差分析，组间比较采用LSD检验。 结果： 野生型PTEN基因及G129E基因在活化大鼠HSC大量表达；对照组与Ad-GFP组HSC多呈星形或多边体形，F-actin重构形成大量粗大的应力纤维，横跨于整个细胞内，细胞周围可见层状伪足；Ad-PTEN组及Ad-G129E组HSC多为梭形，F-actin主要分布于细胞周边，细胞内可见少量纤细的应力纤维，细胞周边层状伪足消失。F-actin的荧光强度：Ad-PTEN组（357.67±13.39）及Ad-G129E组（377.25±14.55）较对照组（961.87±27.33）及Ad-GFP组（954.68±20.71）明显降低（F = 1 783.486，P<0.05）；而Ad-PTEN组与Ad-G129E组、对照组与Ad-GFP组间的差异均无统计学意义（P>0.05）。HSC内Ca(2+)相对浓度：Ad-PTEN组（251.60±90.88）及Ad-G129E组（352.18±146.01）较对照组（1 953.95±132.99）及Ad-GFP组（1 937.57±115.17）明显降低（F = 834.988，P<0.05）；而Ad-PTEN组与Ad-G129E组、对照组与Ad-GFP组间的差异均无统计学意义(P>0.05)。 结论： 过表达的野生型PTEN及其突变体G129E可显著抑制体外活化肝星状细胞骨架蛋白F-actin的形成及细胞骨架的重构、降低了HSC内Ca(2+)浓度；并且，野生型PTEN的作用与其突变体G129E无明显差异。.
Keywords: Calciumion concentration; Cytoskeletal protein; F-actin; Hepatic stellate cells; PTEN.