PTEN过表达及其突变对活化肝星状细胞纤维状肌动蛋白的影响

Effects of wild-type PTEN overexpression and its mutation on F-actin in activated hepatic stellate cells

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Westernblot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（Ca2＋）浓度的变化。多组间比较采用单因素方差分析，组问比较采用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=1783．486，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垆〉0．05）。HSC内Ca2＋相对浓度：Ad—PTEN组（251．60±90．88）及Ad—G129E组（352．18±146．01）较对照组（1953．95±132．99）及Ad-GFP组（1937．57±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泸〉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Ca2＋浓度；并且，野生型PTEN的作用与其突变体G129E无明显差异。

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 isothioeyanate （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 inCa＂ 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-GI29E group had significant reductions in the relative concentration of Ca2＋ 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.