| 徐媛媛,闻广华,张冲,等.选择性上调 pSmad 3C/3L基因对人肝星状细胞增殖及 α平滑肌肌动蛋白、纤维酶原激活物抑制剂?1和Ⅰ型胶原蛋白表达的影响[J].安徽医药,2019,23(12):2351-2355. |
| 选择性上调 pSmad 3C/3L基因对人肝星状细胞增殖及 α平滑肌肌动蛋白、纤维酶原激活物抑制剂?1和Ⅰ型胶原蛋白表达的影响 |
| Effect of selective up?regulation of pSmad 3C/3L on LX?2 cell’s proliferation and α?SMA，PAI?1 and Collagen?I protein expression |
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| DOI：10.3969/j.issn.1009?6469.2019.12.005 |
| 中文关键词: 肝硬化 肝星状细胞 质粒 转染 Smad3蛋白质 转化生长因子 β 脂质体 细胞增殖 印迹法，蛋白质 |
| 英文关键词: Liver cirrhosis Hepatic stellate cells Plasmids Transfection Smad3 Protein Transforming growth factor beta Liposomes Cell proliferation Blotting,western |
| 基金项目:国家自然科学基金面上项目(81573652，81874354) |
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| 中文摘要: |
| 目的观察选择性上调 pSmad 3C/3L基因对人肝星状细胞(LX?2)增殖及 α平滑肌肌动蛋白(α?SMA)、纤维酶原激活物抑制剂 ?1(PAI?1)和 Ⅰ型胶原蛋白(Collagen?Ⅰ)表达的影响。以进一步探究 pSmad 3C/3L在肝纤维化进展中的作用机制。方法采用脂质体转染的技术(FuGENE? HD)向 LX?2细胞转染野生型 Smad3基因(Smad3 WT)、 Smad3连接区磷酸化位点突变的基因(Smad3 EPSM)及 Smad3 C末端磷酸化位点突变的基因(Smad3 3S?A)3种质粒，选择性上调 LX?2细胞中 pSmad 3C/3L的表达水平，以蛋白质印迹法(Western Blot)验证转染效率并检测 LX?2细胞中 α?SMA、PAI?1和 Collagen?Ⅰ的蛋白表达水平。 MTT法检测选择性上调 pSmad 3C/3L对 LX?2细胞增殖能力的影响。结果转化生长因子 ?β1与 LX?2对照组相(TGF?β1)刺激 1h后，比，转染 3种质粒组 Smad3蛋白表达水平均升高［(0.48±0.02)(0.57±0.03)(0.60±0.02)，(0.59±0.03)； P＜0.05］；与 Smad3 WT组相比，转染 Smad3 EPSM质粒组 pSmad3C蛋白表达水平明显，升高［(0.33±0，.02)比(0.44±0.01)P＜0.05］，转染 Smad3 3S? A质粒组 pSmad3L蛋白表达水平明显升高［(0.36±0.01)比(0.42±0.02)，P＜0.05］。 MTT结果显示，转染，Smad3 EPSM质粒可抑制 LX?2细胞增殖，而转染 Smad3 3S?A质粒可促进 LX?2细胞的增殖［吸光度值(0.48±0.03)比(0.93±0.05)P＜0.05］。 TGF?β1刺激 12 h后， α?SMA、PAI?1和 Collagen?Ⅰ在 3种质粒转染组中呈现出差异表达(P＜0.05)。结论转染 3种质，粒成功地选择性上调了 LX?2细胞中 pSmad 3C/3L的表达，选择性高表达 pSmad3L可进一步促进 TGF?β1诱导的细胞增殖反应、促进 α?SMA、PAI?1和 Collagen?Ⅰ的表达，选择性高表达 pSmad3C可抑制细胞增殖反应、降低 α?SMA、PAI?1和 Collagen?Ⅰ的表达；提示 TGF?β1诱导的 Smad3不同位点磷酸化在 LX?2细胞增殖和 α?SMA，PAI?1，Collagen?Ⅰ蛋白表达的调控中发挥重要作用，为肝纤维化的逆转提供了新思路。 |
| 英文摘要: |
| Objective To observe the effect of selective up?regulation of pSmad 3C/3L on LX?2 cell proliferation and the expres?sions of alpha smooth muscle actin(α?SMA)，plasminogen activator inhibitor?1(PAI?1)and Collagen?Ⅰ，so as to further investi? gate the functional mechanism of pSmad 3C/3L in the progression of hepatic fibrosis.Methods Three plasmids，Smad3 WT(wild type Smad3 gene)，Smad3 EPSM(Smad3 link region phosphorylation site mutator gene)and Smad3 3S?A(Smad3 C?terminal phos? phorylation site mutator gene)，were transfected in LX?2 cells by using liposome transfection technique(FuGENE? HD).The pS? mad 3C/3L expression was selectively up?regulated，and Western blot was used to investigate the transfection efficiency and the ex? pressions of α?SMA，PAI?1 Collagen?Ⅰin LX?2 cells.MTT assay was used to detect the effect of selectively up?regulated pSmad 3C/3L on the proliferation of LX?2 cells.Results Compared with the LX?2 control group，after 1 h of transforming growth factor?β1(TGF?β1)stimulation，the levels of Smad3 expression were increased in the three plasmid transfected groups［(0.48±0.02)，(0.57± 0.03)(0.60±0.02)，(0.59±0.03)］(P＜0.05).Compared with the Smad3 WT group，the level of pSmad3C expression was signifi? cantlyin，creased in the Smad3 EPSM group［(0.33±0.02)vs.(0.44±0.01)，P＜0.05］，and the level of pSmad3L was significantly increased in the Smad3 3S?A group［(0.36 ± 0.01)vs.(0.42 ± 0.02)，P＜0.05］.The results of MTT showed that the transfected Smad3 EPSM plasmid inhibited the proliferation of LX?2 cells，while the transfected Smad3 3S?A plasmid promoted the prolifera?tion of LX?2 cells［absorbance value:(0.48±0.03)vs.(0.93±0.05)，P＜0.05］.After 12 h of TGF?β1 stimulation，α?SMA，PAI?1， Collagen?Ⅰ showed different expressions in the three plasmid transfection groups(P＜0.05).Conclusions We succeeded in selec? tively up?regulating pSmad 3C/3L expression in LX?2 cells by transfecting three plasmids.The selective up?regulation of pSmad3Lpromoted cell proliferation induced by TGF?β1and the expressions of α?SMA，PAI?1 and Collagen?Ⅰ in LX?2 cells；the selective up?regulation of pSmad3C inhibited cell proliferation and the expressions of α?SMA，PAI?1 and Collagen?Ⅰ in LX?2 cells.Different site phosphorylation of Smad3 induced by TGF?β1 plays an important role in LX?2 cell proliferation and the regulation of α?SMA，PAI?1，Collagen?Ⅰ expressions，which provides a new idea for the reversal of liver fibrosis. |
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