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[摘要] 目的 转录因子ETS2在器官纤维化的发生及进展中具有重要的作用，研究苦参碱对其在腹膜间皮细胞中表达的影响及其对腹膜纤维化防治的重要意义。 方法 将人腹膜间皮细胞按处理方法分为空白对照组、TGF-β1诱导组、TGF-β1 0.4 mg/mL苦参碱干预组、TGF-β1 0.8 mg/mL苦参碱干预组和TGF-β1 1.2 mg/mL苦参碱干预组，TGF-β1刺激人腹膜间皮细胞并分别予不同浓度的苦参碱干预处理，相对实时荧光定量PCR检测α-SMA和E-cadherin的mRNA表达水平，然后用mRNA-seq分析ETS2的mRNA表达水平，相对实时荧光定量PCR和免疫蛋白印迹验证ETS2的mRNA和蛋白的表达水平。 结果 TGF-β1刺激能上调人腹膜间皮细胞ETS2的mRNA和蛋白的表达水平，上调α-SMA的mRNA表达水平，下调E-cadherin的mRNA表达水平;苦参碱能下调TGF-β1刺激后的ETS2基因mRNA和蛋白的表达水平，下调TGF-β1刺激后α-SMA的mRNA表达水平和上调TGF-β1刺激后的E-cadherin的mRNA表达水平。 结论 苦参碱通过抑制转录因子ETS2的表达阻止人腹膜间皮细胞间充质转化。
[关键词] 苦参碱;ETS2基因;人腹膜间皮细胞;间充质转化
[Abstract] Objective The transcription factor ETS2 plays an important role in the occurrence and progress of organ fibrosis. It is important to study the effect of Matrine on its expression in peritoneal mesothelial cells and its prevention and treatment of peritoneal fibrosis. Methods HPMC were divided into blank control group， TGF-β1-induced group， TGF-β1 0.4 mg/mL matrine intervention group， TGF-β1 0.8 mg/mL matrine intervention group and TGF-β1 1.2 mg/mL matrine intervention group， according to different processing methods. TGF-β1 was applied to stimulate HPMC and treated with matrine intervention of different concentrations. The mRNA expression levels of α-SMA and E-cadherin were detected by relative real-time fluorescent quantitative PCR， then the mRNA expression levels of ETS2 were analyzed by mRNA-seq， and the mRNA and protein expression levels of ETS2 were verified by relative real-time fluorescent quantitative PCR and immunoprotein printing. Results TGF-β1 stimulation can up regulate the expression level of ETS2 mRNA and protein， up regulate the expression level of α-SMA， down regulate the expression level of E-cadherin mRNA. Matrine can down regulate the expression level of ETS2 gene mRNA and protein after TGF-β1 stimulation， down regulate the expression level of α-SMA after TGF-β1 stimulation and up regulate the expression level of E-cadherin after TGF-β1 stimulation. Conclusion Matrine can prevent mesenchymal transformation of HPMC by inhibiting the expression of transcription factor ETS2.
[Key words] Matrine; ETS2 gene; Human peritoneal mesothelial cells; Mesenchymal transformation
人腹膜間皮细胞在终末期肾病（End-stage renal disease，ESRD）患者腹膜透析中具有重要的生物学功能，长期腹膜透析过程中腹膜间皮细胞不断受物理和炎症等刺激会发生间充质转化，最终发展为腹膜纤维化。由此可见，腹膜间皮细胞间充质转化是腹膜纤维化发生发展的早期重要过程[1]。腹膜透析是ESRD患者重要替代疗法之一[2，3]，而腹膜纤维化的发生会导致超滤量下降，患者无法再进行腹膜透析。因此，研究腹膜间皮细胞间充质转化的分子机制并探索抑制其转分化的相关药物，对防治腹膜纤维化具有重要意义。
转化因子ETS2具有E26转换特异性序列，它能与位于靶基因启动子区域的特异性核心GGAA/T序列相互作用，并调控该区域许多基因的转录[4，5]。研究证实ETS2参与多种细胞功能，包括凋亡、增殖、转化、迁移和血管生成[6，7]。此外，还有许多研究报道了ETS2通过调节基质金属蛋白酶（Matrix metalloproteinases，MMPs）和ECM蛋白的表达参与细胞外基质重构及影响肿瘤的发生、创伤的修复和心血管疾病的发生[8-10]。近年来，研究表明ETS2也参与了肺和肾脏等器官纤维化的发生与发展[11，12]。本研究ETS2在TGF-β1诱导的人腹膜间皮细胞间充质转化中的作用分子机制及苦参碱对其表达的影响，为寻求有效抑制腹膜间皮细胞间充质转化的新方法提供科学依据，现报道如下。 1 材料与方法
1.1 材料
人腹膜间皮细胞系（HPMC）购买于广州吉赛科技有限公司，苦参碱购于北京博奥拓达科技有限公司（CAS NO：16837-52-8），ETS2、α-SMA和E-cadherin单克隆抗体购于CST公司。
1.2 方法
1.2.1 实验分组
[关键词] 苦参碱;ETS2基因;人腹膜间皮细胞;间充质转化
[Abstract] Objective The transcription factor ETS2 plays an important role in the occurrence and progress of organ fibrosis. It is important to study the effect of Matrine on its expression in peritoneal mesothelial cells and its prevention and treatment of peritoneal fibrosis. Methods HPMC were divided into blank control group， TGF-β1-induced group， TGF-β1 0.4 mg/mL matrine intervention group， TGF-β1 0.8 mg/mL matrine intervention group and TGF-β1 1.2 mg/mL matrine intervention group， according to different processing methods. TGF-β1 was applied to stimulate HPMC and treated with matrine intervention of different concentrations. The mRNA expression levels of α-SMA and E-cadherin were detected by relative real-time fluorescent quantitative PCR， then the mRNA expression levels of ETS2 were analyzed by mRNA-seq， and the mRNA and protein expression levels of ETS2 were verified by relative real-time fluorescent quantitative PCR and immunoprotein printing. Results TGF-β1 stimulation can up regulate the expression level of ETS2 mRNA and protein， up regulate the expression level of α-SMA， down regulate the expression level of E-cadherin mRNA. Matrine can down regulate the expression level of ETS2 gene mRNA and protein after TGF-β1 stimulation， down regulate the expression level of α-SMA after TGF-β1 stimulation and up regulate the expression level of E-cadherin after TGF-β1 stimulation. Conclusion Matrine can prevent mesenchymal transformation of HPMC by inhibiting the expression of transcription factor ETS2.
[Key words] Matrine; ETS2 gene; Human peritoneal mesothelial cells; Mesenchymal transformation
人腹膜間皮细胞在终末期肾病（End-stage renal disease，ESRD）患者腹膜透析中具有重要的生物学功能，长期腹膜透析过程中腹膜间皮细胞不断受物理和炎症等刺激会发生间充质转化，最终发展为腹膜纤维化。由此可见，腹膜间皮细胞间充质转化是腹膜纤维化发生发展的早期重要过程[1]。腹膜透析是ESRD患者重要替代疗法之一[2，3]，而腹膜纤维化的发生会导致超滤量下降，患者无法再进行腹膜透析。因此，研究腹膜间皮细胞间充质转化的分子机制并探索抑制其转分化的相关药物，对防治腹膜纤维化具有重要意义。
转化因子ETS2具有E26转换特异性序列，它能与位于靶基因启动子区域的特异性核心GGAA/T序列相互作用，并调控该区域许多基因的转录[4，5]。研究证实ETS2参与多种细胞功能，包括凋亡、增殖、转化、迁移和血管生成[6，7]。此外，还有许多研究报道了ETS2通过调节基质金属蛋白酶（Matrix metalloproteinases，MMPs）和ECM蛋白的表达参与细胞外基质重构及影响肿瘤的发生、创伤的修复和心血管疾病的发生[8-10]。近年来，研究表明ETS2也参与了肺和肾脏等器官纤维化的发生与发展[11，12]。本研究ETS2在TGF-β1诱导的人腹膜间皮细胞间充质转化中的作用分子机制及苦参碱对其表达的影响，为寻求有效抑制腹膜间皮细胞间充质转化的新方法提供科学依据，现报道如下。 1 材料与方法
1.1 材料
人腹膜间皮细胞系（HPMC）购买于广州吉赛科技有限公司，苦参碱购于北京博奥拓达科技有限公司（CAS NO：16837-52-8），ETS2、α-SMA和E-cadherin单克隆抗体购于CST公司。
1.2 方法
1.2.1 实验分组