徐静琳　韩颖敏　孔祥静　陶海英

[關键词] 糖尿病肾病;自噬;细胞凋亡;细胞增殖;上皮-间质转化

[中图分类号] R587.24          [文献标识码] A          [文章编号] 1673-9701（2021）17-0029-05

Effect of overexpressed LncRNA H19 on the biological behavior of podocytes of patients with diabetic nephropathy and its related mechanism

XU Jinglin1   HAN Yingmin1   KONG Xiangjing1   TAO Haiying2

1.Department of Nephrology，the First People′s Hospital of Taizhou City in Zhejiang Province， Taizhou   318020， China; 2.Department of Endocrinology，the First People′s Hospital of Taizhou City in Zhejiang Province， Taizhou   318020， China

[Abstract] Objective To investigate the effect of overexpressed lncRNA H19 on the biological behavior of podocytes in patients with diabetic nephropathy and its related mechanism. Methods Mouse podocyte cell line MPC5 was selected. After the incubation，they were divided into the normal group，high glucose group，silencing group and overexpression group，and the four groups of cells were intervened respectively. MTT assay was used to detect cell proliferation. TUNEL assay was used to detect cell apoptosis. Transwell chamber assay was used to detect cell invasion. The cell wound scratch assay was used to detect cell migration. RT-PCR was used to detect the expressions of lncRNA H19，E-cadherin and vimentin. Western blot was used to detect the relative expressions of apoptosis-related proteins PI3K，Akt，mTOR and autophagy-related proteins Beclin-1， PINK1， Parkin. Results At 24 h，48 h and 72 h，compared with the normal group， the other three groups showed higher rate of the cell proliferation and lower rate of apoptosis. Compared with the high glucose group and silencing group，the proliferation rate of cells in overexpression group was higher and the apoptosis rate was lower （P<0.05）. Compared with the normal group，the other three groups had higher invasion index and migration index in cells. Compared with the high glucose group and silencing group，the overexpression group exhibited lower invasion index and migration index in cells（P<0.05）. Compared with the normal group， the expression of E-cadherin in the other three groups was higher， the expression of lncRNA H19 and vimentin was lower，the relative expression of PI3K，Akt and mTOR was lower，and the relative expression of Beclin-1，PINK1 and Parkin was higher. Compared with the high glucose group and silencing group， the expression of E-cadherin in overexpression group was lower， the expression of LncRNA H19 and vimentin was higher，the relative expression of PI3K，Akt and mTOR was higher，and the relative expression of Beclin-1，PINK1 and Parkin was lower （P<0.05）. Conclusion Overexpressed lncRNA H19 can inhibit the apoptosis of podocytes and promote the proliferation， invasion and migration of podocytes in diabetic nephropathy. The mechanism of action may be that the overexpression of lncRNA H19 can target the regulation of the expressions of apoptosis-related proteins and autophagy-related proteins，thus playing a protective role in podocytes in diabetic nephropathy.

[Key words] Diabetic nephropathy; Autophagy; Cell apoptosis; Cell proliferation; Epithelial-mesenchymal transition

糖尿病肾病是一种临床常见的糖尿病慢性并发症，对患者身体健康、生活质量造成严重威胁[1-2]。我国人口老龄化现象不断发展，糖尿病发病率一直居高不下，每年新增糖尿病肾病患者也随之不断上升[3-4]。糖尿病肾病患者主要临床表现为肾功能损伤、蛋白尿。有研究表示，糖尿病肾病患者蛋白尿主要是由于足细胞损伤导致的[5-6]。糖尿病肾病临床治疗难度较高，若病情得不到及时有效的控制，可能会进一步发展为终末期肾病，对患者生命安全造成威胁[7-8]。但目前临床并无特效的糖尿病肾病治疗手段，因此寻找新的糖尿病肾病治疗靶点成为目前临床医学的研究热点。本研究设计高糖刺激小鼠足细胞系MPC5实验，靶向调控LncRNA H19表达，旨在探讨调控LncRNA H19表达对糖尿病肾病足细胞的保护作用，现报道如下。

1 材料与方法

1.1 材料来源

①研究细胞：小鼠足细胞系MPC5（上海弘顺生物科技有限公司）。②主要试剂：小鼠抗兔LncRNA H19抗体（Hyclone公司）;大鼠抗小鼠E-cadherin、vimentin抗体（Invitrogen公司）;小鼠抗大鼠Beclin-1抗体（Gibco公司）;大鼠抗兔PI3K、Akt抗体（BD公司）;大鼠抗小鼠mTOR抗体（Sigma公司）;兔抗小鼠PINK1、Parkin抗体（Selleck公司）。

1.2 细胞培养及分组处理

取冻存的MPC5细胞，40℃环境中火浴处理，充分摇晃均匀后将其置于2 mL培养基（100 g/L青霉素、链霉素及10%胎牛血清）内，进行离心处理，3000 r/min，重悬处理后进行细胞传代，将细胞培养基置于5%CO2培养箱中培养。将MPC5细胞分为正常组、高糖组、沉默组、过表达组。……