【作者】 王莹；

【导师】 马瑞霞；

【作者基本信息】 青岛大学 ， 内科学（肾病）（专业学位）， 2020， 硕士

【摘要】 目的通过构建2型糖尿病(T2DM)动物模型及高糖刺激体外培养足细胞,测定T2DM大鼠模型肾组织、体外培养足细胞中microRNA-26a-5p(miR-26a-5p)、nephrin、瞬时受体电位阳离子通道蛋白6(TRPC6)及凋亡相关蛋白cleaved-caspase-3和bax/bcl-2的表达水平和足细胞凋亡情况,并应用miR-26a-5p mimics、inhibitor转染高糖刺激体外培养的足细胞,观察上述指标的变化,探讨miR-26a-5p对TRPC6介导的糖尿病肾病(diabetic nephropathy,DN)足细胞损伤的作用及其可能机制,为DN足细胞的保护提供新的临床思路和治疗靶点。方法体内实验选取30只SPF级、雄性Wistar大鼠,随机分为两组,其中20只用于构建T2DM大鼠模型,具体方法为采用高糖高脂喂养并联合腹腔注射小剂量链脲佐菌素(streptozotocin,STZ,30 mg/kg),另设正常对照组(NC组)10只。对于建模达到要求的大鼠留取血、尿及肾脏标本,测定两组大鼠24小时尿白蛋白排泄率(UAE)、尿肌酐(Ucr)、内生肌酐清除率(CCr)、空腹胰岛素(FINS)、空腹血糖(FBG)、胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)、血肌酐(Scr)等指标;光镜、电镜观察肾脏病理及足细胞结构改变,采用免疫组织化学方法观察nephrin、TRPC6分布情况;Western blot法测定nephrin、TRPC6、凋亡相关蛋白cleaved-caspase-3和bax/bcl-2的蛋白表达;qRT-PCR测定nephrin、TRPC6、cleaved-caspase-3和bax/bcl-2 mRNA及miR-26a-5p的表达,肾组织足细胞凋亡情况采用TUNEL法检测。体外实验将永生化小鼠足细胞(MPC5)分为高糖组(HG组)、正常糖对照组(NG组)和渗透压处理组(HM组),并对高糖组足细胞应用miR-26a-5p mimics、inhibitor进行体外转染,采用Western blot和qRT-PCR分别测定转染前后nephrin、TRPC6、cleaved-caspase-3和bax/bcl-2蛋白及mRNA的表达水平,流式细胞技术检测足细胞凋亡率。结果1.与NC组相比,T2DM大鼠KW/BM、24h UAE、CCr、FBG、LDL等各生化指标明显升高(均P<0.05),HDL明显下降(P<0.05),伴有明显的胰岛素抵抗;2.光镜下T2DM大鼠肾小球体积肥大,系膜细胞及基质增生,电镜可见基底膜增厚,足突紊乱,融合率明显增加(P<0.05);3.免疫组化结果显示,较NC组大鼠相比,T2DM大鼠肾组织中TRPC6表达明显增加,而nephrin表达明显减低(二者均P<0.05);4.qRT-PCR及Western blot结果显示,DM大鼠肾组织及HG组足细胞中miR-26a-5p、nephrin、bcl-2 m RNA及蛋白表达显著减少(P<0.05),TRPC6、cleaved-caspase-3、bax mRNA及蛋白表达显著增多,TUNEL法及流式细胞术显示高糖条件刺激下足细胞凋亡明显增加(P<0.05);5.体外足细胞经miR-26a-5p mimics转染后nephrin表达较前恢复,TRPC6、cleaved-caspase-3和bax/bcl-2表达较前减少,细胞凋亡明显降低(均P<0.05);而经miR-26a-5p inhibitor转染后,nephrin表达下降,TRPC6、cleaved-caspase-3和bax/bcl-2表达明显增加(均P<0.05)。结论1.高糖刺激可引起足细胞mi R-26a-5p表达明显减少,TRPC6、足细胞凋亡明显增加,特异性增加miR-26a-5p的表达可明显缓解上述改变,抑制miR-26a-5p表达可加重足细胞损伤;2.miR-26a-5p可以通过降低TRPC6表达减少DN足细胞凋亡。更多还原

【Abstract】 Objective To investigate the effect and potential mechanisms of microRNA-26a-5p(miR-26a-5p)on podocyte injury in diabetic nephropathy(DN)by observing the expression of miR-26a-5p,nephrin,transient receptor potential canonical 6(TRPC6),cleaved-caspase-3,bax/bcl-2 and podocyte apoptosis in renal tissue of T2 DM rats and MPC5,providing a new idea about podocyte protection of DN.Methods 20 SPF,8-week male Sprague-Dawley(SD)rats were used for establishing the type 2 diabetic mellitus(T2DM)group by feeding them with high fat and high sugar food for 8 weeks and intraperitoneal injection of low-dose streptozotocin(STZ,30mg/kg).Another 10 normal male SD rats were selected as normal control group(NC).The 24 h urinary albumin excretion rate(UAE),urinary creatinine(Ucr),creatinine clearance rate(CCr)and other major biochemical parameters including the fasting insulin level(FINS),serum creatinine(Scr),fasting blood glucose(FBG),total cholesterol(TC),triglyceride(TG),high density lipoprotein(HDL),low density lipoprotein(LDL)were measured in both groups after establishing the DM models.Light and electron microscope were used to observe the pathology of kidneys and podocyte ultrastructures,respectively.Immunohistochemical method was used to show the distribution of nephrin and TRPC6 protein.To determine the expression of nephrin,TRPC6,apoptotic-associated proteins(cleaved-caspase-3 and bax/bcl-2),western blot was applied.qRT-PCR was used to detect miR-26a-5p and mRNAs of nephrin,TRPC6,cleaved-caspase-3 and bax/bcl-2.Podocyte apoptotic rate was measured using TUNEL method.Cultured immortalized mouse podocytes(MPC5)were divided into three groups: normal glucose group(NG),hypertonic group(HM),high glucose group(HG).Before and after miR-26a-5p mimics and inhibitor transfecting,the expression and m RNAs of nephrin,TRPC6,cleaved-caspase-3 and bax/bcl-2 were detected again using the same methods.Flow cytometry(FCM)was uesd to detect podocyte apoptosis.Results 1.Compared with NC group,rats in DM group exhibited disrupted biochemical conditions,for uAER,CCr,FINS,FBG,TC,TG and LDL were all significantly increased(all P<0.05)while HDL were decreased(P<0.05),along with significant insulin resistance.2.Increased glomeruli volume,mesangial cell proliferation,thickened basement membrane and foot process fusion could be observed by light and electron microscope.3.Immunochemistry results showed a increased expression of TRPC6 and a decreased of nephrin in DM rats than those in NC group(both P<0.05).4.Both qRT-PCR and Western blot results in vivo and in vitro showed a notable increase in the expression of TRPC6,cleaved-caspase-3 and bax/bcl-2 and decrease in the expression of nephrin and miR-26a-5p(P<0.05).TUNEL and flow cytometry indicated higher podocyte apoptosis in DM rats and HG group.5.The expression of TRPC6,cleaved-caspase-3 and bax/bcl-2 were down-regulated and nephrin expression was restored after miR-26a-5p mimics transfection(all P<0.05),while miR-26a-5p inhibitor intervention could aggravate all above changes as the effects of HG on podocytes(all P<0.05).Conclusion 1.Under high-glucose condition,the expression of miR-26a-5p was significantly decreased,whereas TRPC6 and podocyte apoptosis were significantly increased,and mi R-26a-5p mimics transfection significantly ameliorated the above changes.2.In DN,miR-26a-5p can protect podocyte from apoptosis via down-regulating the expression of TRPC6.更多还原