沉默微小RNA-218表达保护STZ诱导的糖尿病大鼠肾组织

发布时间：2018-05-15 05:24

本文选题：微小RNA- + 慢病毒载体　；参考：《中国病理生理杂志》2017年07期

【摘要】：目的:探讨沉默微小RNA-218(microRNA-218,miR-218)表达对链脲佐菌素(streptozotocin,STZ)诱导的糖尿病肾病大鼠肾脏组织的保护作用及其可能机制。方法:采用单次腹腔注射STZ(50 mg/kg)方法制备糖尿病大鼠模型并构建miR-218短发夹RNA(short hairpin RNA,shRNA)慢病毒载体。SD大鼠被随机分为健康对照组、糖尿病模型组、空载慢病毒组及miR-218-shRNA组。于自动生化仪上检测不同时点(4、8和12周)大鼠血糖、24h尿蛋白量、血清肌酐(serum creatinine,SCr)及血尿素氮(blood urea nitrogen,BUN)含量。实时荧光定量PCR(RTqPCR)检测肾脏组织miR-218的表达。RT-qPCR和Western blot检测血红素氧合酶1(heme oxygenase-1,HO-1)、肾病蛋白(nephrin)和p38丝裂原激活的蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)的mRNA及蛋白表达水平。Caspase-3活性检测试剂盒检测caspase-3活性。末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling,TUNEL)法检测肾脏组织细胞凋亡。结果:与健康对照组相比,STZ处理后大鼠miR-218表达水平显著升高。同时模型大鼠的血糖、24 h尿蛋白量、SCr及BUN含量显著升高(P0.05);模型大鼠肾脏组织中HO-1和nephrin的mRNA和蛋白表达水平显著降低,而p38 MAPK蛋白的磷酸化水平显著升高;另外,模型大鼠肾脏组织中的caspase-3活性也显著升高。模型大鼠感染miR-218-shRNA后,miR-218表达水平显著下降并可以显著逆转上述效应。miR-218-shRNA组肾脏组织细胞的凋亡水平显著低于糖尿病模型组及空载慢病毒组。结论:miR-218参与了糖尿病大鼠的肾脏损伤,慢病毒载体沉默其表达能有效抑制肾脏组织细胞的凋亡,提示miR-218可以作为糖尿病肾病的基因治疗靶点。
[Abstract]:Aim: to investigate the protective effect of silencing microRNA-218mRNA-218miR-218 on renal tissue of streptozotocincinin-induced diabetic nephropathy (DN) rats and its possible mechanism. Methods: diabetic rats were induced by single intraperitoneal injection of STZ(50 mg / kg and miR-218 short hairpin RNA(short hairpin RNAs were constructed. SD rats were randomly divided into healthy control group, diabetes model group, no-load lentivirus group and miR-218-shRNA group. 24 hours urine protein, serum creatinine (SCR) and blood urea nitrogen (urea nitrogenn bun) were measured at different time points (4 weeks and 12 weeks). Real-time quantitative PCR was used to detect the expression of miR-218 in renal tissues. RT-qPCR and Western blot were used to detect the expression of heme oxygenase 1(heme oxygenase-1 (HO-1), p38 mitogen-activated protein kinase p38 (p38 mitogen-activated protein kinasep38 MAPK) and the activity of caspase-3. Terminal deoxynucleotidyl transferase-mediated dUTP Nick end labeling terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to detect apoptosis in renal tissue. Results: compared with the control group, the expression of miR-218 increased significantly in rats treated with STZ. At the same time, the contents of SCR and BUN in 24 h urine protein of model rats were significantly increased, while the expression of mRNA and protein of HO-1 and nephrin in renal tissues of model rats was significantly decreased, while the phosphorylation level of p38 MAPK protein was significantly increased. The activity of caspase-3 in kidney tissue of model rats was also significantly increased. The expression of miR-218 decreased significantly after miR-218-shRNA infection in model rats, and the apoptosis level of renal tissue cells in miR-218-shRNA group was significantly lower than that in diabetic model group and no-loaded lentivirus group. Conclusion: 1 miR-218 is involved in renal injury in diabetic rats, and lentivirus vector silencing its expression can effectively inhibit the apoptosis of renal tissue cells, suggesting that miR-218 can be used as a target of gene therapy for diabetic nephropathy.
【作者单位】：西安市中心医院肾脏内科;铜川矿务局中心医院肾脏内科;
【分类号】：R587.2;R692.9

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