CD40 siRNA对MRL／Lpr小鼠狼疮肾炎的治疗作用

摘要

Objective:To observe the effect of CD40 siRNA on the changes in kidney,urinary protein and complement C3 of MRL/Lpr mice and explore its therapy on lupus nephritis.Methods: 16 female MRL/Lpr mice were randomly divided into control group,empty vector group,CD40-siRNA1 group and CD40-siRNA2 group.The vectors expressing siRNA against CD40 were injected by tail veil into MRL/Lpr mice,while MRL/Lpr mice in control group and empty vector group were injected with the same dose of PBS and pGFP-V-RS vector respectively.There were six times injection and every one day.The 24 hours urine output was gathered 24 hours before mice were killed.Fourteen days following administration,these mice were killed,tissue sections of kidney were observed if the signal of siRNA were expressed in kidney.The expression levels of CD40 mRNA and protein in kidney tissue of MRL/Lpr mice were detected by RT-PCR and immunohistochemistry methods respectively.At the same time,the pathological changes of the kidney were observed by haematoxylin-eosin ( HE) staining method.The 24 h urinary protein content was detected using the method of coomassie brilliant blue and the expression levels of complement C3 in serum were detected by Immunoturbidimetric assays.Results:The vector of CD40-siRNA was expressed in kidney of MRL/Lpr.The expression levels of CD40 mRNA and protein in kidney were lower in CD40-siRNA1 group and CD40-siRNA2 group than control group and empty vector group on the 14th day after last injection ( P<0.05).The inflammatory cells infiltration of kidney and some glomerular volume were significantly reduced in CD40-siRNA1 group and CD40-siRNA2 group than control group and empty vector group.The renal tubular swelling was alleviated in CD40-siRNA1 group and CD40-siRNA2 group than control group and empty vector group.The levels of 24 hours urinary protein were lower and the levels of complement C3 were higher in CD40-siRNA1 group and CD40-siRNA2 group than control group and empty vector group ( P<0.05). Conclusion:CD-40 siRNA can suppress the expression levels of CD40 mRNA and protein and decrease inflammatory cells infiltration in kidney of MRL/Lpr.Meanwhile after suppressing expression of CD40 mRNA and protein, it can reduce the content of 24 hours urinary protein and elevate the level of complement C3 in serum,which CD-40 siRNA can delay progress of the disease and protect kidney,so that it has therapy effect on lupus nephritis.%目的：：观察CD40的小干扰RNA( siRNA）对系统性红斑狼疮( SLE）动物模型MRL／Lpr小鼠肾脏、尿蛋白和补体C3的影响，探讨CD40 siRNA对狼疮肾炎的治疗作用。方法：16只MRL／Lpr小鼠随机分为对照组、空载体组、CD40-siRNA1组及CD40-siRNA2组，每组4只，将成功构建的CD40 siRNA表达载体尾静脉注射MRL／Lpr小鼠，对照组和空载体组小鼠分别注射等剂量、等比例的磷酸盐缓冲液( PBS）和pGFP-V-RS空载体。每隔1天1次，共6次。处死前24 h留取24 h的尿量。14 d处死小鼠，肾脏组织切片在荧光显微镜下观察其是否在小鼠肾脏中表达， RT-PCR法检测小鼠肾脏组织中CD40 mRNA的表达水平；免疫组化法检测小鼠肾脏组织中CD40蛋白的表达水平；病理切片苏木精-伊红( HE）染色法观察4组小鼠肾脏的病理变化，考马斯亮蓝染色法检测24 h尿蛋白含量，免疫比浊检测小鼠补体C3的水平。结果：CD40 siRNA表达载体可以在MRL／Lpr小鼠的肾脏中表达。注射后14 d CD40-siRNA1组和CD40-siRNA2组的MRL／Lpr小鼠肾脏组织中CD40 mRNA和蛋白的表达水平明显低于对照组和空载体组，差异有统计学意义( P＜0.05）。 CD40-siRNA1组和CD40-siRNA2组与对照组和空载体组比较肾小球减小，肾小球的浸润的炎性细胞减少，肾小管肿胀程度减轻。 CD40-siRNA1组和CD40-siRNA2组小鼠24 h尿蛋白含量明显下降，而补体C3升高，与对照组和空载体组比较差异有统计学意义( P＜0.05）。结论：MRL／Lpr小鼠注射CD40 siRNA载体后， CD40 mRNA和蛋白的表达水平下降，肾脏中炎性细胞的浸润减少，24 h尿蛋白含量下降，血清中补体C3水平升高，说明CD40 siRNA抑制其mRNA和蛋白后，MRL／Lpr小鼠肾脏炎症反应减轻，尿蛋白含量下降，补体C3升高，提示其可延缓病程的进展，对肾脏起到一定的保护作用，进一步对狼疮肾炎有很好的治疗作用。