靶向骨桥蛋白的小干扰RNA对MRL/Lpr小鼠狼疮肾炎的治疗作用  

The therapeutic effect of osteopontin siRNA on lupus nephritis of MRL/Lpr mice

作　　者：庞春艳[1,2] 张伟 刘媛[1] 常志芳 王永福[1,2] PANG Chunyan;ZHANG Wei;LIU Yuan;CHANG Zhifang;WANG Yongfu(Department of Rheumatology ＆ Institute of Immunology and Rheumatology, First Affiliated Hospital, Baotou Medical College, Baotou 014010, China;Inner Mongolia Key Laboratory of Autoimmunity, Baotou 014010, China)

机构地区：[1]包头医学院风湿免疫研究所包头医学院第一附属医院风湿免疫科,014010 [2]内蒙古自治区自体免疫学重点实验室

出　　处：《免疫学杂志》2018年第5期393-400,共8页Immunological Journal

基　　金：国家自然科学基金(30960354)

摘　　要：目的观察骨桥蛋白(OPN)的小干扰RNA(si RNA)对SLE动物模型MRL/Lpr小鼠24 h尿蛋白、脾组织中Treg和Th17细胞的表达、肾脏的病理改变的影响,探讨OPN si RNA对狼疮肾炎的治疗作用。方法 25只MRL/Lpr小鼠随机分为对照组、空载体组、OPN-si RNA1组、OPN-si RNA2组及OPN-si RNA3组,每组5只,RT-PCR法检测小鼠肾脏组织中OPN m RNA的表达水平;免疫组化法检测小鼠肾脏组织中OPN蛋白的表达水平;考马斯亮蓝染色法检测24 h尿蛋白含量;ELISA法检测小鼠血清中抗ds DNA抗体水平;流式细胞术检测小鼠脾组织中Treg细胞和Th17细胞的表达水平;病理切片苏木精-伊红(HE)染色法观察5组小鼠肾脏的病理变化。结果 OPN si RNA表达载体可以在MRL/Lpr小鼠的肾脏中表达;注射后6周OPN-si RNA1组、OPN-si RNA2组和OPN-si RNA3组中MRL/Lpr小鼠肾脏组织中OPN m RNA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);MRL/Lpr小鼠的24 h尿蛋白含量和抗ds DNA抗体在治疗前5组没有差异,OPN-si RNA作用后2周和4周小鼠24 h尿蛋白含量和抗ds DNA抗体水平下降,随着OPN-si RNA作用时间的延长小鼠24 h尿蛋白含量和抗ds DNA抗体水平逐渐下降,注射后6周MRL/Lpr小鼠的24 h尿蛋白含量和抗ds DNA抗体开始升高,但仍低于对照组和空载体组,差异有统计学意义(P<0.05);OPN-si RNA作用后MRL/Lpr小鼠脾组织中Th17细胞占CD4+T细胞的百分比明显下降,而Treg细胞占CD4+T细胞的百分比上升,与对照组和空载体组比较差异有统计学意义(P<0.05);OPN-si RNA作用后肾小球减小、肾小球的浸润的炎性细胞减少,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OPN si RNA载体后,OPN m RNA和蛋白的表达水平下降,24 h尿蛋白含量和抗ds DNA抗体水平下降,脾组织中Th17细胞的百分比下降,Treg细胞的百分比上升,肾脏中炎性细胞的浸润减少,说明OPN si RNA可调节狼疮小鼠Th17/To explore the therapeutic effects of osteopontin（OPN） si RNA on upus nephritis, we observed the effect of OPN si RNA on 24 h urinary protein, Treg and Th17 cell and pathological changes in kidney of SLE mice.Total of 25 female MRL/Lpr mice were randomly divided into control group, empty vector group, OPN-si RNA1 group, OPN-si RNA2 group and OPN-si RNA3 group. The expression levels of OPN m RNA in kidney tissue of MRL/Lpr mice were detected by RT-PCR, while the expression levels of OPN protein in kidney tissue of MRL/Lpr mice were detected by immunohistochemistry. The 24 h urinary protein level was detected using the method of coomassie brilliant blue; the level of anti-ds DNA antibody in serum was detected by ELISA. The levels of Treg and Th17 cells in spleen were detected by flow cytometry.At the same time, the pathological changes of the kidney were observed by haematoxylin-eosin（HE） staining. Data showed that OPN-si RNA vector could expressed in kidney of MRL/Lpr. The expression levels of OPN m RNA and protein in kidney were lower in OPN-si RNA group than those in control group and empty vector group on the 6 th week after last injection（P0.05）. The levels of 24 hours urinary protein and anti-ds DNA antibody were lower in OPN-si RNA group than those in control group and empty vector group on 2 nd, 4 thand 6 thweek after injection（P0.05）. The level of Treg cell was higher and Th17 cell was lower in OPN-si RNA group, as compared with control group and empty vector group. The inflammatory cell infiltration of kidney and the glomerular volume were significantly reduced in OPN-si RNA group, as compared with control group and empty vector group. In conclusion, OPN si RNA can suppresses the expression levels of OPN m RNA and protein, and decreases inflammatory cells infiltration, subsequently reduces the content of 24 hours urinary protein, the levels of anti-ds DNA antibody and Th17 cells in spleen and elevate the level of Treg cells. OPN si RNA can also modulate the balance of Treg and Th17 cell. S

关 键 词：狼疮肾炎 骨桥蛋白 小干扰RNA 24 h尿蛋白 抗DSDNA抗体 

分 类 号：R593.2[医药卫生—内科学]