PAX2小干扰RNA在UUO大鼠体内的筛选与鉴定

目的梗阻性肾病是由于泌尿道机构和（或）功能改变,阻碍尿液的排出,导致肾实质病理和功能损害的临床综合征。病理学特征主要表现为肾小管萎缩和间质纤维化。文中通过RNA干扰（RNA interference,RNAi）技术,筛选及鉴定有效抑制肾间质纤维化大鼠肾组织PAX2基因表达的小干扰RNA（small interfering RNA,siRNA）序列。方法制作单侧输尿管结扎（unilateral ureteral obstruction,UUO）模型,光镜下观察肾组织病理形态学变化;针对大鼠PAX2 mRNA序列全长（mRNA:NM₀01106361）,设计合成4对siRNA干扰序列和阴性对照序列,为了观察体内转染同时合成FAM标记的阴性对照,以invivo jetPEI体内转染试剂为载体转染至UUO大鼠肾被膜下,利用实时荧光定量PCR及Western blot技术检测肾组织PAX2 mRNA及蛋白表达。结果通过HE和Masson染色观察到UUO模型3 d后肾纤维化表现;UUO模型转染3 d后,荧光显微镜观察到肾小管周围存在绿色荧光,4对干扰序列均有不同程度的抑制肾皮质PAX2 mRNA及蛋白的表达,其中siRNA3位点对PAX2干扰效果最强,mRNA及蛋白抑制分别达到55%和81%。结论成功筛选出1对有效抑制肾间质纤维化大鼠肾小管上皮细胞PAX2表达siRNA,为深入研究肾纤维化的发病机制提供新的手段。

Screening and identification of PAX2-siRNA in the renal tissue of rats with unilateral ureteral obstruction

Objective Obstructive nephropathy is a clinical syndrome of renal parenchymal pathologic and functional injuries caused by structural and/or functional changes of the urinary tract and obstruction of urine discharge,with pathological features of renal tubular atrophy and interstitial fibrosis.This study was to screen and identify siRNA sequences that can effectively inhibit the PAX2 gene expression in the renal tissue of rats with renal interstitial fibrosis.Methods We established a model of UUO（unilateral ureteral obstruction） in Wistar rats,and observed changes in the renal histopathological morphology of the rats under the light microscope.For the PAX2 mRNA sequence（mRNA: NM₀01106361）,we designed 4 pairs of siRNA interference sequences and negative controls,which were transfected into the kidney capsule of the model rats through invivo jetPEI.Then we determined the expressions of PAX2 mRNA and protein in the renal tissue by real time PCR and Western blot.Results HE and Masson staining revealed obvious renal interstitial fibrosis in the UUO rats 3 days after modeling,and fluorescence microscopy exhibited green fluorescence around the renal tubular epithelial cells 3 days after transfection.The expressions of PAX2 mRNA and protein were inhibited in the 4 siRNA interference sequences,mostly strongly in siRNA3,by 55% and 81% respectively.Conclusion We successfully screened a pair of siRNA sequences,which can effectively inhibit the expressions of PAX2 mRNA and protein in the renal tabular cells of the rats with renal interstitial fibrosis.This has provided a new method for further study on the pathogenesis of renal fibrosis.