骨桥蛋白促进腹膜透析相关腹膜纤维化的作用研究  

Effect of osteopontin on promoting peritoneal dialysis-associated fibrosis

作　　者：张玉婷 王宏[1] 谢大洋[1] 张庆涛 周建辉[1] ZHANG Yuting;WANG Hong;XIE Dayang;ZHANG Qingtao;ZHOU Jianhui(Department of Nephrology,the First Medical Center,Chinese PLA General Hospital,National Key Laboratory of Kidney Diseases,National Clinical Research Center for Kidney Diseases,Beijing Key Laboratory of Kidney Diseases Research,Beijing 100853,China)

机构地区：[1]解放军总医院第一医学中心肾脏病医学部,肾脏疾病全国重点实验室,国家慢性肾病临床医学研究中心,肾脏疾病研究北京市重点实验室,北京100853

出　　处：《解放军医学院学报》2024年第8期878-885,共8页Academic Journal of Chinese PLA Medical School

摘　　要：背景腹膜纤维化是腹膜透析(peritoneal dialysis,PD)超滤衰竭最常见的原因,骨桥蛋白(osteopontin,OPN)在很多器官中起促纤维化作用,但其在腹膜透析相关腹膜纤维化中的作用尚未被证实。目的探究骨桥蛋白促进腹膜透析相关腹膜纤维化的作用。方法体外分别使用1.25μg/mL、2.5μg/mL、5μg/mL的骨桥蛋白刺激人腹膜间皮细胞,观察间皮细胞形态。体外培养小鼠巨噬细胞,0.5μg/mL脂多糖(lipopolysaccharide,LPS)刺激,检测骨桥蛋白的表达。对3种siRNA序列进行筛选,用筛选得到的序列合成体内用siRNA。将20只小鼠随机分为0.9%氯化钠注射液对照组、腹膜透析对照组、siRNA载体对照组和siRNA实验组,每组5只。造模方式:0.9%氯化钠注射液对照组,小鼠腹腔注射100 mL/kg 0.9%氯化钠注射液,1次/d;腹膜透析对照组,小鼠腹腔注射100 mL/kg含有4.25%葡萄糖的腹膜透析液,1次/d;siRNA载体对照组和siRNA实验组,在进行每日100 mL/kg含有4.25%葡萄糖的腹膜透析液注射的同时,将5 nmol体内用siRNA空载体或5 nmol体内用siRNA溶解至腹膜透析液中进行腹腔注射,1次/3 d;所有动物模型均造模4周。取小鼠右侧壁腹膜组织,通过病理切片测量壁腹膜厚度、间皮下胶原纤维面积和微血管数量,Western blot检测α-SMA和E-钙黏蛋白表达量,免疫组化进行α-SAM染色,评估腹膜纤维化程度。结果添加外源性骨桥蛋白刺激后,人腹膜间皮细胞排列杂乱,细胞形状变细长,向外延伸出角状,呈间充质样改变。LPS刺激的巨噬细胞中骨桥蛋白表达增加(P<0.05)。siRNA转染后抑制骨桥蛋白表达,其中si-002添加后骨桥蛋白减少更显著(P<0.05)。在腹膜透析对照组中,使用CD68、骨桥蛋白进行免疫荧光共染色,证实骨桥蛋白在巨噬细胞中表达。与腹膜透析对照组相比,siRNA实验组壁腹膜间皮下厚度减小(P<0.05),间皮下微血管数量减少(P<0.05),胶原蛋白沉积面积减小(P<0.05),壁腹膜�Background Peritoneal fibrosis is the most common cause of peritoneal dialysis(PD)ultrafiltration failure.Osteopontin(OPN)plays a pro-fibrotic role in other organs,but its role in peritoneal dialysis-related fibrosis has not been confirmed.Objective To confirm that OPN promotes the development of peritoneal fibrosis in PD.Methods Human peritoneal mesothelial cells were stimulated with 1.25μg/mL,2.5μg/mL,and 5μg/mL OPN in vitro,and the morphology of mesothelial cells was observed.Mouse macrophages were cultured in vitro and stimulated with 0.5μg/mL Lipopolysaccharide(LPS)and the expression of OPN was detected.Three siRNA sequences were screened,and the selected sequences were used to synthesize in vivo siRNA.Twenty mice were randomly divided into saline control group,peritoneal dialysis control group,siRNA vector control group and siRNA experimental group.The modeling method was as follows:100 mL/kg normal saline was injected into the mice in the normal saline group once a day;In the peritoneal dialysis control group,100 mL/kg peritoneal dialysate containing 4.25% glucose was injected into the peritoneal cavity once a day.The siRNA carrier group and the siRNA experimental group were injected with 100 mL/kg of peritoneal dialysate containing 4.25% glucose daily,and at the same time,the empty carrier of 5 nmol in vivo siRNA or the siRNA in vivo was dissolved into the peritoneal dialysate for intraperitoneal injection,once every 3 days.All animal models were constructed for 4 weeks.The right parietal peritoneum of mice was collected,and the peritoneal thickness,subcutaneous collagen fiber area and number of microvessels were measured by pathological section.The expression of α-SMA and e-cadherin were detected by WB andα-SAM staining was performed by immunohistochemistry to evaluate the degree of peritoneal fibrosis.Results After the addition of exogenous OPN,human peritoneal mesothelial cells arranged disorderly,and the cell shape became elongated and extended into angular shape,showing mesenchymal-like changes

关 键 词：腹膜透析 腹膜纤维化 骨桥蛋白 巨噬细胞 小干扰RNA 

分 类 号：R5[医药卫生—内科学]