肾间质纤维化中胶原/DDR2信号活化对肾成纤维细胞增殖和迁移功能影响的实验研究  

An experimental study was conducted to examine the impact of activated collagen/DDR2 signaling on the migration and proliferation of kidney fibroblasts in renal interstitial fibrosis

作　　者：陈惠燕 吴瑶 黄宗炫 卜歆 王庆惠 纪辉涛 陈银珍 赵虎[1] Huiyan Chen;Yao Wu;Zongxuan Huang;Xin Bo;Qinghui Wang;Huitao Ji;Yinzhen Chen;Hu Zhao(Department of General Surgery,Fuzong Clinical Medical College of Fujian Medical University,900th Hospital of Joint Logistics Support Force,PLA,Fuzhou 350025,China;Department of General Surgery,Fuzhou General Teaching Hospital,Fujian University of Traditional Chinese Medicine(900TH Hospital of Joint Logistics Support Force),Fuzhou 350122,China;Department of Biochemistry and Molecular Biology,School of Basic Medicine,Air Force Military Medical University,Xi'an 710032,China)

机构地区：[1]福建医科大学福总临床医学院普通外科,福州350025 [2]福建中医药大学福总教学医院普通外科,福州350122 [3]空军军医大学基础医学院生物化学与分子生物学教研室,西安710032

出　　处：《中华细胞与干细胞杂志（电子版）》2024年第5期294-302,共9页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：国家自然科学基金(82003002);福建省自然科学基金面上项目(2018J01337)。

摘　　要：目的探讨胶原/盘状结构域受体2(DDR2)信号活化对肾间质纤维化进程中肾成纤维细胞增殖和迁移功能的影响。方法采用SPF级雄性C57BL/6小鼠,通过单侧输尿管梗阻(UUO)术建立肾小管间质纤维化模型,并收集病理样本和新鲜组织,进行免疫组化、Western blot等检测。通过培养人肾成纤维细胞,将shDDR2基因慢病毒及其阴性对照慢病毒、OE-DDR2慢病毒及其阴性对照慢病毒分别转染至KFB细胞,经5μg/mL嘌呤霉素筛选后,构建稳转细胞株,并使用浓度为10μg/mL胶原处理48 h。对照组1:shCtrl+胶原组(shDDR2阴性对照,未下调DDR2表达但添加胶原处理);对照组2:Control+胶原组(OE-DDR2阴性对照,未过表达DDR2但添加胶原处理);实验组1:shDDR2+胶原组(DDR2基因敲低并添加胶原处理);实验组2:OE-DDR2+胶原组(DDR2基因过表达并添加胶原处理);实验组3:在实验组2基础上,使用浓度为1μmol/L的达沙替尼处理,所有组别处理时间均为48 h。采用qRT-PCR法检测DDR2 mRNA的表达水平,采用Western blot法检测DDR2蛋白的水平。采用CCK-8和BrdU实验进行细胞增殖检测。采用HE染色和免疫组织化学染色方法观察细胞定位和病理变化。两组间比较采用独立样本t检验,方差不齐性采用t&apos;检验;多组间比较采用单因素方差分析,两两比较组间方差齐时采用LSD-t检验,方差不齐时采用Tamhane&apos;s T2检验。结果DDR2在小鼠肾组织中定位于肾间质成纤维细胞中,且与胶原呈共定位关系。与shCtrl+胶原组相比,sh-DDR2+胶原组的细胞增殖速度(0.28±0.00比0.38±0.01)减缓(P<0.05),Transwell转移[(6.00±1.00比21.66±0.57)个]降低(P<0.05)。与Control+胶原组相比,OE-DDR2+胶原组的细胞增殖速度(0.43±0.01比0.39±0.01)加快(P<0.05),Transwell迁移[(32.66±0.57比22.33±0.57)个]增加(P<0.05),而经达沙替尼处理后,细胞增殖速度(0.36±0.01比0.39±0.01)降低。结论胶原/DDR2信号活化显著促进KFB细胞的增殖及迁移,�Objective To investigate the effect of collagen/DDR2 signaling on the proliferation and migration of renal fibroblasts during renal interstitial fibrosis.Methods A renal tubulointerstitial fibrosis model was established in SPF male C57BL/6 mice via unilateral ureteral obstruction(UUO),and pathological samples and fresh tissues were obtained for Western blotting and immunohistochemistry.Human kidney fibroblasts were cultivated,followed by the transfection of KFB cells with shDDR2 and its negative control lentivirus,OE-DDR2 and its negative control lentivirus,and screening with 5μg/mL puromycin.A stable transfection cell line was then created and exposed to 10μg/mL collagen for 48 hours.Control Group 1:shCtrl+Collagen group(shDDR2 negative control,without downregulation of DDR2 expression but with collagen treatment);Control Group 2:Control+Collagen group(OE-DDR2 negative control,without overexpression of DDR2 but with collagen treatment);Experimental Group 1:shDDR2+Collagen group(DDR2 gene knockdown with collagen treatment);Experimental Group 2:OE-DDR2+Collagen group(DDR2 gene overexpression with collagen treatment);Experimental Group 3:Based on Experimental Group 2,treated with 1μmol/L dasatinib,with all groups being treated for 48 hours.The expression level of DDR2 mRNA was detected by RT-qPCR,and the level of DDR2 protein was assessed by Western blot.Cell proliferation was evaluated using the CCK-8 assay and the BrdU assay.Cell localization and pathological changes were observed by HE staining and immunohistochemical staining methods.Comparisons between two groups were performed using independent samples t-test,and the t&apos;test was used when variances were unequal.For comparisons among multiple groups,one-way ANOVA was applied,with LSD-t test used for pairwise comparisons when variances were homogeneous,and Tamhane&apos;s T2 test was used when variances were heterogeneous.Results DDR2 is localized in renal stromal fibroblasts in mouse kidney tissue,and has a colocalization relationship with collagen.Compa

关 键 词：肾间质纤维化 肌成纤维细胞 胶原 DDR2 治疗靶点 

分 类 号：R692[医药卫生—泌尿科学]