类风湿性关节炎状态下大鼠滑膜细胞P-糖蛋白表达及功能变化研究  

P-glycoprotein expression and functional changes in rat synoviocytes under rheumatoid arthritis condition

作　　者：黄陈[1] 余丹[2] 杨海峰[1] HUANG Chen;YU Dan;YANG Hai-feng(Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China;School of Medicine&Holistic Integrative Medicine,Nanjing University of Chinese Medicine,Nanjing 210023,China)

机构地区：[1]江苏农牧科技职业学院,江苏泰州225300 [2]南京中医药大学医学院整合医学学院,江苏南京210023

出　　处：《中国病理生理杂志》2022年第4期712-719,共8页Chinese Journal of Pathophysiology

基　　金：江苏省自然科学基金项目(No.BK20161368);江苏高校“青蓝工程”资助项目(苏教师[2018]12号);江苏农牧科技职业学院科技创新团队资助项目(No.NSF2021TC02);江苏省“333高层次人才培养工程”资助项目。

摘　　要：目的:研究类风湿性关节炎状态下大鼠滑膜细胞P-糖蛋白(P-glycoprotein,P-gp)表达及功能的变化,并探讨其与炎症的关系。方法:(1)在体实验采用雌性Wistar大鼠,随机分为对照组和造模组(n=12),造模组建立II型胶原诱导的关节炎(collagen-induced arthritis,CIA)模型,Western blot检测大鼠滑膜细胞P-gp的表达。(2)动物细胞体外培养实验采用II型胶原酶法分离培养正常大鼠原代滑膜细胞,分为正常培养液(DMEM)组和巨噬细胞条件培养液(macrophage-conditioned medium,MCM)组,源于RAW 264.7细胞的MCM模拟体内炎症状态,Western blot法检测大鼠滑膜细胞P-gp蛋白表达水平,RT-qPCR法检测大鼠滑膜细胞多药耐药基因1a(multidrug re⁃sistance gene 1a,Mdr1a)和Mdr1b的mRNA表达水平,以罗丹明123(rhodamine 123,Rho123)及氨甲蝶呤(metho⁃trexate,MTX)为探针评价细胞中P-gp的功能。结果:与对照组大鼠相比,CIA造模组大鼠滑膜细胞P-gp表达显著降低(P<0.01);II型胶原酶消化法可以成功获得大鼠原代滑膜细胞,免疫荧光显示第3代滑膜细胞98%以上表达vimentin蛋白;维拉帕米(50和100μmol/L)能显著增加大鼠滑膜细胞中Rho123的浓度(P<0.01);与DMEM组相比,MCM组滑膜细胞Rho123摄取量显著升高(P<0.01),MTX摄取量亦显著升高(P<0.05),分别增加了40%和37%,P-gp蛋白表达水平显著降低(P<0.01),Mdr1a的mRNA表达水平显著降低(P<0.01),Mdr1b的mRNA表达水平无显著差异。结论:类风湿性关节炎状态下大鼠滑膜细胞P-gp表达及功能显著降低,可能与局部炎症微环境有关。AIM:To investigate the changes of P-glycoprotein(P-gp)expression and function in rat synovio⁃cytes under rheumatoid arthritis condition,and to explore its relationship with inflammation.METHODS:(1)For in vi⁃vo experiments,female Wistar rats were randomly divided into control and model groups(n=12).Type II collagen-in⁃duced arthritis(CIA)model was established.The protein expression of P-gp in rat synoviocytes was measured by Western blot.(2)For in vitro experiments,primary synoviocytes from normal rats were isolated by collagenase type II digestion and cultured.The cells were divided into two main groups:normal medium(DMEM)group and macrophage-conditioned medium(MCM)group.The MCM from RAW 264.7 macrophages mimicked the inflammatory condition in vivo.The pro⁃tein expression of P-gp and the mRNA levels of multidrug resistance gene 1a(Mdr1a)and Mdr1b in rat synoviocytes were measured by Western blot and RT-qPCR.Rhodamine 123(Rho123)and methotrexate(MTX)were used as probes to evaluate the function of P-gp in synoviocytes.RESULTS:The P-gp expression in the synoviocytes of the rats with CIA modeling was significantly decreased compared with control rats(P<0.01).Immunofluorescence showed that more than 98%of the synoviocytes at passage 3 expressed vimentin protein.Verapamil(50 and 100μmol/L)significantly increased the concentration of Rho123 in rat synoviocytes(P<0.01).Compared with DMEM group,the synoviocytes in MCM group showed significantly higher Rho123 uptake and MTX uptake,which increased by 40%(P<0.01)and 37%(P<0.05),re⁃spectively.The P-gp protein and Mdr1a mRNA expression in the synovial cells of the rats treated with MCM was signifi⁃cantly decreased(P<0.01).The Mdr1b mRNA level was not significantly different in the synoviocytes of the rats in MCM group compared with DMEM group.CONCLUSION:The expression and function of P-gp in rat synoviocytes under rheu⁃matoid arthritis condition were significantly reduced,which may be related to the local inflammatory microenvironment.

关 键 词：类风湿性关节炎 P-糖蛋白 滑膜细胞 巨噬细胞 

分 类 号：R593.22[医药卫生—内科学] R965[医药卫生—临床医学]