摘要
目的探讨炎性细胞因子与ATP结合盒(ABC)转运蛋白表达调控的相关性。方法建立Ⅱ型胶原诱导的小鼠胶原诱导性关节炎(CIA)模型,将造模成功的14只小鼠于初次免疫后7周处死取材。根据滑膜病理评分将CIA组分为轻度病变组、中重度病变组,选取4只健康小鼠作为正常对照组。通过流式多种蛋白定量技术(CBA)检测血清中IL-1β,IL-2,IL-6,IL-10,TNF-α,IL-17的水平。采用反转录(RT)-PCR检测脾淋巴细胞中的P-糖蛋白(P-gp),乳腺癌耐药蛋白(BCRP),多药耐药相关蛋白1(MRP1)的mRNA表达水平。进一步分析不同炎性细胞因子与耐药蛋白的相关性及其浓度依赖性,选出最具相关性的蛋白进行关节滑膜免疫组织化学定位分析。采用Mann-Whitney U检验,非参数Kruskal-Wallis H和Spearman相关分析。结果① 3组IL-6相比,中重度CIA组[45.03(38.87,64.02) pg/ml]高于轻度组[25.31(15.15,29.27) pg/ml]、正常对照组[7.75(5.14,9.17) pg/ml](Z=14.383,P<0.05);3组TNF-α比较,相较于正常对照组[22.81(20.84,28.17) pg/ml],轻度[45.00(32.76,58.51) pg/ml]、中重度CIA组[45.00(39.78,58.95) pg/ml]均升高(Z=8.375,P<0.05),中重度CIA组与轻度CIA组相比,差异无统计学意义(P>0.05)。②中重度CIA组MRP1 mRNA[2.07(1.77,2.22) pg/ml]高于轻度CIA组[1.32(1.08,1.49) pg/ml]、正常对照组[1.08(0.65,1.30) pg/ml](Z=12.634,P<0.05)。3组P-gp,BCRP的mRNA差异无统计学意义。MRP1与P-gp有相关性(r=0.635,P=0.015)。③ MRP1 mRNA的表达与IL-6呈正相关(r=0.711,P=0.004)。④ MRP1的表达与IL-6水平有一定浓度依赖性。⑤与正常对照组相比,CIA模型组膝关节与踝关节滑膜组织细胞胞质/胞膜呈棕黄色,提示MRP1表达阳性。结论在CIA关节炎模型中,ABC转运蛋白家族中MRP1表达与滑膜病变严重程度相关,且其与血清中炎性因子IL-6水平高度相关。
Objective We investigated the correlation between inflammatory cytokines and drugresi-stant proteins. Methods Fourteen DBA1 mice were successfully induced by collagen and Freund's adjuvant. According to the scores of synovial pathology, the collagen-induced arthritis (CIA) group was divided into mild, moderate-severe groups, another four mice were selected as controls. The mRNA expressions of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance protein 1 (MRP1) in splenic lymphocyte cells were measured by reverse transcription-polymerase chain reaction (RT-PCR). The concentrations of inter-leukin (IL)-1β, IL-2, IL-6, IL-10, umor necrosis factor (TNF)-α, IL-17 in serum were detected by Cytometric Bead Array (CBA). The correlation between different inflammatory cytokines and these proteins were analyzed, then one of the proteins which were most related with cytokines by immunohisto-chemical (IHC) in the synovium was studied. Data were analyzed by the Mann-Whitney U test, Kruskal-Wallis H test and spearman analysis. Results ① Compared with normal controls, the levels of IL-6 and TNF-α in the serum of mild CIA group, moderate-severe CIA group were significantly increased {IL-6 controls:[7.75(5.14, 9.17) pg/ml];mild CIA:[25.31(15.15, 29.27) pg/ml];modeate-severe CIA:[45.03(38.87, 64.02) pg/ml]. TNF-α: controls:[22.81(20.84, 28.17) pg/ml];mild CIA:[45.00(32.76, 58.51) pg/ml];modeate-severe CIA:[45.00(39.78, 8.95) pg/ml]}(Z=14.383, P<0.05;Z=8.375, P<0.05). Compared with the mild CIA group, the level of IL-6 in serum was significantly increased in the moderate-to-severe CIA group (P<0.05), but there was no signigicant difference in the TNF-α level (P>0.05).② In the spleen lymphocytes, there was no significant difference in the mRNA expression level of P-gp and BCRP among the groups, but the mRNA expression level of MRP1 was significantly increased (Z=12.634, P<0.05). Compared with the mild CIA group, the MRP1 mRNA in the moderate-severe CIA group was higher, the difference was significant (Z=12.634, P<0.05). There was a correlation between mRNA expression of MRP1 and P-gp (r=0.635, P=0.015).③ The mRNA expression of MRP1 was positively correlated with IL-6 level (r=0.711, P=0.004).④ The expression of MRP1 in normal group, low-level IL-6 group and high-IL-6 level group were as follows:[1.08(0.65, 1.30)],[1.32(1.08, 1.49)],[2.07(1.77, 2.22)] respectively.⑤ Compared with the controls, the cytoplasm/membrane of the knee and ankle joint synovial tissue in the CIA group was yellowish-brown, which indicated that MRP1 expression was positive. Conclusion In the CIA arthritis model, synovial tissue lesion is not only related to inflammatory cytokines, but also related to MRP1 expression in the ATP-binding cassette (ABC) transport protein family, and it is proved that IL-6 is highly correlated to MRP1.
作者
梁洁
赵向聪
牛红青
穆艳飞
李照华
李小峰
王彩虹
Liang Jie;Zhao Xiangcong;Niu Hongqing;Mu Yanfei;Li Zhaohua;Li Xiaofeng;Wang Caihong
出处
《中华风湿病学杂志》
CAS
CSCD
北大核心
2019年第3期153-159,I0001,共8页
Chinese Journal of Rheumatology
基金
国家自然科学基金(81471618)
山西省重点研发计划(社会发展领域)项目(201803D31-119).
