退行性骨关节炎软骨细胞差异表达蛋白及功能的研究  

Differential proteomic analysis and function study of osteoarthritic chondrocytes

作　　者：宋登新[1] 曹云[1] 何小文[1] 丁徐[1] 黄涛[1] 祁军[2] 

机构地区：[1]上海市第一人民医院宝山分院骨科,200940 [2]武汉华中科技大学同济医学院附属同济医院骨科

出　　处：《中华实验外科杂志》2015年第8期1953-1956,共4页Chinese Journal of Experimental Surgery

基　　金：上海市科学技术委员会科研计划项目课题医学引导项目（124119b0500）

摘　　要：目的分析骨关节炎（OA）与正常人关节软骨细胞差异表达蛋白质图谱，筛选与OA相关的蛋白质并探讨其功能。方法培养OA和正常人关节软骨细胞（OAC和NAC），蛋白质组学比较和筛选OAC和NAC蛋白质表达的差异。构建高迁移率族蛋白1（HMGBl）RNA干扰的真核表达载体Pgenesil-1／HMGBl小干扰RNA（siRNA）转染至OAC中，酶联免疫吸附法（ELISA法）检测转染后OAC上清液中白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）表达。结果体外培养OAC和NAC呈多角形或梭形，甲苯胺蓝染色出现紫色异染颗粒。蛋白质组学研究获得6个在OAC中表达上调的蛋白，分别参与细胞骨架构成、转录调控、胶原合成和物质代谢等过程。成功构建siRNA表达载体Pgenesil-1／HMGBlsiRNA，转染OAC后高迁移率族蛋白1（HMGBl）蛋白含量（0．14±0．03）明显低于对照组（Pgenesil-1／OAC：0．52±0．04，P〈0．05）和空白组（OAC：0．49±0．07，P〈0．05），Pgenesil-1／HMGB1 siRNA／OAC上清液中IL-1β和TNF—α[（25．23±11．39）、（287．36±118．36）ng／L]明显低于Pgenesil-I／OAC[（63.23±5．38）、（655．00±49．27）ng／L]和OAC[（69．71±6．76）、（591．94±75．89）ns／L]，差异有统计学意义（P〈0．05）。结论HMGBl与OA密切相关，下调HMGBl基因，能够明显抑制软骨细胞IL-1β和TNF-α的表达。Objective To investigate differential protein expression profiles of human osteoarthritic chondroeytes （OAC） and normal articular chondrocytes （NAC）, screen osteoarthritis associated proteins and investigate their function. Methods OAC and NAC were cultured in vitro. Proteomies were applied to screen and identify differentially expressed proteins in the OAC and NAC. To construct and identify eukary- oti expression vector containing human Pgenesil - 1/high mobility group box - 1 （ HMGB1 ） small imerfering RNA （siRNA） and the vector was transfected into OAC. Levels of interleukin - 1β （ IL - 1β） and tumor necrosis factor-α （TNF -α） in the supematants were measured by enzyme -linked immunosorbent assay （ELISA）. Results OAC and NAC cultured in vitro were polygonal or fusiform and were stained with purple particles by toluidine blue staining. Six differential expressed proteins, which were correlated with cystoskeleton, transcription regulation, collagen synthesis and cellularmetabolism were identified higher expression in OAC by proteomics. The recombinant plasmid Pgenesil - 1/HMGB1 siRNA was success- fully constructed. The HMGB1 in Pgenesil- 1/HMGB1 siRNA/OAC group （0. 14 ± 0. 03）was significant- ly lower than that in the control group （ Pgenesil - 1/OAC ： 0. 52 ± 0. 04, P 〈 0. 05 ） and blank group （ OAC ： 0. 49 ± 0. 07, P 〈 0. 05 ）. IL - 1β and TNF - α in supematants of the Pgenesil - 1/HMGB1 siRNA/OAC group [ （25.23 ± 11.39）, （287. 36 ± 118. 36） ng/L] were significantly lower than those in Pgenesil-1/OAC group [ （63.23 ±5.38）, （655.00 ±49.27） ng/L] and OAC group [ （69.71 ± 6.76）, （ 591.94 ± 75. 89 ） ng/L] by ELISA, there were significant differences between them （ P 〈 0. 05）. Conclusion HMGB1 is closely related to the Osteoarthritis. Suppress the expression of HMGB1 gene could significantly inhibit IL - 1β and TNF -α in the OAC.

关 键 词：骨关节炎 软骨细胞 蛋白质组学 RNA干扰 高迁移率族蛋白1 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]