CAJ | 학술논문

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(HMGB1) RNA干扰的真核表达载体Pgenesil-1/HMGB1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/HMGB1 siRNA,转染OAC后高迁移率族蛋白1(HMGB1)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-1/OAC[(63.23±5.38)、(655.00±49.27) ng/L]和OAC[(69.71±6.76)、(591.94±75.89) ng/L],差异有统计学意义(P＜0.05).结论 HMGB1与OA密切相关,下调HMGB1基因,能够明显抑制软骨细胞IL-1β和TNF-α的表达.
목적 분석골관절염(OA)여정상인관절연골세포차이표체단백질도보,사선여OA상관적단백질병탐토기공능.방법 배양OA화정상인관절연골세포(OAC화NAC),단백질조학비교화사선OAC화NAC단백질표체적차이.구건고천이솔족단백1(HMGB1) RNA간우적진핵표체재체Pgenesil-1/HMGB1소간우RNA(siRNA)전염지OAC중,매련면역흡부법(ELISA법)검측전염후OAC상청액중백세포개소-1β(IL-1β)화종류배사인자-α(TNF-α)표체.결과 체외배양OAC화NAC정다각형혹사형,갑분알람염색출현자색이염과립.단백질조학연구획득6개재OAC중표체상조적단백,분별삼여세포골가구성、전록조공、효원합성화물질대사등과정.성공구건siRNA표체재체Pgenesil-1/HMGB1 siRNA,전염OAC후고천이솔족단백1(HMGB1)단백함량(0.14±0.03)명현저우대조조(Pgenesil-1/OAC:0.52±0.04,P＜0.05)화공백조(OAC:0.49±0.07,P＜0.05),Pgenesil-1/HMGB1 siRNA/OAC상청액중IL-1β화TNF-α[(25.23±11.39)、(287.36±118.36) ng/L]명현저우Pgenesil-1/OAC[(63.23±5.38)、(655.00±49.27) ng/L]화OAC[(69.71±6.76)、(591.94±75.89) ng/L],차이유통계학의의(P＜0.05).결론 HMGB1여OA밀절상관,하조HMGB1기인,능구명현억제연골세포IL-1β화TNF-α적표체.
Objective To investigate differential protein expression profiles of human osteoarthritic chondrocytes (OAC) and normal articular chondrocytes (NAC),screen osteoarthritis associated proteins and investigate their function.Methods OAC and NAC were cultured in vitro.Proteomics were applied to screen and identify differentially expressed proteins in the OAC and NAC.To construct and identify eukaryoti expression vector containing human Pgenesil-1/high mobility group box-1 (HMGB1) small interfering RNA (siRNA) and the vector was transfected into OAC.Levels of interleukin-1β (IL-1 β) and tumor necrosis factor-α (TNF-α) in the supernatants 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 successfully constructed.The HMGB1 in Pgenesil-1/HMGB1 siRNA/OAC group (0.14 ± 0.03) was significantly 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-1 β and TNF-α in supernatants 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.