逆转录病毒载体介导的白细胞介素-1Ra基因体外转染兔膝关节软骨细胞表达的研究  

Expression of a recombinant retroviral vector containing IL-1Ra cDNA in chondrocytes from rabbit joint knee

作　　者：林树忠[1] 刘君[1] 向川[2] 卫小春[2] 

机构地区：[1]太原市中心医院骨科,030009 [2]山西医科大学第二医院骨科

出　　处：《中国药物与临床》2009年第8期669-671,共3页Chinese Remedies & Clinics

基　　金：国家自然科学基金(30500515);山西省自然科学基金(2006021045)

摘　　要：目的探讨运用重组逆转录病毒载体介导白细胞介素-1受体拮抗剂(IL-1Ra)体外转染兔膝关节软骨细胞,观察其在关节软骨细胞中的表达情况及其对软骨细胞生物学性状的影响。方法将构建含有目的基因的表达载体PLNCX2-IL-1Ra-GFP体外转染到兔膝关节软骨细胞,采用一氧化氮(NO)检测试剂盒检测基因转染对软骨细胞的影响,酶联免疫吸附试验(ELISA)检测细胞培养上清液中hIL-1Ra的表达情况。结果酶切和测序表明构建成功预期的真核表达载体PLNCX2-IL-1Ra-GFP;稳定转染软骨细胞后免疫荧光倒置显微镜观察到绿色荧光,证明转染成功;培养液中NO含量检测发现基因转染组比非基因转染组高,并且差异有统计学意义(P<0.05);ELISA检测发现基因转染组有一定量的hIL-1Ra表达,未转染组与空载体组均无基因表达,基因转染组与非基因转染组间比较差异有统计学意义(P<0.05)。结论逆转录病毒载体PLNCX2介导的IL-1Ra能有效地转染到兔膝关节软骨细胞并获得稳定表达,同时转染后的软骨细胞增生活跃。为目的基因IL-1Ra转染治疗骨关节炎提供了理论基础。Objective To transfect the chondrocytes of the rabbit knee joint using a recombinant retroviral vector containing IL-1Ra cDNA, and to explore for its expression in and effects on the chondrocytes. Methods PLNCX2-IL- 1Ra-GFP containing targeted gene was used to transfect the knee joint chondrocytes from rabbit. The effects of trans- fection on chondrocytes were assessed with NO-detecting kit, and the expression of hIL-1Ra in cell culture supernatant was detected with ELISA. Results Enzyme digestion showed that PLNCX2-IL-1Ra-GFP was constructed successfully. After transfection, immunofluorescence inverted microscopy revealed green fluorescence in the IL-1Ra group, which indicated the successful transfection. Higher level of NO was found in the transfected group than in non-transfected group witlh statistical significance （P〈0.05）. By ELISA, expression of hlL-1Ra was detected in the transfected group but not the non-transfected group and the empty vehicle group （P〈0.05）. Conclusion IL-1Ra mediated by retrovirus PLNCX2 can successfully transfect knee joint chondrocytes of the rabbit and was expressed stably. Meanwhile, the transfected cells showed active proliferation, which may provide a theoretical basis to treat OA with IL-1Ra.

关 键 词：逆转录病毒科 受体 白细胞介素 转染 基因表达 软骨细胞 

分 类 号：R684.3[医药卫生—骨科学] R346[医药卫生—外科学]