肿瘤坏死因子受体2突变载体对巨噬细胞炎症反应的影响

Effect of TNFR2 Mutation Vector on Inflammatory in Macrophages

目的探讨肿瘤坏死因子受体超家族1B(TNFRSF1B)196位基因多态性(T突变为G)与巨噬细胞TNF/TNFR2信号通路介导炎症反应的相关性。方法运用基因重组技术构建真核表达载体pcDNA6.0-TNFR2196Met和pcDNA6.0-TNFR2196Arg,采用脂质体转染法分别转染至巨噬细胞中,转染48 h后使用杀稻瘟菌素抗性筛选4周,建立稳定转染细胞系。将酶消化法培养的巨噬细胞分为四组:空白对照组、pcDNA6.0空质粒组、pcDNA6.0-TNFR2196Met组及pcDNA6.0-TNFR2196Arg组。采用酶切及测序法鉴定重组质粒,RT-PCR检测肿瘤坏死因子受体2(TNFR2)、cIAP1、cIAP2mRNA的变化;Western blot检测p-JNK、cIAP1、cIAP2、TNFR2及核因子κB(NF-κB)的蛋白表达;ELISA检测细胞上清液可溶性TNFR2(sTNFR2)、白细胞介素1β(IL-1β)和白细胞介素6(IL-6)水平。结果酶切测序结果显示成功构建了TNFR2基因196Met和196Arg表达载体。转染到巨噬细胞后,与空白对照组和pcDNA6.0空质粒组比较,pcDNA6.0-TNFR2196Arg组、pcDNA6.0-TNFR2196Met组TNFR2、cIAP1、cIAP2、IL-1β、IL-6和p-JNK的表达增高(P<0.05);与pcDNA6.0-TNFR2196Met组比较,pcDNA6.0-TNFR2196Arg组TNFR2、cIAP1、cIAP2、IL-1β、IL-6和pJNK的表达明显降低(P<0.05),TNFR2196Arg介导的NF-κB活性显著降低。结论成功构建稳定表达TNFR2196Met、TNFR2196Arg载体,TNFR2突变通过TNF/TNFR2信号通路介导炎症反应,可能是参与慢性炎症性疾病的作用机制。

Aim To discuss the correlation of the polymorphism of tumor necrosis factor receptor superfamily 1B （TNFRSFI B） in the 196ed gene （T mutation for G） to the inflammation reaction mediated by the macrophage TNF-TNFR2 signaling pathways. Methods Genetic recombination technology was used to bulid eukaryotic expression vector pcD- NA6. O-TNFR2196ue＇ and pcDNA6. 0-TNFR2196A~, together with pcDN6. 0 being transferred to macrophage by liposome trausfection method respectively. In order to establish stable transfection cell lines, blasticidin was used to select them for 4 weeks after transfected 48 h. Digest the macrophages by pancreatic enzyme and divide them into four groups： control group, pcDNA6.0 empty plasmid group, pcDNA6. 0-TNFR2196Met group and pcDNA6.0-TNFR2~96A~ group. TNFR2, cIAP~, cIAP2 mRNA were detected by RT-PCR, the expression of p-JNK, clAPl , cIAP2, TNFR2 and NF-KB were detec- ted by Western blot, the level of sTNFR2, IL-113 and IL-6 in cell supernatant fluid were detected by ELISA. Results TNFR2 gene 196Mot and 196Arg expression vector were successfully constructed which was verified by enzyme sequencing meth- od. After transfected to marcophages, TNFR2, cIAP~, cIAP2, IL-113, IL-6 and p-JNK expression were increased （P 〈 0. 05） in TNFR2196Ars and TNFR2196Met overexpression group compared with the blank control group and empty plasmid group. Each index decreased obviously in mutant TNFR2196A^s cell group compared with TNF2~96M^t cell group, especially,the reduction of NF-KB activity mediated by TNFR2196A＇~ was significant. Conclusions Successfully building stable ex- pression TNFR2l^Ars and TNFR2196Met vector will lay the foundation for the next inflammation research. The mutation of TNFR2 mediates inflammatory disease through TNF/TNFR2 signaling pathways, which is molecular mechanism of chronic inflammatory disease.