摘要
MCF2L基因突变在骨关节炎的发生与发展过程中扮演重要作用,本研究使用ENSEMBL及COMSIC数据库筛选MCF2L突变位点,构建质粒及腺病毒载体;将小鼠原代滑膜成纤维细胞分为正常对照组,野生型重组腺病毒干预的MCF2L过表达组和突变型重组腺病毒的突变组,检测细胞上清中IL-lβ、MMP-13、PGE2水平及细胞凋亡。通过数据库检测分析发现可将第11位精氨酸作为突变位点;p Yr-ads-4-MCF2L重组质粒及突变型和野生型重组腺病毒包装成功;突变组MCF2L RNA表达是对照组的1.92倍,过表达组是对照组的1.85倍;突变组及过表达组MCF2L蛋白表达水平均显著高于对照组;过表达组上清中PGE2、MMP-13、IL-lβ水平显著低于对照组(p<0.05),突变组上清中PGE2、MMP-13、IL-lβ水平显著高于对照组(p<0.05),且突变组上清中PGE2、MMP-13、IL-lβ水平明显高于过表达组,且差异存在统计学意义(p<0.05);对照组及过表达组细胞凋亡不明显,在1%至10%之间;突变组细胞凋亡率高于对照组及过表达组,凋亡率达60%。滑膜成纤维细胞使用MCF2L基因突变重组腺病毒感染后,PGE2、MMP-13、IL-lβ水平均明显提高,细胞凋亡率明显加大。本研究对MCF2L的单核苷酸多态性对滑膜成纤维细胞的炎症因子及凋亡活性的影响进行分析,探讨骨性关节炎发病过程中MCF2L发挥的作用,探讨MCF2L基因与骨关炎的关系,为临床应用提供依据。
The mutation ofMCF2L gene plays an important role in the occurrence and development of osteoarthri- tis. This research used ENSEMBL and COMSIC database to screen the mutation sites of MCF2L, and to construct plasmid and adenovirus vector. The primary synovial fibroblasts of mice were divided into normal control group, MCF2L over-expression group (intervened by wild type recombinant adenovirus) and mutation group (mutant re- combinant adenovirus) to detect the levels of IL-1β, MMP-13, PGE2 and cell apoptosis in cell supernatant. The results of database analysis indicated that the eleventh arginine could be regarded as the mutation site. pYr-ads-4 MCF2L recombinant plasmid, mutant and wild-type recombinant adenovirus were packaged successfully. The ex- pression ofMCF2L RNA in mutation group was 1.92 times as high as that of control group and the expression of over expression group was 1.92 times as high as that of the control group. The protein expression levels of MCF2Lin mutation group and over-expression group were significantly higher than those of control group. The levels of PGE2, MMP-13, and IL-1βin supernatant in over-expression group were lower than those of the control group significantly (p〈0.05). While the levels ofPGE2, MMP-13, and IL-1βin supernatant in mutation group were sig- nificantly higher than those in control group (p〈0.05), and they were also greatly higher than those in over-expres- sion group significantly (p〈0.05). The apoptosis rate in control group and over-expression group was not obvious, between 1% and 10%. While it was up to 60% in mutation group which was obviously higher than that in control and over-expression group. The level of PGE2, MMP-13 and IL-1β and the apoptosis rate were significantly in- creased after synovial fibroblasts were infected by MCF2L gene mutation recombinant adenovirus. This research studied the effect of single nueleotide polymorphism of MCF2L on the inflammatory factors and apoptotic activity ofsynovial fibroblasts, and analyzed the role of MCF2L in the pathogenesis ofosteoarthritis. We explored the rela- tionship between MCF2L gene and bone infection, and provided references for clinical application.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2017年第6期2137-2142,共6页
Genomics and Applied Biology
基金
泉州市科技项目(非扩髓带锁髓内钉在胫骨开放性骨折的临床应用
No.201101SH01)资助