摘要
目的:探讨组织因子途径抑制物(TFPI-2)与体外角膜基质细胞基质金属蛋白酶(MMPs)表达的关系。方法:体外兔角膜基质细胞原代、传代培养;脂质体介导人类TFPI-2真核表达载体pBos-Cite-neo/TFPI-2转染基质细胞,G418筛选阳性细胞,RT-PCR和Western-blot技术检测转染前后三组角膜基质细胞(转染TFPI-2基因组K-TFPI-2、转染空载体组K-V、未转染组K-P)中TFPI-2mRNA以及相应蛋白质的表达水平;利用明胶酶谱法分析比较转染前后三组角膜基质细胞表达MMPs的活性差异。结果:K-TFPI-2组角膜基质细胞TFPI-2mRNA和蛋白质的表达较K-P和K-V组显著上调(mRNA:0.79±0.02vs0.51±0.03和0.48±0.02,P=0.000和P=0.000;蛋白质:24.5±0.8vs15.5±0.5和14.9±0.9,P=0.000和P=0.000);与K-P和K-V组相比,K-TFPI-2组细胞表达MMP-1,2的活性下降(MMP-1:12.3±0.7vs16.7±1.2和15.9±0.7,P=0.001和P=0.003;MMP-2:15.4±1.3vs18.2±1.1和17.8±1.1,P=0.027和P=0.046)。结论:TFPI-2表达可明显抑制角膜基质细胞表达MMPs的活性,为进一步开展角膜新生血管性疾病的基因治疗提供实验依据。
AIM.-To elucidate the relation between tissue factor pathway inhibitor - 2 （TFPI - 2） expression and the expression of matrix metalloproteinases （MMPs） in keratocytes. METHODS. Primary culture and subculture of rabbit keratocytes were established in vitro. Plasmid vector pBos-Cite-neo/TFPI-2 was transfected into keratocytes with Lipofectamine 2000. After being selected by G418, three groups of cells including TFPI-2 gene transfected cells K-TFPI-2, empty vector transfected cells K-V and non-transfected cells K-P were screened for TFPI- 2 mRNA and protein by reverse transcription-polymerase chain reaction and Western blot analysis, respectively. The activity of MMPs in the three groups of cells was detected by substrate zymography and compared by ANOVA. RESULTS.. Expression of mRNA and protein of TFPI-2 was more in the cells of K-TFPI-2 than in the other cells of K-P and K-V with a significant difference （mRNA..0.79± 0.02 vs0.51±0.03 and 0.48±0.02, P=0.000 and P=0.000 Protein.24.5±0.8 vs 15.5±0.5 and 14.9±0.9, P= 0. 000 and P=0.000）. Compared with the two groups of K-P and K-V, the cells of K-TFPI-2 had a significant decreased activity of MMP1 （12.3±0.7 vs 16.7±1.2 and 15.9±0.7, P=0.001 and P=0.003） and MMP2 （15.4±1.3 vs18.2±1.1 and 17.8± 1.1, P=0.027 and P=0.046）. CONCLUSION; It is suggested that the expression of TFPI-2 may strongly inhibit the activity of MMPs in keratocytes in vitro, which provides an experimental basis for curing CNV with gene therapy.
出处
《国际眼科杂志》
CAS
2013年第4期656-659,共4页
International Eye Science
基金
国家自然科学基金(No.81100664)
武汉大学自主科研项目(No.111091)~~
