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-2 mRNA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-2 mRNA和蛋白质的表达较K-P和K-V组显著上调(mRNA：0.79±0.02 vs 0.51±0.03和0.48±0.02，P=0.000 和 P=0.000； 蛋白质：24.5±0.8 vs 15.5±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±0.7 vs 16.7±1.2 和15.9±0.7，P=0.001和P=0.003； MMP-2 ： 15.4±1.3 vs 18.2±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。

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 vs 0.51±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 vs 18.2±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.