目的 探讨黏着斑激酶(FAK)在肿瘤坏死因子α(TNF-α)上调体外培养人角膜上皮细胞明胶酶活性中的作用.方法 实验研究.体外培养无眼部疾患人角膜上皮细胞,将传至3～4代的细胞暴露于含1 μg/L TNF-α(B组)、10 μg/L TNF-α(C组)、100 μg/L TNF-α(D组)的培养基中,对照组(A组)用含等量磷酸盐缓冲液(PBS)的培养基处理,明胶酶谱分析各组条件培养基中基质金属蛋白酶2和9( MMP-2、MMP-9)的活性,免疫印迹法分析FAK总蛋白表达及磷酸化FAK的表达；运用RNA干扰技术特异性抑制人角膜上皮细胞FAK的表达并重复以上过程.统计学方法使用单因素方差分析,各组间比较采用Tukey HSD检验.结果 明胶酶谱分析:与A相比,B、C、D组MMP-2、MMP-9活性均明显上调,差异有统计学意义(A、B、C、D组MMP-2活性表达分别为:124.06 ±4.06、146.72±5.51、241.18±5.65、389.95±4.44,F=2960.91,P=0.000；MMP-9活性表达分别为:122.78±5.86、165.70±7.90、479.49±6.22、495.88±5.03,F=4937.46,P=0.000),各组间比较差异均有统计学意义(MMP-2各组间比较均为P=0.000；MMP-9各组间比较P=0.000)；免疫印迹分析:C、D组中磷酸化FAK较A组增加,差异有统计学意义(C、D组磷酸化FAK分别为:0.52±0.03、0.61 ±0.06;F=431.03,P=0.000),100 μ.g/L TNF-α组较10 μg/L TNF-α组中磷酸化FAK表达增加(P=0.005)；运用RNA干扰技术特异性抑制人角膜上皮细胞FAK表达后,FAK总蛋白较特异性抑制之前明显下调,B、C、D组MMP-2的活性差异无统计学意义(转染后A、B、C、D组MMP-2分别为:55.13±0.66、55.67±0.43、55.49±0.20、55.91±0.37；F=2.73,P=0.079).10、100μg/L TNF-α干预组中MMP-9少量增加(转染后A、B、C、D组MMP-9分别为:80.48±0.39,81.26±0.62,84.43±0.47,85.56±0.61；F=105.80,P=0.000)但增加幅度较转染前明显减小,D组较C组MMP-9活性增强,差异有统计学意义(P=0.019).仅在100 μg/L TNF-α干预组中检测到FAK磷酸化(0.47±0.05),但表达量较抑制前明显减少(t=5.03,P=0.001).结论 FAK信号在TNF-α上调入角膜上皮细胞明胶酶活性过程中发挥一定作用,针对这一过程干预可能对防治角膜病变有意义.%Objective To investigate the effect of focal adhesional kinase (FAK) on tumor necrosis factor α (TNF-α)-induced MMP-2 and -9 activities in cornea epithelium. Methods Experimental research.The human corneal epithelial cells (HCE) were cultured in vitro. HCEs were incubated with different concentrations of TNF-α for 24 h,including 1 μg/L (group B),10 μg/L (group C) and 100 μg/L (group D).The control group (group A) was incubated with phosphate buffer solution.The activities of MMPs were examined by gelatin zymography and the phosphorylation of FAK was examined hy western blot analysis.FAK was down regulated by FAK siRNA following lipofectamine-mediated transfection in corneal epithelial cells.Down-regulation was confirmed using western blot analysis.Cells cultured with different concentrations of TNF-α ( Groups B to D) and the control group ( group A) was at similar volumes of media.Then the activities of MMP-2 and -9 were examined by gelatin zymography and the phosphorylation of FAK by western blot analysis.Statistical methods adopted one-way ANOVA and Tukey's honestly significant test between each group.Results Gelatin zymography:Activities of MMP-2 and -9 in TNF-α treated groups were greater than those of the control group.The activity of MMP-2 in A,B,C and D groups was 124.06 ±4.06,146.72 ±5.51,241.18 ±5.65 and 389.95 ±4.44,respectively with F =2960.91,P =0.000.The activity of MMP-9 in A,B,C and D groups was 122.78 ± 5.86,165.70 ± 7.90,479.49 ± 6.22 and 495.88 ± 5.03 ( F =4937.46,P =0.000).Significant differences were found in each two groups ( P =0.000).Western blot analysis:the phosphorylation of FAK (p-FAK) in test groups( 10-100 ng/ml) were significantly greater than that in control group( p-FAK of group C and D was 0.52 ±0.03 and 0.61 ±0.06,F =431.03,P =0.000).p-FAK levels in 100 ng/ml group were greater than that in 10 ng/ml group( P =0.005).After down-regulating the protein FAK,TNF-o had no effect on the activity of MMP-2 (The data of MMP-2 were 55.13 ± 0.66,55.67 ± 0.43,55.49 ± 0.20 and 55.91 ± 0.37 in groups A,B,C and D,F =2.73,P =0.079).We detected the increasing activity of MMP-9 in group C,D and p-FAK in group D (The data of MMP-9's activity were 80.48 ± 0.39,81.26 ± 0.62,84.43 ± 0.47,85.56 ± 0.61 in groups A,B,C and D,F=105.80,P =0.000).The activity of MMP-9 in group D was stronger than that from the group C (P =0.019).We just only detected a small quantity of p-FAK in group D(0.47 ±0.05),which was weaker than that before down regulating the protein FAK ( t =5.03,P =0.001 ).Conclusion Our results demonstrate the critical role of FAK in TNF-α induced activity of MMP-2 and -9 in human corneal epithelium cells.Blocking the FAK signaling pathway can reduce the activity of MMP-2 and -9 which may play an important role in prevention and treatment of corneal diseases.
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