uPA-siRNA重组慢病毒载体感染兔软骨细胞对uPA和MMP-3表达的影响

摘要

Objective To construct recombinant lentiviral vector carrying small interfering RNA against uPA gene with express‐ing reporter gene GFP ,and to observe its effect on the expression of uPA and MMP‐3 in chondrocytes .Methods siRNAs against uPA mRNA were designed and chemically synthesized .Recombinant lentiviral vector with uPA‐siRNA and reporter gene GFP was constructed .Rabbit chondrocytes were cultured and randomized into four groups :for uPA‐siRNA transfection group :recombinant lentiviral vector with uPA‐siRNA and GFP gene was transfected into chondrocytes using lipofectamine 2000 ;for empty vector transfection group :empty lentiviral vector was transfected into chondrocytes ;for non transfection group :chondrocytes were cul‐tured without any treatment ;for MMP inhibitor group :chondrocytes were cultured with MMP inhibitor TIMP .The mRNA and protein expression levels of uPA and MMP‐3 of chondrocytes were determined by RT‐PCR and Western blot after 96 h of treatment in all groups respectively .Results Recombinant lentiviral vector carrying uPA‐siRNA and reporter gene GFP was constructed and transfected into primary chondrocytes successfully .The transfection efficiency was more than 85% when multiplicity of infection (MOI) was 100 .The mRNA and protein expression levels of uPA were significantly decreased in uPA‐siRNA transfection group compared to three other groups(P<0 .01) .MMP‐3 mRNA and protein levels were also down‐regulated in uPA‐siRNA transfection group compared to empty‐vector group and non‐transfection group(P<0 .01) .However ,only decreased mRNA level of MMP‐3 was obtaind when compare uPA‐siRNA transfection group to MMP inhibitor group (P<0 .01) .There was no significant difference of MMP‐3 protein level between uPA‐siRNA transfection group and MMP inhibitor group(P>0 .05) .Conclusion Recombinant lenti‐viral vector carrying uPA‐siRNA and GFP gene can transfect primary chondrocytes successfully and inhibit uPA gene expression and protein level as well as MMP‐3 in chondrocytes efficiently .Low level of uPA could down regulate the expression of MMP‐3 .%目的：靶向特异尿激酶型纤溶酶原激活物（uPA）‐siRNA慢病毒表达载体并加载绿色荧光蛋白（GFP）后转染软骨细胞，观察其对软骨细胞表达uPA和基质金属蛋白酶3（MMP‐3）的影响。方法培养软骨细胞并根据实验分为实验组（转染uPA‐siRNA慢病毒载体）、空载体组（转染空慢病毒载体）、空白对照组（未进行任何处理）和 TIMP组（加载 MMP特异性抑制剂TIMP）。实验组：uPA‐siRNA序列经慢病毒包装并加载GFP ，通过Lipofectamine 2000转染入兔软骨细胞；空载体组：将慢病毒载体通过Lipofectamine 2000转染入兔软骨细胞；空白对照组正常培养软骨细胞；药物对照组：培养基中加特异性M M P抑制剂。所有细胞培养96 h后应用逆转录‐聚合酶链反应（RT‐PCR）和蛋白免疫印迹法（Western blot）法分别检测软骨细胞uPA mRNA、MMP‐3 mRNA和蛋白的表达水平。结果慢病毒载体可成功转染到原代软骨细胞中，在感染复数（MOI）为100时转染率达到85％以上。实验组uPA mRNA、uPA蛋白和MMP‐3 mRNA及MMP‐3蛋白的表达水平显著低于空载体组和空白对照组（P＜0．01），而与药物干预组差异无统计学意义（P＞0．05）。结论 uPA‐siRNA慢病毒载体负载GFP可稳定转染软骨细胞并高效抑制uPA基因、蛋白表达，同时对M M P‐3基因、蛋白表达也呈现高效抑制作用。抑制uPA的水平可降低显著M M P‐3表达。