特异性siRNA对大鼠肝星状细胞TIMP-1表达的影响

观察金属蛋白酶组织抑制因子-1（TIMP-1）小干扰RNA真核表达载体转染肝星状细胞株HSC-T6后TIMP-1基因表达的情况。构建携带绿色荧光蛋白（GFP）基因的小干扰RNA真核表达载体pGCsi-U6／TIMP-1 shRNA，利用该载体介导四个特异性和一个非特异性的TIMP-1 shRNA，分别为TIMP-1 shRNA1、TIMP-1 shRNA2、TIMP-1 shRNA3、TIMP-1 shRNA4和TIMP-1 shRNA—NON。采用阳离子脂质体介导法转染HSC-T6细胞，激光共聚焦显微镜观察GFP表达，荧光实时定量PCR方法检测TIMP-1 mRNA表达情况。质粒转染HSC—T6细胞后，激光共聚焦显微镜观察转染组细胞内均见绿色荧光，未转染组未见绿色荧光；mRNA水平检测显示：与正常对照组相比，TIMP-1 shRNA1、TIMP—1 shRNA2和TIMP-1 shRNA4对TIMP-1基因表达较强的抑制作用，尤TIMP-1 shRNA1抑制作用最强（P〈0．01）。pGCsi—U6介导的TIMP-1 shRNA1能更加有效的抑制肝星状细胞中TIMP—1的表达。

Expression of Tissue Inhibitor of Metalloproteinase-1 in Rat Hepatic Stellate Cell Infected by Using Targeted siRNA

To investigate the influence on expression of tissue inhibitor of metalloproteinase-1 （TIMP-1） in rat hepatic stellate cell line HSC-T6 infected by using TIMP-1 siRNA. Four target sequences and one nonspecific sequence were selected to reconstructed plasmids, i. e, pGCsi-U6/TIMP-1shRNA1, pGCsi-U6/TIMP-1shRNA2, pGCsi-U6/ TIMP-1 shRNA3 ,pGCsi-U6/TIMP-1 shRNAd and pGCsi-U6/TIMP-1 shRNA-NON which contained green fluorescent protein（GFP） gene were constructed. Five plasmids were transfected into T6 separately through an oligofectamine package. Laser Scanning Confocal Microscope was used to examine the expression of GFP. The expression of TIMP-1mRNA levels were examined by real time PCR. The expression of GFP was detected in pGCsi-U6/TIMP-1shRNA transfected cells, but was not trailed in nontransfected cells. The mRNA expression of TIMP-1 in shRNA1 ,shRNA2 and shRNA4 were significantly lower than those in control groups, especially TIMP-1 shRNA1 （P 〈0.01 ）. The specific TIMP-1 shRNA1 markedly inhibits the expression of TIMP-1 on the gene levels of HSC-T6.