Cong Min, Wang Ping, Liu Tian-Hui, Xu Yong, Lu Yan, Tang Shu-Zhen, Liu Xiao-Ming, Wang Bao-En, Jia Ji-Dong, You Hong
Liver Research Center, Beijing Friendship Hospital, Capital University of Medical Sciences, Beijing 100050, China.
Zhonghua Gan Zang Bing Za Zhi. 2006 Oct;14(10):742-7.
Elevated tissue inhibitor of metalloproteinase-1 (TIMP-1) expression contributes to excess extracellular matrix in liver fibrosis. This study was designed to construct two recombinant adeno-associated viruses (AAV) carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 (rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo), and then to compare the suppression of TIMP-1 gene expression on rat hepatic stellate cell (HSC)-T6 cells infected by these two types of viruses in vitro.
Antisense RNA amplified by rat HSC-T6 and U6 promoter followed by the annealing siRNA were cloned into the AAV vector (pdl6-95/neo) and packed in 293 cells to construct the recombinants rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo. Rat HSC-T6 cells were infected with these recombinant AAVs and selected by using G418, and real-time PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene in these cells.
The results of PCR, restrictive enzyme digestion and gene sequencing confirmed that the pdl6-95/ANTI-TIMP-1/neo and pdl6-95/siRNA-TIMP-1/neo had been reconstructed successfully. After they had been packed in 293 cells to form rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo, they were used to infect HSC-T6. Thirty days after the infection, the transcription level of TIMP-1 in HSC-T6 cells infected by rAAV/siRNA-TIMP-1/neo decreased dramatically compared with the mock control and normal HSC-T6 cells (P less than 0.01), and the expression level of TIMP-1 gene in HSC-T6 cells decreased significantly (60%), while the transcription and expression level of TIMP-1 in HSC-T6 cells infected by rAAV/ANTI-TIMP-1/neo had no significant difference with mock control and normal HSC-T6 cells (P more than 0.05).
RNA interference can exert a suppression of TIMP-1 gene in rat HSC, and when this function combines with AAV infection, it can suppress the specific gene expression for a long time by chromosomal integration.
金属蛋白酶组织抑制因子-1（TIMP-1）表达升高促使肝纤维化中细胞外基质过多。本研究旨在构建携带TIMP-1反义RNA和小干扰RNA（siRNA）的两种重组腺相关病毒（AAV）（rAAV/ANTI-TIMP-1/neo和rAAV/siRNA-TIMP-1/neo），进而比较这两种病毒体外感染大鼠肝星状细胞（HSC）-T6后对TIMP-1基因表达的抑制作用。
由大鼠HSC-T6和U6启动子扩增的反义RNA经退火后的siRNA克隆至AAV载体（pdl6-95/neo），并在293细胞中包装构建重组体rAAV/ANTI-TIMP-1/neo和rAAV/siRNA-TIMP-1/neo。用这些重组AAV感染大鼠HSC-T6细胞并用G418筛选，进行逆转录后实时PCR及蛋白质印迹检测这些细胞中TIMP-1基因的转录和表达水平。
PCR、限制性酶切及基因测序结果证实pdl6-95/ANTI-TIMP-1/neo和pdl6-95/siRNA-TIMP-1/neo已成功构建。它们在293细胞中包装形成rAAV/ANTI-TIMP-1/neo和rAAV/siRNA-TIMP-1/neo后用于感染HSC-T6。感染30天后，rAAV/siRNA-TIMP-1/neo感染的HSC-T6细胞中TIMP-1的转录水平与模拟对照及正常HSC-T6细胞相比显著降低（P<0.01），HSC-T6细胞中TIMP-1基因的表达水平显著降低（60%），而rAAV/ANTI-TIMP-1/neo感染的HSC-T6细胞中TIMP-1的转录和表达水平与模拟对照及正常HSC-T6细胞无显著差异（P>0.05）。
RNA干扰可抑制大鼠HSC中TIMP-1基因，该功能与AAV感染结合时，可通过染色体整合长时间抑制特定基因表达。
