贮脂细胞TGF-1反义基因转移及对细胞外基质合成的抑制作用

目的探讨ＴＧＦβ１在肝纤维化贮脂细胞激活和细胞外基质产生中的作用。以及对细胞外基质基因的表达和自身ｍＲＮＡ表达的作用。方法将ＴＧＦβ１的基因序列反向插入逆转录病毒载体ｐＬＸＳＮ,构建反义ＴＧＦβ１的逆转录病毒载体ｐＬＡＴＳＮ,用ＰＡ３１７包装细胞包装,获得高滴度的逆转录病毒。将携有反义ＴＧＦβ１ｃＤＮＡ的逆转录病毒载体ｐＬＡＴＳＮ导入人的贮脂细胞株ＬＩ９０内,选择稳定转染的贮脂细胞株培养,应用ＰＣＲ、ＲＴＰＣＲ检测反义基因的表达,用ＥＬＩＳＡ、免疫组织化学和原位杂交等方法检测ＴＧＦβ１和细胞外基质的产生。结果贮脂细胞株ＬＩ９０内有反义ＴＧＦβ１的表达,且其合成分泌ＴＧＦβ１和细胞外基质如ＦＮ、ＣｏｌＡ１降低。结论反义ＴＧＦβ１ＲＮＡ可以抑制贮脂细胞的激活和内源性ＴＧＦβ１和细胞外基质的产生,为抗纤维化基因治疗提供理论基础

TGF-beta 1 antisense gene transfer into ito cells and suppressed extracellular matrix production

OBJECTIVE: To investigate possible role of antisense TGF-beta 1 RNA in the regulation of TGF-beta 1 and ECM production in Ito cells. METHOD: A human transforming growth factor-beta 1(TGF-beta 1) cDNA (1467 bp) was inserted in reverse orientation into the retroviral vector, constructed retroviral vector pLATSN of antisense RNA for TGF-beta 1. A higher-titer, recombinant retroviral vector carried antisense RNA for TGF-beta 1 produced in PA317 packaging cells has been introduced into human Ito cells lines LI90. After selection with G418, resistant colonies were obtained. RESULTS: Stable integration of retrovirus in infectants was shown the presence of antisense RNA was detected by RT-PCR. The expression of TGF-beta 1 protein and the production of extracellular matrix such as FN, Co1A1 were markedly decreased in the antisense TGF-beta 1 transfected cultured cells by ELISA, immunohistochemistry and in situ hybridization. CONCLUSION: Antisense RNA of TGF-beta 1 can be successfully used to inhibit Ito cells activated, endogenous TGF-beta 1 mRNA and extracellular matrix produced, and may provide a basis for the development of anti-fibrosis gene therapy.