摘要
目的探讨ＴＧＦβ１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ＴＧＦβ１的基因序列反向插入逆转录病毒载体ｐＬＸＳＮ，构建反义ＴＧＦβ１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ＴＧＦβ１ｃＤＮＡ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、ＲＴＰＣＲ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ＴＧＦβ１和细胞外基质的产生。结果贮脂细胞株ＬＩ９０内有反义ＴＧＦβ１的表达，且其合成分泌ＴＧＦβ１和细胞外基质如ＦＮ、ＣｏｌＡ１降低。结论反义ＴＧＦβ１ＲＮＡ可以抑制贮脂细胞的激活和内源性ＴＧＦβ１和细胞外基质的产生。
Objective To investigate possible role of antisense TGF β1 RNA in the regulation of TGF β1 and ECM production in Ito cells. Method A human transforming growth factor β1(TGF β1) cDNA (1467 bp) was inserted in reverse orientation into the retroviral vector, constructed retroviral vector pLATSN of antisense RNA for TGF β1. A high titer, recombinant retroviral vector carried antisense RNA for TGF β1 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 β1 protein and the production of extracellular matrix such as FN, ColA1 were markedly decreased in the antisense TGF β1 transfected cultured cells by ELISA, immunohistochemistry and in situ hybridization. Conclusion Antisense RNA of TGF β1 can be successfully used to inhibit Ito cells activated, endogenous TGF β1 mRNA and extracellular matrix produced, and may provide a basis for the development of anti fibrosis gene therapy.
出处
《中华医学杂志》
CAS
CSCD
北大核心
1998年第11期850-852,共3页
National Medical Journal of China
基金
国家自然科学基金
