反义Smad_4基因对贮脂细胞系CFSC生物学特性的影响

Effects of anti-sense Smad_4 gene on the biological characteristics of the fat-storing cell line CFSC

目的 探讨通过反义Smad4基因转移阻断转化生长因子 β1(TGF β1)的信号传导后对贮脂细胞部分生物学特性的影响。方法 分别构建、重组了含有反义Smad4基因序列的复制缺陷型腺病毒表达载体AdvATSmad4和空病毒表达载体Adv0。将两种复制缺陷型腺病毒扩增纯化 ,再分别转入贮脂细胞系CFSC内 ,测定它们生长曲线、3 H掺入率 (3 H TdR)、脯氨酸掺入等部分生物学特性的变化 ,并用逆转录 聚合酶链反应 (RT PCR)、Western印迹和免疫组织化学等方法检测转基因细胞Smad4和细胞外基质的表达。结果 与正常对照组CFSC和转染Adv0的CFSC细胞相比较 ,转染AdvATSmad4的CFSC细胞内有反义Smad4表达 ,其生长曲线、3 H TdR、脯氨酸掺入等均有不同程度的降低 ,且其合成分泌Smad4和细胞外基质减少。结论 反义Smad4基因可以抑制贮脂细胞内源性Smad4的表达 ,抑制贮脂细胞的生长、繁殖和细胞外基质的产生 ,可作为抗肝纤维化基因治疗的选择之一。

Objective To investigate the effects of antisense Smad_4 on the biological characteristics of the fat-storing cell line CFSC. Methods Fat-storing cells of line CFSC from rat with liver fibrosis were cultured and transfected with 50 MOI of recombinant adenoviral vector carrying antisense Smad_4 (AdvATSmad_4) or the control empty adenovirus (Adv0), both produced by 293 packaging cells, respectively. Two, four, and six days after the transfection the cultured cells were collected to undergo trypan blue staining and cell counting. The growth curves were drawn. The presence of antisense Smad_4 was detected by RT-PCR and Western blotting. 3H-TdR was added into the culture media to be co-cultured for 6 hours. Then the cells were collected to examine the 3H-TdR incorporation rate. RT-PCR and immunohistochemistry were used to examine the expression of COL1A1 and type Ⅰcollagen, kinds of extracellular matrix (ECM). Results Compared with the control CFSC and the Adv0-transfeted CFSC cells, the cell growth curve, 3H-TdR incorporation rate, proline incorporation rate, expression of Smad_4, and expression of extracellular matrix were markedly decreased in the AdvATSmad_4-transfected CFSCs. Conclusion The antisense Smad_4 gene inhibits the expression of Smad_4 mRNA and protein, proline incorporation and cell growth, thus down-regulating the production of ECM. Antisense Smad_4 gene may be used as a choice of gene therapy for liver fibrosis.