摘要
目的纤维母细胞是重要的胶原合成细胞，观察转化生长因子β１（ＴＧＦ－β１）对纤维母细胞Ⅰ、Ⅲ型前胶原及胶原酶ｍＲＮＡ表达的影响，以了解ＴＧＦ－β１在肝纤维化中的作用机制。方法首先对含有Ⅰ、Ⅲ型前胶原及胶原酶ｃＤＮＡ片段的质粒进行扩增、提纯及酶切，然后用自由引物延伸法进行地戈辛标记，并对探针进行检测；其次对纤维母细胞进行培养，加用ＴＧＦ－β１孵育后收集细胞并提取细胞总ＲＮＡ，最后将ＲＮＡ点样于尼龙膜上，并与上述探针进行斑点杂交。结果杂交结果显示ＴＧＦ－β能明显促进纤维母细胞Ⅰ型前胶原ｍＲＮＡ的表达（Ｐ＜０．０５），也能促进其Ⅲ型前胶原ｍＲＮＡ的表达（Ｐ＝０．０９）；而对其胶原酶ｍＲＮＡ的表达则起抑制作用（Ｐ＜０．０５）。结论ＴＧＦ－β可以从细胞及分子水平发挥其抗纤维化作用。
Transforming growth factorβ_1(TGF-β_1)is a multifunctional cytokine, it plays an important role in hepatic flbrosis. The aim of the present study is to observe the effects of TGF-β_1 on mRNA expressions of procollagen type Ⅰ,Ⅲ and collagenase of fibroblasts. Methods Plasmids containing procollagen type Ⅰ,Ⅲ and collagenase cDNA fragment, respectively, were amplified. After purification, the plasmids were linearized and the DNA templates were labelled with Dig-dUTP. NIH 3T3 cells(fibroblasts)were cultured in RPMI 640 medium. when the cells grew almost tO confluent,TGF-β_1 was added to the medium in various concentrations. The cells total RNA was extracted with a single-step RNA isolation method.15μg RNA of each sample was spotted onto nylon membrane and allowed the special probes of procollagen type Ⅰ,Ⅲ and collagenase to hybridize with it. Results The result showed that TGF-β_1 could participate the expressions of procollagen type Ⅰ,ⅢmRNA of flbroblasts and meanwhile, it had obvious inhibitory effects on expression of collagenase mRNA of fibroblasts. Conclusions The resuIts suggest that TGF-β_1 has fibrogenetic effect at the transcTiptional level.
出处
《中华肝脏病杂志》
CAS
CSCD
1996年第2期97-100,共4页
Chinese Journal of Hepatology
基金
CMB基金
