GH调控鸡原代肝细胞中GH/IGF-Ⅰ信号通路相关基因mRNA表达的研究

Research on GH regulation of GH/IGF-Ⅰ pathway related gene expression in primary chicken hepatocytes

为研究离体条件下鸡肝脏中表达的GH/IGF-Ⅰ信号通路相关基因受生长激素(GH)调控的mRNA表达情况,以4～5周龄雌性商品代AA肉鸡为试验材料,采用改良的半原位两步灌流法并结合消化法分离肝细胞,进行原代培养;采用SYBR green荧光定量PCR方法,检测GH处理后鸡肝细胞中GH/IGF-Ⅰ信号通路中相关基因IGF-Ⅰ、HNF-1α、HNF-1β、HNF-3β、CIS、SOCS-1、SOCS-2和SOCS-3的表达情况。结果表明:胶原酶消化法获得的肝细胞分离良好,形态完整,铺板4h后完全贴壁,继续培养24h即可用于GH处理试验;GH能引起鸡肝细胞中IGF-Ⅰ、SOCS-2、SOCS-3和CIS基因mRNA表达上调,而对HNF-1α、HNF-1β、HNF-3β、SOCS-1基因mRNA的表达无影响。

In order to investigate roles of growth hormone （GH） in regulating the expression of GH/IGF- I signaling pathway related genes which expressed in liver, we evaluated the response of their mRNAs to GH in primary chicken hepatocytes. Hepatocytes were isolated from female AA chickens （4-5 week-old）, using a modified two-step semi-situ collagenase perfusion method. The cells were subsequently treated with chicken GH for different periods before collection of total RNA. We quantified the expression of IGF- I , HNF-1 α, HNF- 1β, HNF-3β, SOCS-1, SOCS-2, SOCS-3 and CIS genes by SYBR green real periods quantitative PCR. The results showed that newly acquired chicken hepatocytes were round, and most were dispersion under inverted microscope. Four hours after seeding, hepatocytes were firmly attached to the substrate and began to spread. Complete spreading was achieved within 24 h. The expression levels of IGF- I , SOCS-2, SOCS-3 and CIS were up-regulated by GH treatment in chicken hepatocytes, meanwhile, the expression levels of HNF-1α, HNF-1β, HNF-3β and SOCS-1 genes were not regulated by GH.