应用竞争PCR检测丙酸盐对体外培养牛肝细胞丙酮酸羧化酶mRNA水平的影响  

Effect of Propionate on the Expression of Pyruvate Carboxylase mRNA in vitro Culture Bovine Hepatocyte Determinated by Competitive Reverse Transcription Polymerase Chain Reaction

作　　者：徐闯[1] 夏成[1] 刘国文[1] 王哲[1] 李家奎[1] 张乃生[1] 王兴龙[1] 欧阳红生[1] 张永亮[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医学报》2005年第4期397-399,402,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30230620)

摘　　要：根据牛肝细胞内PC(丙酮酸羧化酶)基因组与PCcDNA相比多含有一个内含子序列,在其中再插入一外源DNA序列,从而使最终构建的突变体片段的长度大于PCcDNA的长度,成功构建了PCcDNA的竞争DNA模板,然后应用竞争PCR方法研究了丙酸盐对体外培养新生牛单层肝细胞PCmRNA水平的影响。使单层肝细胞培养液中丙酸钠浓度分别为0、1.5、2.5、3.5、4.5、8.5、11.5mmol/L,处理24h,提取总RNA、逆转录,在同一体系中用相同引物扩增目的带和竞争模板带。结果表明,随着丙酸钠浓度的升高,PCmRNA水平呈上升趋势,提示肝细胞内PCmRNA的表达水平受培养液中丙酸钠浓度的影响。Competive DNA template of pyruvate carboxylase (PC) cDNA was constructed through the methods of intron sequence and insertion sequence. Then, the effect of propionate on the expression of PCmRNA in vitro culture bovine hepatocyte was quantitatively determinated by competitive PCR. Propionate was added into the culture media as various concentration of 0, 1.5, 2.5, 3.5, 4.5, 8.5 and 11.5 mmol/L, cultured for 24 h, and then total RNA was extracted,reverse transcription was done,using the same primer to amplify objective gene and competive gene. Results: PCmRNA level in hepatocyte are enhanced with increase of the propionate concentration. It is concluded that propionate could influence PCmRNA level in liver cell of dairy cow.

关 键 词：丙酮酸羧化酶mRNA 竞争PCR 肝细胞 内含子 

分 类 号：S852.23[农业科学—基础兽医学]