摘要
目的观察大鼠CD90^+Lin^-骨髓细胞诱导分化为肝细胞过程中Klf4、Nanog基因表达及启动子区CpG位点的甲基化变化。方法大鼠CD90^+Lin^-骨髓细胞在体外用25μg/L重组人肝细胞生长因子和0.1 nmol/L地塞米松诱导其分化,在诱导第0、7、14天分别提取细胞DNA和RNA。采用实时荧光定量PCR法检测Klf4、Nanog基因mRNA,甲基化特异性PCR法观察Klf4、Nanog基因启动子区CpG位点甲基化。结果与诱导第0天比较,第7、14天Klf4 mRNA相对表达量降低(P均<0.05);与诱导第0天比较,第7、14天Nanog mRNA相对表达量均降低(P均<0.05)。与诱导第0天比较,诱导第7天Klf4启动子区第1、2个CpG位点甲基化频率升高(P均<0.05);与诱导第7天比较,诱导第14天Klf4启动子区第1个CpG位点甲基化频率升高,第2、3个CpG位点甲基化频率降低,P均<0.05。与诱导第0天比较,诱导第7天Nanog启动子区第1个CpG位点甲基化频率降低,第2个CpG位点甲基化频率升高,P均<0.05;与诱导第7天比较,诱导第14天Nanog启动子区第1个CpG位点甲基化频率升高,第2个CpG位点甲基化频率降低,P均<0.05。结论大鼠CD90^+Lin^-骨髓细胞在体外诱导向肝细胞分化过程中,Klf4、Nanog基因表达随诱导时间增加呈现先下降后回升趋势,两者启动子区CpG位点甲基化程度随诱导时间增加出现不同程度变化;Klf4和Nanog基因启动子区CpG位点的甲基化变化不仅会影响Klf4、Nanog基因的表达,而且起到重要的调控作用。
Objective To observe the expression of Klf4 and Nanog and the promoter region CpG site methylation changes during differentiation of rat bone marrow CD90^＋Lin^-cells to hepatocytes.Methods Rat bone marrow CD90^＋Lin^-cells were induced in vitro by 25 μg/L recombinant human hepatocyte growth factor and 0.1 nmol/L dexamethasone.DNA and RNA were extracted from cells on day 0,7,and 14 of induction.Real-time fluorescent quantitative PCR was used to detect Klf4 and Nanog mRNA expression.Methylation Specific PCR was performed to detect methylation variation of CpG sites in Klf4 and Nanog promoters.Results Compared with the day 0,the relative expression of Klf4 mRNA decreased on day 7 and day 14（P〈0.05）;compared with the day 0,the relative expression of Nanog mRNA decreased on day 7 and day 14（P〈0.05）.Compared with the day 0,the methylation frequency of the first and second CpG sites in Klf4 promoter increased on day 7（P〈0.05）;compared with the day 7,the methylation frequency of the first CpG site in Klf4 promoter increased on day 14,while the second and third CpG sites in Klf4 promoter decreased（P〈0.05）.Compared with the day 0,the methylation frequency of the first site in Nanog promoter decreased in day 7,while the second sites in Nanog promoter increased（P〈0.05）;compared with the day 7,the methylation frequency of the first CpG site in Nanog promoter increased on day 14,while the second CpG site in Nanog promoter decreased（P〈0.05）.Conclusions During differentiation of rat bone marrow CD90^＋Lin^-cells to hepatocytes in vitro,the expression of Klf4 and Nanog first increases and then decreases as time passes,and the CpG sites methylated degrees of both promoters vary as time passes.The methylation of CpG site in the promoter region of Klf4 and Nanog not only affects the expression of Klf4 and Nanog,but also plays an important regulatory role.
作者
刘钦成
张梓朗
潘润华
刘宁
韩晓玉
郭凌宏
王春明
廖彩仙
LIU Qincheng ZHANG Zilang PAN Runhua LIU Ning HAN Xiaoyu GUO Linghong WANG Chunming LIAO Caixian(Nanfang Hospital of Southern Medical University, Guangzhou 510515, China)
出处
《山东医药》
CAS
北大核心
2017年第31期13-16,共4页
Shandong Medical Journal
基金
广东省科技计划项目(2010B080701069)
