【作者】 易宇欣；

【导师】 肖献忠；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 [目的]探讨KLF4对C2C12肌原细胞分化的影响,并初步探讨其可能机制。[方法]①建立稳定表达KLF4和空载体的C2C12细胞株,采用含有10%小牛血清的DMEM培养基在37。C 5%二氧化碳的环境下培养,待细胞进入融合期,采用含有5%灭活马血清的DMEM培基促进细胞分化。在不同时间点观察C2C12细胞中肌管的形成情况,同时采用酶联免疫吸附实验(ELISA)检测C2C12细胞心型肌酸激酶(CKM,一种骨骼肌分化标志物)的含量。②采用cDNA芯片检测KLF4过表达的C2C12细胞中基因表达谱的改变(以空载体细胞为对照)；③用MEDLINE、Genomatix等生物信息学数据库和分析软件对在KLF4过表达的C2C12细胞中表达发生改变的已命名基因进行功能和细胞信号通路分析。[结果]①在马血清刺激下,空载体和KLF4过表达的C2C12细胞均出现明显的肌管形成,但KLF4过表达的C2C12细胞肌管出现时间较转空载体的细胞早,同一时间点形成多核肌管的细胞数目较多,相应的,KLF4过表达的C2C12细胞内心型肌酸激酶(CKM)的含量升高较明显。②KLF4过表达的C2C12细胞中表达发生改变的基因605个；其中下调的基因为250个,已命名基因为155个；上调的基因为355个,已命名基因为255个；其中包含24个细胞周期相关基因。③上述细胞周期相关基因属于5条细胞信号通路。其中最值得关注的是Cdknla、Trp53/p53基因和细胞周期相关的p53信号通路。[结论]KLF4过表达可以促进C2C12肌原细胞的分化。这种促进作用很可能是通过调控下游细胞周期相关基因的表达来实现的。更多还原

【Abstract】 [Objective] To explore the role of KLF4 in the differentiation of C2C12 myogenic cells and preliminarily explore the mechanism. [Method] C2C12 myogenic cells were stably transfected with KLF4 eukaryotic expression constructs or control vector. The cells were grown to confluence and then the culture medium was replaced with DMEM containing 5% heat-inactivated horse serum to induce differentiation. The formation of multinucleated myotubes and measured the concentration of creatine kinase M isozyme (CKM, a differentiation marker for muscle cells)were observed during differentiation. CDNA chips containing 14000 mouse cDNAs were employed to investigate the influence of KLF4 overexpression on the genes expression. The differentially expressed genes after KLF4 transfection were further analyzed using bioinformatics databases and softwares such as MEDLINE and BibioSphere pathway edition. [Results] KLF4 over-expression accelerated the formation of multinucleated myotubes and enhanced the concentration of creatine kinase M isozyme during differentiation.250 genes were down-regulated in KLF4-overexpressed C2C12 cells, of which 155 were known genes; Whereas 355 genes were up-regulated, of which 255 were known genes, including 24 cell cycle-relevent genes.These 24 cell cycle-relevent genes belong to five different signal transduction pathways,among which Cdknla and Trp53/p53 genes and the p53 signal transduction pathway are most important. [Conclusion] KLF4 could accelerate the differentiation of C2C12 myogenic cells induced by horse serum. This role may be related to the regulation of the downstream cell cycle-relevent genes by KLF4.更多还原