Wnt5a基因下调对C2C12肌原细胞增殖与分化的影响

发布时间：2018-02-05 19:36

  本文关键词： Wnt5a C2C12肌原细胞 RNAi 增殖 分化　出处：《大连医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：背景：Wnt家族是调节不同发育过程如增殖、不对称分裂、图式发育和细胞命运决定的信号分子，在肌肉的发育过程中起着重要作用。Wnt5a是Wnt信号家族中的重要成员之一，但尚缺乏其调控肌细胞发育的研究。C2C12肌原细胞系是由C3H小鼠骨骼肌卫星细胞永生化而来的细胞株，常被用做骨骼肌细胞发育的研究对象。 目的：下调Wnt5a在C2C12细胞系中的表达，探讨Wnt5a对C2C12细胞增殖、细胞周期及分化的调控作用。 方法：1、依据Gene Bank中小鼠Wnt5a基因序列设计并合成三条siRNA的DNA模板的两条单链，分别命名为siRNA-1、siRNA-2和siRNA-3，同时设计siRNA-无关序列作为阴性对照（Negative control，NC）序列。以脂质体法转染C2C12细胞，应用SYBR Green实时定量PCR技术检测siRNA转染后的C2C12细胞在mRNA水平的表达，筛选出RNAi效果最好的siRNA序列用于后续的实验。2、以脂质体法转染C2C12细胞，分为实验组（siRNA干扰组）和阴性对照组（NC组）。应用CCK-8试剂盒检测细胞的增殖能力，应用流式细胞仪检测细胞周期。3、加入含2%马血清的分化液使细胞肌向分化，应用SYBR Green实时定量PCR检测细胞分化后生肌调节因子家族成员Myf5、myogenin和MRF4，以及Wnt5a表达水平。应用免疫细胞化学法检测C2C12细胞系肌向分化过程中Myosin的表达，并观察细胞的形态学改变。 结果：1、和NC组及其它各干扰组相比，siRNA-1高效下调了C2C12细胞内Wnt5a的表达水平（96.78%）。2、siRNA-1干扰Wnt5a表达下调后，CCK-8结果显示C2C12细胞增殖无明显变化，无统计学差异（P0.05）；流式细胞仪检测结果显示停留于各期的细胞数未明显增加，无统计学差异（P0.05）。3、siRNA-1干扰Wnt5a表达下调后，在分化的D0天，Wnt5a的表达水平明显下调，有统计学差异（P0.05）；在分化的D2天，Myf5、MRF4的表达水平明显下调，有统计学差异（P0.05）。免疫细胞化学法显示Myosin表达在多核肌管细胞中，胞浆染色强阳性，，在分化的各天，siRNA干扰组和NC组比较细胞的形态无明显的区别。 结论：体外下调Wnt5a基因表达对C2C12细胞系的增殖和细胞周期无显著影响，而肌向诱导分化的C2C12细胞系中Myf5和MRF4等表达水平下调提示了Wnt5a可能对骨骼肌细胞分化具有调控作用。
[Abstract]:Background: the Wnt family is a signaling molecule that regulates different developmental processes such as proliferation asymmetric division schema development and cell fate. Wnt5a is one of the important members of Wnt signaling family. C2C12 myogenic cell line is an immortalized cell line derived from the immortalized skeletal muscle satellite cells of C3H mice, which is often used as the research object of skeletal muscle cell development. Aim: to down-regulate the expression of Wnt5a in C2C12 cell line and investigate the regulation of Wnt5a on the proliferation, cell cycle and differentiation of C2C12 cells. Methods: 1. According to the mouse Wnt5a gene sequence of Gene Bank, two single strands of three DNA templates of siRNA were designed and synthesized. They were named siRNA-1. SiRNA-2 and siRNA-3, siRNA-independent sequences were designed as negative control control. NC12 cells were transfected into C2C12 cells by liposome method. The expression of C2C12 cells transfected with siRNA at mRNA level was detected by real-time quantitative PCR with SYBR Green. The best siRNA sequence of RNAi was selected for further experiment. 2. C2C12 cells were transfected with liposome. CCK-8 kit was used to detect cell proliferation and flow cytometry was used to detect cell cycle. The differentiation fluid containing 2% horse serum was added to induce the differentiation of the cells. Myf5 was detected by SYBR Green real-time quantitative PCR. The expression of myogenin, MRF4, and Wnt5a were detected by immunocytochemistry. The expression of Myosin in C2C12 cell line during myogenic differentiation was detected by immunocytochemistry. The morphological changes of the cells were observed. Results compared with NC group and other interference groups, the expression level of Wnt5a in C2C12 cells was effectively down-regulated by siRNA-1. After down-regulation of Wnt5a expression by siRNA-1 interference, the results of CCK-8 showed that the proliferation of C2C12 cells had no significant change, and there was no statistical difference (P0.05). The results of flow cytometry showed that the number of cells staying at each stage was not significantly increased, and there was no significant difference between the two groups. After the down-regulation of Wnt5a expression, the cells were differentiated on D 0 day. The expression of Wnt5a was significantly down-regulated (P 0.05). The expression level of Myf5nMRF4 was significantly down-regulated in differentiated D _ 2 on D _ 2 day, with a significant difference (P 0.05). Immunocytochemistry showed that Myosin was expressed in multinuclear myotube cells. Cytoplasmic staining was strongly positive, and there was no significant difference in cell morphology between siRNA interference group and NC group. Conclusion: down-regulation of Wnt5a gene expression in vitro has no significant effect on the proliferation and cell cycle of C2C12 cell line. However, the down-regulation of Myf5 and MRF4 in C2C12 cells suggested that Wnt5a might regulate the differentiation of skeletal muscle cells.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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