本文關鍵詞： Wnt5a C2C12肌原細胞 RNAi 增殖 分化　出處：《大連醫(yī)科大學》2012年碩士論文　論文類型：學位論文

【摘要】：背景：Wnt家族是調節(jié)不同發(fā)育過程如增殖、不對稱分裂、圖式發(fā)育和細胞命運決定的信號分子，在肌肉的發(fā)育過程中起著重要作用。Wnt5a是Wnt信號家族中的重要成員之一，但尚缺乏其調控肌細胞發(fā)育的研究。C2C12肌原細胞系是由C3H小鼠骨骼肌衛(wèi)星細胞永生化而來的細胞株，常被用做骨骼肌細胞發(fā)育的研究對象。 目的：下調Wnt5a在C2C12細胞系中的表達，探討Wnt5a對C2C12細胞增殖、細胞周期及分化的調控作用。 方法：1、依據(jù)Gene Bank中小鼠Wnt5a基因序列設計并合成三條siRNA的DNA模板的兩條單鏈，分別命名為siRNA-1、siRNA-2和siRNA-3，同時設計siRNA-無關序列作為陰性對照（Negative control，NC）序列。以脂質體法轉染C2C12細胞，應用SYBR Green實時定量PCR技術檢測siRNA轉染后的C2C12細胞在mRNA水平的表達，篩選出RNAi效果最好的siRNA序列用于后續(xù)的實驗。2、以脂質體法轉染C2C12細胞，分為實驗組（siRNA干擾組）和陰性對照組（NC組）。應用CCK-8試劑盒檢測細胞的增殖能力，應用流式細胞儀檢測細胞周期。3、加入含2%馬血清的分化液使細胞肌向分化，應用SYBR Green實時定量PCR檢測細胞分化后生肌調節(jié)因子家族成員Myf5、myogenin和MRF4，以及Wnt5a表達水平。應用免疫細胞化學法檢測C2C12細胞系肌向分化過程中Myosin的表達，并觀察細胞的形態(tài)學改變。 結果：1、和NC組及其它各干擾組相比，siRNA-1高效下調了C2C12細胞內(nèi)Wnt5a的表達水平（96.78%）。2、siRNA-1干擾Wnt5a表達下調后，CCK-8結果顯示C2C12細胞增殖無明顯變化，無統(tǒng)計學差異（P0.05）；流式細胞儀檢測結果顯示停留于各期的細胞數(shù)未明顯增加，無統(tǒng)計學差異（P0.05）。3、siRNA-1干擾Wnt5a表達下調后，在分化的D0天，Wnt5a的表達水平明顯下調，有統(tǒng)計學差異（P0.05）；在分化的D2天，Myf5、MRF4的表達水平明顯下調，有統(tǒng)計學差異（P0.05）。免疫細胞化學法顯示Myosin表達在多核肌管細胞中，胞漿染色強陽性，，在分化的各天，siRNA干擾組和NC組比較細胞的形態(tài)無明顯的區(qū)別。 結論：體外下調Wnt5a基因表達對C2C12細胞系的增殖和細胞周期無顯著影響，而肌向誘導分化的C2C12細胞系中Myf5和MRF4等表達水平下調提示了Wnt5a可能對骨骼肌細胞分化具有調控作用。
[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.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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