發(fā)布時(shí)間：2018-02-05 19:36

  本文關(guān)鍵詞： Wnt5a C2C12肌原細(xì)胞 RNAi 增殖 分化　出處：《大連醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：背景：Wnt家族是調(diào)節(jié)不同發(fā)育過(guò)程如增殖、不對(duì)稱分裂、圖式發(fā)育和細(xì)胞命運(yùn)決定的信號(hào)分子，在肌肉的發(fā)育過(guò)程中起著重要作用。Wnt5a是Wnt信號(hào)家族中的重要成員之一，但尚缺乏其調(diào)控肌細(xì)胞發(fā)育的研究。C2C12肌原細(xì)胞系是由C3H小鼠骨骼肌衛(wèi)星細(xì)胞永生化而來(lái)的細(xì)胞株，常被用做骨骼肌細(xì)胞發(fā)育的研究對(duì)象。 目的：下調(diào)Wnt5a在C2C12細(xì)胞系中的表達(dá)，探討Wnt5a對(duì)C2C12細(xì)胞增殖、細(xì)胞周期及分化的調(diào)控作用。 方法：1、依據(jù)Gene Bank中小鼠Wnt5a基因序列設(shè)計(jì)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(shí)設(shè)計(jì)siRNA-無(wú)關(guān)序列作為陰性對(duì)照（Negative control，NC）序列。以脂質(zhì)體法轉(zhuǎn)染C2C12細(xì)胞，應(yīng)用SYBR Green實(shí)時(shí)定量PCR技術(shù)檢測(cè)siRNA轉(zhuǎn)染后的C2C12細(xì)胞在mRNA水平的表達(dá)，篩選出RNAi效果最好的siRNA序列用于后續(xù)的實(shí)驗(yàn)。2、以脂質(zhì)體法轉(zhuǎn)染C2C12細(xì)胞，分為實(shí)驗(yàn)組（siRNA干擾組）和陰性對(duì)照組（NC組）。應(yīng)用CCK-8試劑盒檢測(cè)細(xì)胞的增殖能力，應(yīng)用流式細(xì)胞儀檢測(cè)細(xì)胞周期。3、加入含2%馬血清的分化液使細(xì)胞肌向分化，應(yīng)用SYBR Green實(shí)時(shí)定量PCR檢測(cè)細(xì)胞分化后生肌調(diào)節(jié)因子家族成員Myf5、myogenin和MRF4，以及Wnt5a表達(dá)水平。應(yīng)用免疫細(xì)胞化學(xué)法檢測(cè)C2C12細(xì)胞系肌向分化過(guò)程中Myosin的表達(dá)，并觀察細(xì)胞的形態(tài)學(xué)改變。 結(jié)果：1、和NC組及其它各干擾組相比，siRNA-1高效下調(diào)了C2C12細(xì)胞內(nèi)Wnt5a的表達(dá)水平（96.78%）。2、siRNA-1干擾Wnt5a表達(dá)下調(diào)后，CCK-8結(jié)果顯示C2C12細(xì)胞增殖無(wú)明顯變化，無(wú)統(tǒng)計(jì)學(xué)差異（P0.05）；流式細(xì)胞儀檢測(cè)結(jié)果顯示停留于各期的細(xì)胞數(shù)未明顯增加，無(wú)統(tǒng)計(jì)學(xué)差異（P0.05）。3、siRNA-1干擾Wnt5a表達(dá)下調(diào)后，在分化的D0天，Wnt5a的表達(dá)水平明顯下調(diào)，有統(tǒng)計(jì)學(xué)差異（P0.05）；在分化的D2天，Myf5、MRF4的表達(dá)水平明顯下調(diào)，有統(tǒng)計(jì)學(xué)差異（P0.05）。免疫細(xì)胞化學(xué)法顯示Myosin表達(dá)在多核肌管細(xì)胞中，胞漿染色強(qiáng)陽(yáng)性，，在分化的各天，siRNA干擾組和NC組比較細(xì)胞的形態(tài)無(wú)明顯的區(qū)別。 結(jié)論：體外下調(diào)Wnt5a基因表達(dá)對(duì)C2C12細(xì)胞系的增殖和細(xì)胞周期無(wú)顯著影響，而肌向誘導(dǎo)分化的C2C12細(xì)胞系中Myf5和MRF4等表達(dá)水平下調(diào)提示了Wnt5a可能對(duì)骨骼肌細(xì)胞分化具有調(diào)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(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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