FGF13影响小鼠骨骼肌增殖与分化的机制研究

发布时间：2018-06-22 14:03

本文选题：FGF13 + 细胞增殖　；参考：《西北农林科技大学》2016年博士论文

【摘要】：骨骼肌在机体中主要执行收缩和舒张功能,参与完成各种运动。在畜禽类的研究中,骨骼肌的数量和质量被作为衡量畜禽肉品质的主要指标。骨骼肌生长发育是一个相当复杂的过程,包括肌源干细胞分化成单核的成肌细胞、融合形成多核肌管、成熟肌纤维等过程,在这些过程中涉及多个成肌因子的调控。尽管小鼠骨骼肌生长发育的过程研究已较为清楚,但参与调控肌生成的因子还有待进一步的挖掘。FGF13属于FGF亚家族(FHF)的成员,其结构特点是N端不具有自身的信号分泌序列,不能分泌到胞外,作为胞内蛋白发挥作用。FGF13在脑和神经组织中高表达,但有研究发现小鼠腿肌中FGF13基因的表达要比眼外肌高;在培养的单根肌纤维中,检测到FGF13也有较高的表达;在人类的BFLS症中FGF13在病人的脑和骨骼肌中均高表达,因此,我们推测FGF13在骨骼肌生长发育中发挥一定的作用。为验证FGF13在肌肉发育中的作用,首先我们检测FGF13在小鼠各组织器官中特异性表达,并检测FGF13在C2C12成肌细胞增殖和分化过程中的时序表达;构建FGF13的si RNA和超表达载体,分别转染C2C12细胞,采用Real-time qPCR、Western blotting、流式细胞术和免疫荧光染色等细胞分子生物学手段,从细胞形态、mRNA和蛋白水平检测FGF13对C2C12成肌细胞的增殖、分化、肌管的融合以及相关信号通路调控等方面的影响,初步确定FGF13参与肌肉细胞增殖和分化过程。其次,我们构建小鼠的骨骼肌损伤模型,并在肌肉的局部组织注射FGF13的siRNA,采用冰冻切片和组织免疫荧光技术检测肌肉损伤后的修复情况。最后,构建FGF13诱饵重组载体,采用酵母双杂交系统从小鼠的cDNA文库中筛选与FGF13互作的蛋白质,为进一步探索FGF13影响骨骼肌生长发育的信号转导通路提供新线索。获得研究结果如下:1.FGF13基因在脑组织中表达量最高,肌肉、心脏和肺脏等组织处于中等表达水平;在C2C12成肌细胞增殖和分化过程中,发现FGF13在分化第4d表达量达到最高,然后逐渐下降;用si-FGF13转染C2C12成肌细胞后,MyOG和MyHC的表达水平显著增加,肌管形成增多;反之,超表达后,MyOG和MyHC的表达水平显著降低。因此,FGF13抑制C2C12成肌细胞的分化和肌管形成。2.通过在C2C12成肌细胞中超表达FGF13和采用信号通路抑制剂处理的方法,发现超表达FGF13后ERK1/2的磷酸化水平有所提高,ERK1/2信号通路的激活会抑制早期成肌分化;3.Spry1是ERK1/2信号通路的抑制因子,当干扰Spry1的表达,成肌分化能力增强;采用siRNA双敲除方法,发现FGF13通过抑制Spry1的表达而促进ERK1/2磷酸化水平的提高,从而激活ERK1/2信号通路,抑制成肌分化。4.FGF13通过抑制Spry1基因表达使p27基因的表达水平升高,进一步抑制CyclinE蛋白表达水平,最终导致细胞增殖减慢,但并没有影响ERK1/2的磷酸化水平;细胞划痕实验表明,超表达FGF13能够显著抑制C2C12细胞迁移;另一方面,采用BaCl2注射方法损伤骨骼肌,构建肌肉组织损伤模型;注射siFGF13后,局部损伤组织的炎症细胞数量有所减少,成肌相关基因MyOG表达显著增加,表明FGF13在肌肉损伤后的修复过程中发挥负调控的作用。5.构建pGBKT7-mFGF13诱饵蛋白的表达载体,进行酵母双杂交实验,筛选到9个与FGF13相互作用的蛋白质,其中Shcbp1、ARHGAP5可能与FGF13相互结合影响ERK1/2和P38信号通路,从而调节骨骼肌的生长发育过程,但具体的结合形式及功能还需进一步验证。综上所述,FGF13通过下调Spry1的表达,激活ERK1/2信号通路从而抑制C2C12成肌细胞的分化。因此,在成肌分化中FGF13是一个抑制因子。在C2C12成肌细胞的增殖过程中,FGF13也是通过下调Spry1的表达而促进p27基因的表达水平升高,进一步使CyclinE蛋白表达减少,最终导致细胞增殖速度减慢,但ERK1/2信号通路并没有受到影响。运用酵母双杂交系统筛选出9个可能与FGF13相互作用的蛋白质,参与细胞增殖和分化、迁移和运动、自噬、自身免疫以及能量代谢等过程,但具体的功能还需进一步验证。
[Abstract]:Skeletal muscles mainly perform contractile and diastolic functions in the body and participate in various movements. In the study of livestock and poultry, the quantity and quality of skeletal muscles are the main indicators to measure the quality of livestock and poultry meat. The growth and development of skeletal muscles is a rather complicated process, including myogenic stem cells differentiated into mononuclear myoblasts and fusion formation. Nuclear myotubes, mature muscle fibers, and other processes involved in the regulation of several myogenic factors in these processes. Although the study of the growth and development of skeletal muscles in mice is clear, the factors involved in the regulation of muscle generation still need to be further excavated by.FGF13 members of the FGF subfamily (FHF), whose structural characteristics are that the N end does not have their own signal points. The secreting sequences can not be secreted to the extracellular, as intracellular proteins play a role in the expression of.FGF13 in the brain and nerve tissue, but some studies have found that the expression of FGF13 gene in the leg muscles of mice is higher than that of the extraocular muscles; in the single muscle fibers of the culture, the expression of FGF13 is also higher; in human BFLS disease, FGF13 is in the brain and skeletal muscles of the patients. Therefore, we speculate that FGF13 plays a role in the growth and development of skeletal muscle. In order to verify the role of FGF13 in the development of muscle, we first detect the specific expression of FGF13 in various tissues and organs of mice, and detect the time sequence expression of FGF13 in the proliferation and differentiation of C2C12 myoblasts, and construct the Si RNA and overwatch of FGF13. C2C12 cells were transfected, and Real-time qPCR, Western blotting, flow cytometry and immunofluorescence staining were used to determine the effects of FGF13 on the proliferation, differentiation, fusion of myotubes and the regulation of related signal pathways from the cell morphology, mRNA and protein levels, and the preliminary determination of F GF13 participates in the process of proliferation and differentiation of muscle cells. Secondly, we construct a mouse skeletal muscle damage model, and injecting FGF13 siRNA in the local tissue of the muscles, using frozen section and tissue immunofluorescence to detect the repair of muscle injury. Finally, the recombinant vector of FGF13 decoy was constructed and the yeast two hybrid system was used to form a yeast two hybrid system from mice. CDNA library screening and FGF13 interaction proteins provide new clues for further exploring the signal transduction pathway that FGF13 affects the growth and development of skeletal muscle. The results are as follows: the highest expression of 1.FGF13 gene in the brain tissue, muscle, heart and lung tissues at the middle expression level; the proliferation and differentiation process of C2C12 myoblast cells. It was found that the expression of FGF13 reached the highest level in the differentiation 4D, and then decreased gradually; after transfecting C2C12 into myoblasts with si-FGF13, the expression level of MyOG and MyHC increased significantly and the myotube formation increased; on the contrary, the expression level of MyOG and MyHC decreased significantly after overexpression. Therefore, FGF13 inhibited the differentiation of C2C12 myoblasts and the formation of myotube formation.2. through C2C 12 the overexpression of FGF13 in myoblasts and the method of signal pathway inhibitor treatment, it was found that the phosphorylation level of ERK1/2 increased after overexpression of FGF13, and the activation of ERK1/2 signaling pathway inhibited the early myogenic differentiation; 3.Spry1 was a inhibitory factor of ERK1/2 signaling pathway, and when the expression of Spry1 was disturbed, the ability of myogenic differentiation was enhanced; siRNA double was used. It was found that FGF13 promoted the level of phosphorylation of ERK1/2 by inhibiting the expression of Spry1, thus activating the ERK1/2 signaling pathway, inhibiting the differentiation of myogenic differentiation.4.FGF13 by inhibiting the expression of Spry1 gene to increase the expression level of the p27 gene, further inhibiting the level of the expression of CyclinE protein, and eventually causing cell proliferation and slowing down, but there is no shadow. The level of phosphorylation of ERK1/2, cell scratch test showed that overexpression of FGF13 could significantly inhibit the migration of C2C12 cells; on the other hand, BaCl2 injections were used to damage skeletal muscle and to construct a muscle tissue damage model. After siFGF13 injection, the number of inflammatory cells in local injured tissues decreased and the expression of MyOG related genes increased significantly. FGF13 plays a negative regulatory role in the repair of muscle injury after muscle damage,.5. constructs the expression vector of pGBKT7-mFGF13 decoy protein, and carries out yeast two hybrid experiment to screen 9 proteins interacting with FGF13, in which Shcbp1 and ARHGAP5 may interact with FGF13 to influence ERK1/2 and P38 signaling pathways, thus regulating the growth of skeletal muscle. Development, but specific binding forms and functions need further validation. To sum up, FGF13 can inhibit the differentiation of C2C12 myoblasts by downregulating the expression of Spry1 and activating the ERK1/2 signaling pathway. Therefore, FGF13 is an inhibitory factor in the myoblast differentiation. In the proliferation of C2C12 myoblasts, FGF13 is also through the reduction of Spry1. The expression increased the expression level of the p27 gene, further reduced the expression of CyclinE protein and eventually led to the slow growth of cell proliferation, but the ERK1/2 signaling pathway was not affected. The yeast two hybrid system was used to screen 9 proteins that might interact with the FGF13, and to participate in cell proliferation and differentiation, migration and movement, autophagy, and autophagy. However, specific functions need further verification.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q44

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