本文選題：FGF13 + 細(xì)胞增殖�。� 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：骨骼肌在機(jī)體中主要執(zhí)行收縮和舒張功能,參與完成各種運(yùn)動(dòng)。在畜禽類的研究中,骨骼肌的數(shù)量和質(zhì)量被作為衡量畜禽肉品質(zhì)的主要指標(biāo)。骨骼肌生長發(fā)育是一個(gè)相當(dāng)復(fù)雜的過程,包括肌源干細(xì)胞分化成單核的成肌細(xì)胞、融合形成多核肌管、成熟肌纖維等過程,在這些過程中涉及多個(gè)成肌因子的調(diào)控。盡管小鼠骨骼肌生長發(fā)育的過程研究已較為清楚,但參與調(diào)控肌生成的因子還有待進(jìn)一步的挖掘。FGF13屬于FGF亞家族(FHF)的成員,其結(jié)構(gòu)特點(diǎn)是N端不具有自身的信號(hào)分泌序列,不能分泌到胞外,作為胞內(nèi)蛋白發(fā)揮作用。FGF13在腦和神經(jīng)組織中高表達(dá),但有研究發(fā)現(xiàn)小鼠腿肌中FGF13基因的表達(dá)要比眼外肌高;在培養(yǎng)的單根肌纖維中,檢測(cè)到FGF13也有較高的表達(dá);在人類的BFLS癥中FGF13在病人的腦和骨骼肌中均高表達(dá),因此,我們推測(cè)FGF13在骨骼肌生長發(fā)育中發(fā)揮一定的作用。為驗(yàn)證FGF13在肌肉發(fā)育中的作用,首先我們檢測(cè)FGF13在小鼠各組織器官中特異性表達(dá),并檢測(cè)FGF13在C2C12成肌細(xì)胞增殖和分化過程中的時(shí)序表達(dá);構(gòu)建FGF13的si RNA和超表達(dá)載體,分別轉(zhuǎn)染C2C12細(xì)胞,采用Real-time qPCR、Western blotting、流式細(xì)胞術(shù)和免疫熒光染色等細(xì)胞分子生物學(xué)手段,從細(xì)胞形態(tài)、mRNA和蛋白水平檢測(cè)FGF13對(duì)C2C12成肌細(xì)胞的增殖、分化、肌管的融合以及相關(guān)信號(hào)通路調(diào)控等方面的影響,初步確定FGF13參與肌肉細(xì)胞增殖和分化過程。其次,我們構(gòu)建小鼠的骨骼肌損傷模型,并在肌肉的局部組織注射FGF13的siRNA,采用冰凍切片和組織免疫熒光技術(shù)檢測(cè)肌肉損傷后的修復(fù)情況。最后,構(gòu)建FGF13誘餌重組載體,采用酵母雙雜交系統(tǒng)從小鼠的cDNA文庫中篩選與FGF13互作的蛋白質(zhì),為進(jìn)一步探索FGF13影響骨骼肌生長發(fā)育的信號(hào)轉(zhuǎn)導(dǎo)通路提供新線索。獲得研究結(jié)果如下:1.FGF13基因在腦組織中表達(dá)量最高,肌肉、心臟和肺臟等組織處于中等表達(dá)水平;在C2C12成肌細(xì)胞增殖和分化過程中,發(fā)現(xiàn)FGF13在分化第4d表達(dá)量達(dá)到最高,然后逐漸下降;用si-FGF13轉(zhuǎn)染C2C12成肌細(xì)胞后,MyOG和MyHC的表達(dá)水平顯著增加,肌管形成增多;反之,超表達(dá)后,MyOG和MyHC的表達(dá)水平顯著降低。因此,FGF13抑制C2C12成肌細(xì)胞的分化和肌管形成。2.通過在C2C12成肌細(xì)胞中超表達(dá)FGF13和采用信號(hào)通路抑制劑處理的方法,發(fā)現(xiàn)超表達(dá)FGF13后ERK1/2的磷酸化水平有所提高,ERK1/2信號(hào)通路的激活會(huì)抑制早期成肌分化;3.Spry1是ERK1/2信號(hào)通路的抑制因子,當(dāng)干擾Spry1的表達(dá),成肌分化能力增強(qiáng);采用siRNA雙敲除方法,發(fā)現(xiàn)FGF13通過抑制Spry1的表達(dá)而促進(jìn)ERK1/2磷酸化水平的提高,從而激活ERK1/2信號(hào)通路,抑制成肌分化。4.FGF13通過抑制Spry1基因表達(dá)使p27基因的表達(dá)水平升高,進(jìn)一步抑制CyclinE蛋白表達(dá)水平,最終導(dǎo)致細(xì)胞增殖減慢,但并沒有影響ERK1/2的磷酸化水平;細(xì)胞劃痕實(shí)驗(yàn)表明,超表達(dá)FGF13能夠顯著抑制C2C12細(xì)胞遷移;另一方面,采用BaCl2注射方法損傷骨骼肌,構(gòu)建肌肉組織損傷模型;注射siFGF13后,局部損傷組織的炎癥細(xì)胞數(shù)量有所減少,成肌相關(guān)基因MyOG表達(dá)顯著增加,表明FGF13在肌肉損傷后的修復(fù)過程中發(fā)揮負(fù)調(diào)控的作用。5.構(gòu)建pGBKT7-mFGF13誘餌蛋白的表達(dá)載體,進(jìn)行酵母雙雜交實(shí)驗(yàn),篩選到9個(gè)與FGF13相互作用的蛋白質(zhì),其中Shcbp1、ARHGAP5可能與FGF13相互結(jié)合影響ERK1/2和P38信號(hào)通路,從而調(diào)節(jié)骨骼肌的生長發(fā)育過程,但具體的結(jié)合形式及功能還需進(jìn)一步驗(yàn)證。綜上所述,FGF13通過下調(diào)Spry1的表達(dá),激活ERK1/2信號(hào)通路從而抑制C2C12成肌細(xì)胞的分化。因此,在成肌分化中FGF13是一個(gè)抑制因子。在C2C12成肌細(xì)胞的增殖過程中,FGF13也是通過下調(diào)Spry1的表達(dá)而促進(jìn)p27基因的表達(dá)水平升高,進(jìn)一步使CyclinE蛋白表達(dá)減少,最終導(dǎo)致細(xì)胞增殖速度減慢,但ERK1/2信號(hào)通路并沒有受到影響。運(yùn)用酵母雙雜交系統(tǒng)篩選出9個(gè)可能與FGF13相互作用的蛋白質(zhì),參與細(xì)胞增殖和分化、遷移和運(yùn)動(dòng)、自噬、自身免疫以及能量代謝等過程,但具體的功能還需進(jìn)一步驗(yàn)證。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q44

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10 劉愛麗;采用酵母雙雜交技術(shù)篩選熱激反應(yīng)相關(guān)剪接因子的互作蛋白[D];華中師范大學(xué);2016年

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