本文關(guān)鍵詞：IGF1剪接變異體IGF1Ec對成肌細胞生物學(xué)功能的影響及相關(guān)機理研究　出處：《重慶大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： IGF1Ec IGF1 MGF E IGF1-24 C2C12細胞

【摘要】：胰島素樣生長因子1(Insulin-like growth factor1,IGF1)是分泌的細胞生長因子,對身體的正常生長、發(fā)育和維持非常重要。研究發(fā)現(xiàn)由肌肉拉伸或電刺激造成的機械損傷、運動誘發(fā)的肌肉損傷以及注射布比卡因等都會誘導(dǎo)IGF1發(fā)生可變剪接,其剪接變異體IGF1Ec的表達會迅速增加。有研究指出IGF1Ec羧基末端(C端)的24個氨基酸多肽(又稱為MGF E肽)參與肌肉、神經(jīng)、心臟等疾病的修復(fù)治療,被認(rèn)為是極有潛力的組織工程生長因子。然而現(xiàn)有關(guān)IGF1Ec結(jié)構(gòu)與功能的研究不是十分深入,并且MGF E肽對肌肉功能的研究還存在諸多分歧和爭議。因此,我們嘗試對IGF1剪接變異體IGF1Ec的結(jié)構(gòu)和功能進行深入系統(tǒng)的研究。本研究比較了IGF1Ec、IGF1和MGF E肽對成肌細胞增殖、分化和遷移的影響,系統(tǒng)闡明了IGF1Ec不同結(jié)構(gòu)區(qū)域和細胞功能的關(guān)系,對IGF1Ec、IGF1和MGF E肽在肌肉組織修復(fù)再生中的應(yīng)用具有重要意義;另一方面,利用GST-Pulldown聯(lián)合質(zhì)譜分析技術(shù)初步獲得了IGF1Ec和MGF E肽的相互作用蛋白,為進一步探尋IGF1Ec和MGF E肽新的細胞膜受體和下游信號通路奠定基礎(chǔ);在機理研究的同時,利用點突變PCR技術(shù)對IGF1Ec基因序列進行優(yōu)化,提高了IGF1Ec蛋白在大腸桿菌BL21(DE3)中的產(chǎn)量,為工業(yè)化大量生產(chǎn)IGF1Ec蛋白提供了技術(shù)改進方案。本論文主要研究內(nèi)容及結(jié)論如下:(1)IGF1Ec基因中存在的大腸桿菌BL21(DE3)稀有密碼子極大地阻礙了IGF1Ec蛋白在該菌株中的表達。利用點突變PCR和重疊PCR的技術(shù)對IGF1Ec的基因序列進行了優(yōu)化,構(gòu)建了兩個原核表達載體pGEX-4T-1/IGF1Ec(Mut54-56)和pGEX-4T-1/IGF1Ec(Mut-total)。這兩個載體在BL21(DE3)中目的蛋白IGF1Ec的表達量顯著高于pGEX-4T-1/IGF1Ec在Rosetta(DE3)中的表達。在獲得IGF1Ec后,我們進一步在成骨細胞中檢驗其生物活性。結(jié)果顯示低濃度的IGF1Ec即可顯著增強成骨細胞增殖和遷移能力,并且一定程度上抑制成骨細胞分化水平。因此我們獲得的IGF1Ec具有較高的生物活性。(2)比較了IGF1Ec、IGF1和MGF E肽對成肌細胞C2C12的行為的影響。實驗結(jié)果顯示a.IGF1Ec和IGF1可以促進細胞的增殖,而MGF E肽則對細胞增殖沒有影響;b.IGF1Ec、IGF1和MGF E肽都能促進成肌細胞肌管融合,但IGF1Ec的促進效率明顯高于IGF1;c.IGF1Ec、IGF1和MGF E肽都能夠促進遷移,但IGF1Ec和MGF E肽促遷移能力明顯高于IGF1。隨后我們用抑制劑封閉IGF1受體(IGF1Receptor,IGF1R),對其信號通路進行了研究。實驗結(jié)果顯示,對于細胞增殖效應(yīng),IGF1Ec和IGF1都是通過IGF1R激活介導(dǎo)的;對于細胞遷移效應(yīng),IGF1只通過IGF1R激活介導(dǎo),而IGF1Ec和MGF E肽除了通過IGF1R激活介導(dǎo),還存在其他受體參與。因此,我們認(rèn)為IGF1Ec的不同結(jié)構(gòu)區(qū)域具有不同的細胞生物學(xué)功能:氨基端(N端)的IGF1的序列,主要影響細胞增殖;C端的MGF E的序列,主要影響細胞分化和遷移。(3)設(shè)計合成了重組蛋白IGF1-24。該重組蛋白包括N端IGF1的氨基酸序列和C端的MGF E肽氨基酸序列兩部分。通過比較重組蛋白IGF1-24和IGF1Ec對成肌細胞C2C12細胞行為的影響,發(fā)現(xiàn)IGF1-24對細胞的增殖、分化、遷移等細胞行為的作用同IGF1Ec非常相似,并且引起的生物學(xué)效應(yīng)對IGF1R依賴性也是基本相同。因此,我們的實驗結(jié)果進一步證明IGF1Ec對細胞行為的影響是通過N端IGF1的序列和C端MGF E肽的序列共同完成的。(4)對IGF1Ec在細胞內(nèi)和細胞外存在形式的研究結(jié)果顯示:細胞內(nèi)只檢測到IGF1Ec蛋白一條帶,表明IGF1Ec在細胞內(nèi)沒有被蛋白酶裂解;在細胞外同樣只檢測到IGF1Ec一條帶,表明分泌出胞外的IGF1Ec同樣沒有被蛋白酶裂解。因此,我們認(rèn)為IGF1Ec并不是通過裂解為成熟IGF1和E肽來發(fā)揮功能,而是作為一個整體蛋白來發(fā)揮功能。(5)通過GST-Pulldown聯(lián)合LC-MS/MS質(zhì)譜分析的方法檢測了分別與IGF1Ec和MGF E肽相互作用的蛋白,發(fā)現(xiàn)其中有46個只與IGF1Ec和MGF E肽相互作用,而不和IGF1相互作用。利用GOEAST和DAVID基因功能分析軟件對這些蛋白進行了生物信息學(xué)分析,結(jié)果顯示這些蛋白參與調(diào)控細胞的許多生物學(xué)過程,如蛋白合成、蛋白轉(zhuǎn)運、細胞遷移、細胞連接和細胞分化等。
[Abstract]:Insulin-like growth factor 1 (Insulin-like growth, factor1, IGF1) is a secreted growth factor, the normal growth of the body, development and maintenance is very important. The study found that by muscle tension or electrical stimulation caused by mechanical damage, exercise-induced muscle injury and injection of bupivacaine can induce the expression of IGF1 alternative splicing and splicing variant IGF1Ec will rapidly increase. Studies have shown that IGF1Ec (C) of the C-terminal polypeptide of 24 amino acids (also known as MGF E peptide) in muscle, nerve repair, treatment of heart disease, is considered to be a potential growth factor in tissue engineering. However the structure and function of IGF1Ec is not very deep, and also many differences and disputes exist on MGF E peptide on muscle function. Therefore, we try to structure and function of IGF1 spliced variant IGF1Ec in-depth system research Study. This study compared the IGF1Ec, IGF1 and MGF of E peptide on myoblast proliferation, differentiation and migration of the affected system, clarify the relationship between IGF1Ec, different structural regions and cell function of IGF1Ec, IGF1 and MGF has important significance for application of E peptide in muscle tissue repair and regeneration; on the other hand, the use of GST-Pulldown combined with mass spectrometry to obtain the preliminary interaction between IGF1Ec protein and MGF E peptide, which lays a foundation for the further study of the IGF1Ec and MGF E peptide new cell membrane receptors and the downstream signaling pathway in the mechanism research; at the same time, the use of point mutations on the IGF1Ec gene sequence of PCR optimization techniques, improved IGF1Ec protein in Escherichia coli BL21 (DE3) in production, provides the technology for industrialized mass production of IGF1Ec protein improvement program. The main research contents and conclusions of this paper are as follows: (1) IGF1Ec gene in Escherichia coli BL21 (DE3) of rare codons Greatly hindered the expression of IGF1Ec protein in this strain. The point mutation PCR and overlapping PCR technology gene sequences of IGF1Ec were optimized to construct two prokaryotic expression vector pGEX-4T-1/IGF1Ec (Mut54-56) and pGEX-4T-1/IGF1Ec (Mut-total). The two vectors in BL21 (DE3) expression of the target protein IGF1Ec the pGEX-4T-1/IGF1Ec was significantly higher than that in Rosetta (DE3). The expression in IGF1Ec, we further to test the biological activity of bone cells. The results showed that low concentration of IGF1Ec can significantly enhance osteoblast proliferation and migration, and to some extent inhibit osteoblast differentiation level. So we get IGF1Ec has a high biological activity. (2) compared with IGF1Ec, IGF1 and MGF effect of E peptide on myoblast C2C12 behavior. The experimental results showed that a.IGF1Ec and IGF1 can promote cell proliferation, and MGF peptide E It has no effect on cell proliferation; b.IGF1Ec, IGF1 and MGF E peptide can promote myoblast fusion and myotube, but IGF1Ec promote efficiency was significantly higher than that of IGF1; c.IGF1Ec, IGF1 and MGF E peptide can promote migration, but the IGF1Ec and MGF E peptide promoting migration capacity was significantly higher than that of IGF1. and then we used inhibitors blocking the IGF1 receptor (IGF1Receptor, IGF1R), the signal pathway was studied. Experimental results show that the effect of cell proliferation, IGF1Ec and IGF1 are activated by IGF1R mediated cell migration; the effect of IGF1 through IGF1R mediated by activation of IGF1Ec and MGF, and E peptide except through IGF1R mediated activation, there are other receptors participation. Therefore, we believe that the different structure region of IGF1Ec cells with different biological functions: the N-terminal sequence of IGF1 (N), the main effect of cell proliferation; sequence C end MGF E, the main effect of cell differentiation and migration (3). The design and synthesis of the two part of the MGF E peptide amino acid sequence and amino acid sequence of the recombinant C protein IGF1-24. of the recombinant protein including the N end of the IGF1. Through the comparison of recombinant IGF1-24 protein and the effect of IGF1Ec on myoblast C2C12 cells act, found that the proliferation of IGF1-24 on cell differentiation, cell migration behaviors with IGF1Ec too similar to the biological effect and cause of IGF1R dependence is basically the same. Therefore, our experimental results further demonstrate the effect of IGF1Ec on cell behavior by N end IGF1 sequence and C sequence MGF E end peptide dotogether. (4) the presence of IGF1Ec in the form of intracellular and extracellular results display: inside the cell was detected with a IGF1Ec protein showed that IGF1Ec was not protease cleavage in cells; extracellular also detected a band of IGF1Ec, showed that the secretion of extracellular IGF1Ec of the same Not by protease cleavage. Therefore, we believe that IGF1Ec is not by cleavage into mature IGF1 and E peptide to function as a whole protein function. (5) by GST-Pulldown method combined with LC-MS/MS mass spectrometry detection respectively with IGF1Ec and MGF mutual E peptide protein, found that there are 46 only with IGF1Ec and MGF E peptide interactions with IGF1 interaction. To analyze the bioinformatics of these proteins using GOEAST and DAVID gene showed many biological functions, these proteins participate in the regulation of cell processes, such as protein synthesis, protein transport, cell migration, cell junction and cell differentiation.

【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R329.2

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