IGF1剪接变异体IGF1Ec对成肌细胞生物学功能的影响及相关机理研究

发布时间：2018-01-01 20:41

  本文关键词：IGF1剪接变异体IGF1Ec对成肌细胞生物学功能的影响及相关机理研究　出处：《重庆大学》2016年博士论文　论文类型：学位论文

  更多相关文章： IGF1Ec IGF1 MGF E IGF1-24 C2C12细胞

【摘要】：胰岛素样生长因子1(Insulin-like growth factor1,IGF1)是分泌的细胞生长因子,对身体的正常生长、发育和维持非常重要。研究发现由肌肉拉伸或电刺激造成的机械损伤、运动诱发的肌肉损伤以及注射布比卡因等都会诱导IGF1发生可变剪接,其剪接变异体IGF1Ec的表达会迅速增加。有研究指出IGF1Ec羧基末端(C端)的24个氨基酸多肽(又称为MGF E肽)参与肌肉、神经、心脏等疾病的修复治疗,被认为是极有潜力的组织工程生长因子。然而现有关IGF1Ec结构与功能的研究不是十分深入,并且MGF E肽对肌肉功能的研究还存在诸多分歧和争议。因此,我们尝试对IGF1剪接变异体IGF1Ec的结构和功能进行深入系统的研究。本研究比较了IGF1Ec、IGF1和MGF E肽对成肌细胞增殖、分化和迁移的影响,系统阐明了IGF1Ec不同结构区域和细胞功能的关系,对IGF1Ec、IGF1和MGF E肽在肌肉组织修复再生中的应用具有重要意义;另一方面,利用GST-Pulldown联合质谱分析技术初步获得了IGF1Ec和MGF E肽的相互作用蛋白,为进一步探寻IGF1Ec和MGF E肽新的细胞膜受体和下游信号通路奠定基础;在机理研究的同时,利用点突变PCR技术对IGF1Ec基因序列进行优化,提高了IGF1Ec蛋白在大肠杆菌BL21(DE3)中的产量,为工业化大量生产IGF1Ec蛋白提供了技术改进方案。本论文主要研究内容及结论如下:(1)IGF1Ec基因中存在的大肠杆菌BL21(DE3)稀有密码子极大地阻碍了IGF1Ec蛋白在该菌株中的表达。利用点突变PCR和重叠PCR的技术对IGF1Ec的基因序列进行了优化,构建了两个原核表达载体pGEX-4T-1/IGF1Ec(Mut54-56)和pGEX-4T-1/IGF1Ec(Mut-total)。这两个载体在BL21(DE3)中目的蛋白IGF1Ec的表达量显著高于pGEX-4T-1/IGF1Ec在Rosetta(DE3)中的表达。在获得IGF1Ec后,我们进一步在成骨细胞中检验其生物活性。结果显示低浓度的IGF1Ec即可显著增强成骨细胞增殖和迁移能力,并且一定程度上抑制成骨细胞分化水平。因此我们获得的IGF1Ec具有较高的生物活性。(2)比较了IGF1Ec、IGF1和MGF E肽对成肌细胞C2C12的行为的影响。实验结果显示a.IGF1Ec和IGF1可以促进细胞的增殖,而MGF E肽则对细胞增殖没有影响;b.IGF1Ec、IGF1和MGF E肽都能促进成肌细胞肌管融合,但IGF1Ec的促进效率明显高于IGF1;c.IGF1Ec、IGF1和MGF E肽都能够促进迁移,但IGF1Ec和MGF E肽促迁移能力明显高于IGF1。随后我们用抑制剂封闭IGF1受体(IGF1Receptor,IGF1R),对其信号通路进行了研究。实验结果显示,对于细胞增殖效应,IGF1Ec和IGF1都是通过IGF1R激活介导的;对于细胞迁移效应,IGF1只通过IGF1R激活介导,而IGF1Ec和MGF E肽除了通过IGF1R激活介导,还存在其他受体参与。因此,我们认为IGF1Ec的不同结构区域具有不同的细胞生物学功能:氨基端(N端)的IGF1的序列,主要影响细胞增殖;C端的MGF E的序列,主要影响细胞分化和迁移。(3)设计合成了重组蛋白IGF1-24。该重组蛋白包括N端IGF1的氨基酸序列和C端的MGF E肽氨基酸序列两部分。通过比较重组蛋白IGF1-24和IGF1Ec对成肌细胞C2C12细胞行为的影响,发现IGF1-24对细胞的增殖、分化、迁移等细胞行为的作用同IGF1Ec非常相似,并且引起的生物学效应对IGF1R依赖性也是基本相同。因此,我们的实验结果进一步证明IGF1Ec对细胞行为的影响是通过N端IGF1的序列和C端MGF E肽的序列共同完成的。(4)对IGF1Ec在细胞内和细胞外存在形式的研究结果显示:细胞内只检测到IGF1Ec蛋白一条带,表明IGF1Ec在细胞内没有被蛋白酶裂解;在细胞外同样只检测到IGF1Ec一条带,表明分泌出胞外的IGF1Ec同样没有被蛋白酶裂解。因此,我们认为IGF1Ec并不是通过裂解为成熟IGF1和E肽来发挥功能,而是作为一个整体蛋白来发挥功能。(5)通过GST-Pulldown联合LC-MS/MS质谱分析的方法检测了分别与IGF1Ec和MGF E肽相互作用的蛋白,发现其中有46个只与IGF1Ec和MGF E肽相互作用,而不和IGF1相互作用。利用GOEAST和DAVID基因功能分析软件对这些蛋白进行了生物信息学分析,结果显示这些蛋白参与调控细胞的许多生物学过程,如蛋白合成、蛋白转运、细胞迁移、细胞连接和细胞分化等。
[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.

【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R329.2

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