GLP-1串联体体外表达与大鼠小肠原代上皮细胞的分离培养研究

发布时间：2018-05-14 06:34

本文选题：GLP-1多聚体 + 2型糖尿病　；参考：《天津医科大学》2017年硕士论文

【摘要】：糖尿病(Diabetes)是一种终身性代谢性疾病,其发病原因众多,常表现为慢性高血糖。2013年,国际糖尿病联合会(Internatinal Diabetes Federation(IDF))公布,全球罹患糖尿病的患者达到了惊人的3.83亿。尽快糖尿病本身没有很大的危害,但是长期的血糖增高会损伤大血管、微血管,进而威胁到脑、心、周围神经、肾、眼睛等的正常功能,而且随之而来的并发症也会极大影响人类的健康。糖尿病的发病机制复杂,当前现有的治疗手段尚达不到治愈的效果,随着糖尿病患者数量的日益增多,对于它们的研究已经成为当前医学领域的热点。由哺乳动物肠道L细胞分泌的胰高血糖素样肽(glucagon-like peptide-1,GLP-1)是一种多肽类激素,该多肽由31个氨基酸组成。由于它可以促进胰岛素在高血糖状态下的分泌,所以GLP-1在2型糖尿病的治疗中备受关注。但是机体内存在的二肽基肽酶(DPPⅣ)能够使GLP-1迅速降解,这极大地限制了GLP-1的生理功能。为了提升GLP-1在体内的生理功能,需要对GLP-1的结构进行优化或者提高GLP-1在体内的表达产量,从而抵抗DPP-IV的降解。本研究通过构建GLP-1串联重复多聚体来提高GLP-1在体外的表达产量,从而使其能够更长时间地抵抗DPPⅣ的降解作用,最终提升GLP-1在体内的生理功能。在本研究中,我们分别构建了p ET-22b-GLP4,GLP8,GLP12和GLP16的表达载体,在大肠杆菌BL21(D3)中转化后利用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。结果显示,GLP4的表达量最高,GLP8次之,而GLP12和GLP16几乎不表达。除了基因本身的结构和表达系统的效率外,诱导温度、诱导时间以及诱导剂浓度对GLP-1多聚体的表达量都有影响,通过实验我们发现,GLP-1串联体的表达量在26℃,诱导剂IPTG浓度为0.6m M时最高,并且GLP-1多聚体体外表达的最佳诱导时间为8小时。在上述获得的优化条件之下,我们最终确定了该串连体在体外原核表达系统中的最适合拷贝数为4。近期研究表明,小肠也同样具有分泌胰岛素的功能,而且在发育过程中,小肠上皮细胞和胰岛内分泌细胞具有共同的起源,这就暗示我们,处在发育阶段的小肠上皮细胞具有诱导分化成胰岛素分泌细胞的潜能。为了深入研究小肠上皮细胞与胰岛素分泌之间的相关性,本研究第二部分分离了原代培养的大鼠胚胎期小肠上皮细胞。通过对细胞的形态学进行观察,我们发现培养过程中的细胞符合上皮细胞的形态学特征。而且,对培养的细胞进行免疫荧光染色后发现,培养的细胞表达角蛋白8(小肠上皮细胞表达标志物),进一步确定了细胞的来源。基于这些研究,为了方便未来对小肠上皮细胞的改造,我们进一步构建了hr GFP慢病毒载体,并成功感染大鼠小肠上皮细胞。结论:本研究成功地对GLP-1多聚体进行了体外的诱导表达,确定了GLP-1串联体的最佳表达条件以及最适合的拷贝数,为GLP-1多聚体的体内研究打下了坚实的基础。同时,大鼠胚胎期小肠上皮细胞培养体系的建立,为下一阶段的功能学以及作用机制的研究提供了可能。
[Abstract]:Diabetes (Diabetes) is a life-long metabolic disease with many causes, often characterized by chronic hyperglycemia in.2013, and the International Diabetes Federation (Internatinal Diabetes Federation (IDF)) announced that the global diabetic patients have reached an astonishing 383 million. Increased sugar can damage the large blood vessels, microvessels, and then threaten the normal functions of the brain, heart, peripheral nerves, kidney and eyes, and the accompanying complications will greatly affect human health. The pathogenesis of diabetes is complex, and the current treatment means can not be cured, with the increasing number of diabetics, with the increasing number of diabetic patients, Their research has become a hot spot in the current medical field. The glucagon-like peptide-1 (GLP-1), which is secreted by L cells in the intestinal tract of mammals, is a polypeptide hormone, which is composed of 31 amino acids. Because it can promote insulin secretion in hyperglycemia, GLP-1 is used in the treatment of type 2 diabetes. The two peptidyl peptidase (DPP IV) existing in the body can degrade GLP-1 rapidly, which greatly restricts the physiological function of GLP-1. In order to improve the physiological function of GLP-1 in the body, it is necessary to optimize the structure of GLP-1 or improve the expression of GLP-1 in the body to resist the degradation of DPP-IV. GLP-1 tandem repeats are constructed to increase the expression of GLP-1 in vitro, thus making it able to resist the degradation of DPP IV for a longer time and ultimately improve the physiological function of GLP-1 in the body. In this study, we constructed the expression vectors of P ET-22b-GLP4, GLP8, GLP12 and GLP16 respectively, and then converted to Escherichia coli in BL21 (D3). The expression of isopropyl - beta -D- Thioglucoside (IPTG) was induced. The results showed that the expression of GLP4 was the highest, GLP8 was the highest, and GLP12 and GLP16 were almost not expressed. In addition to the structure of the gene itself and the efficiency of the expression system, the induction temperature, induction time and inducer concentration had an influence on the expression of GLP-1 polymer, and we sent it through experiments. At present, the expression of GLP-1 series is at 26 C, the concentration of inducer IPTG is 0.6m M, and the optimal induction time of GLP-1 polypolymer expression in vitro is 8 hours. Under the optimized conditions obtained above, we finally determined that the most suitable copy number of the string in the in vitro prokaryotic expression system is 4.. The sample has the function of secreting insulin, and in the process of development, the epithelial cells of the small intestine and the islet endocrine cells have a common origin. This suggests that the small intestinal epithelial cells in the developmental stage have the potential to induce differentiation into insulin secreting cells. In the second part of this study, the primary cultured rat embryonic small intestinal epithelial cells were isolated. By observing the morphology of the cells, we found that the cells in the culture process conformed to the morphological characteristics of the epithelial cells. Moreover, the cultured cells were detected by immunofluorescence staining, and the cultured cells expressed keratin 8 (on the small intestine). In order to facilitate the transformation of the small intestinal epithelial cells in the future, we have further constructed the HR GFP lentivirus vector and successfully infected the rat small intestinal epithelial cells. Conclusion: This study successfully induced the expression of the GLP-1 polymer in vitro and determined the GLP The optimal expression conditions of -1 and the most suitable copy number have laid a solid foundation for the study of GLP-1 polymers in vivo. At the same time, the establishment of the culture system of small intestinal epithelial cells in the embryonic stage of the rat is possible for the study of the next stage of function and the mechanism of action.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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