本文選題：GLP-1多聚體 + 2型糖尿病　； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：糖尿病(Diabetes)是一種終身性代謝性疾病,其發(fā)病原因眾多,常表現(xiàn)為慢性高血糖。2013年,國際糖尿病聯(lián)合會(huì)(Internatinal Diabetes Federation(IDF))公布,全球罹患糖尿病的患者達(dá)到了驚人的3.83億。盡快糖尿病本身沒有很大的危害,但是長期的血糖增高會(huì)損傷大血管、微血管,進(jìn)而威脅到腦、心、周圍神經(jīng)、腎、眼睛等的正常功能,而且隨之而來的并發(fā)癥也會(huì)極大影響人類的健康。糖尿病的發(fā)病機(jī)制復(fù)雜,當(dāng)前現(xiàn)有的治療手段尚達(dá)不到治愈的效果,隨著糖尿病患者數(shù)量的日益增多,對于它們的研究已經(jīng)成為當(dāng)前醫(yī)學(xué)領(lǐng)域的熱點(diǎn)。由哺乳動(dòng)物腸道L細(xì)胞分泌的胰高血糖素樣肽(glucagon-like peptide-1,GLP-1)是一種多肽類激素,該多肽由31個(gè)氨基酸組成。由于它可以促進(jìn)胰島素在高血糖狀態(tài)下的分泌,所以GLP-1在2型糖尿病的治療中備受關(guān)注。但是機(jī)體內(nèi)存在的二肽基肽酶(DPPⅣ)能夠使GLP-1迅速降解,這極大地限制了GLP-1的生理功能。為了提升GLP-1在體內(nèi)的生理功能,需要對GLP-1的結(jié)構(gòu)進(jìn)行優(yōu)化或者提高GLP-1在體內(nèi)的表達(dá)產(chǎn)量,從而抵抗DPP-IV的降解。本研究通過構(gòu)建GLP-1串聯(lián)重復(fù)多聚體來提高GLP-1在體外的表達(dá)產(chǎn)量,從而使其能夠更長時(shí)間地抵抗DPPⅣ的降解作用,最終提升GLP-1在體內(nèi)的生理功能。在本研究中,我們分別構(gòu)建了p ET-22b-GLP4,GLP8,GLP12和GLP16的表達(dá)載體,在大腸桿菌BL21(D3)中轉(zhuǎn)化后利用異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo)表達(dá)。結(jié)果顯示,GLP4的表達(dá)量最高,GLP8次之,而GLP12和GLP16幾乎不表達(dá)。除了基因本身的結(jié)構(gòu)和表達(dá)系統(tǒng)的效率外,誘導(dǎo)溫度、誘導(dǎo)時(shí)間以及誘導(dǎo)劑濃度對GLP-1多聚體的表達(dá)量都有影響,通過實(shí)驗(yàn)我們發(fā)現(xiàn),GLP-1串聯(lián)體的表達(dá)量在26℃,誘導(dǎo)劑IPTG濃度為0.6m M時(shí)最高,并且GLP-1多聚體體外表達(dá)的最佳誘導(dǎo)時(shí)間為8小時(shí)。在上述獲得的優(yōu)化條件之下,我們最終確定了該串連體在體外原核表達(dá)系統(tǒng)中的最適合拷貝數(shù)為4。近期研究表明,小腸也同樣具有分泌胰島素的功能,而且在發(fā)育過程中,小腸上皮細(xì)胞和胰島內(nèi)分泌細(xì)胞具有共同的起源,這就暗示我們,處在發(fā)育階段的小腸上皮細(xì)胞具有誘導(dǎo)分化成胰島素分泌細(xì)胞的潛能。為了深入研究小腸上皮細(xì)胞與胰島素分泌之間的相關(guān)性,本研究第二部分分離了原代培養(yǎng)的大鼠胚胎期小腸上皮細(xì)胞。通過對細(xì)胞的形態(tài)學(xué)進(jìn)行觀察,我們發(fā)現(xiàn)培養(yǎng)過程中的細(xì)胞符合上皮細(xì)胞的形態(tài)學(xué)特征。而且,對培養(yǎng)的細(xì)胞進(jìn)行免疫熒光染色后發(fā)現(xiàn),培養(yǎng)的細(xì)胞表達(dá)角蛋白8(小腸上皮細(xì)胞表達(dá)標(biāo)志物),進(jìn)一步確定了細(xì)胞的來源�；谶@些研究,為了方便未來對小腸上皮細(xì)胞的改造,我們進(jìn)一步構(gòu)建了hr GFP慢病毒載體,并成功感染大鼠小腸上皮細(xì)胞。結(jié)論:本研究成功地對GLP-1多聚體進(jìn)行了體外的誘導(dǎo)表達(dá),確定了GLP-1串聯(lián)體的最佳表達(dá)條件以及最適合的拷貝數(shù),為GLP-1多聚體的體內(nèi)研究打下了堅(jiān)實(shí)的基礎(chǔ)。同時(shí),大鼠胚胎期小腸上皮細(xì)胞培養(yǎng)體系的建立,為下一階段的功能學(xué)以及作用機(jī)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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 江偉華;劉益麗;江明鋒;;大腸桿菌外源蛋白表達(dá)載體穩(wěn)定性的研究進(jìn)展[J];生物技術(shù)通報(bào);2014年05期

2 范翠英;馮利興;樊金玲;果德安;劉璇;;重組蛋白表達(dá)系統(tǒng)的研究進(jìn)展[J];生物技術(shù);2012年02期

3 金玉翠;申傳安;柴家科;;胰高血糖素樣肽1的結(jié)構(gòu)及生物學(xué)作用研究進(jìn)展[J];解放軍醫(yī)學(xué)雜志;2011年08期

4 王慧;李宏亮;李光偉;;人GLP-1類似物——2型糖尿病治療新篇章[J];藥品評價(jià);2008年11期

5 劉瑞;胡仁明;王慶華;;GLP-1類似物:糖尿病預(yù)防和治療的新型藥物[J];中國糖尿病雜志;2008年08期

6 馬義;周天鴻;;胰高血糖素樣肽1的研究及應(yīng)用進(jìn)展[J];中國組織工程研究與臨床康復(fù);2007年29期

7 傅勇;李蓬秋;;GLP-1抗糖尿病作用研究進(jìn)展[J];實(shí)用糖尿病雜志;2007年03期

，

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