發(fā)布時(shí)間：2018-05-05 10:15

  本文選題：糖尿病 + 胰腺干細(xì)胞��； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：糖尿病是目前世界上對(duì)公眾健康危害最嚴(yán)重的一類慢性疾病,主要由胰島素分泌不足或機(jī)體不能有效利用胰島素引起,臨床表現(xiàn)為高血糖,可引發(fā)心臟病、腎功能衰竭、中風(fēng)、下肢截肢、神經(jīng)損傷及視力損傷等嚴(yán)重并發(fā)癥。糖尿病的核心問題在于胰島β細(xì)胞的缺損。豬胰腺干細(xì)胞(porcine pancreatic stem cells,pPSCs)是由胎豬胰腺組織中分離出來的成體干細(xì)胞,該細(xì)胞可以定向誘導(dǎo)分化為胰島β樣細(xì)胞。因?yàn)樨i的胰島素結(jié)構(gòu)與人相近,所以經(jīng)pPSCs誘導(dǎo)分化得到的胰島β樣細(xì)胞有望用于臨床糖尿病的治療。自噬是真核生物中一種進(jìn)化上高度保守的細(xì)胞機(jī)制,是用于降解、回收利用細(xì)胞內(nèi)生物大分子和受損細(xì)胞器的過程,對(duì)維持細(xì)胞及組織器官的穩(wěn)態(tài)有非常重要的作用。有研究表明,自噬在維持胰腺中胰島β細(xì)胞的數(shù)量、功能及維持血糖平衡中發(fā)揮重要作用。干細(xì)胞自我更新和分化的過程需要對(duì)細(xì)胞內(nèi)蛋白和細(xì)胞器數(shù)量進(jìn)行嚴(yán)格控制,自噬能快速有效地降解細(xì)胞內(nèi)酶和轉(zhuǎn)錄因子等物質(zhì),因此,自噬在干細(xì)胞自我更新和分化中也發(fā)揮重要作用。但是到目前為止,自噬對(duì)胰腺干細(xì)胞的影響仍不清楚。本實(shí)驗(yàn)探究了自噬在pPSCs增殖及向胰島β樣細(xì)胞分化中的作用及機(jī)理。實(shí)驗(yàn)一方面用穩(wěn)定表達(dá)EGFP-LC3B的豬胰腺干細(xì)胞系(pPSCs-EGFP-LC3B)實(shí)時(shí)監(jiān)測細(xì)胞內(nèi)自噬的變化,最終證明饑餓誘導(dǎo)的自噬可以促進(jìn)pPSCs的增殖。另一方面,發(fā)現(xiàn)自噬水平在pPSCs向胰島β樣細(xì)胞誘導(dǎo)分化后期升高,抑制自噬會(huì)影響pPSCs的誘導(dǎo)分化效率。本實(shí)驗(yàn)為進(jìn)一步揭示pPSCs增殖及分化機(jī)理奠定了理論基礎(chǔ)。1.自噬能促進(jìn)pPSCs增殖首先,通過激光共聚焦觀察和Western blotting實(shí)驗(yàn),我們對(duì)pPSCs-EGFP-LC3B的自噬水平進(jìn)行了評(píng)估,發(fā)現(xiàn)血清饑餓4 h可以誘導(dǎo)該細(xì)胞發(fā)生自噬,添加氯喹可以抑制自噬的發(fā)生。接著,BrdU染色、流式細(xì)胞術(shù)檢測細(xì)胞周期及Western blotting實(shí)驗(yàn)的結(jié)果表明,與正常培養(yǎng)的pPSCs相比,無血清誘導(dǎo)的自噬組內(nèi)pPSCs的Brd U陽性率高,處于S期的細(xì)胞比例高,細(xì)胞內(nèi)PCNA的蛋白表達(dá)水平較高,說明細(xì)胞增殖速度加快,加入氯喹抑制自噬后,這些指標(biāo)都降低,細(xì)胞增殖速度減慢。同時(shí),我們發(fā)現(xiàn)血清饑餓誘導(dǎo)自噬發(fā)生的同時(shí),活性β-catenin表達(dá)量增高、入核明顯,經(jīng)典Wnt信號(hào)通路被激活,于是我們推測自噬對(duì)pPSCs的促增殖作用可能受經(jīng)典Wnt信號(hào)通路調(diào)控。2.自噬是pPSCs誘導(dǎo)分化為胰島β樣細(xì)胞的必要條件首先,我們用實(shí)驗(yàn)室已經(jīng)建立的誘導(dǎo)分化體系對(duì)pPSCs進(jìn)行誘導(dǎo),并成功得到了DTZ染色陽性,在mRNA水平高表達(dá)胰島素、Glut2、NKX6.1、MafA,表達(dá)胰島素和C肽蛋白并且能對(duì)高糖應(yīng)答分泌胰島素的胰島β樣細(xì)胞。其后,通過對(duì)處于誘導(dǎo)分化第0、1、3、4、6、9天的細(xì)胞樣進(jìn)行Western blotting檢測,我們發(fā)現(xiàn)從誘導(dǎo)的第4天開始細(xì)胞內(nèi)自噬一直維持在較高水平。第4天是由貼壁誘導(dǎo)換為懸浮誘導(dǎo)的時(shí)間點(diǎn),在第4天添加氯喹抑制自噬,細(xì)胞不能聚團(tuán)得到胰島樣細(xì)胞團(tuán),在第5天添加氯喹抑制自噬,得到的胰島樣細(xì)胞團(tuán)不能對(duì)高糖產(chǎn)生應(yīng)答并分泌胰島素。與此同時(shí),我們發(fā)現(xiàn)誘導(dǎo)過程中,細(xì)胞內(nèi)自噬水平的變化與活性β-catenin的變化呈正相關(guān)關(guān)系,自噬活性增強(qiáng)時(shí),活性β-catenin的表達(dá)顯著增加,抑制自噬,活性β-catenin也在一定程度受到抑制。據(jù)此,我們推測自噬對(duì)pPSCs誘導(dǎo)分化的影響可能與經(jīng)典Wnt信號(hào)通路有關(guān)。
[Abstract]:Diabetes is the most serious chronic disease of the public health in the world. It is mainly caused by insufficient secretion of insulin or the inability of the body to use insulin effectively. The clinical manifestation is hyperglycemia, which can cause serious complications such as heart disease, renal failure, stroke, lower limb amputation, nerve injury and visual impairment. The problem lies in the defect of islet beta cells. Porcine pancreatic stem cells (pPSCs) is a adult stem cell isolated from the pancreatic tissue of fetal pigs. This cell can be induced to differentiate into islet beta like cells. Because the insulin structure of pigs is similar to human, the pancreatic islet beta cells obtained by pPSCs induced differentiation are hopeful. The treatment of clinical diabetes. Autophagy is an evolutionary highly conserved cell mechanism in eukaryotes, used for degradation, recycling of intracellular macromolecules and damaged organelles, and plays an important role in maintaining the homeostasis of cells and tissues. Quantity, function and maintenance of blood glucose balance play an important role. The process of self renewal and differentiation of stem cells requires strict control of the number of intracellular proteins and organelles, and autophagy can quickly and effectively degrade substances such as enzymes and transcription factors. Therefore, autophagy also plays an important role in the self-renewal and differentiation of stem cells. So far, the effect of autophagy on pancreatic stem cells is still unclear. The experiment explored the role and mechanism of autophagy in the proliferation of pPSCs and the differentiation of pancreatic beta like cells. On the one hand, the changes of autophagy in the cell line of the porcine pancreatic stem cell line (pPSCs-EGFP-LC3B), which are stable expression of EGFP-LC3B, are monitored in real time. Autophagy can promote the proliferation of pPSCs. On the other hand, it is found that autophagy increases at the late stage of differentiation from pPSCs to islet beta like cells, and inhibition of autophagy may affect the induced differentiation of pPSCs. This experiment provides a theoretical basis for further revealing the mechanism of pPSCs proliferation and differentiation:.1. autophagy can promote pPSCs proliferation first, through laser confocal view. In the Western blotting experiment, we evaluated the autophagy level of pPSCs-EGFP-LC3B. It was found that serum starvation 4 h could induce autophagy, and chloroquine could inhibit the occurrence of autophagy. Then, BrdU staining, flow cytometry detection of cell cycle and Western blotting experiment showed that it was with normal pPSCs phase. The positive rate of pPSCs Brd U in the autophagy group was higher than that in the serum-free group. The proportion of cells in the S stage was high and the protein expression level of PCNA in the cells was higher, indicating that the cell proliferation speed was accelerated. After adding chloroquine to inhibit autophagy, these indexes were reduced and the cell proliferation rate slowed. The expression of sex beta -catenin is higher and the nucleation is obvious. The classical Wnt signaling pathway is activated. Therefore, we speculate that autophagy may be regulated by the classical Wnt signaling pathway to regulate.2. autophagy as a necessary condition for pPSCs induced differentiation into islet beta like cells. We use the induced differentiation system established in the laboratory to induce pPSCs. Guided, and successfully obtained DTZ staining positive, high expression of insulin, Glut2, NKX6.1, MafA, expression of insulin and C peptide protein at mRNA level and high glucose response secreting insulin islet like cells. Subsequently, we detected the Western blotting detection by the cell samples in the induced differentiation day 0,1,3,4,6,9, we found that from the fourth days of induction. Autophagy at the beginning of the cell was maintained at a high level. The fourth day was changed from adherent induction to suspension induced time. In the fourth day, chloroquine was added to inhibit autophagy, and the cells could not get the group of islet like cell groups. In the fifth day, chloroquine was added to inhibit autophagy. The obtained islet like cell group could not respond to high glucose and secrete insulin. At the same time, we found that there was a positive correlation between the changes of autophagy and the changes of active beta -catenin in the induction process. When the autophagic activity was enhanced, the expression of active beta -catenin increased significantly, and the inhibition of autophagy was suppressed to a certain extent. Accordingly, we speculated that the effect of autophagy on the induced differentiation of pPSCs might be associated with the effect of the autophagy. The canonical Wnt signaling pathway is related.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.1

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本文編號(hào)：1847293