自噬在猪胰腺干细胞增殖及向胰岛β样细胞分化中的作用及机理

发布时间：2018-05-05 10:15

本文选题：糖尿病 + 胰腺干细胞　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：糖尿病是目前世界上对公众健康危害最严重的一类慢性疾病,主要由胰岛素分泌不足或机体不能有效利用胰岛素引起,临床表现为高血糖,可引发心脏病、肾功能衰竭、中风、下肢截肢、神经损伤及视力损伤等严重并发症。糖尿病的核心问题在于胰岛β细胞的缺损。猪胰腺干细胞(porcine pancreatic stem cells,pPSCs)是由胎猪胰腺组织中分离出来的成体干细胞,该细胞可以定向诱导分化为胰岛β样细胞。因为猪的胰岛素结构与人相近,所以经pPSCs诱导分化得到的胰岛β样细胞有望用于临床糖尿病的治疗。自噬是真核生物中一种进化上高度保守的细胞机制,是用于降解、回收利用细胞内生物大分子和受损细胞器的过程,对维持细胞及组织器官的稳态有非常重要的作用。有研究表明,自噬在维持胰腺中胰岛β细胞的数量、功能及维持血糖平衡中发挥重要作用。干细胞自我更新和分化的过程需要对细胞内蛋白和细胞器数量进行严格控制,自噬能快速有效地降解细胞内酶和转录因子等物质,因此,自噬在干细胞自我更新和分化中也发挥重要作用。但是到目前为止,自噬对胰腺干细胞的影响仍不清楚。本实验探究了自噬在pPSCs增殖及向胰岛β样细胞分化中的作用及机理。实验一方面用稳定表达EGFP-LC3B的猪胰腺干细胞系(pPSCs-EGFP-LC3B)实时监测细胞内自噬的变化,最终证明饥饿诱导的自噬可以促进pPSCs的增殖。另一方面,发现自噬水平在pPSCs向胰岛β样细胞诱导分化后期升高,抑制自噬会影响pPSCs的诱导分化效率。本实验为进一步揭示pPSCs增殖及分化机理奠定了理论基础。1.自噬能促进pPSCs增殖首先,通过激光共聚焦观察和Western blotting实验,我们对pPSCs-EGFP-LC3B的自噬水平进行了评估,发现血清饥饿4 h可以诱导该细胞发生自噬,添加氯喹可以抑制自噬的发生。接着,BrdU染色、流式细胞术检测细胞周期及Western blotting实验的结果表明,与正常培养的pPSCs相比,无血清诱导的自噬组内pPSCs的Brd U阳性率高,处于S期的细胞比例高,细胞内PCNA的蛋白表达水平较高,说明细胞增殖速度加快,加入氯喹抑制自噬后,这些指标都降低,细胞增殖速度减慢。同时,我们发现血清饥饿诱导自噬发生的同时,活性β-catenin表达量增高、入核明显,经典Wnt信号通路被激活,于是我们推测自噬对pPSCs的促增殖作用可能受经典Wnt信号通路调控。2.自噬是pPSCs诱导分化为胰岛β样细胞的必要条件首先,我们用实验室已经建立的诱导分化体系对pPSCs进行诱导,并成功得到了DTZ染色阳性,在mRNA水平高表达胰岛素、Glut2、NKX6.1、MafA,表达胰岛素和C肽蛋白并且能对高糖应答分泌胰岛素的胰岛β样细胞。其后,通过对处于诱导分化第0、1、3、4、6、9天的细胞样进行Western blotting检测,我们发现从诱导的第4天开始细胞内自噬一直维持在较高水平。第4天是由贴壁诱导换为悬浮诱导的时间点,在第4天添加氯喹抑制自噬,细胞不能聚团得到胰岛样细胞团,在第5天添加氯喹抑制自噬,得到的胰岛样细胞团不能对高糖产生应答并分泌胰岛素。与此同时,我们发现诱导过程中,细胞内自噬水平的变化与活性β-catenin的变化呈正相关关系,自噬活性增强时,活性β-catenin的表达显著增加,抑制自噬,活性β-catenin也在一定程度受到抑制。据此,我们推测自噬对pPSCs诱导分化的影响可能与经典Wnt信号通路有关。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

【参考文献】