【作者】 李华青；

【导师】 赵宝珍；

【作者基本信息】 山西医科大学 ， 内科内分泌学， 2010， 博士

【摘要】 2型糖尿病(T2DM)是由遗传和环境共同作用而引起的一组以糖代谢紊乱为主要表现的临床综合征,由于胰岛素分泌、作用或两者同时存在缺陷引起碳水化合物、脂肪、蛋白质、水和电解质等代谢紊乱,可以引起多个系统器官同时损伤的慢性并发症,是致残致死的主要原因。目前的治疗以降糖为主,一定程度上能够改善胰岛素抵抗、代谢综合征,但不能根治糖尿病,尤其是对包括胰岛细胞在内的多器官组织的损伤疗效甚微,间充质干细胞(MSCs)因其具有损伤修复和多向分化潜能,成为糖尿病及其并发症治疗领域的研究热点。研究显示,骨髓间充质干细胞(BMSCs)移植后对大多数组织损伤的修复起效快,具有直接的修复能力,课题组前期的研究结果显示,BMSCs对T2DM多种器官组织的损伤具有良好的直接修复作用,对T2DM并发症及一般状况的改善非常明显,对胰岛的修复也有一定的作用,但是短期内还未能观察到胰岛功能的完全恢复,也未见到同类报道。同时我们看到,BMSCs还具有一定的改善胰岛素抵抗及降糖作用,但是短期内直接降糖作用较弱。杨卫红等的研究显示,高糖可以抑制BMSCs的增殖,促进BMSCs的凋亡,我们的研究也显示,高糖高脂培养的BMSCs凋亡增加,高糖高脂还可以导致β细胞分泌胰岛素的能力下降。因此,如果无辅助的治疗,移植入体内的BMSCs在发挥作用时可能会受到T2DM病因的影响,而且在T2DM病因的作用下不利于BMSCs向胰岛细胞分化,单独使用BMSCs治疗还具有一定的局限性。Exendin-4是从蜥蜴唾液腺分离出来的GLP-1 (glucagon-like peptide-1)的类似物,具有较长的血浆半衰期和较强的生物活性。它能够针对T2DM的多个病理生理靶点发挥降糖效应,改善胰腺局部的微环境,同时能够促进胰岛细胞的再生及干细胞向胰岛细胞的分化。但是,它进入体内发挥作用相对较慢,刺激胰岛细胞再生需较长周期,而且对于全身并发症的改善是一种间接的作用,依赖体内的干细胞体系,BMSCs与Exendin-4在T2DM及其并发症的治疗上具有很好的互补作用。为探索一条副作用低的、有效的治疗T2DM胰岛细胞损伤的方法,本研究课题将探讨在BMSCs移植的过程中联合使用Exendin-4是否有利于BMSCs进入体内更好地发挥作用,Exendin-4在体外是否能够促进BMSCs向胰岛素分泌细胞(IPCs)的分化,Exendin-4是否能够促进BMSCs在体内分化为IPCs, BMSCs是否通过促进胰岛β细胞再生,减少β细胞凋亡发挥治疗作用,联合使用Exendin-4是否能协同增强BMSCs的治疗作用。各部分研究结果分别简述如下。一、正常大鼠BMSCs的分离培养和鉴定采用全骨髓贴壁培养法分离、培养、纯化大鼠BMSCs,观察细胞在光镜下的生长特征并记录其生长曲线,对所获得的细胞进行成骨及成脂肪细胞诱导分化,流式细胞仪分析其表面标记(CD34, CD44, CD45, CD71, CD105)。结果显示,BMSCs呈梭形、漩涡状生长,BMSCs成骨和成脂诱导可以使BMSCs出现钙盐沉积和脂滴,表达成骨细胞、脂肪细胞特征,表面标志鉴定CD34、CD45呈阴性,CD44、CD71、CD105呈阳性,确定所获细胞绝大多数为BMSCs。本部分实验建立了大鼠BMSCs的体外培养体系,为后续的干细胞研究提供了稳定的细胞模型。二、Exendin-4在BMSCs转分化为胰岛样细胞中作用的体外研究本部分研究中我们采用两步法诱导BMSCs分化为胰岛样细胞,同时加入不同浓度的Exendin-4 (5、10、20、40、80nM),观察其对分化的影响。双硫腙染色鉴定胰岛样细胞团,免疫细胞化学染色法对胰岛素分泌细胞进行鉴定,用放射免疫分析法观察诱导后细胞对葡萄糖刺激试验的反应,Real time-PCR检测胰岛β细胞相关基因Ngn3、PDX-1、Insl及Glut2的表达。结果表明,在Exendin-4作用下,BMSCs向胰岛样细胞转化增多,体外培养细胞上清液中胰岛素含量明显升高,胰岛β细胞相关基因Ngn3、PDX-1、Insl及Glut2 mRNA的表达增加,在一定范围内呈剂量依赖性。三、Exendin-4在BMSCs移植治疗T2DM中的作用建立T2DM大鼠的模型,分为正常对照组、T2DM模型组、BMSCs移植组、Exendin-4处理组及BMSCs+Exendin-4治疗组,将Brdu标记的BMSCs从大鼠尾静脉移植入T2DM模型大鼠体内,观察不同的干预措施对各组大鼠的体重、血糖、血脂及血浆胰岛素、脂联素水平的影响,并通过免疫组化、免疫荧光双标技术观察BMSCs在大鼠胰腺组织的分布及分化情况。结果表明,BMSCs能够迁移至T2DM模型大鼠胰腺组织,并转化为胰岛素分泌细胞。BMSCs移植的同时使用Exendin-4可以更好地降低血糖,增加大鼠胰腺组织中胰岛素染色阳性区域的面积,改善β细胞功能。四、Exendin-4在BMSCs移植治疗T2DM中作用的机制研究探讨BMSCs移植治疗T2DM的机制,TUNEL方法观察各组胰岛细胞的凋亡情况,Real-time PCR与Western blot方法检测各组胰腺组织PDX-1与胰岛素mRNA及蛋白的表达,免疫组化与Western blot方法检测各组胰腺组织Caspase3与Bax的蛋白水平。结果显示,BMSCs移植能够通过增加胰岛PDX-1与胰岛素mRNA及蛋白的表达促进胰岛β细胞的再生,同时能够减少胰岛Caspase3与Bax的蛋白水平,减少胰岛细胞的凋亡,联合使用Exendin-4能够协同增强治疗效果。结论1.通过全骨髓贴壁法,成功分离、培养了大鼠BMSCs,通过对该细胞生物学特性的观察及应用流式细胞检测其表面抗原,证实了所得到的贴壁细胞为BMSCs,为后续的实验提供了充足的BMSCs和良好的实验基础。2.体外研究结果显示Exendin-4可以促进BMSCs分化为功能性胰岛样细胞,该细胞DTZ染色阳性,经葡萄糖刺激能够分泌一定数量的胰岛素,并能表达胰岛细胞的相关基因Ngn3、PDX-1、Insl及Glut2。Exendin-4的促分化作用在一定范围内呈剂量依赖性。3.利用高糖高脂饮食加小剂量注射STZ的方法成功建立T2DM大鼠模型,该T2DM模型具有肥胖、高血糖、血脂异常、高胰岛素血症及胰岛素抵抗的特点,接近人类T2DM的发病特征,可以用于T2DM的实验研究。4.同种异体BMSCs移植能够在一定程度上治疗大鼠T2DM,移植的BMSCs能够迁移至T2DM大鼠的胰腺组织,并分化为胰岛素分泌细胞,同时应用Exendin-4,能够更好地降低T2DM大鼠的血糖,增加胰岛β细胞胰岛素染色阳性区域的面积,改善β细胞功能。5.BMSCs移植通过增加PDX-1与胰岛素的表达,促进β细胞再生,同时,能够下调Caspase3、Bax的表达,减少β细胞的凋亡,联合Exendin-4能够协同增强BMSCs移植治疗T2DM的作用,为T2DM的细胞治疗提供了新的思路。更多还原

【Abstract】 Type 2 diabetes mellitus (T2DM) is a clinical syndrome mainly represented glucose metabolic disorder caused by genetic and environmental elements. Insulin secretion, effect or both defects lead to metabolic disorders included carbohydrates, fats, protein, water, and electrolytes, et al. T2DM can cause multiple system organ damaged chronic complications which is the major cause of disability or death. The current treatment is mainly to lower blood sugar, to a extent, it can improve insulin resistance and metabolic syndrome, but can not cure diabetes, especially for multi-organ tissue included islet cells damage has little effect. Mesenchymal stem cells (MSCs) which have repair ability and multi-differentiation potential have become the research focus in the area of diabetes and its complications treatment.Studies have shown that bone marrow-derived mesenchymal stem cells (BMSCs) transplantation has fast onset of action in most tissue damage repair, with a direct repair capacity. Our previous research showed that BMSCs have a good direct repair in multiple organ tissue injury in T2DM, it is very clear to improve the complications and overall situation in T2DM, BMSCs also have a certain role in islet restoration, but not yet observed in the short term islet function fully restored, also not seen the same reports. At the same time, we have seen BMSCs have a certain degree of improving insulin resistance and the hypoglycemic effect, but their direct hypoglycemic effect is weaker in short-term. YANG Wei-hong’s study has shown that high glucose can inhibit the proliferation of BMSCs and promote their apoptosis, our studies also show that high glucose and fat increase apoptosis of BMSCs, also can lead to (3-cell insulin secretion capacity decline. Therefore, if no supplementary treatment, the role of BMSCs in T2DM body may be influenced by etiology, under the influence of etiology of T2DM, it is not conducive for BMSCs to differentiate to islet cells, BMSCs alone treatment has some limitations.Exendin-4 is a GLP-1 (glucagon-like peptide-1) analogues extracted from lizard salivary glands, with a longer plasma half-life and strong biological activity. It can play a hypoglycemic effect through a number of pathophysiological targets for T2DM, improve pancreatic local micro-environment at the same time, it also can promote the regeneration of islet cells and stem cells differentiation to islet cells. However, it is relatively slow to play a role, it will take longer cycles to stimulate islet cell regeneration, but also for the improvement of systemic complications is an indirect role, dependent on the body’s stem cell system, BMSCs and Exendin-4 has a very good complementary in the T2DM treatment. In order to explore a lower side effect, effective treatment method for pancreatic islet dysfunction in T2DM, this research will explore whether Exendin-4 helps BMSCs play a better role in the process of BMSCs transplantation, whether Exendin-4 in vitro can promote the differentiation of BMSCs to the insulin-producing-cells (IPCs), whether Exendin-4 in the body can promote BMSCs to differentiate into IPCs, whether BMSCs play a therapeutic effect is through the promotion of pancreaticβ-cell regeneration, reducingβ-cell apoptosis, the joint use of Exendin-4 is able to enhance the synergistic therapeutic effect of BMSCs. The various parts of the findings are summarized below, respectively.1.Isolation and identification of normal rats BMSCsBMSCs were isolated and cultured by whole bone marrow adherent culture method, the cells were observed under light microscope, their growth characteristics were recorded by growth curve, obtained BMSCs were induced to differentiate into the bone and fat cells, their surface markers (CD34, CD44, CD45, CD71, CD 105) were analysised by flow cytometry. The results showed that, BMSCs grew spindle-shaped whirlpool, they could make calcium deposits and lipid droplets in the course of osteogenic and adipogenic differentiation, BMSCs appeared to express osteoblast and fat cells characteristics, their identification of surface markers CD34, CD45 were negative, CD44, CD71 and CD105 were positive, the vast majority of obtained cells were BMSCs. This part we established a rat BMSCs cultured system, provided a stable cell model for the follow-up of stem cell research.2.Exendin-4 on rat BMSCs transdifferentiation into islet-like cells in vitroIn this section study, we used two-step method to induce BMSCs differentiate into islet-like cells, different concentrations of Exendin-4 (5,10,20,40,80nM) were added to observe its effect on differentiation. Dithizone staining were used to identify islet-like cell clusters, insulin-producing cells were identified through immune staining, the induced cells response to glucose stimulation were observed by radioimmunoassay, Real time-PCR were used to detect the expression of isletβ-cell-related genes Ngn3, PDX-1, Insl and Glut2. The results showed that islet-like cell transformation increased under Exendin-4, supernatant insulin levels were significantly higher, isletβ-cell-related genes Ngn3, PDX-1, Insl and Glut2 mRNA expression increased, within a certain range in a dose-dependent.3.The effect of Exendin-4 on BMSCs transplantation for treatment of T2DMT2DM rats were randomly divided into normal control group, T2DM model group, BMSCs transplantation group, Exendin-4 treatment group and BMSCs+Exendin-4 group, while Brdu labeled BMSCs transplanted from the rat tail vein to the rats body with T2DM, the effects of different interventions on the body weight, blood glucose, blood lipids, plasma insulin and adiponectin levels were observed, distribution and differentiation of BMSCs in rat pancreatic tissue were observed by immunohistochemistry and double-labeled immunofluorescence technique. The results showed that, BMSCs can migrate to the T2DM rat pancreatic tissue, and induce into insulin-producing cells. The use of Exendin-4 in BMSCs transplantation process can significantly reduce blood sugar, increase insulin positive area expression in rat pancreatic tissue, improveβ-cell function.4.Mechanism study of Exendin-4 on BMSCs transplantation for treatment of T2DMTo explore mechanism of BMSCs transplantation for the treatment of T2DM, TUNEL were observed in each group of islet cell apoptosis, Real-time PCR and Western blot methods were used to detect PDX-1 and insulin mRNA and protein expression in each group pancreatic tissues, islet Caspase3 and Bax protein levels were detected by immunohistochemistry and Western blot method. The results showed that, BMSCs transplantation increased islet PDX-1 and insulin mRNA and protein expression, promoted regeneration of islet P-cells, while able to reduce islet Caspase3 and Bax protein levels, reduce the apoptosis of islet cells, the joint use of Exendin-4 can enhance the therapeutic effect.Conclusions1.Through the whole bone marrow adherent methods, successfully isolated and cultured rat BMSCs, the obtained cells were confirmed as BMSCs through the biological characteristics and application of flow cytometry, for the follow-up experiments provided sufficient BMSCs products and a good experimental basis.2.In vitro studies showed that Exendin-4 can effectively promote BMSCs to differentiate into functional islet-like cells, these cells were positive in DTZ staining, they could secret insulin after glucose stimulation, they also express P-cell-related genes such as Ngn3,PDX-1,Ins1 and Glut2. The role of Exendin-4 on BMSCs differenciation was in a dose-dependent within a certain range.3.The use of high-sugar high-fat diet plus low-dose injection of STZ method successfully established rat model of T2DM, the T2DM model had obesity, hyperglycemia, dyslipidemia, hyperinsulinemia and insulin resistance characteristics, closed to human T2DM pathogenesis, they could be used to T2DM experimental research.4.Allogeneic BMSCs transplantation can treat rats T2DM, transplanted BMSCs can migrate to the T2DM rat pancreatic tissue and to differentiate into insulin-producing cells, while application of Exendin-4, can significantly reduce the T2DM rats blood glucose, increase insulin secretion, improve P-cell function.5. BMSCs transplantation increased islet PDX-1 and insulin mRNA and protein expression, promoted regeneration of islet P-cells, while able to reduce islet Caspase3 and Bax protein levels, reduce the apoptosis of islet cells, the joint use of Exendin-4 can enhance the therapeutic effect, this study gives a new insight into the cells treatment of T2DM.更多还原