大鼠骨髓间充质干细胞促进胰岛细胞再生的研究

发布时间：2018-04-25 21:25

本文选题：骨髓间充质干细胞 + 胰岛细胞再生　；参考：《中国医科大学》2010年硕士论文

【摘要】： 背景及目的糖尿病(Diabetes mellitus)是一种以高血糖为主要特征严重威胁人类健康的疾病。全球每年有5%死亡是由于糖尿病的原因,据WHO估计若不采取有效措施,未来10年每年因糖尿病而死亡的人数会增加50%。其中1型糖尿病是由于自身免疫病造成的Langerhans内的p细胞的缺失,而2型糖尿病是由于组织对胰岛素的不敏感和β细胞分泌胰岛素的减少。目前,临床上主要是采用注射胰岛素治疗1型及部分2型糖尿病,但治疗效果未尽如人意。最近对患者成功移植胰岛或者β细胞,为攻克糖尿病提供了新的希望。但是,胰岛来源不足是目前最大的难题。干细胞作为一类具有多向分化潜能的细胞,已经逐渐成为人们寻找胰岛β细胞替代物的新资源。利用胚胎干细胞体外诱导胰岛素分泌细胞为治疗糖尿病提供了希望,但诱导胚胎干细胞分化比率低,诱导过程复杂,具有免疫排斥反应且易成瘤,距离临床应用还需要克服很多技术上的困难。成体干细胞具有跨系分化的能力,如肝干细胞可以分化为胰腺p细胞,神经干细胞能够分化为血细胞等。骨髓间充质干细胞(Bone Marrow Mesenchymal stem cells, MSCs)是一类具有向多种细胞分化潜能的细胞,是一种理想的组织工程种子细胞,易于提取,分离及扩增,在特定的条件下,可以分化为骨、软骨、肌肉等中胚层组织细胞,还可以跨胚层分化为神经细胞等。体外诱导MSCs分化为胰岛素分泌细胞,为治疗糖尿病提供了新的策略,但仍然面临分化过程复杂、分化比率低等困难。有研究报道,移植骨髓可以缓解糖尿病症状,在一定程度上降低了糖尿病的高血糖。有研究表明小鼠骨髓来源的干细胞(c-kit+)可以启动内源的胰腺的增殖,该项研究成果认为供体的内皮细胞(PECAM+)参与了胰腺细胞的再生。同种异体移植小鼠、大鼠的MSCs可以修复多种组织损伤,如心肌缺血、肾、肺、脊髓损伤等,然而其修复机理仍不清楚。在成年小鼠与成人中胰岛的增殖是通过已存在的胰岛β细胞产生新的β细胞,而不是通过干细胞分化的途径。移植MSCs缓解糖尿病高血糖症状是否与胰岛的增殖有关,尚未见报道。 Cdk4基因是调节细胞的增殖一种细胞周期调节蛋白,通过与D-cyclin形成复合物指导细胞由G1期进入到S期。研究认为Cdk4基因是胰岛β细胞的再生、增殖与衰老的重要的调节因子。因而MSCs是否通过Cdk4基因促进胰岛细胞的再生缓解糖尿病高血糖症状,需要进一步的研究。文献报道人与小鼠的胰岛在体外可以去分化成胰岛前体细胞.NRSF/REST (neuron restrictive silencing factor/repressor element silencing transciption factor,NRSF／REST)基因在成熟的神经细胞与胰岛细胞中不表达,只在它们的前体细胞中表达。胰岛的增殖是否通过去分化为REST表达阳性细胞来完成,MSCs是否通过这种途径促进胰岛细胞的再生缓解糖尿病高血糖症状尚不清楚。本课题组前期工作发现体外共培养胰腺细胞与MSCs,可以诱导MSCs分化为胰岛样细胞。移植MSCs到STZ诱导的糖尿病大鼠体内观察到糖尿病症状的改善,血糖的降低。本研究通过体内和体外实验两部分来探讨MSCs是否促进胰岛细胞的再生以及胰岛中Cdk4与REST基因表达情况。方法 1、大鼠骨髓间充质干细胞(MSCs)的分离培养和鉴定(1)无菌条件下取大鼠股骨骨髓细胞离心做原代培养(2)根据细胞的贴壁性能不同选取贴壁不牢的细胞继续传代培养(3)流式细胞鉴定CD14,CD34,CD44,CD45,CD73,CD90的表达 2、慢病毒转染大鼠骨髓间充质干细胞(MSCs)(1)带有GFP标签的慢病毒质粒的包装(2)慢病毒转染MSCs 3、大鼠糖尿病模型的建立及移植转染的大鼠骨髓间充质干细胞(1)一次性腹腔注射链脲菌素建立大鼠糖尿病模型(2)监测大鼠血糖以确定建模是否成功(3)尾静脉注射转染的大鼠MSCs(4)检测移植后糖尿病大鼠的空腹血糖的变化 4、胰岛素免疫荧光染色评估胰岛大小并进行软件分析(1)获得大鼠的胰腺冰冻切片(2)利用胰岛素免疫荧光染色评估胰岛的大小(3)利用软件分析不同处理组胰岛大小的差异 5、大鼠骨髓间充质干细胞(MSCs)分别与胰岛素分泌细胞系(INS1)、胰岛细胞共培养(1)大鼠胰岛细胞原代培养(2)分隔培养的方法将MSCs分别和INS1细胞、胰岛细胞共培养(3)Real-time PCR检测细胞周期调控基因Cdk4和REST的表达情况实验结果 1、分离培养大鼠MSCs,并经流式细胞术鉴定为CD14(-),CD34(-),CD45(-),CD44(+),CD73(+),CD90(+)。 2、移植细胞第11天检测到移植组血糖显著比对照组低,但此后血糖值又再次升高,与对照组无显著差异。 3、在胰腺组织中发现带有GFP标签的MSCs,移植的MSCs植入到胰腺组织内部,并得到一定的增殖,但MSCs自身并不表达胰岛素。 4、移植组胰岛荧光面积显著大比对照组,体内损伤的胰岛细胞获得再生。 5、MSC分别与INS1细胞、胰岛细胞共培养后,INS1细胞、胰岛的Cdk4基因分别高表达2.281、1.869倍,即共培养后促进了INS1细胞与胰岛p细胞增殖。 6、MSC与胰岛细胞共培养后,使胰岛REST表达量提高1.608倍。结论 1、移植MSCs短期可降低STZ诱导的糖尿病模型大鼠的高血糖。 2、MSC与胰岛细胞共培养后,上调了胰岛Cdk4、REST的表达。
[Abstract]:Background and purpose
Diabetes (Diabetes mellitus) is a disease that seriously threatens human health with high blood sugar. 5% deaths worldwide are due to diabetes. According to WHO estimates, if no effective measures are taken, the number of people dying of diabetes in the next 10 years is increased by 50%., of which type 1 diabetes is due to the L of autoimmune diseases. The loss of P cells in angerhans, and type 2 diabetes is due to tissue insensitivity to insulin and the decrease of insulin secretion in beta cells. Currently, the clinical use of insulin injection is mainly used to treat type 1 and type 2 diabetes, but the effect of the treatment is not satisfactory. New hope is provided. However, the insufficient source of islets is the biggest problem at present.
As a class of cells with multiple differentiation potential, stem cells have gradually become a new resource for the search for pancreatic beta cell substitutes. The use of embryonic stem cells in vitro to induce insulin secreting cells provides a hope for the treatment of diabetes, but the induction of embryonic stem cell differentiation is low, the induction process is complex, and the immune rejection reaction is easy and easy. It is also necessary to overcome many technical difficulties. Adult stem cells have the ability to differentiate into P cells, such as liver stem cells can differentiate into pancreatic P cells, and neural stem cells can differentiate into blood cells. Bone marrow mesenchymal stem cells (Bone Marrow Mesenchymal stem cells, MSCs) are a class of cell differentiation to multiple cell differentiation. The potential cell, an ideal tissue engineering seed cell, is easy to extract, isolate and amplify. Under specific conditions, it can differentiate into mesodermal tissue cells, such as bone, cartilage, muscle and so on. It can also differentiate into nerve cells across the embryo layer. In vitro, MSCs is induced to differentiate into Isle secreting cells, which provides a new strategy for the treatment of diabetes. However, it is reported that transplantation of bone marrow can alleviate the symptoms of diabetes and reduce the hyperglycemia of diabetes to a certain extent. Studies have shown that the bone marrow stem cells (c-kit+) of mice can initiate endogenous pancreatic proliferation. (PECAM+) participate in the regeneration of pancreatic cells. Allograft mice, rat MSCs can repair a variety of tissue damage, such as myocardial ischemia, kidney, lung, spinal cord injury and so on, but its repair mechanism is still unclear. In adult mice and adults, the proliferation of islets in adult and adult islet beta cells produce new beta cells, not through dry. The way of cell differentiation. Whether transplantation of MSCs to alleviate the symptoms of hyperglycemia in diabetes is related to islet proliferation has not been reported.
The Cdk4 gene is a cell cycle regulating protein that regulates the proliferation of cells by forming a complex with D-cyclin to guide cells from G1 to S. The study suggests that the Cdk4 gene is an important regulator of the regeneration, proliferation and senescence of islet beta cells. Therefore, MSCs has been used to promote the regeneration of islet cells by the Cdk4 basis to alleviate the high level of diabetes. The symptoms of blood sugar need further study.
It is reported that the islets of human and mouse can be differentiated into islet precursor cells.NRSF/REST (neuron restrictive silencing factor/repressor element silencing transciption factor, NRSF / REST), which are not expressed in mature nerve cells and islet cells, only in their precursor cells. The proliferation of islets is the proliferation of islet cells. It is not clear whether or not MSCs can promote the regeneration of islet cells to alleviate the symptoms of diabetic hyperglycemia by using this pathway as a REST expression positive cell.
In our previous work, we found that co culture of pancreatic cells and MSCs in vitro could induce MSCs to differentiate into islet like cells. The improvement of diabetes symptoms and the decrease of blood glucose were observed in the diabetic rats induced by MSCs to STZ. This study was to explore whether MSCs promotes the regeneration of islet cells by two parts in vivo and in vitro. The expression of Cdk4 and REST gene in islets of islets.
Method
1, the isolation, culture and identification of rat bone marrow mesenchymal stem cells (MSCs) (1) the bone marrow cells of the rat femur were centrifuged for the primary culture under aseptic conditions (2) to select the cells with different adherence to the cells to continue the passage culture (3) flow cytometry (3) to identify the expression of CD34, CD44, CD45, CD73, CD90.
2, lentivirus transfected rat bone marrow mesenchymal stem cells (MSCs) (1) packaged with lentiviral plasmid with GFP tag (2) lentivirus transfected MSCs.
3, establishment of rat diabetes model and transplantation of rat bone marrow mesenchymal stem cells (1) intraperitoneal injection of streptozotocin to establish diabetic rat model (2) to monitor rats' blood glucose to determine whether the model was successful (3) MSCs (4) transfected by tail vein (4) to detect the change of fasting blood glucose in diabetic rats after transplantation
4, insulin immunofluorescence staining was used to assess islet size and software analysis (1) the frozen section of the pancreas of rats was obtained (2) using insulin immunofluorescence staining to assess the size of islets (3) using software to analyze the difference of islet size in different treatment groups
5, rat bone marrow mesenchymal stem cells (MSCs) and insulin secreting cell line (INS1), islet cells co culture (1) rat islet cells primary culture (2) isolation and culture method, MSCs and INS1 cells, islet cells were co cultured (3) Real-time PCR to detect the expression of Cdk4 and REST of the cell cycle regulation gene.
experimental result
1, isolation and culture of rat MSCs and identification of CD14 (-), CD34 (-), CD45 (-), CD44 (+), CD73 (+), CD90 (+) by flow cytometry.
2, on the eleventh day after transplantation, the blood glucose level of the transplantation group was significantly lower than that of the control group, but the blood glucose level increased again thereafter, and there was no significant difference between the transplanted group and the control group.
3, in the pancreatic tissue, GFP labeled MSCs was found. The transplanted MSCs was implanted into the pancreatic tissue and proliferating, but MSCs itself did not express insulin.
4, the islet fluorescence area in the transplantation group was significantly larger than that in the control group, and the injured islet cells were regenerated.
5, after co culture of INS1 cells and islet cells, the Cdk4 genes of INS1 cells and islets were highly expressed as 2.281,1.869 times, that is, after co culture, the proliferation of INS1 cells and islet P cells was promoted.
6, the co expression of MSC and islet cells increased the expression of REST in pancreatic islets by 1.608 times.
conclusion
1, transplantation of MSCs can reduce the hyperglycemia of STZ induced diabetic rats in a short time.
2, co culture of MSC and islet cells increased the expression of Cdk4 and REST in islets.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329.2~8

【引证文献】