不同微环境对骨髓间充质干细胞分化为产胰岛素细胞的影响

发布时间：2018-04-29 21:46

  本文选题：骨髓间充质干细胞 + 产胰岛素细胞　； 参考：《第四军医大学》2008年硕士论文

【摘要】： 糖尿病是常见的慢性疾病,全世界患病率约6%。患者常需要终身注射胰岛素治疗,但胰岛素注射疗法对血糖的控制并不理想,无法阻止糖尿病肾病、冠心病、糖尿病眼病等并发症的发生和发展。最近几年胰岛移植的成功表明了通过补充缺乏的β细胞可以治愈糖尿病。但供体来源缺乏和移植排斥阻碍了胰岛细胞移植的广泛应用。 干细胞作为一类具有极强自我更新及多向分化潜能的细胞,已经逐渐成为人们寻找胰岛β细胞替代物的新的细胞资源。由于骨髓间充质干细胞(MSCs)具有多向分化和很强的增殖能力,具有用于细胞治疗的巨大潜能。它们可以分化为神经细胞、脂肪细胞、平滑肌细胞、骨骼肌细胞、心肌细胞、软骨细胞和产胰岛素细胞等。但是由骨髓间充质干细胞诱导分化而来的产胰岛素细胞(IPCs),其胰岛素分泌量不足正常胰腺β细胞的1%。如何促进MSCs分化为较成熟的IPCs成为亟待解决的关键问题。目前认为其分化机制,主要是微环境因素通过一定的信号转导通路传递,转录因子抑制或激活,启动关键基因表达的结果。 本研究的目标是:探索不同微环境下对大鼠MSCs体外分化为IPCs的影响,在糖尿病大鼠体内微环境下MSCs是否还可以分化为IPCs,为糖尿病细胞替代治疗提供理想的种子细胞。 大鼠MSCs的体外分离培养和生物学特征:利用贴壁法分离纯化大鼠MSCs,传代培养于含10%胎牛血清的DMEM培养液中;通过倒置显微镜、电镜、免疫细胞化学分析大鼠MSCs的生物学特征。结果显示原代和传代培养MSCs均保持较强的增殖能力,形态为均一的成纤维样细胞。超微结构显示了干细胞的幼稚特征,免疫细胞化学显示CD34阴性,CD44阳性。结论:采用我们的方法,可以获得生长稳定,扩增较快和纯度较高的MSCs。 不同微环境对大鼠MSCs体外分化为IPCs的影响:采用第三代MSCs,用不同的微环境进行诱导,对照组诱导剂为含有角朊细胞生长因子、胰岛素铁硒传递蛋白(ITS)、尼克酰胺的无血清DMEM/F12培养基、实验组在对照组基础上添加胰腺条件培养液;对诱导后细胞进行光、电镜观察,双硫腙和免疫细胞化学等进行鉴定,并行体外葡萄糖刺激实验,测定细胞分泌胰岛素及C-肽功能。结果表明,实验组及对照组均可诱导分化为IPCs,但实验组分化而成的IPCs在数量及功能上均好于对照组。结论:大鼠胰腺提取物模拟的微环境能促进大鼠MSCs体外诱导分化为较高质量的IPCs。 在糖尿病大鼠体内微环境下MSCs分化为IPCs的潜能:通过腹腔注射链脲佐菌素(STZ)诱导健康SD大鼠建立糖尿病模型;将MSCs及由MSCs诱导分化来的IPCs注射到糖尿病模型大鼠的肾包膜下,检测移植前后血糖、体重、尿量、生存时间变化,当移植大鼠血糖开始下降后,行荷移植物肾切除术去除移植物,继续观察血糖变化,看是否有血糖反跳,以评价其治疗糖尿病的效果,结合标本免疫组织化学染色,观察移植入的MSCs在糖尿病大鼠体内是否分化为IPCs。结果:将MSCs及IPCs移植到大鼠肾包膜下后,大鼠的血糖水平下降,糖尿病症状得到控制,生存时间明显延长。免疫组化显示移植入的MSCs分化为胰岛素染色阳性细胞。结论:在糖尿病大鼠体内微环境下MSCs可以分化为IPCs。 综上所述,大鼠MSCs在不同的体外及体内微环境下均可特异诱导分化为IPCs,而大鼠胰腺提取物模拟的微环境能促进大鼠MSCs体外诱导分化为IPCs;将大鼠MSCs和IPCs移植入糖尿病大鼠模型体内能够明显改善糖尿病症状,延长其生存时间。虽然骨髓间充质干细胞在安全而有效地运用于临床治疗之前还有许多基本的问题有待解决,但它为我们解决移植治疗糖尿病中供体缺乏问题方面提供了一条新的途经。
[Abstract]:Diabetes is a common chronic disease, the worldwide prevalence of about 6%. patients often needs life-long insulin therapy, but insulin injection therapy is not ideal for blood glucose control, and it can not prevent diabetic nephropathy, coronary heart disease, diabetes and other complications. The lack of beta cells can cure diabetes, but the lack of donor sources and graft rejection have hindered the wide application of islet cell transplantation.
Stem cells, as a class of cells with strong self renewal and pluripotent differentiation potential, have gradually become a new cell resource for the search for substitutes for islet beta cells. Bone marrow mesenchymal stem cells (MSCs) have a multidirectional differentiation and strong proliferation ability, and have great potential for cell therapy. They can be differentiated into nerve cells. Cells, adipocytes, smooth muscle cells, skeletal myocytes, cardiomyocytes, chondrocytes, and insulin producing cells. But the insulin producing cells (IPCs) induced by bone marrow mesenchymal stem cells (MSCs), the insulin secretion of the normal pancreatic beta cells is less than the 1%. of the normal pancreatic beta cells, and it is an urgent problem to promote the differentiation of MSCs into a mature IPCs. The key problem is that the mechanism of differentiation is that the microenvironmental factors pass through a certain signal transduction pathway, the transcription factor is suppressed or activated, and the result of the key gene expression is started.
The aim of this study is to explore the effect of different microenvironment on the differentiation of rat MSCs into IPCs in vitro, and whether MSCs can also differentiate into IPCs in the microenvironment of diabetic rats and provide ideal seed cells for the replacement therapy of diabetic cells.
The isolation and culture of rat MSCs in vitro and biological characteristics: the rat MSCs was isolated and purified by adhesion method and cultured in DMEM culture solution containing 10% fetal bovine serum. The biological characteristics of MSCs were analyzed by inverted microscope, electron microscopy and immunocytochemical analysis. The results showed that both primary and subculture MSCs maintained strong proliferative ability and morphology. The ultrastructure showed the naive characteristics of the stem cells, and the immunocytochemistry showed CD34 negative and CD44 positive. Conclusion: we can obtain stable growth, faster amplification and higher purity of MSCs. by using our method.
The effect of different microenvironment on the differentiation of MSCs in vitro to IPCs: third generation MSCs was used to induce different microenvironment. The inducers in the control group were keratinocyte growth factor, insulin iron selenium transferrin (ITS), and serum free DMEM/F12 medium of nikamide, and the experimental group was added to the control group on the basis of the pancreatic condition culture solution. After induction, the cells were identified by light, electron microscopy, dithizone and immunocytochemistry, and in vitro glucose stimulation test, and the cell secretion of insulin and C- peptide were measured. The results showed that the experimental group and the control group could be induced to differentiate into IPCs, but the differentiated IPCs in the experimental group was better in quantity and function than in the control group. The simulated microenvironment of rat pancreatic extract can promote the differentiation of rat MSCs into high quality IPCs. in vitro.
The potential of MSCs to differentiate into IPCs in the microenvironment of diabetic rats: induced diabetic model in healthy SD rats by intraperitoneal injection of streptozotocin (STZ); MSCs and MSCs induced IPCs were injected into the renal capsule of diabetic rat model, and the blood glucose, weight, urine volume and survival time were detected, and the transplantation was large. After the blood glucose began to decrease, the graft nephrectomy was performed to remove the graft, continue to observe the blood sugar changes and see if there was a blood glucose rebound to evaluate the effect of the treatment of diabetes. Combined with specimen immunohistochemical staining, whether the transplanted MSCs was differentiated into IPCs. in diabetic rats: MSCs and IPCs were transplanted into the kidney of rats. After subcapsular, the blood glucose level of the rats decreased, the diabetic symptoms were controlled and the survival time was prolonged obviously. The immunohistochemical staining showed that the transplanted MSCs was differentiated into insulin positive cells. Conclusion: MSCs can be differentiated into IPCs. in the microenvironment of diabetic rats.
To sum up, the rat MSCs can differentiate into IPCs specifically in different in vitro and in vivo microenvironment, and the microenvironment simulated by the rat pancreas extract can promote the differentiation of rat MSCs into IPCs in vitro, and the transplantation of MSCs and IPCs into the rat model of diabetic rats can obviously improve the symptoms of diabetes and prolong its survival time. There are many basic problems to be solved before bone marrow mesenchymal stem cells are safely and effectively used for clinical treatment, but it provides a new way for us to solve the problem of donor deficiency in the treatment of diabetes.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R657.5;R329

【参考文献】

相关期刊论文 前10条

1 郭丽,尹飞,王晓丽,孟洪琪,凌翎,呼合塔娜,范洪学;骨髓间充质干细胞向神经元的诱导分化[J];吉林大学学报(医学版);2003年01期

2 刘雅娟;李秀东;陈强;凌翎;刘丽珠;范洪学;;骨髓间充质干细胞体外诱导分化为胰岛样细胞及其对坏死胰腺组织的修复[J];吉林大学学报(医学版);2005年06期

3 杨巍;罗春元;于春雷;王莉;许丽芳;李一;;不同剂量STZ诱导小鼠糖尿病模型的发病机制[J];吉林大学学报(医学版);2006年03期

4 邓方阁;张秀英;王心蕊;马英智;李玉林;;体外模拟心肌微环境中人骨髓间充质干细胞向心肌细胞的分化[J];吉林大学学报(医学版);2007年02期

5 傅文玉,路艳蒙,朴英杰;人骨髓间充质干细胞的分化与端粒酶活性[J];第一军医大学学报;2001年11期

6 王运涛;骨髓间充质干细胞分离培养的研究进展[J];国外医学.生物医学工程分册;2002年04期

7 周毅;测定胰腺干细胞分子标志的临床意义[J];国外医学(消化系疾病分册);2001年03期

8 漆白文;喻爱喜;张功礼;潘国栋;施永彦;程鑫华;余永桂;;体外共培养诱导骨髓间充质干细胞向雪旺细胞分化[J];武汉大学学报(医学版);2007年04期

9 李烨,汪保和;骨髓间充质干细胞生物学特性的研究进展[J];医学临床研究;2004年06期

10 郑杰;王培训;周联;;骨髓间充质干细胞分离及条件培养基在其培养扩增中的作用[J];广州中医药大学学报;2006年04期

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