体外定向诱导干细胞分化为胰岛样细胞

发布时间：2018-03-13 00:38

本文选题：胎儿　切入点：骨髓间充质干细胞　出处：《青岛大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的:为糖尿病细胞替代治疗提供理想的种子细胞,分别行胎儿骨髓间充质干细胞(MSCs)、胎儿胰腺源性干细胞(PSCs)以及构建人自体基因的胚胎干细胞(SCNT-hES)向胰岛样细胞的定向诱导分化,动态观察诱导分化细胞形态的时空变化及阶段特异性抗原表达,对终末分化细胞进行定性鉴定及功能检测。方法:(1)取自然流产胎儿的股骨、胫骨等长骨骨髓,采用全骨髓培养法,按1x10~6/ml的密度接种至含10%胎牛血清(FBS)的DMEM培养基中,5%CO_2箱中孵育48h后换液,每3d换液1次,细胞贴壁生长呈成纤维样,1:3传代继续扩增培养,取3-8代MSCs予以2—巯基乙醇、EGF、bFGF、B27及尼克酰胺分三阶段定向诱导分化为胰岛样细胞。(2)同时取胎儿胰腺体组织,机械破碎,胶原酶及胰酶消化,离心后将单细胞悬液种植于含10%FBS及1XB27的高糖DMEM培养基中,48h后培养液改换为含2%FBS及1XB27的DMEM-F12培养液,1:2消化传代。观察加ITS-A及EGF后细胞自发分化为神经元及胰岛样结构,同时予以EGF、bFGF、B27及尼克酰胺分两阶段定向诱导分化。(3)取山东省干细胞工程技术研究中心自行建立的体细胞核移植胚胎干细胞系(SCNT-hES),采用六阶段诱导分化:(一)用含15%FBS及2—巯基乙醇的高糖DMEM预分化3d;(二)用含15%FBS的高糖DMEM悬浮培养6d形成类胚体(EBs);(三)EBs加入含ITS-A和纤维结合素的DMEM-F12贴壁培养6d;(四)将细胞消化接种于含N2、B27及bFGF的DMEM-F12中扩增胰前体细胞6d;(五)撤掉bFGF加入尼克酰胺促进胰岛细胞形成;(六)胰岛细胞进一步成熟。对三种不同来源的各期细胞分别进行形态学观察、双硫腙染色、免疫细胞组织化学及共聚焦显微镜免疫荧光鉴定nestin、PDX-1、insulin及glucagon的表达,并用电化学发光法定量测定胰岛素分泌量。结果:(1)胎儿MSCs于接种后24h即可见到贴壁生长的成纤维样细胞,8—9d达到80%融合,其后进入对数生长期,细胞倍增时间为30h,诱导过程中细胞逐渐变圆,表达nestin、PDX-1、insulin和glucagon,双硫腙染色见棕红色细胞团,MSCs分化得到的胰岛样细胞胞浆胰岛素分泌量为81.3±23.6uu/ml,有一定程度葡萄糖依赖性
[Abstract]:Objective: to provide the ideal seed cells for the treatment of diabetic cell replacement, and to induce differentiation into islet like cells of fetal bone marrow mesenchymal stem cells (MSCs), fetal pancreatic stem cells (PSCs) and human autologous gene derived embryonic stem cells (SCNT-hESs), respectively. The temporal and spatial changes of differentiation cells and the expression of stage specific antigen were observed dynamically. The terminal differentiation cells were identified qualitatively and their functions were detected. Methods the bone marrow of femur and tibia of abortive fetus was taken. The bone marrow of femur and tibia was cultured by whole bone marrow culture method. The bone marrow was inoculated into the DMEM medium containing 10% fetal bovine serum (FBS) at the density of 1 x 10 / ml, and incubated in box 5CO 2 for 48 hours and then changed solution once every 3 days. The cells grew in fibroblast like 1: 3 and then expanded and cultured. The 3-8 passages of MSCs were treated with 2-mercaptoethanol, EGFbFGFB27 and nicotinamide in three stages to induce differentiation into islet like cells. Meanwhile, the fetal pancreatic tissue was extracted, and the tissue of fetal pancreas was mechanically broken. Collagenase and trypsin digestion, After centrifugation, the single cell suspension was planted in high sugar DMEM medium containing 10s and 1XB27 for 48 h. The culture medium was replaced with DMEM-F12 medium containing 2S and 1XB27 after 48 hours. The cells differentiated spontaneously into neurons and islet like structures after adding ITS-A and EGF. At the same time, EGFNbFGFB27 and nicotinamide were used to induce differentiation in two stages. (3) SCNT-hESN, a somatic cell nuclear transfer embryonic stem cell line established by Shandong Stem Cell Engineering Research Center, was obtained. Pre-differentiation of S and 2-mercaptoethanol with high sugar DMEM for 3 days (2) suspension culture of high sugar DMEM containing 15s for 6 days to form embryoid DMEM (3) EBs added with DMEM-F12 containing ITS-A and fibronectin for 6 days (4) cells digested and inoculated with N2B27 and bFGF. (5) removing bFGF and adding nicotinamide to promote the further maturation of islet cells. Dithizone staining, immunohistochemistry and confocal microscopy immunofluorescence were used to detect the expression of PDX-1 insulin and glucagon in Neisin, and the amount of insulin secretion was measured by electrochemiluminescence. Results Fetal MSCs could reach 80% fusion at 8-9 days after inoculation, then entered logarithmic growth period, the cell doubling time was 30h, and the cells became round during induction. The expression of PDX-1insulin and glucagon.Dithizone staining showed that the cytoplasmic insulin secretion of islet like cells was 81.3 卤23.6uU / ml, which was dependent on glucose to some extent.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2

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