人骨髓间充质干细胞向光感受器细胞诱导分化的实验研究

发布时间：2018-01-26 02:53

本文关键词： 骨髓间充质干细胞分化光感受器细胞　出处：《福建医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的:探索骨髓间充质干细胞(BMSCs)在体外定向分化成为视网膜光感受器细胞所需的微环境,为临床上治疗视网膜变性疾病提供新思路。方法:无菌条件下自5位健康成年自愿骨髓捐赠者髂前上棘采集骨髓,采用淋巴细胞分离液密度梯度离心法分离纯化获得BMSCs进行原代培养及传代培养,培养传代至第3代的BMSCs行流式细胞术鉴定,将第3代的BMSCs以含10%FBS的DMEM-LG培养基中加入碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)及脑源性神经营养因子(BDNF)三种因子首先进行第一阶段向神经前体细胞诱导分化,对照组不用任何因子诱导,只用含10%FBS的DMEM-LG培养基培养。应用免疫细胞化学检测细胞诱导后巢蛋白及微管相关蛋白-2的表达情况,连续检测不同诱导时间巢蛋白的阳性表达率,当诱导至巢蛋白阳性表达率达到最高时更换诱导因子,向培养基中加入色素上皮衍生因子(PEDF)及牛磺酸(Taurine)进行第二阶段诱导2w,用免疫细胞化学及RT-PCR方法检测诱导后细胞视紫红质的表达情况。结果:实验组诱导BMSCs第3d开始免疫细胞化学能检测到巢蛋白表达,第12d巢蛋白阳性表达率达到最高,达(90.9±2.6)%, 14d时巢蛋白阳性率减低为(75.5±3.7)%。微管相关蛋白-2在诱导第6d检测到阳性表达。第12d诱导因子更换为PEDF及Taurine继续诱导2w后,免疫细胞化学方法检测到有视紫红质表达,第2w视紫红质阳性率为(20.7±3.8)%,对照组均未检测到视紫红质表达。在诱导第2w后采用RT-PCR方法检测到诱导细胞有视紫红质表达;对照组未见视紫红质表达。结论:体外采用分阶段诱导BMSCs,第一阶段应用因子bFGF、EGF及BDNF进行向神经前体细胞诱导分化,BMSCs能够成功向神经前体细胞分化,呈现神经元细胞样形态,表达神经前体细胞标志物巢蛋白及神经元细胞标志物微管相关蛋白。第二阶段应用因子PEDF和Taurine能在体外诱导BMSCs表达光感受器细胞标志物视紫红质,结果显示BMSCs在体外特定微环境的作用下能够向表达光感受器细胞特异性标志物的细胞方向分化,表明分化的细胞在某种程度上具有光感受器细胞类似特征。这为临床上治疗视网膜变性疾病提供新思路。
[Abstract]:Objective: to explore the microenvironment of bone marrow mesenchymal stem cells (BMSCs) to differentiate into retinal photoreceptor cells in vitro, and to provide a new idea for the clinical treatment of retinal degeneration. Methods: bone marrow was collected from the anterior superior iliac spine of 5 healthy adult voluntary bone marrow donors under aseptic condition. BMSCs was isolated and purified by density gradient centrifugation of lymphocytes. The primary culture and passage culture were carried out. The BMSCs from the culture to the third generation was identified by flow cytometry. The third passage of BMSCs was added to the DMEM-LG medium containing 10s with basic fibroblast growth factor (bFGF). Epidermal growth factor (EGF) and brain-derived neurotrophic factor (BDNF) were firstly induced into neural precursor cells in the first stage, but no factors were used in the control group. The expression of nestin and microtubule-associated protein -2 was detected by immunocytochemistry in DMEM-LG medium containing 10s. The positive expression rate of nestin was continuously detected at different induction time, and the induction factor was replaced when the positive expression rate of nestin reached the highest level. The pigment epithelium-derived factor (PEDF) and taurine Taurine were added to the culture medium for 2 weeks. The expression of rhodopsin was detected by immunocytochemistry and RT-PCR. Results: the expression of nestin was detected by immunocytochemistry on the 3rd day after induction of BMSCs in the experimental group, and the positive rate of nestin was the highest (90.9 卤2.6%) on the 12th day. The positive rate of nestin decreased to 75.5 卤3.7 at 14 days. The positive expression of microtubule-associated protein -2 was detected on the 6th day after induction, and the induction factor was replaced by PEDF and Taurine for 2 weeks on the 12th day. The expression of rhodopsin was detected by immunocytochemistry. The positive rate of rhodopsin was 20.7 卤3.8% at the 2nd week. The expression of rhodopsin was not detected in the control group. After 2 weeks of induction, the expression of rhodopsin was detected by RT-PCR assay. No rhodopsin expression was found in the control group. Conclusion: BMSCs were induced in vitro by stepwise induction, and differentiation into neural progenitor cells was induced by bFGFEGF and BDNF in the first stage. BMSCs can successfully differentiate into neural precursor cells and present neuronal cell-like morphology. Expression of nestin, a neural precursor marker, and microtubule-associated protein, a neuronal marker. In the second stage, factors PEDF and Taurine could induce BMSCs to express photoreceptor cell labeling in vitro. The volunteers, rhodopsin. The results showed that BMSCs could differentiate into cells expressing specific markers of photoreceptor cells under specific microenvironment in vitro. It is suggested that differentiated cells have similar characteristics of photoreceptor cells to some extent, which provides a new idea for the clinical treatment of retinal degeneration.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1

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