大鼠视网膜匀浆上清液体外诱导骨髓间充质干细胞分化为神经元样细胞实验研究

发布时间：2018-05-06 08:07

本文选题：骨髓间充质干细胞 + 诱导分化　；参考：《天津医科大学》2008年博士论文

【摘要】： 目的随着眼科临床和基础研究的进步,白内障、角膜病等可得到有效控制和治疗,但一些致盲性视网膜疾患如视网膜色素变性、年龄相关性黄斑变性、青光眼视神经视网膜病变等采用目前的方法无法逆转。目前青光眼视神经保护的策略是在有效降低眼压的基础上,针对视神经损害的不同环节,利用不同的药物,保护患者视功能。此外,通过移植细胞使视网膜细胞更新是治疗神经变性疾病的另一种方法,逐渐受到关注。用于移植的细胞有脑源性神经干细胞、视网膜祖细胞、骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)等。本课题旨在探讨体外诱导大鼠BMSCs向神经细胞分化的可能性和条件,为青光眼视神经视网膜损害及其它视网膜退行性疾病的治疗提供种子细胞奠定实验基础。方法本课题选用大鼠骨髓间充质干细胞,根据其取材方便,便于体外扩增,可自身取材,来源丰富,免疫源性低等特性,对BMSCs进行体外诱导为神经元细胞进行研究。本研究包括2部分: 1.大鼠BMSCs的培养和鉴定采用贴壁筛选法获得BMSCs,体外增殖纯化,获得纯化干细胞。流式细胞仪细胞鉴定。 2.BMSCs经bFGF预诱导后,以β-巯基乙醇(β-BM)和视网膜匀浆上清液为诱导剂进行诱导。用倒置相差显微镜观察细胞形态,细胞免疫组织化学法检测诱导后各组细胞巢蛋白(Nestin),神经丝蛋白(NF),神经元特异烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)等特异性标志物的表达。结果 1.大鼠BMSCs的分离、培养和鉴定 (1)细胞分离培养通过贴壁法能获得纯度较高的BMSCs,原代培养3天后细胞呈纺锤形。7天后细胞呈团簇、集落状生长。细胞传代后,24h细胞大部分贴壁,传代后细胞增殖旺盛,仍呈梭形。传代过多细胞增殖减慢,可见到一些扁平,宽大的细胞。 (2)流式细胞仪检测细胞表面标志表达CD90、CD29、CD44,不表达CD34、CD45、CD31。 (3)多向分化潜能在含有诱导剂的培养基中,BMSCs可在体外分化成为成骨细胞、脂肪细胞。 (4)10%血清浓度为BMSCs生长最佳浓度。 (5)不同传代细胞增殖能力不同,P_1和P_3代细胞强于P_8代细胞。 2.体外神经分化 (1)β-BM体外诱导BMSCs分化加入5 mmol/Lβ-BM1h左右开始细胞质向核收缩,胞体变小,变圆,透亮,并出现细胞突起,随时间延长部分细胞胞体收缩,细胞突起变细、变长,呈双极及多极,但数量不多。5h后更多细胞出现类神经细胞改变,细胞突起相互交织。少数细胞悬浮。诱导1h后Nestin阳性率高于NSE和NF,差异有显著性(P＜0.01);诱导5h后Nestin阳性率低于NSE和NF,差异有显著性(P＜0.01)。NSE阳性率1h和5h,差异无显著性(P＞0.05)。Nestin:1h高于5h,差异有显著性(P＜0.01)。NF:1h低于5h,差异有显著性(P＜0.01) 加入10 mmol/Lβ-BM诱导30 min左右细胞开始出现神经元细胞样形态,但较多细胞悬浮。诱导1h后阳性率NSE＞NF＞Nestin,差异有显著性(P＜0.001),诱导5h后Nestin阳性率低于NSE和NF,差异有显著性(P＜0.01)。NSE和NF:阳性率1h低于5h(P＜0.01),Nestin则相反。各组GFAP和对照组抗体染色阳性率在任何时间较其它抗体明显低,差异有显著性(P＜0.01)。 (2)视网膜匀浆上清液诱导BMSCs向神经元细胞分化两组不同浓度视网膜匀浆上清液均能诱导BMSCs向神经细胞分化,与β-BM相比作用比较缓和,3天为分化高峰。诱导细胞存活时间1周以上。大鼠视网膜匀浆上清液诱导24h后,不同浓度组Nestin阳性率均明显高于NSE和NF,差异有显著性(P＜0.01)。诱导3d后Nestin抗体染色阳性细胞数量减少,大部分神经元样细胞NSE和NF染色阳性,差异有显著性(P＜0.01)。诱导7d后,Nestin抗体染色阳性细胞明显减少,大部分神经元样细胞NSE和NF染色阳性,差异有显著性(P＜0.01)。 NSE与NF:诱导24h比3d和7d阳性细胞率低,差异有显著性(P＜0.01)。3d和7d差异无显著性(P＞0.05)。高浓度组阳性细胞率高,差异有显著性(P＜0.01)。 Nestin:随诱导时间延长阳性率逐渐降低,各组差异均有显著性(P＜0.01)。高浓度组阳性细胞率高,差异有显著性(P＜0.01)。各组GFAP和对照组抗体染色阳性率在任何时间较其它抗体明显低,差异有显著性(P＜0.01)。结论 1.β-BM在体外可诱导BMSCs分化为神经元样细胞,5mmol/L浓度为最佳浓度。诱导细胞早期表达神经干细胞标志物Nestin,以后表达成熟神经元标志物NF,NSE,不表达胶质细胞标志物GFAP。诱导细胞存活时间短。 2.视网膜匀浆上清液可在体外诱导BMSCs分化为神经元样细胞;诱导细胞表达标志物与β-BM诱导细胞相似。匀浆液作用缓和,细胞存活时间长,但分化率较低。
[Abstract]:objective
With the progress of clinical and basic research in the ophthalmology, cataracts and keratopathy can be effectively controlled and treated, but some blind retinal diseases such as retinitis pigmentosa, age related macular degeneration, and glaucomatous retinopathy can not be reversed. On the basis of effective reduction of intraocular pressure (IOP), different drugs are used to protect the visual function of the patients in different links of optic nerve damage. In addition, retinal cell renewal is another way to treat neurodegenerative diseases through transplantation of cells. Bone marrow mesenchymal stem cells (BMSCs) and so on.
The purpose of this study is to explore the possibility and conditions for inducing the differentiation of BMSCs into neural cells in vitro, and to provide the experimental basis for the treatment of retinal damage of glaucomatous optic nerve and other retinal degenerative diseases.
Method
In this study, rat bone marrow mesenchymal stem cells (MSCs) were used in vitro. According to their convenience and convenience in extracorporeal expansion, the bone marrow mesenchymal stem cells were easily obtained, rich in source, and low immunogenic characteristics. The BMSCs was induced in vitro to study the neuron cells. This study includes 2 parts:
The culture and identification of BMSCs in 1. rats
BMSCs was obtained by adherence screening method. The cells were purified and purified by flow cytometry.
After bFGF preinduction, 2.BMSCs was induced by beta mercapto ethanol (beta -BM) and the supernatant of retina homogenate. Cell morphology was observed by inverted phase contrast microscope. Cell nestin (Nestin), neurofilament (NSE), glial fibrillary acidic protein (GF), and glial fibrillary acidic protein (GF) were detected by cell immunohistochemistry. AP) the expression of the specific markers.
Result
Isolation, culture and identification of BMSCs in 1. rats
(1) cell isolation and culture
After 3 days of primary culture, the cells displayed a high purity of BMSCs. After 3 days of primary culture, the cells showed a cluster of spindle shaped.7 cells and colonies. After the passage of the cells, most of the cells were adhered to the wall, and the cells proliferated vigorously after the passage, and the cells were still spindle shaped.
(2) detection of cell surface markers by flow cytometry
Express CD90, CD29, CD44, do not express CD34, CD45, CD31.
(3) multidirectional differentiation potential
In the medium containing inducers, BMSCs can differentiate into osteoblasts and adipocytes in vitro.
(4) the concentration of 10% serum was the best concentration of BMSCs growth.
(5) the proliferation ability of cells from different passages is different. The cells of P_1 and P_3 are stronger than those of P_8 cells.
2. in vitro nerve differentiation
(1) induction of BMSCs differentiation in vitro by beta -BM
After adding 5 mmol/L beta -BM1h, the cytoplasm began to shrink to the nucleus, the cell body became smaller, round and bright, and the cell protruded, and the cell bodies contracted with time, the cell protruding became thinner and longer, and the cells were bipolar and multipolar, but more cells appeared to change and the cell protruding interlaced after the number of.5h. A few cells suspended. After 1h, the positive rate of Nestin was higher than that of NSE and NF (P < 0.01), and the positive rate of Nestin was lower than NSE and NF (P < 0.01). There was no significant difference (P < 0.01). There was no significant difference (> 0.05).
After the addition of 10 mmol/L beta -BM, the cells of about 30 min cells began to appear cell like morphology, but more cell suspension. The positive rate of NSE > NF > Nestin after induction of 1H was significant (P < 0.001). The positive rate of Nestin was lower than NSE and NF. The difference was significant (0.01) and the positive rate was lower than that of 0.01.
The positive rates of antibody staining in GFAP and control groups were significantly lower than those in other groups at any time (P < 0.01).
(2) the supernatant of retinal homogenate induces BMSCs to differentiate into neurons.
The two groups of different concentrations of retina homogenate could induce BMSCs to differentiate into nerve cells. Compared with beta -BM, the effect was relatively mild, and the 3 day was the peak of differentiation. The survival time of the cells was more than 1 weeks. After the induction of 24h in the rat retinal homogenate supernatant, the positive rates of Nestin in the different concentration groups were significantly higher than that of NSE and NF (P < 0.01). After 3D, the number of positive cells with Nestin antibody staining decreased, and most of the neuron like cells were positive for NSE and NF staining (P < 0.01). After induced 7d, the positive cells of Nestin antibody staining positive cells were significantly reduced, and most of the neuron like cells were positive for NSE and NF staining, the difference was significant (P < 0.01).
The rate of NSE and NF: induced 24h was lower than that of 3D and 7d positive cells. The difference was significant (P < 0.01) and there was no significant difference between.3d and 7d (P > 0.05). The positive cell rate of high concentration group was high, and the difference was significant (P < 0.01).
The positive rate of Nestin: gradually decreased with the induction time, and there was significant difference in each group (P < 0.01). The rate of positive cells in high concentration group was high, and the difference was significant (P < 0.01).
The positive rates of antibody staining in GFAP and control groups were significantly lower than those in other groups at any time (P < 0.01).
conclusion
1. beta -BM can induce BMSCs to differentiate into neuron like cells in vitro, and the concentration of 5mmol/L is the best concentration. It induces the early expression of neural stem cell marker Nestin, and then expresses the marker of mature neuron NF, NSE, and does not express the glial marker GFAP. to induce cell survival time.
2. the supernatant of the retina homogenate can induce BMSCs to differentiate into neuron like cells in vitro, and the induced cell expression marker is similar to that induced by beta -BM. The effect of homogenate fluid is mild, the cell survival time is long, but the differentiation rate is low.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

【参考文献】