IGF-1体外诱导人脐带间充质干细胞向神经样细胞分化

发布时间：2018-07-09 09:28

本文选题：人脐带间充质干细胞 + 胰岛素样生长因子-1　；参考：《河北医科大学》2012年硕士论文

【摘要】：目的：对人脐带间充质干细胞（human umbilical cord mesenchymalstem cells, hUMSCs）生物学特性进行鉴定，探讨胰岛素样生长因子-1（insulin-like growth factor-1，IGF-1）在体外定向诱导hUMSCs向神经细胞分化的作用和条件，为hUMSCs神经分化以及临床应用提供理论和实验基础。方法：取足月妊娠剖宫产健康胎儿的脐带，以D-Hank’s充分冲洗，剔除脐动脉和脐静脉，将剩余的脐带间质组织切割成1mm3大小的组织块，0.2%胶原酶Ⅱ消化，在含有20%胎牛血清（FBS）、2ng/ml表皮细胞生长因子（EGF）、25mM左旋谷氨酰胺（L-Glu）、100U/ml青霉素，100μg/ml链霉素的DMEM/F12中培养。观察原代培养细胞的形态学变化，当细胞达80％～90％汇合时，加入0.25%胰酶-1mM EDTA消化细胞，并传代。收集消化后的细胞，采用流式细胞术行细胞表型检测，包括CD73、CD90和CD105、CD19、CD34、CD45、CD11和组织相容性抗原HLA-DR(MHC-II)，以抗鼠IgG1-PE和IgG1-FITC作为同型对照。原代hUMSCs按1:1的比例传代后是为P1代，倒置显微镜下观察形态变化，细胞达到90%以上融合时按1:2或1:3比例进行传代，绘制细胞生长曲线。取P4代生长状态良好的hUCMSCs，分为四组：25ng/ml组、50ng/ml组、100ng/ml组和对照组。以3×104/孔的密度接种于4个多聚赖氨酸包被的六孔板中（每个六孔板为一组），待细胞融合达到70-80%时，三个六孔板分别换为含有25ng/ml、50ng/ml、100ng/ml IGF-1的无血清低糖DMEM/F12培养基的诱导液，为实验组；另一六孔板为对照组，除不加IGF-1外其余均同实验组。镜下观察细胞形态变化，每2小时在高倍显微镜视野（100倍）下进行观察12小时，随机选取10个视野，拍照并计数典型神经样细胞形态（有明显的两个及以上突起形成，细胞体屈光性强，类似神经元样细胞）的细胞数及总细胞数，用以计算分化率，结果以均数±标准差表示。比较不同浓度IGF-1诱导hUCMSCs的分化率，选取分化率高且稳定的一组诱导后8h用免疫细胞化学技术检测星形胶质细胞标志（GFAP）、神经元标志（NSE）和神经干细胞标志（Nestin），拍照记录计数阳性细胞，计算阳性细胞率结果以均数±标准差表示。结果：原代细胞传代24h后，大部分细胞逐渐贴壁，细胞呈菱形、三角形、椭圆形等形态；随着培养时间的延长，渐变为长梭形，细胞数量逐渐增多。培养7-8d时细胞融合到90%左右，低倍镜下可以看到hUCMSCs呈漩涡状排列。按照1:2的比例进行再传代，连续传代到P15代，hUCMSCs仍保持旺盛的生长能力。流式细胞检测P3、P5和P10代细胞的分子标志物，结果显示各代均表达CD73、CD90和CD105，而不表达CD34、CD45CD19、CD11b和HLA-DR。加入IGF诱导液后30min，IGF-1为50ng/ml和100ng/ml组有个别细胞开始发生形态改变，25ng/ml组细胞开形态改变不明显。光学显微镜下可见：胞体收缩变圆，伸出指状突起，细胞体折光度增强；随着诱导时间的延长，，细胞进一步收缩，突起较前增长，1h后个别细胞形成双极突起以100ng/ml组变化细胞数量最多同时也可以看到该组内有少量细胞死亡漂浮于培养基上。4h后50ng/ml和100ng/ml组细胞折光度增大，突起较前更加细长、增多，多在2至5条，有的细胞逐渐形成多级突起，25ng/ml组变化不明显；8h后50ng/ml和100ng/ml组多个相临的细胞逐渐连接形成树突样网络，25ng/ml组细胞形态改变较前不明显；随诱导时间延长，分化的细胞形态越来越典型，但死亡、漂浮的细胞也逐渐增多，以100ng/ml组死亡的细胞最多；24h后100ng/ml组细胞大量死亡，仅有少量细胞存活且细胞透亮度降低；50ng/ml组存活细胞表现出更为典型的神经细胞样改变，这些细胞的胞体折光度增大，细胞质向细胞核收缩，形成双极、多极样突起；有的呈放射状伸展，突起伸长更加明显，可以看到2、3级突起但同时死亡细胞也逐渐增多，漂浮于培养基表面。免疫组化染色可见大量GFAP、NSE和Nestin阳性表达的神经样细胞，其中GFAP和NSE阳细胞着色较深，Nestin阳性细胞着色较浅；免疫荧光染色也显示其GFAP、NSE，Nestin呈阳性表达；对照组免疫组化和免疫荧光染色只有极少量的细胞表达GFAP，而不表达NSE和Nestin。结论:hUCMSCs由脐带中分离纯化后可以在体外稳定传代，并保持较高的增殖能力；hUCMSCs表达间充质干细胞表面标志CD73、CD90和CD105，不表达造血细胞表面标志CD34、CD45、CD19、CD11b和组织相容性抗原HLA-DR （MHC-Ⅱ）；IGF-1在体外可诱导hUCMSCs分化成神经样细胞，并表达神经胶质细胞表面标志（GFAP）、神经元标志（NSE）神经干细胞标志（Nestin），以IGF-1浓度为50ng/ml组最为明显。
[Abstract]:Objective: to identify the biological characteristics of human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymalstem cells (hUMSCs) and hUMSCs), and to explore the use and conditions of insulin like growth factor -1 (insulin-like growth factor-1, IGF-1) to induce the differentiation of hUMSCs to neural cells in vitro. Theoretical and experimental basis.
Methods: the umbilical cord of the healthy fetus of the term pregnancy was taken, the umbilical artery and the umbilical vein were washed with D-Hank 's, the umbilical cord and umbilical veins were removed, the remaining umbilical cord interstitial tissue was cut into 1mm3 size tissue block, 0.2% collagenase II was digested, with 20% fetal bovine serum (FBS), 2ng/ml epidermal cell growth factor (EGF), 25mM L-glutamine (L-Glu), 100U/ml. Penicillin, cultured in DMEM/F 12 of 100 g/ml streptomycin, observed the morphological changes of the primary cultured cells. When the cells reached 80% to 90% confluence, 0.25% trypsin -1mM EDTA was added to the cells to digest the cells.
Cells after digestion were collected and cell phenotypes were detected by flow cytometry, including CD73, CD90 and CD105, CD19, CD34, CD45, CD11 and histocompatibility antigen HLA-DR (MHC-II), with anti mouse IgG1-PE and IgG1-FITC as the same type.
The primary hUMSCs was transmitted to the P1 generation after 1:1, and the morphological changes were observed under the inverted microscope. When the cells reached more than 90% fusion, the cell growth curve was plotted according to the proportion of 1:2 or 1:3.
HUCMSCs of good growth state of P4 generation was divided into four groups: group 25ng/ml, 50ng/ml group, 100ng/ml group and control group. The density of 3 x 104/ holes was inoculated in 4 polylysine coated six orifice plates (each group of six orifice plates), and when the cell fusion reached 70-80%, three six pore plates were replaced with 25ng/ml, 50ng/ml, 100ng/ml IGF-1, without blood. The induction fluid of DMEM/F12 culture medium with low glucose was used as the experimental group; the other 16 orifice plates were in the control group. The morphological changes of the cells were observed in the same group except without IGF-1. The morphological changes were observed under the microscope (100 times) for 12 hours at the high power microscope (100 times), and 10 fields were randomly selected to take pictures and count the typical neurolike cell morphology. Two or more protrusions were formed, cell body diopter was strong, the number of cells like neuron like cells and the total number of cells were used to calculate the differentiation rate. The results were expressed in the mean number of standard deviation. The differentiation rate of hUCMSCs induced by different concentrations of IGF-1 was compared, and a group of high and stable differentiation rates was selected to detect astrocytes by immunocytochemical technology. Cell markers (GFAP), neuron markers (NSE) and neural stem cell markers (Nestin) were taken to record counting positive cells. The results of the calculated positive cell rate were expressed as mean standard deviation.
Results: after the primary cell passage of 24h, most of the cells gradually adhered to the wall, and the cells were rhombic, triangular, oval and so on. With the prolongation of the culture time, the cells gradually changed into long spindle shape, and the number of cells gradually increased. The cells fused to about 90% when the 7-8d was cultivated, and the whirlpool arrangement could be seen under the low magnification mirror. The retransmission of the cells according to the proportion of 1:2 was retransmitted. Generation, continuous passage to P15 generation, hUCMSCs still maintained strong growth ability. Flow cytometry detected the molecular markers of P3, P5 and P10 generation cells. The results showed that all generations expressed CD73, CD90 and CD105, but did not express CD34, CD45CD19, CD11b, and HLA-DR..
In the group of 50ng/ml and 100ng/ml, there were some cells in the group of 50ng/ml and 100ng/ml after the addition of IGF. The morphological changes of the cells in the group of 50ng/ml and 100ng/ml were changed. The morphology of the cells in the 25ng/ml group was not obvious. The optical microscope showed that the contraction of the cell body became round, the finger shaped protuberance was extended, the cell body fold increased, and the cells further contracted with the time of induction, and the protuberance was increased by 1 After h, a few cells formed a bipolar protuberance to change the number of cells in the 100ng/ml group at the same time. At the same time, a small amount of cell death in the group was found to float on the medium of.4h. After.4h, the refraction degree of the cells in the 50ng/ml and 100ng/ml groups increased. The protuberances were more slender and more elongated than before, and the number of cells was 2 to 5. Some cells gradually formed multistage protuberances, and the 25ng/ml group changed unidentified. After 8h, the cells of 50ng/ml and 100ng/ml groups were connected gradually to form a dendrite like network, and the morphological changes of the 25ng/ml group were not obvious. With the prolongation of the induction time, the differentiated cells became more and more typical, but the cell died, the floating cells increased gradually, and the cells died in the 100ng/ml group were the most; the 100ng/ml group after 24h was larger than the 24h group. In the 50ng/ml group, the survival cells showed a more typical nerve cell like change, the cell body refraction of these cells increased, the cytoplasm contracted to the nucleus and formed a bipolar, multipolar protuberance; some were radially extended, the protuberance was more obvious, and the 2,3 protuberance could be seen. But at the same time, the number of dead cells increased gradually and floating on the surface of the culture medium. A large number of GFAP, NSE and Nestin positive neurons were found in the immunofluorescent staining cells, in which GFAP and NSE positive cells were coloured more deeply and the Nestin positive cells were coloured relatively shallow; immunofluorescence staining also showed that the positive expression of GFAP, NSE, Nestin was expressed in the immunofluorescence staining; and the immunofluorescence of the control group was positive. Immunofluorescence staining showed that only a few cells expressed GFAP but not NSE and Nestin..
Conclusion: after isolation and purification of hUCMSCs from the umbilical cord, it can be passaged in vitro and maintain high proliferation ability. HUCMSCs expressed CD73, CD90 and CD105 on the surface of mesenchymal stem cells, which do not express the hematopoietic cell surface markers CD34, CD45, CD19, CD11b and histocompatibility antigen HLA-DR (MHC- II), and IGF-1 can be induced to differentiate in vitro. Neurocyte like cells, and expression of glial cell surface markers (GFAP), neuron marker (NSE) neural stem cell marker (Nestin), the IGF-1 concentration is the most obvious in the 50ng/ml group.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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