VEGF-A与SB-431542联合诱导SHEDs分化为Ecs的初步研究

发布时间：2018-05-17 01:01

本文选题：SHEDs + VEGF-A　；参考：《山东大学》2017年硕士论文

【摘要】：目的:体外获取和分离人脱落乳牙牙髓干细胞并采用胰酶消化、有限稀释法对其纯化,培养并分析干细胞干性;采用VEGF-A与SB-431542联合诱导人脱落乳牙牙髓干细胞,初步探讨其分化为内皮细胞的高效性,为研究牙源性干细胞高效分化内皮细胞提供一种新思路。方法:1、人脱落乳牙牙髓干细胞的获取、分离、纯化、培养及细胞干性鉴定选取山东大学口腔医学院儿牙科因滞留而拔除的健康乳前牙,在无菌状态下获取滞留乳牙牙髓组织,并采用胰酶消化法获取单细胞的悬液,有限稀释法进行分离、纯化。倒置显微镜下观察人脱落乳牙牙髓干细胞原代、传代细胞形态和生长状况。流式细胞仪检测分析CD45、CD73、CD90、CD105表面抗原阳性表达情况。选取第三代处于生长对数期的人脱落乳牙牙髓干细胞,体外成脂诱导液诱导,向脂肪组织分化。观察细胞形态变化及胞内成脂情况,4周后油红-0染色。选取第三代人脱落乳牙牙髓干细胞,体外成骨诱导液诱导培养,第1周和第3周分别行碱性磷酸酶和茜素红染色,检测是否有矿化结节的形成,进一步鉴定人脱落乳牙牙髓干细胞多向分化能力。2、体外VEGF-A与SB-431542联合诱导人脱落乳牙牙髓干细胞向内皮细胞分化:将处在生长对数期的第三代人脱落乳牙牙髓干细胞以3×103个/cm2分别接种于α-MEM、VEGF-A、VEGF-A+SB-431542联合培养基中,隔天更换一次培养液,分别提取第7天、第14天的上述细胞,RT-PCR、Western blot分别检测三组实验内皮细胞相关基因、蛋白表达情况。统计分析:所有实验组一式三份进行,所有数值以平均值±标准偏差(SD)来表示。使用单因素方差分析确定是否具有统计学显著性差异,并将具有统计学意义的阈值设定为P0.05。结果:1、酶消化法和有限稀释法获得了纯的人脱落乳牙牙髓干细胞,显微镜下人脱落乳牙牙髓干细胞排列紧密,呈集落状生长。流式细胞仪检测细胞表面抗原CD45阳性检出率为2.8%,CD73阳性检出率为99.8%,CD90阳性检出率为99.5%,CD105阳性检出率为94.2%。人脱落乳牙牙髓干细胞经成脂诱导液诱导4周后,油红-0染色阳性细胞内呈现橙红色颗粒。成骨诱导液诱导,有钙化结节逐渐形成,茜素红染色呈现橘红色或深红色;ALP染色细胞胞浆呈现均匀的蓝色。2、实时定量逆转录聚合酶链反应(RT-qPCR)结果显示:诱导7天、14天无论是单纯的VEGF-A组还是VEGF-A+SB-431542组,均有特异性内皮细胞分子(VEGFR-2、VEGFR-1、EphrinB2 和 Tie-2)的表达。诱导 7 天后,与单纯 VEGF-A组相比,用VEGF-A和SB-431542联合培养的人脱落乳牙牙髓干细胞显示出更高的VEGFR-2表达(P0.05),VEGFR-1、CD31也呈现出相似的高表达状态。与单独的VEGF-A组相比,人脱落乳牙牙髓干细胞在含有VEGF-A和SB-431542培养基中连续培养14天后内皮细胞相关基因(VEGFR-2、VEGFR-1、EphrinB2和Tie-2)表达显着增加(P0.05)。Western blot检测相应蛋白的表达情况:培养7天和14天,VEGF-A组及VEGF-A+SB-431542组均能产生内皮细胞的特异性蛋白(CD31和VEGFR-2)。但无论是第7天还是第14天,联合组诱导分化的内皮细胞特异性蛋白表达量均高于VEGF-A单纯组。结论:1、体外获取、分离并采用酶消化法和有限稀释法可以获得纯的人脱落乳牙牙髓干细胞。流式细胞术检测人脱落乳牙牙髓干细胞表达早期的间充质干细胞标志物,人脱落乳牙牙髓干细胞可以向成骨和成脂诱导分化,具有多向分化潜能,因而具有干细胞干性。2、VEGF-A单纯诱导人脱落乳牙牙髓干细胞可以向内皮细胞分化,但VEGF-A与SB-4315412联合诱导则可以显著增强这一过程。
[Abstract]:Objective: to obtain and isolate human deciduous dental pulp stem cells from human deciduous teeth in vitro and to use trypsin digestion. The purification, culture and analysis of stem cells were carried out by the finite dilution method. VEGF-A and SB-431542 were used to induce human deciduous dental pulp stem cells, and the efficiency of differentiation into endothelial cells was preliminarily studied to study the high efficiency of odontogenic stem cells differentiation. The skin cells provide a new idea. Methods: 1, the extraction, isolation, purification, culture and dry identification of the dental pulp stem cells from the deciduous teeth were selected to select the healthy anterior teeth extracted by the dental dental hospital of Shandong University and obtain the retained deciduous teeth in the aseptic state, and the single cell suspension was obtained by trypsin digestion. The finite dilution method was used to separate and purify. The original cells of the pulp stem cells of the deciduous teeth and the cell morphology and growth status were observed under the inverted microscope. The positive expression of the surface antigen of CD45, CD73, CD90 and CD105 was detected by flow cytometry. The third generations of human deciduous dental pulp stem cells in deciduous teeth and in vitro lipid inducer were selected. Induction, differentiation to adipose tissue. Observe cell morphological changes and intracellular fat formation. After 4 weeks, oil red -0 staining. Third generations of deciduous dental pulp stem cells were selected and cultured in vitro. Alkaline phosphatase and alizarin red staining were performed for first and third weeks, respectively, to detect the formation of mineralized nodules, and to further identify human abscission. The multiple differentiation ability of the pulp stem cells was.2, and in vitro VEGF-A and SB-431542 combined to induce the human deciduous tooth pulp stem cells to differentiate into endothelial cells. The third generation of deciduous dental pulp stem cells in the logarithmic period of growth will be inoculated with 3 x 103 /cm2 respectively in the joint culture medium of alpha -MEM, VEGF-A, VEGF-A +SB-431542, and replaced every other day. The cells were extracted for seventh days and fourteenth days respectively, RT-PCR, and Western blot were used to detect the endothelial cells related genes and protein expression in the three groups respectively. Statistical analysis: all experimental groups were carried out in three copies, and all the values were expressed with mean mean standard deviation (SD). The statistical significance was determined by single factor variance analysis. The threshold of statistical significance was set as P0.05. results: 1, the pure human deciduous dental pulp stem cells were obtained by enzyme digestion and finite dilution method. Under the microscope, the stem cells of the deciduous teeth and dental pulp stem cells were close and colony-shaped. The positive detection rate of the cell surface antigen CD45 was detected by flow cytometry, and the positive detection of CD73 was positive. The rate of expetion was 99.8%, the positive rate of CD90 was 99.5%, and the positive rate of CD105 positive was 4 weeks after the induction of 94.2%. human deciduous tooth pulp stem cells. The positive cells in the oil red -0 staining were orange red granules. The calcified nodules were formed gradually, alizarin red staining showed orange red or deep red, and ALP stained cell cytoplasm. A homogeneous blue.2 and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) showed that the expression of specific endothelial cell molecules (VEGFR-2, VEGFR-1, EphrinB2 and Tie-2) was induced for 7 days, 14 days in both simple VEGF-A and VEGF-A+SB-431542 groups. After 7 days of inducement, compared with the simple VEGF-A group, VEGF-A and SB-431542 were used. The cultured human deciduous dental pulp stem cells showed a higher VEGFR-2 expression (P0.05), VEGFR-1, and CD31 also showed a similar state of high expression. Compared with the single VEGF-A group, the human deciduous dental pulp stem cells were cultured for 14 days in the VEGF-A and SB-431542 culture medium for endothelial cell related genes (VEGFR-2, VEGFR-1, EphrinB2, and EphrinB2). Tie-2) expression significantly increased (P0.05).Western blot detection of the expression of corresponding protein: 7 days and 14 days of culture, VEGF-A and VEGF-A+SB-431542 groups could produce specific proteins (CD31 and VEGFR-2) of endothelial cells. However, the specific protein expression of endothelial cells induced by the combined group was higher than VEGF-A, no matter seventh days or fourteenth days. Conclusion: 1, the pure human deciduous dental pulp stem cells can be obtained by enzyme digestion and finite dilution method in vitro. Flow cytometry is used to detect the early mesenchymal stem cell markers in human deciduous dental pulp stem cells, and the human deciduous tooth pulp stem cells can differentiate into osteogenesis and lipid. As a result, the stem cells have dry.2, and VEGF-A can differentiate into endothelial cells by simple induction of human deciduous tooth pulp stem cells, but the combination of VEGF-A and SB-4315412 can significantly enhance this process.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781

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