脑活素及法舒地尔诱导骨髓间充质干细胞增殖分化的研究
发布时间：2018-12-12 14:10
【摘要】： 目的： 研究大鼠骨髓间充质干细胞(BMSCs)体外分离、培养和扩增方法,使其保持干细胞特性--自我更新和多向分化潜能；并探讨脑活素及法舒地尔体外诱导骨髓间充质干细胞增殖分化为神经元样细胞的可能性和条件,为神经系统损伤和神经退行性疾病的治疗提供良好的理论基础和实验依据。 方法： 本课题选取150g左右SD大鼠股骨和胫骨骨髓做骨髓间充质干细胞的分离培养,采用全骨髓直接贴壁法获得原代大鼠骨髓BMSCs,差速贴壁结合消化控制法纯化细胞。MTT法检测BMSCs生长最佳血清浓度及生长曲线。流式细胞仪细胞检测BMSCs生长周期及表面抗原CD34、CD44、CD29、CD90、CD45和CD31的表达。BMSCs经bFGF预诱导后,使用脑活素和法舒地尔诱导骨髓间充质干细胞,并用倒置相差显微镜观察记录细胞的形态学变化；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)等神经细胞特异性标志物的表达情况。 结果： 1.大鼠BMSCs的分离、培养和鉴定 (1)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3代后仍增殖旺盛呈梭形。 (2)10%血清浓度为BMSCs生长最佳浓度。 (3)从传代细胞生长曲线可见：第1-2天为细胞生长潜伏期,第3-4天为对数增长期,6天以后细胞的生长进入平台期,符合干细胞的生长规律。 (4)细胞周期分析显示：处于G0/G1期的细胞为93.99%,处于非增殖状态,S期的细胞为5.46%,G2/M0期的细胞为0.54%。G0/G1期占整个细胞群的比例达93.99%,同时也说明了BMSCs的高分化潜能。 (5)流式细胞仪检测细胞表面标志：表达CD90、CD29、CD44,不表达CD34、CD45、CD31。 2.体外诱导BMSCs后的形态学变化 (1)脑活素体外诱导BMSCs增殖分化 以10ng/mlbFGF预诱导24小时后,细胞形态较预诱导前未发生改变。接着以脑活素诱导后,细胞生长状态较未加入脑活素前好,细胞数量增多,且具有统计学意义(P0.05),但细胞形态改变不明显,细胞收缩,出现三角形样细胞,但未见明显细胞突起。 (2)法舒地尔体外诱导BMSCs增殖分化 法舒地尔诱导组诱导6小时后细胞形态逐渐发生变化,出现双极形、多极形和锥形细胞,呈神经元样细胞。诱导24小时后,可见较多神经元样细胞,突触逐渐形成并增多,细胞突起相互交织呈现网络状连接。诱导48小时后,细胞突触发生断裂溶解,网络状连接消失,细胞数量减少。 3免疫组织化学染色结果 对照组Nestin、NSE、NF、GFAP抗体均未见阳性染色。脑活素组中免疫组化可见少数Nestin、NSE、NF、GFAP染色阳性细胞,与空白对照组相比,无显著性差异(P0.05)。 法舒地尔诱导实验组：诱导6小时后Nestin、NSE、NF、GFAP抗体染色呈阳性反应,与脑活素组及空白对照组相比,有显著性差异(P0.05)。诱导24小时后Nestin、NF、GFAP抗体染色阳性率高于6小时,有显著性差异(P0.05)；NSE阳性率低于6小时,无显著性差异(P0.05)。诱导48小时后,Nestin、NF、GFAP阳性率高于24小时,有显著性差异(P0.05)；NSE阳性率低于24小时,无显著性差异(P0.05)。 Nestin、NF与GFAP:随诱导时间延长阳性率逐渐增高,各组差异均有显著性(P0.01)。 NSE：阳性细胞率6小时48小时24小时,差异无显著性(P0.05)。 结论： 通过贴壁筛选法体外分离、培养,传代后可获得纯度较高的骨髓间充质干细胞。脑活素诱导BMSCs后细胞形态改变不明显,胞体收缩,出现三角形样细胞,但未见明显细胞突起；法舒地尔在体外可诱导BMSCs分化为神经元样细胞,免疫组化显示诱导细胞表达Nestin、NSE、NF、GFAP等神经细胞特异性标记物。为进一步研究BMSCs应用于神经系统损伤和神经退行性疾病的细胞治疗提供了良好的实验基础。
[Abstract]:Purpose: To study the isolation, culture and amplification of bone marrow mesenchymal stem cells (BMSCs) in rats to keep the characteristics of stem cells--self-renewal and multi-orientation To explore the potential and condition of the proliferation and differentiation of mesenchymal stem cells into neuron-like cells in vitro, and to provide a good theoretical basis for the treatment of nervous system injury and neurodegenerative diseases. Basis for inspection Methods: The bone marrow of the left and right SD rats and the bone marrow of the tibia were selected to be isolated and cultured. The bone marrow BMSCs and the differential apposed of the bone marrow of the primary rat were obtained by using the full-bone marrow direct attachment method. Purification of cells by digestion control method. The growth of BMSCs was detected by MTT assay. Best serum concentration and growth curve. The growth cycle of BMSCs and surface antigen CD34, CD44, CD29, CD90, and C were detected by flow cytometry. Expression of D45 and CD31. After the BMSCs were pre-induced with bFGF, the bone marrow mesenchymal stem cells were induced by using the brain activin and the method, and the morphological changes of the cells were observed with an inverted phase-contrast microscope. Nerve cells such as neurofilament protein (NF), neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), and the like specificity The expression of the marker. 1. Isolation, culture and identification of BMSCs in rats (1) BMSCs with higher purity can be obtained by the method of apposition, and the primary culture 3 The cells were in the shape of a spindle after the day. The cells were in a group after 7 days. cluster, colony-like growth. (2) The concentration of 10% serum was the best concentration of BMSCs. (3) From the growth curve of the passage cells, the incubation period of the cells was 1-2 days, and the third-fourth day was the cell growth latent period. The cell cycle analysis showed that the cells in the G0/ G1 phase were 90.99%, the cells in the non-proliferation state, the S-phase cells were 5.46%, and the cells in the G2/ M0 stage were 0.54%. G0/ G1 The proportion of the whole cell population was 90.99%, and the high differentiation potential of BMSCs was also described. (5) Flow cytometry was used to detect the cells. Surface markers: expression of CD90, CD29, C D44, not expressing CD34, CD45, CD 31.2. Morphological changes (1) brain activity after induction of BMSCs in vitro In vitro, the proliferation and differentiation of BMSCs were pre-induced by 10 ng/ ml bFGF for 24 hours, and the cell morphology was not changed before the pre-induction. and had statistical significance (P0.05), but the cells The morphological changes were not clear, the cells were contracted, triangular-like cells were present, but no significant cell projections were found. (2) in-vitro induction of the method The proliferation and differentiation of BMSCs induced a gradual change in the morphology of the cells after 6 hours of induction, and the two-pole, multipolar, in that form of a neuron-like cell, in the form of a neuron-like cell, more neuron-like cells are visible after 24 h of induction. The synapses gradually form and increase, fine. The cells of the cells were in the form of a network-like connection. After 48 hours of induction, the cells of the cells were broken. in that control group, Nestin, NSE, NF and GFAP were all of the control group. No positive staining was found. The positive cells of Nestin, NSE, NF and GFAP were found in the brain activin group, and there was no significant difference compared with the blank control group (P0.05). The results showed that in the experimental group, the staining of Nestin, NSE, NF and GFAP was positive in 6 hours, and there was a significant difference (P0.05). The positive rate of Nestin, NF and GFAP was higher than 6 hours after 24 hours, and there was a significant difference (P0.05). The positive rate of NSE was lower than 6 hours without significant difference (P0.05). After 48 hours, the positive rate of Nestin, NF and GFAP was higher than 24 hours, and there was a significant difference (P0.05). The positive rate of E was lower than 24 hours without significant difference (P0.05)., NF The positive rate of GFAP and GFAP was increased with the time of induction, and there was a significant difference in each group (P0.001). 1). NSE: The rate of positive cells was 6 h, 48 h and 24 h, and the difference was not significant (P0.05). Conclusion: The bone marrow mesenchymal stem cells with high purity can be obtained by in vitro isolation, culture and passage of the adherent screening method. After Cs, the morphological changes of the cells were not obvious, and the cells were collected. In vitro, BMSCs can be induced to differentiate into neuron-like cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2374716
[Abstract]:Purpose: To study the isolation, culture and amplification of bone marrow mesenchymal stem cells (BMSCs) in rats to keep the characteristics of stem cells--self-renewal and multi-orientation To explore the potential and condition of the proliferation and differentiation of mesenchymal stem cells into neuron-like cells in vitro, and to provide a good theoretical basis for the treatment of nervous system injury and neurodegenerative diseases. Basis for inspection Methods: The bone marrow of the left and right SD rats and the bone marrow of the tibia were selected to be isolated and cultured. The bone marrow BMSCs and the differential apposed of the bone marrow of the primary rat were obtained by using the full-bone marrow direct attachment method. Purification of cells by digestion control method. The growth of BMSCs was detected by MTT assay. Best serum concentration and growth curve. The growth cycle of BMSCs and surface antigen CD34, CD44, CD29, CD90, and C were detected by flow cytometry. Expression of D45 and CD31. After the BMSCs were pre-induced with bFGF, the bone marrow mesenchymal stem cells were induced by using the brain activin and the method, and the morphological changes of the cells were observed with an inverted phase-contrast microscope. Nerve cells such as neurofilament protein (NF), neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), and the like specificity The expression of the marker. 1. Isolation, culture and identification of BMSCs in rats (1) BMSCs with higher purity can be obtained by the method of apposition, and the primary culture 3 The cells were in the shape of a spindle after the day. The cells were in a group after 7 days. cluster, colony-like growth. (2) The concentration of 10% serum was the best concentration of BMSCs. (3) From the growth curve of the passage cells, the incubation period of the cells was 1-2 days, and the third-fourth day was the cell growth latent period. The cell cycle analysis showed that the cells in the G0/ G1 phase were 90.99%, the cells in the non-proliferation state, the S-phase cells were 5.46%, and the cells in the G2/ M0 stage were 0.54%. G0/ G1 The proportion of the whole cell population was 90.99%, and the high differentiation potential of BMSCs was also described. (5) Flow cytometry was used to detect the cells. Surface markers: expression of CD90, CD29, C D44, not expressing CD34, CD45, CD 31.2. Morphological changes (1) brain activity after induction of BMSCs in vitro In vitro, the proliferation and differentiation of BMSCs were pre-induced by 10 ng/ ml bFGF for 24 hours, and the cell morphology was not changed before the pre-induction. and had statistical significance (P0.05), but the cells The morphological changes were not clear, the cells were contracted, triangular-like cells were present, but no significant cell projections were found. (2) in-vitro induction of the method The proliferation and differentiation of BMSCs induced a gradual change in the morphology of the cells after 6 hours of induction, and the two-pole, multipolar, in that form of a neuron-like cell, in the form of a neuron-like cell, more neuron-like cells are visible after 24 h of induction. The synapses gradually form and increase, fine. The cells of the cells were in the form of a network-like connection. After 48 hours of induction, the cells of the cells were broken. in that control group, Nestin, NSE, NF and GFAP were all of the control group. No positive staining was found. The positive cells of Nestin, NSE, NF and GFAP were found in the brain activin group, and there was no significant difference compared with the blank control group (P0.05). The results showed that in the experimental group, the staining of Nestin, NSE, NF and GFAP was positive in 6 hours, and there was a significant difference (P0.05). The positive rate of Nestin, NF and GFAP was higher than 6 hours after 24 hours, and there was a significant difference (P0.05). The positive rate of NSE was lower than 6 hours without significant difference (P0.05). After 48 hours, the positive rate of Nestin, NF and GFAP was higher than 24 hours, and there was a significant difference (P0.05). The positive rate of E was lower than 24 hours without significant difference (P0.05)., NF The positive rate of GFAP and GFAP was increased with the time of induction, and there was a significant difference in each group (P0.001). 1). NSE: The rate of positive cells was 6 h, 48 h and 24 h, and the difference was not significant (P0.05). Conclusion: The bone marrow mesenchymal stem cells with high purity can be obtained by in vitro isolation, culture and passage of the adherent screening method. After Cs, the morphological changes of the cells were not obvious, and the cells were collected. In vitro, BMSCs can be induced to differentiate into neuron-like cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
【参考文献】
相关期刊论文 前2条
1 项鹏,夏文杰,张丽蓉,陈振光,张秀明,李艳,李树浓;碱性成纤维生长因子等诱导间质干细胞分化为神经元样细胞的研究[J];中华神经科杂志;2002年03期
2 刘爱军,项平,黄锦桃,李海标;碱性成纤维细胞生长因子对大鼠骨髓间质干细胞增殖的影响[J];中山大学学报(医学科学版);2004年06期
，本文编号：2374716
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2374716.html
最近更新
教材专著
