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
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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(P<0.05),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(P>0.05)。
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(P<0.05)。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(P<0.05)；NSE阳性率低于6小时,无显著性差异(P>0.05)。诱导48小时后,Nestin、NF、GFAP阳性率高于24小时,有显著性差异(P<0.05)；NSE阳性率低于24小时,无显著性差异(P>0.05)。
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(P<0.01)。
NSE：阳性细胞率6小时>48小时>24小时,差异无显著性(P>0.05)。
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。
Objective:To study the method to maintain stem cell characteristics - self-renewal, and multi-directional differentiation potential in its isolation, culture and amplification for rat bone marrow mesenchymal stem cells (BMSCs), and to investigate the possibility and the proper condition that BMSCs could be proliferated and differentiated into neuron neuron-like cells by cerebrolysin,fsudil. To provide a theoretical and experimental basis for the BMSCs therapy for nervous system injuries and neurodegenerative diseases.
Method:BMSCs were separated from rats bone marrow through femoral and tibial by plastic adherence methods and purified by controlling the time of digestion combied with adhesion separation.To investigate the optimal serumlevels and growth curve by MTT Assay.To investigate growth cycle and cell surface antigen CD34, CD44, CD29, CD90, CD45 and CD31 expression of BMSCs by flow cytometry.In vitro the BMSC were preindueed by bFGF and then induced by cerebrolysin and fasudil.The morphological changes were observed under phase contrast microscope, the specific markers of induced cells were identified by immunocytochemically with Nestin, NF, NSE, GFAP.
Results:
1.Isolation, culture and identification of BMSCs
(1)Pure BMSCs were successfully obtained by adherence sieving from rat bone. After culturing primary BMSCs for 3 days, BMSCs were characterized by spindle-shaped spperearence and proliferated rapidly for 7 days, the cells were cluster, colony-like growth. Cells after 3 passage still on spindle.
(2) 10%FBS of growth BMSCs is optimal serum levels.
(3)Growth curve of passage cells showed that: the first 1-2d is latency, the first 3-4d is the logarithmic growth phase,6d after the cells enter the plateau phase.
(4) Cell growth cycle analysis:cells in G0/G1 phase of 93.99%, S phase cells were 5.46%, G2/M0 0.54% of the cells. G0/G1 phase proportion of the total cell population reached 93.99%, while suggested that most of cells are not in period of proliferation.
(5)Cell surface markers by flow cytometry:BMSCs expressed CD90, CD29, CD44. In contrast, no expression of the hematopoietic lineage marker CD34, CD45, CD31.
2. Morphological changes after BMSCs induced
(1)proliferation and differentiation of BMSCs induced by cerebrolysin
lOng/mlbFGF pre-induction of 24h, the cells did not change. Then after exposed to cerebrolysin, the cell growth state was better than before and number of cells increased, and was statistically significant (P＜0.05). However, no obvious morphological changes, cell shrinkage, triangle-like cells appeared, but no obvious cell processes.
(2) Proliferation and differentiation of BMSCs induced by fasudil
Fasudil induced group after 6h induced morphological changes gradually emerged bipolar shape, multipolar shape and conical cells present neuron-like cells.24h after induction, showing that more neuron-like cells, synapse formation and the gradual increase in cell processes are intercrossed present network-like connection.48h after induction, cell synapse fracture dissolution, the network-like connections disappear, the number of cells decreased.
3.Immunocytochemical staining
The control group showed no positive staining of the four antibodies. cerebrolysin group immunohistochemistry shows that a small number of Nestin, NSE, NF, GFAP positive cells, compared with the control group (P> 0.05).
Fasudil induced experimental groups:6h after induction of Nestin, NSE, NF, GFAP antibody staining was positive (P<0.05).24h after induction the positiverate of Nestin, NF, GFAP were higher than 6h (P＜0.05); NSE-positive rate was lower than 6h (P> 0.05).48h after induction, Nestin, NF, GFAP-positive rate is higher than 24h (P<0.05); but the positive rate of NSE was in contrast (P> 0.05).
Nestin, NF and GFAP:With the prolongation of induction time positive rate increased gradually (P<0.01).
NSE:The positive rate of the 24h group was lower than those of the 6h and the 48h group, no significant difference(P> 0.05).
Conclusion:
High purity BMSCs were successfully obtained by screening adherence in vitro.After induction by cerebrolysin, no obvious morphological changes were found. It was after induction by fasudil that neuron like cells were induced to differentiate from BMSCs in vitro. By immunohistochemical analysis, these induced cells expressed neural cell-specific markers such as Nestin, NSE, NF, GFAP. It suggested that BMSCs might be used as nervel seed cells for stem cell therapy for brain injury and neurodegenerative diseases.
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(P<0.05),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(P>0.05)。
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(P<0.05)。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(P<0.05)；NSE阳性率低于6小时,无显著性差异(P>0.05)。诱导48小时后,Nestin、NF、GFAP阳性率高于24小时,有显著性差异(P<0.05)；NSE阳性率低于24小时,无显著性差异(P>0.05)。
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(P<0.01)。
NSE：阳性细胞率6小时>48小时>24小时,差异无显著性(P>0.05)。
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。
Objective:To study the method to maintain stem cell characteristics - self-renewal, and multi-directional differentiation potential in its isolation, culture and amplification for rat bone marrow mesenchymal stem cells (BMSCs), and to investigate the possibility and the proper condition that BMSCs could be proliferated and differentiated into neuron neuron-like cells by cerebrolysin,fsudil. To provide a theoretical and experimental basis for the BMSCs therapy for nervous system injuries and neurodegenerative diseases.
Method:BMSCs were separated from rats bone marrow through femoral and tibial by plastic adherence methods and purified by controlling the time of digestion combied with adhesion separation.To investigate the optimal serumlevels and growth curve by MTT Assay.To investigate growth cycle and cell surface antigen CD34, CD44, CD29, CD90, CD45 and CD31 expression of BMSCs by flow cytometry.In vitro the BMSC were preindueed by bFGF and then induced by cerebrolysin and fasudil.The morphological changes were observed under phase contrast microscope, the specific markers of induced cells were identified by immunocytochemically with Nestin, NF, NSE, GFAP.
Results:
1.Isolation, culture and identification of BMSCs
(1)Pure BMSCs were successfully obtained by adherence sieving from rat bone. After culturing primary BMSCs for 3 days, BMSCs were characterized by spindle-shaped spperearence and proliferated rapidly for 7 days, the cells were cluster, colony-like growth. Cells after 3 passage still on spindle.
(2) 10%FBS of growth BMSCs is optimal serum levels.
(3)Growth curve of passage cells showed that: the first 1-2d is latency, the first 3-4d is the logarithmic growth phase,6d after the cells enter the plateau phase.
(4) Cell growth cycle analysis:cells in G0/G1 phase of 93.99%, S phase cells were 5.46%, G2/M0 0.54% of the cells. G0/G1 phase proportion of the total cell population reached 93.99%, while suggested that most of cells are not in period of proliferation.
(5)Cell surface markers by flow cytometry:BMSCs expressed CD90, CD29, CD44. In contrast, no expression of the hematopoietic lineage marker CD34, CD45, CD31.
2. Morphological changes after BMSCs induced
(1)proliferation and differentiation of BMSCs induced by cerebrolysin
lOng/mlbFGF pre-induction of 24h, the cells did not change. Then after exposed to cerebrolysin, the cell growth state was better than before and number of cells increased, and was statistically significant (P＜0.05). However, no obvious morphological changes, cell shrinkage, triangle-like cells appeared, but no obvious cell processes.
(2) Proliferation and differentiation of BMSCs induced by fasudil
Fasudil induced group after 6h induced morphological changes gradually emerged bipolar shape, multipolar shape and conical cells present neuron-like cells.24h after induction, showing that more neuron-like cells, synapse formation and the gradual increase in cell processes are intercrossed present network-like connection.48h after induction, cell synapse fracture dissolution, the network-like connections disappear, the number of cells decreased.
3.Immunocytochemical staining
The control group showed no positive staining of the four antibodies. cerebrolysin group immunohistochemistry shows that a small number of Nestin, NSE, NF, GFAP positive cells, compared with the control group (P> 0.05).
Fasudil induced experimental groups:6h after induction of Nestin, NSE, NF, GFAP antibody staining was positive (P<0.05).24h after induction the positiverate of Nestin, NF, GFAP were higher than 6h (P＜0.05); NSE-positive rate was lower than 6h (P> 0.05).48h after induction, Nestin, NF, GFAP-positive rate is higher than 24h (P<0.05); but the positive rate of NSE was in contrast (P> 0.05).
Nestin, NF and GFAP:With the prolongation of induction time positive rate increased gradually (P<0.01).
NSE:The positive rate of the 24h group was lower than those of the 6h and the 48h group, no significant difference(P> 0.05).
Conclusion:
High purity BMSCs were successfully obtained by screening adherence in vitro.After induction by cerebrolysin, no obvious morphological changes were found. It was after induction by fasudil that neuron like cells were induced to differentiate from BMSCs in vitro. By immunohistochemical analysis, these induced cells expressed neural cell-specific markers such as Nestin, NSE, NF, GFAP. It suggested that BMSCs might be used as nervel seed cells for stem cell therapy for brain injury and neurodegenerative diseases.
引文
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