体外诱导大鼠骨髓间充质干细胞向GABA能神经元分化
摘要
骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)作为来源于中胚层的具有多向分化潜能的成体干细胞,具有容易进行分离纯化培养且能避开自体移植的免疫障碍,同时还突破了神经干细胞(neural stem cell, NSC)和胚胎干细胞(embryonic stem cell, ESC)研究的伦理束缚等等鲜明特点,近来成为干细胞研究的热点趋势。BMSCs在适当条件下可向成骨细胞、成软骨细胞、脂肪细胞、神经细胞等方向分化,尤其向神经细胞方向分化的趋势为中枢神经系统损伤病人带来了新的福音。目前研究表明BMSCs对大鼠癫痫模型有一定的疗效,γ-氨基丁酸(gama aminobutyric acid , GABA)能神经元损伤作为癫痫的关键发病因素,BMSCs是否具有分化为GABA能神经元的能力还未有明确报道,同时体外诱导分化的GABA能神经元的功能性还有待进一步研究。本论文在前面学者研究NSC和BMSCs分化的基础上,结合GABA能神经元自身诱导分化的特点,成功在体外将BMSCs诱导分化为GABA能神经元。尽管目前研究显示其向GABA能神经元分化效率和相应功能有限,但此研究的初步结果为临床应用BMSCs治疗癫痫奠定了一定的理论基础,为颅脑病变的细胞移植替代治疗开辟了新的方向。本论文从BMSCs的分离培养、BMSCs的诱导分化两个方面作以探索性研究:
1、BMSCs的分离培养。
目的获取纯化的BMSCs。方法用10ml无菌注射器吸取PBS冲洗鼠龄4-6w、体重100g左右的SD大鼠股骨骨髓腔,收集5ml左右的骨髓液,缓慢沿试管壁加入到等体积的Percol(l1.073g/ml)分离液中,而后以2500rpm的速度高速离心20min,谨慎吸取试管内液体交界面处的薄层细胞转移到含PBS的试管中,吹打后以1000rpm的速度离心5min,倒掉PBS然后在细胞中加入含10%FBS的DNEM/F12,吹打均匀后以1×106个/ml的浓度转移移到40ml的塑料培养瓶中培养,即为原代细胞。静止培养3天后先半量换液,以后正常换液,大约10-12天待细胞铺满瓶底70%左右时传代培养,获得第1代细胞。以此类推。通过形态学观察和免疫组化法分析获取的细胞。结果细胞培养48h左右即开始贴壁,形态多呈梭形,原代细胞呈巢状生长。传代后细胞增殖迅速,培养液中有少量悬浮细胞,到第2代后细胞形态单一,基本达到纯化。同时对纯化细胞作免疫组化分析发现其表面标志抗体的表达情况为CD29(91. 3±5.0) %、CD105( 72. 1±6.3 ) %和CD34 (2.4±0.8) %。结论通过大鼠骨髓的分离培养,在第2代即可得到比较纯化的BMSCs。此结论为BMSCs的诱导分化提供了有利前提。
2、体外诱导大鼠BMSCs向GABA能神经元分化。
目的探讨BMSCs向GABA能神经元的分化趋向。方法取生长状态良好的第二代BMSCs添加NSC培养基培养一周,而后加入10 ng/ml脑源性神经营养因子(brain-derived neurotrophic factor , BDNF)和10 ng/ml骨形成蛋白-2(bone morphogenetic protein-2, BMP-2)诱导3-4天,再以1μmol/L全反式维甲酸(all transretinoic acid, ATRA)诱导3-4天完成分化。观察细胞形态学在诱导分化中的变化,免疫荧光和Western blot鉴定分化后细胞并进行统计学分析。结果细胞在添加NSC培养基3天后即开始出现变化,到一周时细胞明显变圆,透光性增强。添加BDNF和BMP-2后细胞开始出现突起,至诱导分化完成时细胞拥有明显的胞体和突起,呈典型的神经元样细胞形态。免疫荧光发现神经细胞抗体阳性:Nestin( 27.3±5.6 ) %、NSE (37.6±4.6) %和GAD67 (18.7±5.7 ) %。Western blot分析显示,经诱导分化的细胞GAD67和NSE的蛋白条带相对灰度值明显高于未诱导分化细胞的灰度值。结论BMSCs在体外有向GABA能神经元方向分化的趋势,在添加BMP-2趋势更为明显。上述结果为癫痫的细胞移植治疗奠定了一定的理论基础。
Bone marrow mesenchymal stem cells(BMSCs), as the somatic stem cells derived from the mesoderm which have multi-directional differentiation potential , easily purified from the culture and can avoid the immune barriers to transplantation, and break through the ethical constraints of NSC and ESC research, recently have become a hot trend in stem cells study. Under appropriate conditions BMSCs can differentiating into bone cells, cartilage cells, fat cells, neural cells and so on, especially the direction of the trend of differentiating into neural cells which has brought new gospel for patients who are central nervous system injured in clinical work. Moreover recent studies have shown that BMSCs have some effects on rat epilepsy model, gama aminobutyric acid(GABA)ergic neurons as a key pathogenic factors to epilepsy happened, whether BMSCs own the ability of differentiating into GABAergic neurons have not a definite coverage, while the function of the induced GABAergic neurons in vitro remains to be further studied. In this paper, resort to the previous scholars about studying the differentiation of NSC and BMSCs and characteristics of self-induced differentiation of GABAergic neurons, successfully induced the BMSCs to GABAergic in vitro. Although studies have shown that the efficiency and corresponding function of the differentiated GABAergic neurons is limited, the initial results of this research has laid a theoretical foundation for the treatment of epilepsy and opened a new direction of the cell transplantation for brain lesions. In this paper, the two aspects of isolation and culture of BMSCs, induced differentiation of BMSCs were researched as following:
1. Isolation and culture of BMSCs.
Objective To obtain purified BMSCs. Methods took 10ml sterile syringes to wash rat bone marrow cavity with PBS towards the SD rats age of 4-6 week, weighing about 100g, collected about 5ml of bone marrow fluid in the tube, along the tube wall slowly added an equal volume of Percoll (1.073g/ml) separation of liquid, then to take the speed of 2500rpm to high-speedly centrifuge 20min, carefully swabbed the thin interface of the cells, transferred the cells to tube containing PBS, blew the cells with pipette and centrifuged with 1000rpm about 5min, drained the PBS and added DNEM/F12 containing 10% FBS, transferred to the 40ml plastic bottle after wind and percussion uniform and got the primary cultured cells. Static cultured for 3 days to change the first half of the amount of fluid, the normal exchange of medium later, about 10-12 days closely 70% of bottom of the bottle was covered with cells and then passaged to obtain the first generation of the cells, so did the later passage. Obtained cells with morphological observation and immunohistochemical analysis. Results Separated cells began to attach the wall around 48h, most morphology was spindle, primary cells were nested growth. The cells proliferated rapidly after passage and found a few amount of suspended cells in the medium, the second generation of the cells became purified generally. Meanwhile immunohistochemical analysis showed the situation of surface marker antibodies for CD29 (91.3±5.0)%, CD105 (72.1± 6.3)%, and CD34 (2.4±0.8)%. Conclusion Isolation and culture of rat bone marrow can be obtained the relatively purified BMSCs at the second generation. This conclusion provided a favorable prerequisite for the induction of differentiation of BMSCs.
2. Induce rat bone marrow mesenchymal stem cells differentiate into GABAergic neurons in vitro.
Objective To explore the trend of BMSCs differentiate into GABAergic neurons. Methods Added neural stem cells(NSC) medium to the favorable conditions of BMSCs for 1 week, then induced with 10ng/ml brain-derived neurotrophic factor(BDNF) and 10ng/ml bone morphogenetic protein-2(BMP-2) for 3-4 days, next cultured with 1μmol/L all trans retinoic acid(ATRA) for 3-4 days to complete the differentiation. Observed the morphological change of the induced cells, analyzed with Immunofluorescence and Western blot to the differentiated cells, statistical disposal to the last results. Results The cells’morphology began to change in the NSC culture medium after three days, existed a clear spherical body for about 1 week and increasing transparent. The cells began to appear bulge after adding BDNF and BMP-2, clear body and processes following the differentiation completed, showing a typical neuron-like cell morphology. Immunofluorescence manifested the positive neural cells: Nestin( 27.3±5.6 ) %, NSE (37.6±4.6) % and GAD67 (18.7±5.7 ) %. Western blot analysis showed that the relative gray value of protein bands of GAD67 and NSE was significantly higher than non-induced differentiated cells. Conclusion BMSCs had the trend of differentiating into GABAergic neurons in vitro, even more pronounced trend adding in BMP-2. The results of this research laid a certain theoretical basis for BMSCs transplantation for the treatment of epilepsy.
1、BMSCs的分离培养。
目的获取纯化的BMSCs。方法用10ml无菌注射器吸取PBS冲洗鼠龄4-6w、体重100g左右的SD大鼠股骨骨髓腔,收集5ml左右的骨髓液,缓慢沿试管壁加入到等体积的Percol(l1.073g/ml)分离液中,而后以2500rpm的速度高速离心20min,谨慎吸取试管内液体交界面处的薄层细胞转移到含PBS的试管中,吹打后以1000rpm的速度离心5min,倒掉PBS然后在细胞中加入含10%FBS的DNEM/F12,吹打均匀后以1×106个/ml的浓度转移移到40ml的塑料培养瓶中培养,即为原代细胞。静止培养3天后先半量换液,以后正常换液,大约10-12天待细胞铺满瓶底70%左右时传代培养,获得第1代细胞。以此类推。通过形态学观察和免疫组化法分析获取的细胞。结果细胞培养48h左右即开始贴壁,形态多呈梭形,原代细胞呈巢状生长。传代后细胞增殖迅速,培养液中有少量悬浮细胞,到第2代后细胞形态单一,基本达到纯化。同时对纯化细胞作免疫组化分析发现其表面标志抗体的表达情况为CD29(91. 3±5.0) %、CD105( 72. 1±6.3 ) %和CD34 (2.4±0.8) %。结论通过大鼠骨髓的分离培养,在第2代即可得到比较纯化的BMSCs。此结论为BMSCs的诱导分化提供了有利前提。
2、体外诱导大鼠BMSCs向GABA能神经元分化。
目的探讨BMSCs向GABA能神经元的分化趋向。方法取生长状态良好的第二代BMSCs添加NSC培养基培养一周,而后加入10 ng/ml脑源性神经营养因子(brain-derived neurotrophic factor , BDNF)和10 ng/ml骨形成蛋白-2(bone morphogenetic protein-2, BMP-2)诱导3-4天,再以1μmol/L全反式维甲酸(all transretinoic acid, ATRA)诱导3-4天完成分化。观察细胞形态学在诱导分化中的变化,免疫荧光和Western blot鉴定分化后细胞并进行统计学分析。结果细胞在添加NSC培养基3天后即开始出现变化,到一周时细胞明显变圆,透光性增强。添加BDNF和BMP-2后细胞开始出现突起,至诱导分化完成时细胞拥有明显的胞体和突起,呈典型的神经元样细胞形态。免疫荧光发现神经细胞抗体阳性:Nestin( 27.3±5.6 ) %、NSE (37.6±4.6) %和GAD67 (18.7±5.7 ) %。Western blot分析显示,经诱导分化的细胞GAD67和NSE的蛋白条带相对灰度值明显高于未诱导分化细胞的灰度值。结论BMSCs在体外有向GABA能神经元方向分化的趋势,在添加BMP-2趋势更为明显。上述结果为癫痫的细胞移植治疗奠定了一定的理论基础。
Bone marrow mesenchymal stem cells(BMSCs), as the somatic stem cells derived from the mesoderm which have multi-directional differentiation potential , easily purified from the culture and can avoid the immune barriers to transplantation, and break through the ethical constraints of NSC and ESC research, recently have become a hot trend in stem cells study. Under appropriate conditions BMSCs can differentiating into bone cells, cartilage cells, fat cells, neural cells and so on, especially the direction of the trend of differentiating into neural cells which has brought new gospel for patients who are central nervous system injured in clinical work. Moreover recent studies have shown that BMSCs have some effects on rat epilepsy model, gama aminobutyric acid(GABA)ergic neurons as a key pathogenic factors to epilepsy happened, whether BMSCs own the ability of differentiating into GABAergic neurons have not a definite coverage, while the function of the induced GABAergic neurons in vitro remains to be further studied. In this paper, resort to the previous scholars about studying the differentiation of NSC and BMSCs and characteristics of self-induced differentiation of GABAergic neurons, successfully induced the BMSCs to GABAergic in vitro. Although studies have shown that the efficiency and corresponding function of the differentiated GABAergic neurons is limited, the initial results of this research has laid a theoretical foundation for the treatment of epilepsy and opened a new direction of the cell transplantation for brain lesions. In this paper, the two aspects of isolation and culture of BMSCs, induced differentiation of BMSCs were researched as following:
1. Isolation and culture of BMSCs.
Objective To obtain purified BMSCs. Methods took 10ml sterile syringes to wash rat bone marrow cavity with PBS towards the SD rats age of 4-6 week, weighing about 100g, collected about 5ml of bone marrow fluid in the tube, along the tube wall slowly added an equal volume of Percoll (1.073g/ml) separation of liquid, then to take the speed of 2500rpm to high-speedly centrifuge 20min, carefully swabbed the thin interface of the cells, transferred the cells to tube containing PBS, blew the cells with pipette and centrifuged with 1000rpm about 5min, drained the PBS and added DNEM/F12 containing 10% FBS, transferred to the 40ml plastic bottle after wind and percussion uniform and got the primary cultured cells. Static cultured for 3 days to change the first half of the amount of fluid, the normal exchange of medium later, about 10-12 days closely 70% of bottom of the bottle was covered with cells and then passaged to obtain the first generation of the cells, so did the later passage. Obtained cells with morphological observation and immunohistochemical analysis. Results Separated cells began to attach the wall around 48h, most morphology was spindle, primary cells were nested growth. The cells proliferated rapidly after passage and found a few amount of suspended cells in the medium, the second generation of the cells became purified generally. Meanwhile immunohistochemical analysis showed the situation of surface marker antibodies for CD29 (91.3±5.0)%, CD105 (72.1± 6.3)%, and CD34 (2.4±0.8)%. Conclusion Isolation and culture of rat bone marrow can be obtained the relatively purified BMSCs at the second generation. This conclusion provided a favorable prerequisite for the induction of differentiation of BMSCs.
2. Induce rat bone marrow mesenchymal stem cells differentiate into GABAergic neurons in vitro.
Objective To explore the trend of BMSCs differentiate into GABAergic neurons. Methods Added neural stem cells(NSC) medium to the favorable conditions of BMSCs for 1 week, then induced with 10ng/ml brain-derived neurotrophic factor(BDNF) and 10ng/ml bone morphogenetic protein-2(BMP-2) for 3-4 days, next cultured with 1μmol/L all trans retinoic acid(ATRA) for 3-4 days to complete the differentiation. Observed the morphological change of the induced cells, analyzed with Immunofluorescence and Western blot to the differentiated cells, statistical disposal to the last results. Results The cells’morphology began to change in the NSC culture medium after three days, existed a clear spherical body for about 1 week and increasing transparent. The cells began to appear bulge after adding BDNF and BMP-2, clear body and processes following the differentiation completed, showing a typical neuron-like cell morphology. Immunofluorescence manifested the positive neural cells: Nestin( 27.3±5.6 ) %, NSE (37.6±4.6) % and GAD67 (18.7±5.7 ) %. Western blot analysis showed that the relative gray value of protein bands of GAD67 and NSE was significantly higher than non-induced differentiated cells. Conclusion BMSCs had the trend of differentiating into GABAergic neurons in vitro, even more pronounced trend adding in BMP-2. The results of this research laid a certain theoretical basis for BMSCs transplantation for the treatment of epilepsy.
引文
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