大鼠胚胎前脑神经干细胞培养及植入脑挫伤灶的形态学观察
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摘要
脑挫伤(cerebral contusion)常常引起神经细胞大量死亡和严重的神经功能障碍。一般说来,中枢神经系统缺乏再生能力。研究认为治疗脑挫伤的关键是:移植外源性细胞来补充或替代损失的神经细胞;促进残存的神经细胞存活及轴突生长;重建功能性突触。神经干细胞(neural stem cells, NSCs)可以在受体中枢神经系统内补充或替代损失的细胞;参与神经结构的重建;改善损伤局部的微环境;促进神经再生。因此,NSCs移植治疗脑挫伤具有良好的应用前景。在本研究中,我们探讨体外分离大鼠胚胎前脑NSCs的方法,原代和传代培养方法和NSCs的鉴定;观察NSCs培养特性、分化机理及诱导条件,进一步探讨将NSCs植入大鼠脑挫伤灶周边区成活、迁移和分化情况。
1.大鼠胚胎前脑神经干细胞体外培养分化的研究
实验于2008年11月至2009年10月在河北北方学院实验中心完成。清洁级,E 16d的Wistar孕鼠(由中国医学科学院实验动物研究所提供)在无菌条件下取其胚胎,分离出前脑,制备单细胞悬液,以1×106·ml-1和1×108·ml-1分别接种于含N2的DMEM/F12培养瓶中,加入终浓度为20ng·ml-1表皮细胞生长因子(epidermal growth factor, EGF)和10ng·ml-1碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)刺激其克隆生长。诱导分化实验分为悬浮培养实验和爬片培养实验。a悬浮培养实验分为三组:体积分数10%胎牛血清(fetal bovine serum,FBS) +20ng·ml-1 EGF +10ng·ml-1 bFGF,10%FBS,20%FBS,在上述诱导条件下刺激其分化。b爬片培养实验分为三组:多聚赖氨酸铺板组、明胶铺板组和无铺板组,采用体积分数为20% FBS刺激分化。研究采用免疫细胞化学和间接免疫细胞荧光染色法检测NSCs巢蛋白(nestin)、胶质纤维酸性蛋白(glial fibrillary acid protein, GFAP)、神经元特异性烯醇化酶(neuron-specific enolase, NSE)和微管相关蛋白2(microtubule-associated protein 2, MAP-2)。结果成功从胎鼠前脑分离出神经干细胞,巢蛋白表达阳性。a悬浮培养实验在不同诱导条件下,20%FBS组促分化作用略强于10% FBS组(P>0.05)。10% FBS组促分化作用强于10%FBS +20ng·ml-1 EGF +10ng·ml-1 bFGF组(P<0.05)。NSCs分化为神经元及神经胶质细胞。b爬片培养实验中,多聚赖氨酸铺板组和明胶铺板组贴壁后分化为神经元及神经胶质细胞的能力强于无铺板组(P<0.01)。多聚赖氨酸铺板组略强于明胶铺板组(P>0.05)。神经谱系标记物中的GFAP,NSE和MAP-2免疫细胞化学及间接免疫细胞荧光染色均阳性。结论:大鼠胚胎前脑NSCs可以通过体外培养获得,且具有增殖能力和多向分化潜能。20%血清组NSCs分化显著,10%FBS+EGF +bFGF组NSCs分化不显著。多聚赖氨酸和明胶作为细胞贴壁支持物可以提高分化细胞数量,而且多为星形胶质细胞。
2.神经干细胞在大鼠脑挫伤灶周边区成活和迁移
本研究利用体外无血清培养技术,加EGF和bFGF刺激大鼠胚胎源性前脑NSCs克隆增殖。传1代后,NSCs在培养过程中加入终浓度为6μg·ml-1 5-溴脱氧尿嘧啶核苷(5-bromodeoxyuridine, BrdU)。标记BrdU的NSCs,用免疫细胞化学和间接免疫细胞荧光染色检测其增殖能力。研究采用改进的Fenney’s自由落体脑挫伤模型,于致伤1d后,将NSCs用立体定位法移植到鼠脑挫伤灶边缘皮层内,免疫组化及免疫荧光双染法观察移植后1,7,14,21d NSCs在挫伤灶周边成活,迁移和分化情况。结果,移植后1,7,14,21d,损伤灶BrdU阳性细胞数目逐渐减少;BrdU阳性细胞在损伤灶周边散在分布,并向损伤皮层下迁移。损伤后灶区GFAP表达上调,并在时间上具有一定规律性。结论:本研究结果显示,大鼠胚胎前脑获得的NSCs在异体移植后,能够在受体大鼠脑损伤灶周边区存活和迁移;形态学上,其显示出与脑组织整合的特点。
The death of a large quantities of cells and the serious neuraldysfunction syndrome are usually caused by the cerebral contusion.Generally speaking, the central nervous system lacks the ability ofregeneration. The study concludes that the key to treatment of braincontusion is to transplant the exogenous cells for supplying or replacingloss of the nerve cells inorder to promote the survival of the nerve cells andthe growth of axons and reconstruct the functional synapse. Thetransplantation of neural stem cells (NSCs) has good application prospectsto treat brain contusion. In this study, we investigated the methods ofisolating the NSCs of the fetal rats forebrain in vitro, the methods inprimary and subculture cultivation and the identification of the NSCs. TheNSCs characteristics, the mechanisms of differentiation and the conditionsof induction were observed. Furthermove, we explored survival, migrationand differentiation of NSCs transplanted into rat cerebral contusion focus.
Part One: A study of cultivation and differentiation of fetal ratsforebrain NSCs in vitro
The experiment was completed at Experimental Center of Hebei NorthUniversity from November 2008 to October 2009. Cleanliness level,embryonic 16d and pregnant Wistar rats were provided by the AnimalLaboratory of the Medical Sciences Institute of the Chinese Academy.Under aseptic conditions, we obtained fetal rats. NSCs were isolated fromfetal rats forebrain to prepare single cell suspension. The cell density of 1×106·ml-1 and 1×108·ml-1 was inoculated in culture bottle containing theDMEM/F12 medium and N2 additives, with a final concentration of20ng·ml-1 epidermal growth factor (EGF) and 10ng·ml-1 basic fibroblastgrowth factor (bFGF) to stimulate clonal growth. The inducingdifferentiation experiment included two parts. A. The suspensioncultivation test was divided into three groups: the volume fraction of 10%fetal bovine serum (FBS) +20ng·ml-1 EGF +10ng·ml-1 bFGF, 10%FBS,20%FBS. In these conditions, NSCs were induced differentiation. B. Theadherent cultivation test was divided into three groups: deckingpoly-L-lysine group, decking gelatin group and non-decking group. NSCswere stimulated differentiation with the volume fraction of 20% FBS.Nestin, glial fibrillary acid protein (GFAP), neuron-specific enolase (NSE)and microtubule-associated protein 2 (MAP-2) were detected withimmunocytochemistry and indirect immunofluorescence staining. As aresult, NSCs were successfully isolated from fetal rats forebrain, and thenand showed nestin-positive antigen. In inducing conditions of thesuspension cultivation test, the promoting differentiation effect of 20% FBSgroup was slightly stronger than 10% FBS group (P>0.05); the promotingdifferentiation effect of 10% FBS group was stronger than 10% FBS+20ng·ml-1 EGF +10ng·ml-1 bFGF group (P<0.05). NSCs weredifferentiated into neurons and glial cells. In the adherent cultivation test,the anchorage-dependent ability of decking poly-L-lysine group anddecking gelatin group to differentiate into neurons and glial cells wasstronger than not-decking group (P<0.01). Decking poly-L-lysine groupwas a little stronger than decking gelatin group (P>0.05). GFAP, NSE andMAP-2 were detected positive by immunocytochemistry and indirectimmunofluorescence staining.
Conclusion:NSCs were rich in the fetal rats forebrain, and they couldbe obtained in vitro cultivation. They had the proliferative capacity and theproficiency of multi-differentiation. At the same time, the effect of promoting differentiation was very strong in 20% FBS group. It was notsignificant in 10% FBS + EGF + bFGF group. In the inductingdifferentiation of NSCs, the poly-L-lysine and the gelatin, as the supportivematerial of adherent cultivation, played a role in raising the quantity of celldifferentiation, and NSCs were mostly differentiated into astrocytes.
Part Two: The survival, migration and differentiation of NSCs aroundthe rat cerebral contusion focus region.
The study used the serum-free cultivation technology, plus EGF andbFGF, to stimulate the growth and the proliferation of embryo-derivedNSCs. They were cultivated two passage in vitro, marked by 6μg·ml-15-bromodeoxyuridine (BrdU) and detected by immunocytochemistry andindirect immunofluorescence staining, to confirm the ability ofproliferation. Then we produced an improved model Fenney's free-fallcerebral contusion by Wistar rats. After transplanted 1d, NSCs weretransplanted into the edge of the rat cerebral contusion focus cortex bystereotactic localization. Transplanted NSCs were detected byimmunohistochemical and immunofluorescence double staining method,and the survival, migration and differentiation of them had been observedaround the rat cerebral contusion focus region in the post-transplant 1d, 7d,14d, 21d. Results: The number of BrdU-positive cells around the ratcerebral contusion focus region reduced gradually. BrdU-positive cellsscattered around the cerebral contusion focus and migrated to thesubcortical region. Besides, GFAP-positive cells increased with someregularity around cerebral contusion focus region after injury.
Conclusion: The results of this study suggest that forebrain NSCs afterallogenic transplantation can survive and migrate around the rat's cerebralcontusion focus region, and they shows the characteristics ofmorphological integration with the brain.
1.大鼠胚胎前脑神经干细胞体外培养分化的研究
实验于2008年11月至2009年10月在河北北方学院实验中心完成。清洁级,E 16d的Wistar孕鼠(由中国医学科学院实验动物研究所提供)在无菌条件下取其胚胎,分离出前脑,制备单细胞悬液,以1×106·ml-1和1×108·ml-1分别接种于含N2的DMEM/F12培养瓶中,加入终浓度为20ng·ml-1表皮细胞生长因子(epidermal growth factor, EGF)和10ng·ml-1碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)刺激其克隆生长。诱导分化实验分为悬浮培养实验和爬片培养实验。a悬浮培养实验分为三组:体积分数10%胎牛血清(fetal bovine serum,FBS) +20ng·ml-1 EGF +10ng·ml-1 bFGF,10%FBS,20%FBS,在上述诱导条件下刺激其分化。b爬片培养实验分为三组:多聚赖氨酸铺板组、明胶铺板组和无铺板组,采用体积分数为20% FBS刺激分化。研究采用免疫细胞化学和间接免疫细胞荧光染色法检测NSCs巢蛋白(nestin)、胶质纤维酸性蛋白(glial fibrillary acid protein, GFAP)、神经元特异性烯醇化酶(neuron-specific enolase, NSE)和微管相关蛋白2(microtubule-associated protein 2, MAP-2)。结果成功从胎鼠前脑分离出神经干细胞,巢蛋白表达阳性。a悬浮培养实验在不同诱导条件下,20%FBS组促分化作用略强于10% FBS组(P>0.05)。10% FBS组促分化作用强于10%FBS +20ng·ml-1 EGF +10ng·ml-1 bFGF组(P<0.05)。NSCs分化为神经元及神经胶质细胞。b爬片培养实验中,多聚赖氨酸铺板组和明胶铺板组贴壁后分化为神经元及神经胶质细胞的能力强于无铺板组(P<0.01)。多聚赖氨酸铺板组略强于明胶铺板组(P>0.05)。神经谱系标记物中的GFAP,NSE和MAP-2免疫细胞化学及间接免疫细胞荧光染色均阳性。结论:大鼠胚胎前脑NSCs可以通过体外培养获得,且具有增殖能力和多向分化潜能。20%血清组NSCs分化显著,10%FBS+EGF +bFGF组NSCs分化不显著。多聚赖氨酸和明胶作为细胞贴壁支持物可以提高分化细胞数量,而且多为星形胶质细胞。
2.神经干细胞在大鼠脑挫伤灶周边区成活和迁移
本研究利用体外无血清培养技术,加EGF和bFGF刺激大鼠胚胎源性前脑NSCs克隆增殖。传1代后,NSCs在培养过程中加入终浓度为6μg·ml-1 5-溴脱氧尿嘧啶核苷(5-bromodeoxyuridine, BrdU)。标记BrdU的NSCs,用免疫细胞化学和间接免疫细胞荧光染色检测其增殖能力。研究采用改进的Fenney’s自由落体脑挫伤模型,于致伤1d后,将NSCs用立体定位法移植到鼠脑挫伤灶边缘皮层内,免疫组化及免疫荧光双染法观察移植后1,7,14,21d NSCs在挫伤灶周边成活,迁移和分化情况。结果,移植后1,7,14,21d,损伤灶BrdU阳性细胞数目逐渐减少;BrdU阳性细胞在损伤灶周边散在分布,并向损伤皮层下迁移。损伤后灶区GFAP表达上调,并在时间上具有一定规律性。结论:本研究结果显示,大鼠胚胎前脑获得的NSCs在异体移植后,能够在受体大鼠脑损伤灶周边区存活和迁移;形态学上,其显示出与脑组织整合的特点。
The death of a large quantities of cells and the serious neuraldysfunction syndrome are usually caused by the cerebral contusion.Generally speaking, the central nervous system lacks the ability ofregeneration. The study concludes that the key to treatment of braincontusion is to transplant the exogenous cells for supplying or replacingloss of the nerve cells inorder to promote the survival of the nerve cells andthe growth of axons and reconstruct the functional synapse. Thetransplantation of neural stem cells (NSCs) has good application prospectsto treat brain contusion. In this study, we investigated the methods ofisolating the NSCs of the fetal rats forebrain in vitro, the methods inprimary and subculture cultivation and the identification of the NSCs. TheNSCs characteristics, the mechanisms of differentiation and the conditionsof induction were observed. Furthermove, we explored survival, migrationand differentiation of NSCs transplanted into rat cerebral contusion focus.
Part One: A study of cultivation and differentiation of fetal ratsforebrain NSCs in vitro
The experiment was completed at Experimental Center of Hebei NorthUniversity from November 2008 to October 2009. Cleanliness level,embryonic 16d and pregnant Wistar rats were provided by the AnimalLaboratory of the Medical Sciences Institute of the Chinese Academy.Under aseptic conditions, we obtained fetal rats. NSCs were isolated fromfetal rats forebrain to prepare single cell suspension. The cell density of 1×106·ml-1 and 1×108·ml-1 was inoculated in culture bottle containing theDMEM/F12 medium and N2 additives, with a final concentration of20ng·ml-1 epidermal growth factor (EGF) and 10ng·ml-1 basic fibroblastgrowth factor (bFGF) to stimulate clonal growth. The inducingdifferentiation experiment included two parts. A. The suspensioncultivation test was divided into three groups: the volume fraction of 10%fetal bovine serum (FBS) +20ng·ml-1 EGF +10ng·ml-1 bFGF, 10%FBS,20%FBS. In these conditions, NSCs were induced differentiation. B. Theadherent cultivation test was divided into three groups: deckingpoly-L-lysine group, decking gelatin group and non-decking group. NSCswere stimulated differentiation with the volume fraction of 20% FBS.Nestin, glial fibrillary acid protein (GFAP), neuron-specific enolase (NSE)and microtubule-associated protein 2 (MAP-2) were detected withimmunocytochemistry and indirect immunofluorescence staining. As aresult, NSCs were successfully isolated from fetal rats forebrain, and thenand showed nestin-positive antigen. In inducing conditions of thesuspension cultivation test, the promoting differentiation effect of 20% FBSgroup was slightly stronger than 10% FBS group (P>0.05); the promotingdifferentiation effect of 10% FBS group was stronger than 10% FBS+20ng·ml-1 EGF +10ng·ml-1 bFGF group (P<0.05). NSCs weredifferentiated into neurons and glial cells. In the adherent cultivation test,the anchorage-dependent ability of decking poly-L-lysine group anddecking gelatin group to differentiate into neurons and glial cells wasstronger than not-decking group (P<0.01). Decking poly-L-lysine groupwas a little stronger than decking gelatin group (P>0.05). GFAP, NSE andMAP-2 were detected positive by immunocytochemistry and indirectimmunofluorescence staining.
Conclusion:NSCs were rich in the fetal rats forebrain, and they couldbe obtained in vitro cultivation. They had the proliferative capacity and theproficiency of multi-differentiation. At the same time, the effect of promoting differentiation was very strong in 20% FBS group. It was notsignificant in 10% FBS + EGF + bFGF group. In the inductingdifferentiation of NSCs, the poly-L-lysine and the gelatin, as the supportivematerial of adherent cultivation, played a role in raising the quantity of celldifferentiation, and NSCs were mostly differentiated into astrocytes.
Part Two: The survival, migration and differentiation of NSCs aroundthe rat cerebral contusion focus region.
The study used the serum-free cultivation technology, plus EGF andbFGF, to stimulate the growth and the proliferation of embryo-derivedNSCs. They were cultivated two passage in vitro, marked by 6μg·ml-15-bromodeoxyuridine (BrdU) and detected by immunocytochemistry andindirect immunofluorescence staining, to confirm the ability ofproliferation. Then we produced an improved model Fenney's free-fallcerebral contusion by Wistar rats. After transplanted 1d, NSCs weretransplanted into the edge of the rat cerebral contusion focus cortex bystereotactic localization. Transplanted NSCs were detected byimmunohistochemical and immunofluorescence double staining method,and the survival, migration and differentiation of them had been observedaround the rat cerebral contusion focus region in the post-transplant 1d, 7d,14d, 21d. Results: The number of BrdU-positive cells around the ratcerebral contusion focus region reduced gradually. BrdU-positive cellsscattered around the cerebral contusion focus and migrated to thesubcortical region. Besides, GFAP-positive cells increased with someregularity around cerebral contusion focus region after injury.
Conclusion: The results of this study suggest that forebrain NSCs afterallogenic transplantation can survive and migrate around the rat's cerebralcontusion focus region, and they shows the characteristics ofmorphological integration with the brain.
引文
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