骨髓基质细胞定向分化为神经干细胞及自体移植治疗中脑损伤研究
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摘要
研究背景
中脑是脑干的重要组成部分之一，结构复杂，内有许多核团和纤维束。中脑损伤是脑干损伤中的一种特殊类型，大约50％～70％的脑干损伤合并有不同程度的中脑损伤。脑干损伤(primary brain stem injury)是一种致命性损伤，死亡率超过44.1％～62.5％，存活者重残率高达60％以上，其中植物生存者超过1／3。如何使包括创伤性脑干损伤、脑中风等疾病引起的持续性植物状态(PVS)以及创伤性脊髓损伤所致截瘫等在内的一系列中枢神经系统疾病得到妥善治疗，使其造成的后果和并发症减少到最低程度，一直是神经内外科医生和神经科学工作者奋斗的目标。
近年来的研究成果表明，神经系统损伤具有修复的潜能，因为发现了成体神经组织内仍然存在有可分裂增殖的神经干细胞。因此，对内源性神经干细胞进行人工干预调控，促使其继续增殖和分化；或进行神经干细胞移植，以促进中枢神经系统损伤的修复等，是目前治疗神经系统损伤及退行性疾病的最新和最有希望的方法。骨髓基质细胞多潜能性的发现和向神经细胞方向定向分化的成功，为神经干细胞的来源增加了一个新的途径，也为解决“自体源神经干细胞”移植方案中细胞数量不足的问题提供了很好的思路。
通过建立体外、体内实验模型等手段，对骨髓基质细胞的体外扩增、定向分化方案，及移植细胞在脑内的存活、迁移、整合情况等方面进行研究探索，有利于自体干细胞移植技术的规范化开展和推广应用。
第一部分
目的
1．建立骨髓基质细胞快速扩增方案，为骨髓基质细胞作为“种子细胞”在医学和组织工程学领域的广泛应用创造条件。
第一军医大学博士研究生学位论文
2．建立骨髓基质细胞向神经干细胞定向分化的体外模型，探索骨髓
基质细胞向神经干细胞定向分化的可行性，为“骨髓源神经干细胞”的
临床应用提供理论依据。
方法
1．无菌穿刺抽取骨髓，密度梯度离心，粘附法提取骨髓基质细胞。
然后在 D－MEM” 培养基（lml／孔）内加 bFGF（10ng／inlX EGF
（ ong／ml），进行扩增培养。待 ZWk左右，细胞接合后，胰蛋白酶消化。
收集细胞；配成 10‘个活细胞／ml的浓度（工作浓度）备用，或进行 50
倍稀释后继续扩增培养。
2．将工作浓度细胞接种于 96孔板，分别将 bFGF、EGF按不同终
浓度（单独或联合）加人培养板内。孵育6h后2％戊二醛固定，记数、
比较不同浓度bFGF、EGF对BMSCs粘附特性的影响。
3．用 a．MTT法和 b．细胞集落记数法检测分析不同终浓度（单独或
联合）bFGF、EGF分别在实验第 IX 24小时、3 X 24小时、5 X 24小时、
7x犯小时4个时间点对＊＊肌s的存活和增殖能力的影响。
4．SABC法鉴定不同浓度RA、BDNF、GDNF（单独或联合）情况
下，第 IX 24小时、3 X 24小时、7 X 24小时三个时间点细胞分化情况。
一抗分别为：鼠抗－巢蛋白（Nestin）、兔抗－神经元特异稀醇化酶（NSE）。
鼠抗－胶质纤维酸性蛋白（GFAP人 二抗为羊抗兔／或鼠 IgG。DAB显色。
结果：
1．骨髓细胞原代培养6h内有成纤维细胞样细胞贴壁生长，48h去
除非粘附细胞后，粘附细胞有大、中、小三种形态；加入bFGF、EGF
扩增培养24h后见细胞分裂相；培养72h有克隆形成：至2＿k时，大型
的成纤维样细胞铺爬满培养板底部。
二．传代后观察可见细胞均匀散在分布，约 10－14d后细胞接合，大
部分细胞的形态属成纤维样（同前），仍有小型和中型细胞。
3．bFGF、EGF对BMSCS粘附特性均有促进作用；bFGF对BMSCS
的粘附促进作用优于EGF。二者联合应用优于单独使用。
4．bFGF、EGF对BMSCS存活及增殖均有促进作用；bFGF的促进
存活作用优于EGF。EGF的促进增殖作用优于bFGF。二者联合应用优
于单独使用。
3
第一军医大学博士研究生学位论文
5．RA、BDNF、GDNF均可促进BMSCS向神经干细胞方向分化。
三者连合应用优于单独使用。RA、BDNF、GDNF不但可以促进BMSCS
向神经干细胞方向分化，而且可以促进神经干细胞向成熟神经细胞方向
分化。加入 RA、BDNF及 GDNF进行诱导分化培养第 10d时即可见到
有神经细胞（神经元、胶质细胞）样细胞形成。
结论
1．骨髓基质细胞形态多样，至少有H种形态。不但在适当条件下可
以快速扩增，而且在适当条件下可以向神经细胞方向分化。
2．bFGF、EGF对BMSCS的粘附、存活和增殖均有促进作用。bFGF
Back Ground
The midbrain is a main part of brain stem. Its superior part is diencephalon, and lower part is pons. The mesencephalon is complicatedly composed of much nucleus and fibrous bands. Midbrain injury is a special type of brain stem injuries, which is often accompanied injuries of other brain parts. About 50%~70% of brain stem injuries accompanied various midbrain injuries. Primary brain stem injury is a very seriously fatal injuries. Its mortality is as high as 44.1%~62.5%, and its disability rate of survivor reach to 60%, and vegetable state patients surpass 1/3 of them. How to make the diseases of central nervous system, include PVS, paraplegia etc., obtain appropriate therapy, to recover well, and to make its outcomes, sequelaes and complications decreasing to minimum level, has been constantly the target of all neurologists, surgeons and neuroscientists.
The investigations of recent years manifest that nervous system have the auto prosthetic potential. It has been discovered that in adult nervous system remained little neural stem cells, which could proliferate in some situations. Therefore, the latest and hopeful methods, for the treatment of trauma and regression diseases of nervous system, are through artificial intervention and controlling of the endogenous neural stem cells re-proliferation and differentiation, or proceeded to the transplantation with neural stem cells to promote the plerosis of the central nervous system injuries.
The discovery of the multi-potential of bone marrow stromal cells and the successful oriented differentiation towards neural cells, which increase a new source of neural stem cells, and also provide a new idea for resolving the problems of the quantity and resource of neural stem cells in the auto-transplantation project.
By means of in vitro and in vivo model, to investigate the projects of the purification, amplification and differentiation and the situations of the survival, migration, integration of bone marrow stromal cells, which is useful
for the standardized develop and apply of the "neural stem cells derived from bone marrow stromal cells" in clinic.
Part One
Objective
1. To establish the method of high-speed bone marrow stromal cells amplification in vitro, for the extensive applying of bone marrow stromal cells serving as "seed cells" in the fields of medical science and tissues engineering.
2. To set up model of bone marrow stromal cells orientation differentiation into neural stem cells in vitro, and to quest the feasibility of differentiating into neural stem cells from bone marrow stromal cells, and to provide theory base for the clinical application of neural stem cells derived from bone marrow stromal cells.
Methods
1. Isolation and amplification culture of bone marrow stromal cells Acquired bone marrow under aseptic condition; Density gradient
centrifugation; Adherence Technique to acquire bone marrow stromal cells; Then added bFGF(10ng/ml), EGF(10ng/ml) into D-MEM/F12 culture medium(l ml/hole); Two weeks later, trypsin method digested, and collected cells. The harvested cells was produced into 104 alive cells/ml density for later using; Then the cells was diluted 50 times with D-MEM/F12 and continued to amplification.
2. Influence of bFGF and EOF on adhesiveness of BMSCs
Added different end concentration bFGF and EGF alone or associatively, into culture plate after work cells had been seeded into 96 hole tissue-culture plate and hatched for 6 hours; Then BMSCs cells were fixed with 2% glutaraldehyde and counted. There is difference between the effects of bFGF and EGF on BMSCs adhesiveness.
3. Effect of bFGF and EGF on survival and proliferation of BMSCs Determined the effects of bFGF and EGF on the level of survival and
proliferation of BMSCs by routine MTT and clone count methods at 1@24,3 @24h, 5@24h, 7@24h.
4. The influence of RA, BDNF and GDNF on oriented differentiation of BMSCs
SABC Technique was used to identify the cells state of differentiation under various concentrations of RA, BDNF,
中脑是脑干的重要组成部分之一，结构复杂，内有许多核团和纤维束。中脑损伤是脑干损伤中的一种特殊类型，大约50％～70％的脑干损伤合并有不同程度的中脑损伤。脑干损伤(primary brain stem injury)是一种致命性损伤，死亡率超过44.1％～62.5％，存活者重残率高达60％以上，其中植物生存者超过1／3。如何使包括创伤性脑干损伤、脑中风等疾病引起的持续性植物状态(PVS)以及创伤性脊髓损伤所致截瘫等在内的一系列中枢神经系统疾病得到妥善治疗，使其造成的后果和并发症减少到最低程度，一直是神经内外科医生和神经科学工作者奋斗的目标。
近年来的研究成果表明，神经系统损伤具有修复的潜能，因为发现了成体神经组织内仍然存在有可分裂增殖的神经干细胞。因此，对内源性神经干细胞进行人工干预调控，促使其继续增殖和分化；或进行神经干细胞移植，以促进中枢神经系统损伤的修复等，是目前治疗神经系统损伤及退行性疾病的最新和最有希望的方法。骨髓基质细胞多潜能性的发现和向神经细胞方向定向分化的成功，为神经干细胞的来源增加了一个新的途径，也为解决“自体源神经干细胞”移植方案中细胞数量不足的问题提供了很好的思路。
通过建立体外、体内实验模型等手段，对骨髓基质细胞的体外扩增、定向分化方案，及移植细胞在脑内的存活、迁移、整合情况等方面进行研究探索，有利于自体干细胞移植技术的规范化开展和推广应用。
第一部分
目的
1．建立骨髓基质细胞快速扩增方案，为骨髓基质细胞作为“种子细胞”在医学和组织工程学领域的广泛应用创造条件。
第一军医大学博士研究生学位论文
2．建立骨髓基质细胞向神经干细胞定向分化的体外模型，探索骨髓
基质细胞向神经干细胞定向分化的可行性，为“骨髓源神经干细胞”的
临床应用提供理论依据。
方法
1．无菌穿刺抽取骨髓，密度梯度离心，粘附法提取骨髓基质细胞。
然后在 D－MEM” 培养基（lml／孔）内加 bFGF（10ng／inlX EGF
（ ong／ml），进行扩增培养。待 ZWk左右，细胞接合后，胰蛋白酶消化。
收集细胞；配成 10‘个活细胞／ml的浓度（工作浓度）备用，或进行 50
倍稀释后继续扩增培养。
2．将工作浓度细胞接种于 96孔板，分别将 bFGF、EGF按不同终
浓度（单独或联合）加人培养板内。孵育6h后2％戊二醛固定，记数、
比较不同浓度bFGF、EGF对BMSCs粘附特性的影响。
3．用 a．MTT法和 b．细胞集落记数法检测分析不同终浓度（单独或
联合）bFGF、EGF分别在实验第 IX 24小时、3 X 24小时、5 X 24小时、
7x犯小时4个时间点对＊＊肌s的存活和增殖能力的影响。
4．SABC法鉴定不同浓度RA、BDNF、GDNF（单独或联合）情况
下，第 IX 24小时、3 X 24小时、7 X 24小时三个时间点细胞分化情况。
一抗分别为：鼠抗－巢蛋白（Nestin）、兔抗－神经元特异稀醇化酶（NSE）。
鼠抗－胶质纤维酸性蛋白（GFAP人 二抗为羊抗兔／或鼠 IgG。DAB显色。
结果：
1．骨髓细胞原代培养6h内有成纤维细胞样细胞贴壁生长，48h去
除非粘附细胞后，粘附细胞有大、中、小三种形态；加入bFGF、EGF
扩增培养24h后见细胞分裂相；培养72h有克隆形成：至2＿k时，大型
的成纤维样细胞铺爬满培养板底部。
二．传代后观察可见细胞均匀散在分布，约 10－14d后细胞接合，大
部分细胞的形态属成纤维样（同前），仍有小型和中型细胞。
3．bFGF、EGF对BMSCS粘附特性均有促进作用；bFGF对BMSCS
的粘附促进作用优于EGF。二者联合应用优于单独使用。
4．bFGF、EGF对BMSCS存活及增殖均有促进作用；bFGF的促进
存活作用优于EGF。EGF的促进增殖作用优于bFGF。二者联合应用优
于单独使用。
3
第一军医大学博士研究生学位论文
5．RA、BDNF、GDNF均可促进BMSCS向神经干细胞方向分化。
三者连合应用优于单独使用。RA、BDNF、GDNF不但可以促进BMSCS
向神经干细胞方向分化，而且可以促进神经干细胞向成熟神经细胞方向
分化。加入 RA、BDNF及 GDNF进行诱导分化培养第 10d时即可见到
有神经细胞（神经元、胶质细胞）样细胞形成。
结论
1．骨髓基质细胞形态多样，至少有H种形态。不但在适当条件下可
以快速扩增，而且在适当条件下可以向神经细胞方向分化。
2．bFGF、EGF对BMSCS的粘附、存活和增殖均有促进作用。bFGF
Back Ground
The midbrain is a main part of brain stem. Its superior part is diencephalon, and lower part is pons. The mesencephalon is complicatedly composed of much nucleus and fibrous bands. Midbrain injury is a special type of brain stem injuries, which is often accompanied injuries of other brain parts. About 50%~70% of brain stem injuries accompanied various midbrain injuries. Primary brain stem injury is a very seriously fatal injuries. Its mortality is as high as 44.1%~62.5%, and its disability rate of survivor reach to 60%, and vegetable state patients surpass 1/3 of them. How to make the diseases of central nervous system, include PVS, paraplegia etc., obtain appropriate therapy, to recover well, and to make its outcomes, sequelaes and complications decreasing to minimum level, has been constantly the target of all neurologists, surgeons and neuroscientists.
The investigations of recent years manifest that nervous system have the auto prosthetic potential. It has been discovered that in adult nervous system remained little neural stem cells, which could proliferate in some situations. Therefore, the latest and hopeful methods, for the treatment of trauma and regression diseases of nervous system, are through artificial intervention and controlling of the endogenous neural stem cells re-proliferation and differentiation, or proceeded to the transplantation with neural stem cells to promote the plerosis of the central nervous system injuries.
The discovery of the multi-potential of bone marrow stromal cells and the successful oriented differentiation towards neural cells, which increase a new source of neural stem cells, and also provide a new idea for resolving the problems of the quantity and resource of neural stem cells in the auto-transplantation project.
By means of in vitro and in vivo model, to investigate the projects of the purification, amplification and differentiation and the situations of the survival, migration, integration of bone marrow stromal cells, which is useful
for the standardized develop and apply of the "neural stem cells derived from bone marrow stromal cells" in clinic.
Part One
Objective
1. To establish the method of high-speed bone marrow stromal cells amplification in vitro, for the extensive applying of bone marrow stromal cells serving as "seed cells" in the fields of medical science and tissues engineering.
2. To set up model of bone marrow stromal cells orientation differentiation into neural stem cells in vitro, and to quest the feasibility of differentiating into neural stem cells from bone marrow stromal cells, and to provide theory base for the clinical application of neural stem cells derived from bone marrow stromal cells.
Methods
1. Isolation and amplification culture of bone marrow stromal cells Acquired bone marrow under aseptic condition; Density gradient
centrifugation; Adherence Technique to acquire bone marrow stromal cells; Then added bFGF(10ng/ml), EGF(10ng/ml) into D-MEM/F12 culture medium(l ml/hole); Two weeks later, trypsin method digested, and collected cells. The harvested cells was produced into 104 alive cells/ml density for later using; Then the cells was diluted 50 times with D-MEM/F12 and continued to amplification.
2. Influence of bFGF and EOF on adhesiveness of BMSCs
Added different end concentration bFGF and EGF alone or associatively, into culture plate after work cells had been seeded into 96 hole tissue-culture plate and hatched for 6 hours; Then BMSCs cells were fixed with 2% glutaraldehyde and counted. There is difference between the effects of bFGF and EGF on BMSCs adhesiveness.
3. Effect of bFGF and EGF on survival and proliferation of BMSCs Determined the effects of bFGF and EGF on the level of survival and
proliferation of BMSCs by routine MTT and clone count methods at 1@24,3 @24h, 5@24h, 7@24h.
4. The influence of RA, BDNF and GDNF on oriented differentiation of BMSCs
SABC Technique was used to identify the cells state of differentiation under various concentrations of RA, BDNF,
引文
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34．李秀森，郭子宽，杨靖清等．骨髓间充质干细胞的生物学特性．解放军医学杂志，25(5)：346-348，2000．
35. Gronthos,Stewart K, Gravess E, et al. Integrin expression and function on human osteoblast-like cslls [J]. J Bone Miner Res, 12:1189-1194, 1997.
36. Boyan BD, Hummert TS, Deand D, etal. Role of material surfaces in regulating bone and cartilage cell response [J]. Biomaterials, 17:137-143, 1996.
37．周序珑，邓漪平．碱性成纤维细胞生长因子对内皮细胞在聚氨酯材料上粘附的影响[J]，生物医学工程学杂志，15(3)：214-217，1998．
38. Sieber-Blum M, Zhang JM J. Growth factor action in neural crest cell diversification. Anat, 191:493-499, 1997.
39. Pincus DW, Keyoung HM, Harrison Restelli C, et al. Fibroblast growth factor-2/brain-derived neurotrophic factor-associated maturation of new neurons generated from adult human subependymal cells. Ann Neurol, 43(5):576-585, 1998.
40. Prockop DJ. Marrow stromalcells as stem cells for nonhematopoietic tissues. Science, 276(5309):71-74, 1997.
41. Eglitis MA, Mezey E. Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc Natl Acad Sci USA, 94:4080-4085, 1997.
42. Li M, Pevny L, Lovell-Badge R, et al. Generation of purified neural precursors from embryonic stem cells by lineage selection. Current biology, 8:971-976, 1998.
43. Pincus DW, Keyoung HM, Harrison Restelli C, et al. Fibroblast growth factor-2/brain-derived neurotrophic factor-associated maturation of new neurons generated from adult human subependymal cells. Ann Neurol, 43(5):576-585,1998.
44. Meng X, Lindahl M, Hyvonen ME, et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science, 287:1489-1493, 2000.
45. Donahue LM, Webster DR, Martinez I, et al: Decreased gap-junctional communication associated with segregation of the neuronal phenotype in the RT4 cell-line family. Cell Tissue Res, 292(1):27-35, 1998.
46. Jacques TS, Relvas JB, Nishimura S, et al: Neural precursor cell chain migration and division are regulated through different betal integrins. Development, 125(16):3167-77, 1998.
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