骨髓基质干细胞移植治疗大鼠脊髓损伤及其体外向神经细胞诱导分化的实验研究
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摘要
脊髓损伤(spinal cord injury,SCI)是一种严重威胁人类生命和健康的常见疾病。近年来,由交通事故、高处坠落和运动损伤等因素导致的SCI患者数量日渐增多,多发生于青壮年人群中。大部分患者因SCI而导致身体的终生残疾,给社会和家庭带来沉重的经济负担。目前人类还没有找到针对SCI治疗的行之有效的方法,现阶段在临床上常用的方法是在损伤后早期静脉使用甲基强的松龙和外科手术治疗,但是治疗效果不佳。因此,寻找针对SCI有效的治疗方法,确实能够在SCI后可以有效的减轻患者脊髓损伤程度,甚至促进损伤神经轴突的再生,重建脊髓的功能,一直是神经科学领域的研究热点。近年来随着组织工程技术研究的兴起和对SCI后的病理改变、机理认识不断深入的基础上,科学家们提出采用组织工程技术治疗脊髓损伤的思路,为脊髓损伤后的救治提供了新的方法。骨髓基质干细胞(bone marrow stromal cells, BMSCs)是骨髓造血系统中的非造血干细胞,具有容易获取、体外增殖快和具有多向分化潜能的特点,成为组织工程研究领域的种子细胞之一。
本实验研究包括:1.在成年大鼠脊髓损伤区内移植BMSCs,观察BMSCs对脊髓损伤区神经纤维的保护和再生修复促进作用。2.体外观察嗅鞘细胞条件培养液对BMSCs的诱导分化作用。
实验一BMSCs在骨髓造血过程中为血细胞的生成和分化提供适宜的微环境。研究表明BMSCs在体外培养过程中,具有很强的增殖能力和多向分化潜能,在体外特定的诱导条件下不仅可以分化为骨、软骨和韧带等多种细胞,也可以向神经系统细胞分化。此外,BMSCs在生长和增殖过程中可以分泌一些营养因子,如脑源性神经生长因子(BDNF)、血管内皮生长因子(VEGF)等。SCI后脊髓损伤区将出现组织结构破坏、细胞凋亡坏死,损伤区囊性空洞形成等一系列复杂的病理生理变化。因此我们尝试利用BMSCs的生物学特性,移植入损伤区内,替代脊髓坏死凋亡的细胞,来达到对损伤区脊髓神经纤维的保护和再生修复促进作用。
采用细胞贴壁培养法获得同种异体BMSCs,Hoechst标记后,采用微量注射器移植入脊髓挤压损伤大鼠的损伤间隙内。于2、4周后取材制作切片,HE和免疫组织化学染色,采用BBB评分观察运动功能恢复情况。结果证实:BMSCs可以在脊髓损伤区间内存活、迁移和向神经细胞分化,能够有效的阻止脊髓损伤囊性空洞的形成,对SCI后损伤区神经纤维有保护作用,甚至可以促进神经纤维的再生和修复,但BBB评分结果各组之间在各时间点没有统计学差别。因此认为BMSCs可以参与大鼠脊髓损伤后的修复重建过程,但还不足以促进其运动功能恢复。
实验二嗅神经鞘细胞(olfactory ensheathing cells,OECs)是一种仅存在于嗅神经系统的细胞,能终生产生维持神经元存活和轴突延长的神经营养因子及神经轴突促进因子,包括NGF、BDNF、GDNF等多种神经营养因子和一些表面粘附分子。而研究证实BMSCs具有多向分化潜能,因此,我们尝试利用OECs在培养过程中分泌的神经营养因子和表面粘附分子,来诱导BMSCs向神经细胞分化,为接下来研究诱导后的BMSCs与仅纯化的BMSCs移植,在SCI损伤修复中有无差异提供实验理论依据。
选择生长状态良好的OECs,取其培养液,无菌条件下离心后,收集上清液即为OECs条件培养液。将已纯化传代培养第3代的BMSCs,按103～104/cm2接种于24孔培养板中,分为两组:A组为条件培养组,加入OECs条件培养液共培养,B组为空白对照组,加入原BMSCs培养液。诱导后观察BMSCs的形态学变化和免疫组织化学染色鉴定。结果A组BMSCs在诱导后出现胞体缩小,可见突起出芽生长,突起细长。B组细胞仍呈长梭形,无明显形态学变化。免疫组织化学染色A组呈神经细胞标志物阳性,MAP-2和GFAP阳性细胞的比率分别为55.2%和23.4%。因此证实嗅鞘细胞条件培养液可以诱导BMSCs向神经系统细胞分化。
本实验成功的获得了大鼠的骨髓基质干细胞,将其应用于大鼠脊髓挤压损伤的基础治疗研究和体外向神经系统细胞诱导分化的实验研究。成功的观察到BMSCs,在大鼠体内参与脊髓损伤后神经纤维的修复重建过程及其在体外向神经系统细胞的诱导分化过程。为将来临床上应用BMSCs治疗脊髓损伤及其它神经系统疾病提供了理论依据。
Spinal cord injury (SCI) is a kind of serious disease that can threaten man- kind life and healthy. For the past few years, the patients with SCI to increase gradually by traffic accident, high crash and sports injury. And the incidence is usually high in the youth and the adult. Many of the SCI people result in perma- nent disability, and bring about a huge burden to their family and the society. But nowadays there are no effective and useful methods for SCI in clinical, except for the high-dose intravenous administration of methylprednisolone and surgical in- tervention. Therefore, finding a way to effectively reduce the lesion and even to make the injured axonal regenerate has been being a hot spot in neurobiology re- search all the time. With the development of tissue engineering and the pathology, mechanism cognition of SCI, scientists are raising a new way to cure SCI by tissue engineering. Bone marrow stromal cells (BMSCs) are not haemopoietic stem cells in hematopoietic system. They are one of ideal kind of seed cells of tissue engineering for their easy to obtain, proliferate soon and multi-directional differentiation. In our experiments, we investigated the effect of BMSCs when transplanted into rats after SCI on morphologically for nerve fiber and to explore the differentiation of BMSCs by olfactory ensheathing cells (OECs) conditioned medium.
In our first experiment, we used the spinal cord compressive injury model. We transplanted BMSCs into the interspace of the spinal cord lesion site to in- vestigate the promoting effects on axonal regeneration in adult rats. BMSCs can provide eligible microenvironment in the process of haemocytes’formation and differentiation. BMSCs can not only differentiate into bone, cartilage, tendon and ligament under specified inductions, but also can differentiate into neural cells and dendritic cells. During the process of growth and proliferation, BMSCs can secrete some nutrition factors, such as brain-derived neurotrophic factor (BDNF) and vascular endothelial cell growth factor(VEGF). After SCI the spinal cord results in a series of tissue disorganization, apoptosis, necrosis and cavitation formation. We tried to protect nerve fiber by transplanting BMSCs to SCI rats. BMSCs were obtained by adherent culture, and then they were labeled by hoechst and transplanted into interspace of the spinal cord lesion site by microinjector. Four weeks later, we found that BMSCs can survival, migration and differentiate into neural cells. They also can effectively interrupt the formation of cavitation and provide protection for axonal survival and regeneration, but there are no difference (P>0.05) among three groups by BBB scale.
In our second experiment, we explored the differentiation of BMSCs by OECs conditioned medium. OECs can secrete some nutrition factors, such as nerve growth factor(NGF), BDNF and glial cell derived neurotrophic factor (GDNF) in their lifetime. Other nutrition factors, such as surface adhesion secreted at the same time. OECs only existed in olfactory nerve system. BMSCs have the ability to multi-directional differentiation. So OECs conditioned medium which contain the nutrition factors maybe have the ability to induce BMSCs differentiate into neural cells. In order to study the different results between the induced and non-induced BMSCs when transplanted into the spinal cord injury rats. We obtained the purified OECs by selective culture medium and cultured in the fibemectin coated plate. After 9~12 days culture, we collected and made OECs conditioned medium, then it being co-cultured with purified BMSCs which are 3th generation. The differentiation of BMSCs were observed under inverted microscope and were identified by immumofluorescence method. After 72h’s induction, we found that BMSCs can acquire neuronal morphology and were being immunopositive for neural markers.
On the whole, our experiment found that it is useful to transplant BMSCs for axon regeneration and prevent cavity formation after SCI, and also found that neu -ronal differentiation of BMSCs can be obtained by OECs conditioned medium in vitro.
本实验研究包括:1.在成年大鼠脊髓损伤区内移植BMSCs,观察BMSCs对脊髓损伤区神经纤维的保护和再生修复促进作用。2.体外观察嗅鞘细胞条件培养液对BMSCs的诱导分化作用。
实验一BMSCs在骨髓造血过程中为血细胞的生成和分化提供适宜的微环境。研究表明BMSCs在体外培养过程中,具有很强的增殖能力和多向分化潜能,在体外特定的诱导条件下不仅可以分化为骨、软骨和韧带等多种细胞,也可以向神经系统细胞分化。此外,BMSCs在生长和增殖过程中可以分泌一些营养因子,如脑源性神经生长因子(BDNF)、血管内皮生长因子(VEGF)等。SCI后脊髓损伤区将出现组织结构破坏、细胞凋亡坏死,损伤区囊性空洞形成等一系列复杂的病理生理变化。因此我们尝试利用BMSCs的生物学特性,移植入损伤区内,替代脊髓坏死凋亡的细胞,来达到对损伤区脊髓神经纤维的保护和再生修复促进作用。
采用细胞贴壁培养法获得同种异体BMSCs,Hoechst标记后,采用微量注射器移植入脊髓挤压损伤大鼠的损伤间隙内。于2、4周后取材制作切片,HE和免疫组织化学染色,采用BBB评分观察运动功能恢复情况。结果证实:BMSCs可以在脊髓损伤区间内存活、迁移和向神经细胞分化,能够有效的阻止脊髓损伤囊性空洞的形成,对SCI后损伤区神经纤维有保护作用,甚至可以促进神经纤维的再生和修复,但BBB评分结果各组之间在各时间点没有统计学差别。因此认为BMSCs可以参与大鼠脊髓损伤后的修复重建过程,但还不足以促进其运动功能恢复。
实验二嗅神经鞘细胞(olfactory ensheathing cells,OECs)是一种仅存在于嗅神经系统的细胞,能终生产生维持神经元存活和轴突延长的神经营养因子及神经轴突促进因子,包括NGF、BDNF、GDNF等多种神经营养因子和一些表面粘附分子。而研究证实BMSCs具有多向分化潜能,因此,我们尝试利用OECs在培养过程中分泌的神经营养因子和表面粘附分子,来诱导BMSCs向神经细胞分化,为接下来研究诱导后的BMSCs与仅纯化的BMSCs移植,在SCI损伤修复中有无差异提供实验理论依据。
选择生长状态良好的OECs,取其培养液,无菌条件下离心后,收集上清液即为OECs条件培养液。将已纯化传代培养第3代的BMSCs,按103～104/cm2接种于24孔培养板中,分为两组:A组为条件培养组,加入OECs条件培养液共培养,B组为空白对照组,加入原BMSCs培养液。诱导后观察BMSCs的形态学变化和免疫组织化学染色鉴定。结果A组BMSCs在诱导后出现胞体缩小,可见突起出芽生长,突起细长。B组细胞仍呈长梭形,无明显形态学变化。免疫组织化学染色A组呈神经细胞标志物阳性,MAP-2和GFAP阳性细胞的比率分别为55.2%和23.4%。因此证实嗅鞘细胞条件培养液可以诱导BMSCs向神经系统细胞分化。
本实验成功的获得了大鼠的骨髓基质干细胞,将其应用于大鼠脊髓挤压损伤的基础治疗研究和体外向神经系统细胞诱导分化的实验研究。成功的观察到BMSCs,在大鼠体内参与脊髓损伤后神经纤维的修复重建过程及其在体外向神经系统细胞的诱导分化过程。为将来临床上应用BMSCs治疗脊髓损伤及其它神经系统疾病提供了理论依据。
Spinal cord injury (SCI) is a kind of serious disease that can threaten man- kind life and healthy. For the past few years, the patients with SCI to increase gradually by traffic accident, high crash and sports injury. And the incidence is usually high in the youth and the adult. Many of the SCI people result in perma- nent disability, and bring about a huge burden to their family and the society. But nowadays there are no effective and useful methods for SCI in clinical, except for the high-dose intravenous administration of methylprednisolone and surgical in- tervention. Therefore, finding a way to effectively reduce the lesion and even to make the injured axonal regenerate has been being a hot spot in neurobiology re- search all the time. With the development of tissue engineering and the pathology, mechanism cognition of SCI, scientists are raising a new way to cure SCI by tissue engineering. Bone marrow stromal cells (BMSCs) are not haemopoietic stem cells in hematopoietic system. They are one of ideal kind of seed cells of tissue engineering for their easy to obtain, proliferate soon and multi-directional differentiation. In our experiments, we investigated the effect of BMSCs when transplanted into rats after SCI on morphologically for nerve fiber and to explore the differentiation of BMSCs by olfactory ensheathing cells (OECs) conditioned medium.
In our first experiment, we used the spinal cord compressive injury model. We transplanted BMSCs into the interspace of the spinal cord lesion site to in- vestigate the promoting effects on axonal regeneration in adult rats. BMSCs can provide eligible microenvironment in the process of haemocytes’formation and differentiation. BMSCs can not only differentiate into bone, cartilage, tendon and ligament under specified inductions, but also can differentiate into neural cells and dendritic cells. During the process of growth and proliferation, BMSCs can secrete some nutrition factors, such as brain-derived neurotrophic factor (BDNF) and vascular endothelial cell growth factor(VEGF). After SCI the spinal cord results in a series of tissue disorganization, apoptosis, necrosis and cavitation formation. We tried to protect nerve fiber by transplanting BMSCs to SCI rats. BMSCs were obtained by adherent culture, and then they were labeled by hoechst and transplanted into interspace of the spinal cord lesion site by microinjector. Four weeks later, we found that BMSCs can survival, migration and differentiate into neural cells. They also can effectively interrupt the formation of cavitation and provide protection for axonal survival and regeneration, but there are no difference (P>0.05) among three groups by BBB scale.
In our second experiment, we explored the differentiation of BMSCs by OECs conditioned medium. OECs can secrete some nutrition factors, such as nerve growth factor(NGF), BDNF and glial cell derived neurotrophic factor (GDNF) in their lifetime. Other nutrition factors, such as surface adhesion secreted at the same time. OECs only existed in olfactory nerve system. BMSCs have the ability to multi-directional differentiation. So OECs conditioned medium which contain the nutrition factors maybe have the ability to induce BMSCs differentiate into neural cells. In order to study the different results between the induced and non-induced BMSCs when transplanted into the spinal cord injury rats. We obtained the purified OECs by selective culture medium and cultured in the fibemectin coated plate. After 9~12 days culture, we collected and made OECs conditioned medium, then it being co-cultured with purified BMSCs which are 3th generation. The differentiation of BMSCs were observed under inverted microscope and were identified by immumofluorescence method. After 72h’s induction, we found that BMSCs can acquire neuronal morphology and were being immunopositive for neural markers.
On the whole, our experiment found that it is useful to transplant BMSCs for axon regeneration and prevent cavity formation after SCI, and also found that neu -ronal differentiation of BMSCs can be obtained by OECs conditioned medium in vitro.
引文
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