【作者】 邓均；

【导师】 粟永萍；

【作者基本信息】 第三军医大学 ， 军事预防医学， 2008， 博士

【摘要】 颅脑创伤是战时和平时常发生的严重伤类,救治尤其困难,伤亡和伤残率为严重创伤之首。据报道,全世界每年至少有1千万颅脑创伤病人。在美国,每年有1.4百万颅脑创伤病人,死亡达5万人。我国每年有百万以上的严重颅脑创伤病人,其中死亡约10万人,而存活伤员多并发伤残。可见,颅脑创伤已成为全球严重的公共卫生问题。如何降低死亡率、减少伤残率是创伤医学领域一直攻克的难题,取得了一些进展,特别是通过多种手段综合救治,在一定程度上减轻或控制全身性损害的发生发展,降低了死亡率。但对促进脑组织再生修复、神经功能恢复尚未取得突破性进展,主要原因是神经细胞再生极为困难。以往认为神经元细胞是终末分化的功能细胞,失去增殖能力。脑组织或脊髓组织损伤后的修复,主要由神经胶质细胞增殖修复,缺乏相应神经元再生,这种不完全性再生导致神经功能丧失,形成智残或肢残的严重后果。随着干细胞研究的不断深入,从脑组织中分离出神经干细胞,体外培养有增殖活性,并可向神经元细胞诱导分化,说明神经组织中存在少量具增殖活性的干细胞,可以通过体内途径增强其增殖并诱导向神经元细胞分化,可能有利于损伤组织的结构修复和功能康复。也可以利用神经干细胞具有体外增殖能力的特性,在体外将少量神经干细胞扩增并定向诱导后移植到损伤部位,或静脉移植随血液循环到达损伤部位促进其修复。但在临床应用中则难以实施,因为不可能从自体取材分离神经干细胞,获得异体神经干细胞亦十分困难,胚胎干细胞涉及伦理学问题,加之异体细胞移植的免疫排斥问题、体外扩增后的细胞成瘤风险等问题远未解决。然而,成体干细胞具多向分化潜能的理论认识,为再生医学、创伤医学等研究领域开创了新局面,拓展了新思路,奠定了新途径。不少文献报道,骨髓间充质干细胞(mesenchymal stem cells, MSCs)具有可获得性(自体或异体来源)、体外大量扩增性和体外诱导的多向分化性(成骨分化、成肌分化、成脂分化、成神经细胞分化等),本单位前期研究还证实,将绿色荧光蛋白(GFP)转基因小鼠骨髓细胞静脉移植给受10Gyγ线全身照射的同种小鼠(多脏器损伤模型),移植的GFP阳性骨髓细胞广泛存在于受体小肠、肝、脑、皮肤等多种组织脏器,通过形态学结合免疫组化分析,有的移植细胞已转化为小肠上皮细胞、肝细胞和神经元样细胞,又将原代神经元细胞培养上清液加入MSCs培养体系,发现MSCs可转化表达神经元细胞的分子标志。综合体内体外结果分析认为,MSCs通过血液循环定植于损伤局部,并受局部壁龛微环境因素和体液因素调节分化为局部功能细胞。而生理情况下,MSCs大量存在骨髓,循环血液中仅有少量,如果采取措施动员骨髓中的MSCs进入血液循环,使定植于损伤局部的MSCs数量增加,并在微环境因素调节下分化为功能细胞,必将有利于创伤(包括颅脑创伤)组织修复和功能恢复。利用干细胞的生物学特性,切入创伤救治研究,探索促进创伤组织修复的实用新途径并阐明相关机理,对揭示干细胞分化受微环境调控机制和推进临床创伤救治具有重要意义。因此,本课题第一部分建立从大鼠、人等不同种属外周血分离培养MSCs的方法,以成纤维细胞集落形成单位(colony forming units-fibroblast,CFU-F)技术定量分析动物体内注射粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)后,是否增加外周血MSCs数量,以明确G-CSF的动员效应,进一步分析经动员进入外周血的MSCs是否保留骨髓MSCs的分子标志和多向分化潜能,尤其是阐明能否向神经元细胞分化,有无神经元细胞的电生理活动。第二部分自行改进制作颅脑撞击伤致伤装具,复制严重颅脑创伤小鼠模型(50g×30cm撞击力),设计3种G-CSF剂量分别注射给3组致伤小鼠,观测动物14天内的累积死亡率,参照Bederson等评分方法和Shapira等评分方法对致伤小鼠分别进行神经行为学评分和运动功能评分的动态观测,从整体水平评估G-CSF动员骨髓细胞参与严重颅脑创伤的救治作用。由于G-CSF不仅可以动员骨髓MSCs进入血液循环,也可动员造血干祖细胞或其它细胞进入血液循环,参与颅脑创伤修复主要是哪类细胞?创伤局部主要是什么因素吸引循环细胞定植?定植后的细胞在原位能否转化为神经细胞?这些问题还鲜未阐明。故设计第三部分实验,Western blot方法检测对MSCs具趋化作用的基质源性细胞因子-1(stromal-derived factor-1,SDF-1)表达量的变化,采用经典方法将GFP转基因骨髓细胞分为MSCs细胞、造血干祖细胞和其它细胞3类,分别移植给颅脑创伤小鼠,以确定参与颅脑创伤修复的细胞类型,用双标(GFP和NeuN等神经细胞标志分子)免疫组化鉴定移植细胞的转化类型,获如下主要结果与结论。1.对不同种属进行外周血MSCs分离培养,成功率不同,其直接原因是生理条件下外周血中存在的MSCs量少导致的。在本实验条件下,我们可以稳定的直接分离培养出大鼠外周血的MSCs。2.通过CFU-F分析,证实G-CSF可以有效对动员大鼠骨髓和外周血MSCs(接近4倍),动员的外周血MSCs特性鉴定实验显示:其形态上和骨髓MSCs一致;并阳性表达MSCs的标志分子:CD44、CD73、CD90和CD106,不表达造血系的标志分子:CD31和CD45;并具有成骨、成脂和成神经元的分化能力。证实其为真正的MSCs。3.体外实验证实,动员的MSCs可以在β-巯基乙醇、神经元原代培养上清液和组合诱导剂(含生长因子和化学分子的鸡尾酒似的混合诱导剂)诱导下向神经元分化,表达神经元标志分子:NF、NSE和NeuN。在神经元原代培养上清液的诱导下,动员的MSCs还可出现功能上的改变:类似神经元的内向钠电流。4.确定撞击小鼠头部的撞击力为50g×30cm,建立稳定的小鼠严重颅脑创伤模型,为下一步的实验提供研究平台。5.通过在不同时相点观察创伤小鼠的神经行为学和运动功能的改变、累积死亡率及病理切片,发现注射G-CSF后,严重颅脑创伤小鼠在神经行为学和运动功能上有显著改善,累计死亡率显著降低,从整体水平上证实G-CSF对严重颅脑创伤小鼠具有救治效应。并证实在本实验条件下,G-CSF的最佳动员剂量为20μg/kg.d。6.将GFP转基因C57BL/6小鼠骨髓细胞通过贴壁培养和磁珠分选分成3种细胞:MSCs、造血干细胞、其它类细胞,流式细胞仪检测证实分选效果比较好。将分选的细胞移植到严重颅脑创伤小鼠,结果表明:在大脑组织中未发现GFP标记的造血干细胞和其它类细胞的定植。GFP标记的MSCs移植颅脑创伤小鼠后5d、10d、15d和20d,均在大脑发现GFP阳性细胞的表达。证实骨髓细胞中是MSCs参与了小鼠严重颅脑创伤的修复。7. Western blot检测撞击伤小鼠创位不同时相点的SDF-1的表达,结果显示,小鼠严重颅脑创伤后,创位脑组织的SDF-1高表达,显著高于正常脑组织的SDF-1。在撞击伤后7d,SDF-1的表达达到高峰。Western blot检测撞击伤小鼠伤后第7d不同组织SDF-1的表达,结果显示,小鼠严重颅脑创伤第7d后,创位脑组织的SDF-1高表达,显著高于其他组织的SDF-1。其他组织中心脏的SDF-1表达最低。证实小鼠脑部创伤后可高表达SDF-1分子,吸引、趋化动员的MSCs向脑损伤部位移行、定植。8. NeuN为神经元的特异标志分子,在核上表达。因此,用NeuN为一抗进行荧光免疫组化,可观察到脑组织神经元的分布情况。以荧光双标免疫组化实验来观察GFP标记的MSCs在脑创位向神经元(表达特异标志分子NeuN)的分化。结果显示,第10d和20d的标本都可见同时表达NeuN分子和GFP分子的移植细胞,证实移植的GFP标记MSCs在脑创位向神经元分化,从而参与严重颅脑创伤的修复。9.以荧光双标免疫组化实验观察MSCs在严重颅脑创伤小鼠的创位向神经胶质细胞的分化。激光共聚焦显微镜结果显示,第7d、12d和20d的标本都可见同时表达GFAP分子和GFP分子的移植细胞,表明移植的MSCs(GFP标记)还可通过在脑创位向神经胶质细胞分化而促进严重颅脑创伤的修复。10.基于上述研究结果,我们提出,对严重颅脑创伤伤员,采用G-CSF可有效动员骨髓间充质干细胞进入血液循环,循环中的间充质干细胞受损伤部位高表达SDF-1的吸引趋化而定植,定植的间充质干细胞可在局部微环境因素诱导下转化为神经元细胞和神经胶质细胞参与损伤组织修复,尤其有利于功能恢复。此方法实用性强,有良好的临床应用前景。更多还原

【Abstract】 Traumatic brain injury (TBI) is a kind of serious injury happened in war time and peace time. Its treatment remains a great challenge, and the casualty and disable rate is the highest among serious trauma. At least 10 million patients with TBI worldwide are serious enough to result in death or hospitalization annually. In the United States, an average of 1.4 million TBI occur each year, and 50,000 deaths. There are about 1 million TBI patients every year in our country, and 100,000 of them can not survive the injury, and the most survivals are disabled, causing big burden on individuals and society as well. How to decrease the death rate and disable rate is a long-lasting tough problem in traumatic medicine. There are some advancement in the manipulation of TBI patients recent years, especailly by the combination of different countermeasures, which can lessen or prevent the further systemic damage and lower the death rate. However, the progress on promoting the regeneration of encephal tissue and the recovery of nerve function because nerve cells are difficult in regeneration and the stem cells only are confined to certain areas in brain.Neurons were considered previously a terminal differentiated functional cell, and loss of reproductive activity. Brain and spinal cord injury were repaired main by glial cell replacement, but not by neurons. This unsatisfied reapir would result in neural functional incapacity, and disable in intellegence or motion. Pliles of Studies have confirmed that neural stem cells can be isolated from brain tissue, amplified in vitro, and induced to differentiate into neurons. So it is possible for neural stem cells in brain tissue to differentiate into neuron in vivo and promote the histological construction repair and functional rehabilitation in injured tissue. After amplification and committed differentiation induction in vitro, neural stem cells can be transplanted into injury site to promote damage repair. In fact, it is almost impossible to get human neural stem cells for thraputic amplification and differntiation induction in vitro. The idea of adopting embryonic stem cells has evoked contradiction in ethics in addition to the technique problems such as allograft-caused immunologic rejection and the risk of neoplastic tramsformation during in vitro amplification. These remianed problems need to be solved before the application of stem cell transplantation in clinic.However, the multippotential differentiation, the plasticity, of adult stem cells has laid the foundation for new strategy and methods in the research interest of regeneration medicine and traumatic medicine. Increasing studies have reported that mesenchymal stem cells (MSCs) posses the characteristics, such as easy acquirement, (autoallergic or variant), feasible amplification in vitro and multipotential differentiation (to differentiate into osteocytes, muscle cells, adipocytes and neural cells). In our previous study, bone marrow cells from green fluorescent protein(GFP) transgenic mice were transplanted intravenously into homogenic mice with total-body irradiation of 10 Gyγray, and GFP-positive cells were found in intestine, liver, brain, skin of the recipients, confirming that the transplanted cells have differentiated or de-differntiated into enterocytes, hepatic cells and neurons. We also demonstrated that MSCs treated by supernatants of primary neural cell cultures expressed markers of neurons. From above findings, we drew the conclusion that MSCs can be recruited to the injury site through blood circulation and indued by the microenvironment (niche and humoral factors) to differentiate into specific tissue cells. Under physiological condition, the quantity of circulating MSCs that derived from bone marrow is too small to show theraputic effect. It is possible to increase the amount of MSCs recruited to injury site if take measures to mobilize MSCs from bone marrow to peripheral blood, and subsequently promote trauma tissue (include TBI) repair and function recovery. To explore the new way for trauma tissue repair and its mechanism will reveal the differentiation mechanism of MSCs regulated by microenvironment and facilitate clinic trauma care.In first part of this study, peripheral blood MSCs from rat and human were isolated and cultured. Colony forming units-fibroblastc (CFU-F) assay was performed to evaluate the mobilization effect on MSCs by granulocyte colony-stimulating factor (G-CSF). The morphology, differentiation potential, especially to neuron, and immunophenotype of PB-derived adherent cells were detected. In second part of this study, the experimental mouse model of serious TBI was established (impact force 50g in weight and 30cm in high of free fall). the effect of MSCs mobilized by G-CSF on the trauma recovery of different severities was evaluated by recording neuroethology scores (reference Bederson), motor function scores (reference Shapira) and the animal mortality rate. As G-CSF could not only mobilize bone marrow MSCs but also haemopoietic stem cell, which population of cells participate trauma repair need to be clearified. What was the main factor to attracte circulating cells to the injury site? Could the cells recruit to injury site of brain transform to neuron? So we designed third part study, the expression of SDF-1 was detected by Western blot, bone marrow cells of GFP mice were fractionated into MSCs, HSCs and non-MSCs/HSCs cells by adherent culture and magnetic bead cell sorting. The fractionated cells were transplanted into TBI mice to find out which cell population of cells participate in trauma repair. The differentiation/transdifferentiation of MSCs labeled by GFP in situ in trauma site were detected by double immunofluorescence. Main results and conlusion were as follow:1. PB MSCs were isolated and cultureed successfully or unsuccessfully from different genus, it concerned with many factor, especially a small number of circulating MSCs in BP. Under our experiment condition, rat’s PB MSCs could been isolated and cultured steadily.2. we confirmed that G-CSF mobilized BM-derived MSCs, especially PB-derived MSCs (almost 4-fold increase) by CFU-F assay. PB-derived adherent cells formed a homogeneous cell layer that closely resembled BM MSCs. These cells were positive for marker molecule of MSCs: CD44, CD73, CD90, and CD106, but were negative for marker molecule of systema haemale: CD31 and CD45. These cells could be induced to differentiate into bone, lipoids and nerve cells. PB-derived adherent cells were considered bona fide MSCs in light of their morphology, differentiation potential, and immunophenotype.3. MSCs mobilized by G-CSF were induced to differentiate into neurons withβ-mercap- toethanol, supernatants of nerve cell cultures and con-antileptic (contain growth factor and chemical molecular), and express nerve cell markers NF, NSE, and NeuN. These MSCs could appear introvert sodium current after inducing with supernatants of nerve cell cultures.4. The experiment mouse model of serious TBI was established (impact force: 50g×30cm). It provided a experiment platform for research. 5. By research neuroethology scores, motor function scores and pathological section, result confirmed that G-CSF could significantly promote injury repair of serious TBI mice, and the mortality rate of mice after trauma was significantly degraded. Under our experiment condition, neuroethology and motor function scores were determined at different phase point after G-CSF mobilization, it was confirmed that the optimization mobilization dose was 20μg/kg.d.6. We fractionated bone marrow cells of GFP mice into MSCs, HSCs, and non-MSCs/HSCs cells by adherent culture and using magnetic beads, detect result by flow cytometer showed separation effect was good. Transplanted these cell types into trauma mice, We confirmed no HSCs or non-MSCs/HSCs cells labeled by GFP planted in injury sites, but MSCs labeled by GFP could plant in injury sites on 5d, 10d, 15d and 20d after trauma. Hence, MSCs mobilized by G-CSF promoted trauma repair.7. The expression of SDF-1 was detected by Western blot at trauma sites on different phase point. Result confirmed the expression of SDF-1 at trauma sites was significantly higher than that of normal brain tissue, and the expression of SDF-1 approached peak after trauma on 7d. The expression of SDF-1 was detected by Western blot in different tissue on 7d. Result confirmed the expression of SDF-1 at trauma sites was significantly higher than that of other tissue, and the expression of SDF-1 was lowest in heart. It could result in MSCs migrated to and planted in trauma sites.8. As the idio-marker molecule of neuron, NeuN is express on neuron nucleus. So we carried out fluorescence immunocytochemically, and got the disposition condition of neuron at brain tissue. MSCs labeled by GFP planted in trauma sites were found by double immunofluorescence to express the neuron marker NeuN on 10d and 20d. These findings confirmed that MSCs could directly differentiate into neurons at brain trauma sites to promot trauma repair.9. MSCs labeled by GFP planted in trauma sites were found by double immuno- fluorescence to express the glial cell marker GFAP on 7d, 12d and 20d. These findings suggest that MSCs can directly differentiate into glial cell at brain trauma sites to promot trauma repair.10. In view of above study, we considered G-CSF could mobilize TBI patients’MSCs from bone marrow to PB, SDF-1 high expressed at trauma sites could result in MSCs migrated to and planted in trauma sites, and planting MSCs could be induced by microenvironment factor to directly differentiate into neuron and glial cell for promot trauma repair, especial for functional recovery. This method is very practical, and has a favourable prospect for clinic application.更多还原