不同途径自体移植骨髓基质细胞治疗兔创伤性脑损伤
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摘要
创伤性脑损伤(Traumatic brain injury,TBI),是由外伤引起的脑组织损害。在交通事故伤等外伤所导致的死亡中,TBI的死亡率居于首位,严重威胁着人民的生命安全。同时,TBI的致残率也非常高,伤后多出现偏瘫失语等功能障碍,甚至持续性植物状态,给家庭和社会带来了沉重负担。因此,TBI的救治是人类社会面临的一项重要课题。
但是,目前为止,传统的治疗方法对于TBI的救治效果还不很理想。近年来,随着组织工程和成体干细胞研究的不断进展,成体干细胞移植替代受损神经细胞或神经功能的治疗方法越来越受到研究人员的重视。
骨髓基质细胞(Bone marrow stromal cells,BMSCs)是成体干细胞的一种,是骨髓中除造血干细胞外的未完全分化的原始细胞,能分化成多种类型的间充质干细胞,在合适的条件下,也能跨胚层向神经细胞分化。BMSCs移植在啮齿类动物TBI模型的治疗研究证明BMSCs能够促进脑损伤的恢复。同时,BMSCs获取方便,来源充足,可进行自体移植,避免了胚胎干细胞移植所带来的伦理争议和免疫排斥反应等问题。因此,BMSCs成为治疗TBI的最理想的种子细胞之一。
目前,在成体干细胞移植治疗脑损伤的实验研究中,细胞移植的途径主要有以下几种:注射入脑实质内、注射入侧脑室内和静脉注射等,以及较少见的动脉注射,注射入腹腔内和注射入枕大池内等。经由这些途径的细胞移植,可能存在加重脑损伤、进入损伤部位的细胞数少,以及治疗效果欠佳等问题。因此,有必要寻找一条微创、进入损伤部位的细胞数多且治疗效果较理想的移植途径。
近年来,在脊髓损伤(Spinal cord injury,SCI)的干细胞移植治疗研究中,有研究者发现经腰穿移植的干细胞能够到达颈髓的损伤部位,且有助于神经功能障碍的修复,同时少量的细胞可以进入脑组织内。这就提示了,在中枢神经系统损伤的情况下,腰穿移植的干细胞可以迁移到相对距离较远的中枢神经系统的其他部位,也很有可能迁移到TBI部位。那么,腰穿移植也就有可能是BMSCs移植治疗TBI的一个较理想的途径。
本研究以BMSCs为移植细胞,在体外培养增殖,并以腺病毒转染绿色荧光蛋白(Adeno-virus transfected green fluorescent protein,Ad-GFP)标记,检测体外培养的BMSCs的生长特性,建立了重锤击落伤所导致的中度脑损伤兔TBI模型,并通过静脉注射和腰穿注射等途径进行BMSCs移植,在不同的时间点评估实验动物运动功能障碍的恢复情况,并于经心脏灌注固定后获取脑损伤处的病理组织,检测标记细胞,将不同途径移植治疗的效果进行比较。
第一章、原子力显微镜在体外培养的兔骨髓基质细胞和免疫球蛋白胶体金结构观察中的应用研究
目的:探索原子力显微镜(Atomic force microscope,AFM)在骨髓基质细胞(BMSCs)表面超微结构及生物大分子标记物检测中的作用,为BMSCs的鉴定和示踪提供更多依据,同时明确AFM在生命科学领域中的应用前景。方法:取兔股骨骨髓,密度梯度离心法获取BMSCs,以DMEM/F12培养基进行体外培养,对原代培养的BMSCs,分别在第1、7、14天以2.5%戊二醛固定15分钟,再用双蒸水清洗3次,在空气中自然晾干后,进行AFM接触模式扫描。用0.5mmol/L氯化钠溶液将免疫球蛋白胶体金(IgG-gold)原液(浓度1mg/ml)稀释至1μg/ml。再调pH值为7.0。吹打2次后,吸取10μL加到新鲜解离的云母片表面,空气中自然晾干,再用去离子水滴加表面清洗2遍,在空气中自然晾干后,室温下进行AFM接触模式扫描,以AFM自带的软件对扫描图像进行测算,并对所得到的数据进行分析校正。结果:BMSCs在不同的培养阶段都具备粘附特征,都具有相应的粘附结构,该结构位于细胞周缘,呈条纹状结构,相邻的该结构之间近乎平行,远端均插入到细胞外基质的深部。IgG-gold分子的构象为“椭圆球状”结构,其三维径线长度分别为22.0nm×9.0nm×5.7nm,分子表面积为2000±973nm~2,体积为8100±5717nm~3,表面粗糙度为1.089±0.109。结论:AFM扫描提示BMSCs的粘附特征具有相应的形态结构基础。IgG-gold分子具有均一稳定的特征性“椭圆球状”构象。AFM可以用于观察BMSCs表面的超微结构,及以纳米尺度来测定生物大分子的结构,在生命科学研究领域中有着很好的应用前景。
第二章、绿色荧光蛋白、溴脱氧尿嘧啶和羧基荧光素二醋酸盐琥珀酰亚胺酯标记体外培养的兔骨髓基质细胞的实验研究
目的:明确腺病毒转染绿色荧光蛋白(Ad-GFP)、溴脱氧尿嘧啶(5-bromodeoxyuridine,BrdU)和羧基荧光素二醋酸盐琥珀酰亚胺酯(5,6-carboxyfluorescein diacetate succinimidyl ester,CFSE)对体外培养的兔BMSCs标记的可行性,为自体移植后细胞示踪作准备。方法:通过培养HEK293细胞,经过多次传代和反复冻溶,体外扩增纯化Ad-GFP,分别以Ad-GFP、BrdU和CFSE标记体外培养的BMSCs。以免疫荧光法检测BrdU的标记率,以流式细胞仪检测Ad-GFP和CFSE的标记率。以CCK-8法检测标记后细胞的生长曲线,以流式细胞仪检测标记后的细胞周期和凋亡率。并制作重锤击落兔脑损伤模型,自体移植4周后,以4%多聚甲醛经兔心脏灌注,获取兔脑病理标本,以免疫荧光法或直接在荧光显微镜下观察脑损伤灶切片处的标记细胞。结果:BrdU的标记率为(73.3±3.6)%;GFP的标记率为(81.6±5.7)%;CFSE的标记率为(99.1±0.6)%。四组细胞均于第3d至第5d处于快速生长期,对各个时间点上的吸光度值进行t检验,提示差异无显著意义(P＞0.05)。各种方法标记后的BMSCs和未标记的BMSCs相似,其绝大部分细胞(＞85%)处于G0/G1期,而S期的细胞较少(＜6%)。各组标记后细胞的凋亡率与未标记细胞的凋亡率相近,均较低(约3%),统计学上差异均无显著意义(P＞0.05)。腰穿自体移植BMSCs 4w后,三种方法标记的细胞均可在脑损伤处发现。移植的细胞主要位于损伤灶的周缘,损伤中心很少见到细胞。对侧相应大脑皮层处未见标记的移植细胞。而在双侧侧脑室的脉络膜和室管膜处可见少量移植细胞。GFP标记的细胞位于损伤灶深缘,在荧光显微镜下,发出较强的绿色荧光。CFSE标记细胞移植的脑组织切片中,可发现临近移植细胞的脑组织也存在相对较弱的绿色荧光,这是由于CFSE渗漏所致,BrdU标记的细胞主要也位于脑损伤灶的周缘及半阴影区。结论:Ad-GFP、BrdU和CFSE均可用于标记BMSCs,对于体外培养的BMSCs的细胞周期、凋亡率和生长特性没有显著影响。CFSE标记法存在渗漏的问题,BrdU标记法的检测较繁琐,且存在假阳性的问题,而Ad-GFP标记的细胞易于检测示踪。
第三章、不同途径自体移植骨髓基质细胞治疗兔创伤性脑损伤
目的:建立一个比较稳定的兔TBI模型;明确不同途径BMSCs自体移植对于兔创伤性脑损伤的治疗效果,探索干细胞移植的微创方法,并明确是否存在最优化的移植途径。方法:实验动物随机分为5组,每组9只,以TBI后腰穿鞘内注射自体移植BMSCs组和耳缘静脉内注射自体移植BMSCs组为治疗组,以TBI后腰穿鞘内注射PBS组、正常兔腰穿鞘内注射自体移植BMSCs组和TBI后自然恢复组为对照组。BMSCs体外培养10天,并以Ad-GFP标记72h后,无菌条件下收集2×10~6个兔BMSCs,悬浮于60μl的无菌PBS溶液中。麻醉后,于兔右侧额顶部开颅,以重锤击落伤方法打击兔右侧大脑皮层运动区,打击力度为30×40g.cm,建立中度脑损伤的兔TBI模型。分别于损伤后24h,进行BMSCs自体移植,分别于移植后24h、1周、2周和4周进行兔运动功能障碍评估,并于移植后1周、2周和4周每组随机取2只兔,以4%多聚甲醛经心脏灌注固定后,取脑标本进行冰冻病理切片,并在荧光显微镜下进行细胞计数。结果:在治疗组中,运动功能障碍恢复都优于对照组,其中以腰穿移植组的运动功能障碍恢复更加明显;损伤部位细胞数由多到少依次为脑实质内移植组、腰穿移植组、对侧侧脑室内移植组和右侧耳缘静脉移植组。结论:建立了较稳定的中度脑损伤兔TBI模型。经上述途径进行的BMSCs移植均有助于TBI兔运动功能的恢复,其中腰穿移植组的效果最理想。发现了一种微创、高效移植BMSCs治疗TBI的较理想的途径。
Traumatic brain injury (TBI) is the brain tissue damage caused by trauma. Among the death caused by vehicle accidents, the mortality ratio of TBI is second to none. So that TBI is a vital threat to the human beings. At the same time, the mutilation ratio of TBI is also very high. After TBI, the neurological dysfunction like hemiplegia, even persistent vegetable state (PVS) is often encountered. Thus, heavy burden is put on the family and the society. Therefore, TBI treatment is a serious topic of our world.
But up to now, the traditional measures have brought little light to treatment of TBI. In the recent years, the method is enheartening the researchers that the transplanted adult stem cells can serve to substitute the injured neural cells or the damaged neural functions.
Bone marrow stromal cells (BMSCs), a member of the family of adult stem cells, are the incompletely differentiated primitive cells in the bone marrow other than haemopoietic stem cells (HSCs). They can differentiate into many kinds of mesenchymal stem cells. And under certain condition, they can transdifferentiate into neural cells. It has been demonstrated that BMSCSs transplantation can improve the rehabilitation from neurological dysfunction in TBI models in rodents. And moreover, BMSCs can be easily and sufficiently harvested. Furthermore, autologous transplantation can be accompanied when the choice of seed-cells is BMSCs. By the way, ethical debate and immunological rejection caused by embryonic stem cells (ESCs) transplantation can be avoided. Therefore, BMSCs have developed into the optimal seed-cells for cell based therapy of TBI.
At present, the ways that cells are delivered in the treatment of experimental TBI by adult stem cells transplantation are as following: injected into parenchyma of the cerebrum, injected into the lateral ventricle, injected into the remote vein, and relatively minor used methods like injected into artery, injected into abdominal cavity and injected into cisterna magna. Cells transplantation by these ways might have some shortcomings, such as aggravating brain injury, sending fewer cells to the injured site, and providing unsatisfied therapeutics. Thus, it's essential for us to create a new way for cells delivery that has many advantages compared to the traditional methods, such as bringing minimal invasion, sending more cells to the injured sits, and providing satisfied therapeutics.
In the recent years, some researchers have detected that stem cells implanted through lumbar puncture could migrate to the injured cervical cord, and even more a few implanted cells could be identified in the brain tissues. And the neurological function was improved after cells based therapy in these studies. Thus, the idea is illuminated that lumbar puncture delivery might be a relatively optimal pathway for TBI therapy with BMSCs transplantation.
In this study, BMSCs cultured in vitro, were the seed cells. And they were marked by the adeno-virus transfected green fluorescent protein (Ad-GFP). The characteristics of the cultured BMSCs were identified. Then TBI models of New Zealand rabbits were developed. The marked BMSCs were injected into the parenchyma of the cerebrum, the lateral ventricle, the vein and the subarachnoid cavity by lumbar puncture, respectively. The motor dysfunction was evaluated at several time points after cells transplantation. And the injured tissues were harvested for pathological examination. Finally, the results were compared within different groups.
Part one: Investigation of BMSCs cultured in vitro and the configuration of goat anti-mouse IgG gold conjugate with atomic force microscopy
Objective: To detect the adhesion device and its structure of bone marrow stromal cell, and to warrant the isolation and differentiation of the cell. To identify the configuration of goat anti-mouse IgG gold conjugate (IgG-gold).
Methods: Bone marrows were harvested from the femurs of rabbits. And BMSCs were obtained with density gradient centrifugation. After cultured in primary for 1day, 7 days and 14 days, BMSCs were fixed with 2.5% glutaraldehyde for 5 minutes. Then the cells were cleansed thrice with double evaporated water. After the cells were open-air dried, they were scanned with atomic force microscope under contact mode respectively. The molecules of IgG-gold affiliated on the new cleaved mica were scanned with atomic force microscope (AFM) under the physiological environment. The data was analyzed with the statistic software in the AFM.
Results: Adhesion was the characteristic of bone marrow stromal cells in the given stage of their lives. And correspondingly every cell had the adhesion device. The configuration of IgG-gold molecule is an "ellipsoidal sphere" sized as 22.0nm×9.0nm×5.7nm.
Conclusions: Atomic force microscope had special predominance in observation of the ultrastructure of cell surface. Bone marrow stromal cells had adhesion device with corresponding structure. IgG-gold has a steady uniform characteristic conformation. AFM can play an important role in detecting biomacromolecules at a nanometer scale resolution.
Part two: study on green fluorescent protein, 5-bromodeoxyuridine and 5,6-carboxyfluorescein diacetate succinimidyl ester marking BMSCs in rabbits
Objective: To detect the applicability of BrdU, CFSE and GFP serving as labels of MSCs of rabbits.
Methods: BMSCs of rabbits were labeled with BrdU, CFSE and GFP respectively. The efficiency of the three markers was quantified. Cell cycle and apoptosis ratio of labeled BMSCs were revealed, the growth curves were delineated, so as to identify whether the markers cast an influence on the cell growth characteristics in vitro. Furthermore, the traumatic brain injury model was introduced. And the labeled BMSCs were transplanted by means of lumbal puncture. 4 weeks later, the animals were sacrificed and the pathological sections underwent fluoroscopy.
Results: The efficiency of BrdU labeling was 73.3%, that of GFP was 81.6%, and that of CFSE was 99.1%. The three markers had no effects on the growth characteristics of BMSCs of rabbits cultured in vitro. The transplanted labeled cells were detected at the site of the injured area. And CFSE leakage was encountered.
Conclusions: BrdU, CFSE and GFP can serve as a label of BMSCs of rabbits. Each of BrdU and CFSE has its limitations.
Part three: Autologous transplantation of bone marrow stromal cells for treatment of traumatic brain injury in rabbits
Objective: To establish a stable TBI model in rabbits. To identify the therapeutics of treatment of TBI with autologous BMSCs transplantation through different pathways in rabbits. To explore a minimally invasive method for stem cell transplantation. And to clarify whether the optimizing pathway of stem cell transplantation does exist or not.
Methods: 45 New Zealand rabbits were randomly divided into five groups equally. BMSCs were injected into right ear-edge vein, subarachnoid cavity through lumbar puncture in one of the two treatment groups after TBI respectively. PBS was injected into subarachnoid cavity through lumbar puncture in another group. And BMSCs were injected into subarachnoid cavity through lumbar puncture in the last group without TBI. The last two groups together with the group receiving no engraftment after TBI served as control group. 72h after marked with Ad-GFP, 2×106BMSCs cultured for 10d in vitro, were harvested and suspended in 60μl sterile PBS. Moderate TBI models of rabbits were established. The cells were autologously implanted 24h after TBI. The motor dysfunction was assessed at 24h, 1w, 2w and 4w after cells transplantation respectively. And 2 models in each group were sacrificed at 1w, 2w and 4w after cells transplantation through cardio-perfusion with 4% paraformaldehyde (PFA). Then the brain specimen underwent frozen section. And the labeled cells were counted under fluorescence microscope.
Results: The recover of motor dysfunction in treated groups were better than that in the control groups. And the rehabilitation of motor function in the group of BMSCs transplantation by lumbar puncture was more outstanding. The transplanted cells found at the injured sites were not the same. And from many to few, they are by turns: the group of transplantation by mtraparenchymal injection, by lumbar puncture, by intraventricle injection, by intravenous injection.
Conclusions: A stable moderate TBI model of rabbits was established. BMSCs transplantation through various pathways can improve the motor dysfunction rehabilitation after TBI in rabbits. Among the several commonly used methods, the therapeutics of BMSCs injection into subarachnoid cavity by lumbar puncture is optimized. An effective and minimally invasive pathway for BMSCs transplantation to treat TBI has been explored.
但是,目前为止,传统的治疗方法对于TBI的救治效果还不很理想。近年来,随着组织工程和成体干细胞研究的不断进展,成体干细胞移植替代受损神经细胞或神经功能的治疗方法越来越受到研究人员的重视。
骨髓基质细胞(Bone marrow stromal cells,BMSCs)是成体干细胞的一种,是骨髓中除造血干细胞外的未完全分化的原始细胞,能分化成多种类型的间充质干细胞,在合适的条件下,也能跨胚层向神经细胞分化。BMSCs移植在啮齿类动物TBI模型的治疗研究证明BMSCs能够促进脑损伤的恢复。同时,BMSCs获取方便,来源充足,可进行自体移植,避免了胚胎干细胞移植所带来的伦理争议和免疫排斥反应等问题。因此,BMSCs成为治疗TBI的最理想的种子细胞之一。
目前,在成体干细胞移植治疗脑损伤的实验研究中,细胞移植的途径主要有以下几种:注射入脑实质内、注射入侧脑室内和静脉注射等,以及较少见的动脉注射,注射入腹腔内和注射入枕大池内等。经由这些途径的细胞移植,可能存在加重脑损伤、进入损伤部位的细胞数少,以及治疗效果欠佳等问题。因此,有必要寻找一条微创、进入损伤部位的细胞数多且治疗效果较理想的移植途径。
近年来,在脊髓损伤(Spinal cord injury,SCI)的干细胞移植治疗研究中,有研究者发现经腰穿移植的干细胞能够到达颈髓的损伤部位,且有助于神经功能障碍的修复,同时少量的细胞可以进入脑组织内。这就提示了,在中枢神经系统损伤的情况下,腰穿移植的干细胞可以迁移到相对距离较远的中枢神经系统的其他部位,也很有可能迁移到TBI部位。那么,腰穿移植也就有可能是BMSCs移植治疗TBI的一个较理想的途径。
本研究以BMSCs为移植细胞,在体外培养增殖,并以腺病毒转染绿色荧光蛋白(Adeno-virus transfected green fluorescent protein,Ad-GFP)标记,检测体外培养的BMSCs的生长特性,建立了重锤击落伤所导致的中度脑损伤兔TBI模型,并通过静脉注射和腰穿注射等途径进行BMSCs移植,在不同的时间点评估实验动物运动功能障碍的恢复情况,并于经心脏灌注固定后获取脑损伤处的病理组织,检测标记细胞,将不同途径移植治疗的效果进行比较。
第一章、原子力显微镜在体外培养的兔骨髓基质细胞和免疫球蛋白胶体金结构观察中的应用研究
目的:探索原子力显微镜(Atomic force microscope,AFM)在骨髓基质细胞(BMSCs)表面超微结构及生物大分子标记物检测中的作用,为BMSCs的鉴定和示踪提供更多依据,同时明确AFM在生命科学领域中的应用前景。方法:取兔股骨骨髓,密度梯度离心法获取BMSCs,以DMEM/F12培养基进行体外培养,对原代培养的BMSCs,分别在第1、7、14天以2.5%戊二醛固定15分钟,再用双蒸水清洗3次,在空气中自然晾干后,进行AFM接触模式扫描。用0.5mmol/L氯化钠溶液将免疫球蛋白胶体金(IgG-gold)原液(浓度1mg/ml)稀释至1μg/ml。再调pH值为7.0。吹打2次后,吸取10μL加到新鲜解离的云母片表面,空气中自然晾干,再用去离子水滴加表面清洗2遍,在空气中自然晾干后,室温下进行AFM接触模式扫描,以AFM自带的软件对扫描图像进行测算,并对所得到的数据进行分析校正。结果:BMSCs在不同的培养阶段都具备粘附特征,都具有相应的粘附结构,该结构位于细胞周缘,呈条纹状结构,相邻的该结构之间近乎平行,远端均插入到细胞外基质的深部。IgG-gold分子的构象为“椭圆球状”结构,其三维径线长度分别为22.0nm×9.0nm×5.7nm,分子表面积为2000±973nm~2,体积为8100±5717nm~3,表面粗糙度为1.089±0.109。结论:AFM扫描提示BMSCs的粘附特征具有相应的形态结构基础。IgG-gold分子具有均一稳定的特征性“椭圆球状”构象。AFM可以用于观察BMSCs表面的超微结构,及以纳米尺度来测定生物大分子的结构,在生命科学研究领域中有着很好的应用前景。
第二章、绿色荧光蛋白、溴脱氧尿嘧啶和羧基荧光素二醋酸盐琥珀酰亚胺酯标记体外培养的兔骨髓基质细胞的实验研究
目的:明确腺病毒转染绿色荧光蛋白(Ad-GFP)、溴脱氧尿嘧啶(5-bromodeoxyuridine,BrdU)和羧基荧光素二醋酸盐琥珀酰亚胺酯(5,6-carboxyfluorescein diacetate succinimidyl ester,CFSE)对体外培养的兔BMSCs标记的可行性,为自体移植后细胞示踪作准备。方法:通过培养HEK293细胞,经过多次传代和反复冻溶,体外扩增纯化Ad-GFP,分别以Ad-GFP、BrdU和CFSE标记体外培养的BMSCs。以免疫荧光法检测BrdU的标记率,以流式细胞仪检测Ad-GFP和CFSE的标记率。以CCK-8法检测标记后细胞的生长曲线,以流式细胞仪检测标记后的细胞周期和凋亡率。并制作重锤击落兔脑损伤模型,自体移植4周后,以4%多聚甲醛经兔心脏灌注,获取兔脑病理标本,以免疫荧光法或直接在荧光显微镜下观察脑损伤灶切片处的标记细胞。结果:BrdU的标记率为(73.3±3.6)%;GFP的标记率为(81.6±5.7)%;CFSE的标记率为(99.1±0.6)%。四组细胞均于第3d至第5d处于快速生长期,对各个时间点上的吸光度值进行t检验,提示差异无显著意义(P＞0.05)。各种方法标记后的BMSCs和未标记的BMSCs相似,其绝大部分细胞(＞85%)处于G0/G1期,而S期的细胞较少(＜6%)。各组标记后细胞的凋亡率与未标记细胞的凋亡率相近,均较低(约3%),统计学上差异均无显著意义(P＞0.05)。腰穿自体移植BMSCs 4w后,三种方法标记的细胞均可在脑损伤处发现。移植的细胞主要位于损伤灶的周缘,损伤中心很少见到细胞。对侧相应大脑皮层处未见标记的移植细胞。而在双侧侧脑室的脉络膜和室管膜处可见少量移植细胞。GFP标记的细胞位于损伤灶深缘,在荧光显微镜下,发出较强的绿色荧光。CFSE标记细胞移植的脑组织切片中,可发现临近移植细胞的脑组织也存在相对较弱的绿色荧光,这是由于CFSE渗漏所致,BrdU标记的细胞主要也位于脑损伤灶的周缘及半阴影区。结论:Ad-GFP、BrdU和CFSE均可用于标记BMSCs,对于体外培养的BMSCs的细胞周期、凋亡率和生长特性没有显著影响。CFSE标记法存在渗漏的问题,BrdU标记法的检测较繁琐,且存在假阳性的问题,而Ad-GFP标记的细胞易于检测示踪。
第三章、不同途径自体移植骨髓基质细胞治疗兔创伤性脑损伤
目的:建立一个比较稳定的兔TBI模型;明确不同途径BMSCs自体移植对于兔创伤性脑损伤的治疗效果,探索干细胞移植的微创方法,并明确是否存在最优化的移植途径。方法:实验动物随机分为5组,每组9只,以TBI后腰穿鞘内注射自体移植BMSCs组和耳缘静脉内注射自体移植BMSCs组为治疗组,以TBI后腰穿鞘内注射PBS组、正常兔腰穿鞘内注射自体移植BMSCs组和TBI后自然恢复组为对照组。BMSCs体外培养10天,并以Ad-GFP标记72h后,无菌条件下收集2×10~6个兔BMSCs,悬浮于60μl的无菌PBS溶液中。麻醉后,于兔右侧额顶部开颅,以重锤击落伤方法打击兔右侧大脑皮层运动区,打击力度为30×40g.cm,建立中度脑损伤的兔TBI模型。分别于损伤后24h,进行BMSCs自体移植,分别于移植后24h、1周、2周和4周进行兔运动功能障碍评估,并于移植后1周、2周和4周每组随机取2只兔,以4%多聚甲醛经心脏灌注固定后,取脑标本进行冰冻病理切片,并在荧光显微镜下进行细胞计数。结果:在治疗组中,运动功能障碍恢复都优于对照组,其中以腰穿移植组的运动功能障碍恢复更加明显;损伤部位细胞数由多到少依次为脑实质内移植组、腰穿移植组、对侧侧脑室内移植组和右侧耳缘静脉移植组。结论:建立了较稳定的中度脑损伤兔TBI模型。经上述途径进行的BMSCs移植均有助于TBI兔运动功能的恢复,其中腰穿移植组的效果最理想。发现了一种微创、高效移植BMSCs治疗TBI的较理想的途径。
Traumatic brain injury (TBI) is the brain tissue damage caused by trauma. Among the death caused by vehicle accidents, the mortality ratio of TBI is second to none. So that TBI is a vital threat to the human beings. At the same time, the mutilation ratio of TBI is also very high. After TBI, the neurological dysfunction like hemiplegia, even persistent vegetable state (PVS) is often encountered. Thus, heavy burden is put on the family and the society. Therefore, TBI treatment is a serious topic of our world.
But up to now, the traditional measures have brought little light to treatment of TBI. In the recent years, the method is enheartening the researchers that the transplanted adult stem cells can serve to substitute the injured neural cells or the damaged neural functions.
Bone marrow stromal cells (BMSCs), a member of the family of adult stem cells, are the incompletely differentiated primitive cells in the bone marrow other than haemopoietic stem cells (HSCs). They can differentiate into many kinds of mesenchymal stem cells. And under certain condition, they can transdifferentiate into neural cells. It has been demonstrated that BMSCSs transplantation can improve the rehabilitation from neurological dysfunction in TBI models in rodents. And moreover, BMSCs can be easily and sufficiently harvested. Furthermore, autologous transplantation can be accompanied when the choice of seed-cells is BMSCs. By the way, ethical debate and immunological rejection caused by embryonic stem cells (ESCs) transplantation can be avoided. Therefore, BMSCs have developed into the optimal seed-cells for cell based therapy of TBI.
At present, the ways that cells are delivered in the treatment of experimental TBI by adult stem cells transplantation are as following: injected into parenchyma of the cerebrum, injected into the lateral ventricle, injected into the remote vein, and relatively minor used methods like injected into artery, injected into abdominal cavity and injected into cisterna magna. Cells transplantation by these ways might have some shortcomings, such as aggravating brain injury, sending fewer cells to the injured site, and providing unsatisfied therapeutics. Thus, it's essential for us to create a new way for cells delivery that has many advantages compared to the traditional methods, such as bringing minimal invasion, sending more cells to the injured sits, and providing satisfied therapeutics.
In the recent years, some researchers have detected that stem cells implanted through lumbar puncture could migrate to the injured cervical cord, and even more a few implanted cells could be identified in the brain tissues. And the neurological function was improved after cells based therapy in these studies. Thus, the idea is illuminated that lumbar puncture delivery might be a relatively optimal pathway for TBI therapy with BMSCs transplantation.
In this study, BMSCs cultured in vitro, were the seed cells. And they were marked by the adeno-virus transfected green fluorescent protein (Ad-GFP). The characteristics of the cultured BMSCs were identified. Then TBI models of New Zealand rabbits were developed. The marked BMSCs were injected into the parenchyma of the cerebrum, the lateral ventricle, the vein and the subarachnoid cavity by lumbar puncture, respectively. The motor dysfunction was evaluated at several time points after cells transplantation. And the injured tissues were harvested for pathological examination. Finally, the results were compared within different groups.
Part one: Investigation of BMSCs cultured in vitro and the configuration of goat anti-mouse IgG gold conjugate with atomic force microscopy
Objective: To detect the adhesion device and its structure of bone marrow stromal cell, and to warrant the isolation and differentiation of the cell. To identify the configuration of goat anti-mouse IgG gold conjugate (IgG-gold).
Methods: Bone marrows were harvested from the femurs of rabbits. And BMSCs were obtained with density gradient centrifugation. After cultured in primary for 1day, 7 days and 14 days, BMSCs were fixed with 2.5% glutaraldehyde for 5 minutes. Then the cells were cleansed thrice with double evaporated water. After the cells were open-air dried, they were scanned with atomic force microscope under contact mode respectively. The molecules of IgG-gold affiliated on the new cleaved mica were scanned with atomic force microscope (AFM) under the physiological environment. The data was analyzed with the statistic software in the AFM.
Results: Adhesion was the characteristic of bone marrow stromal cells in the given stage of their lives. And correspondingly every cell had the adhesion device. The configuration of IgG-gold molecule is an "ellipsoidal sphere" sized as 22.0nm×9.0nm×5.7nm.
Conclusions: Atomic force microscope had special predominance in observation of the ultrastructure of cell surface. Bone marrow stromal cells had adhesion device with corresponding structure. IgG-gold has a steady uniform characteristic conformation. AFM can play an important role in detecting biomacromolecules at a nanometer scale resolution.
Part two: study on green fluorescent protein, 5-bromodeoxyuridine and 5,6-carboxyfluorescein diacetate succinimidyl ester marking BMSCs in rabbits
Objective: To detect the applicability of BrdU, CFSE and GFP serving as labels of MSCs of rabbits.
Methods: BMSCs of rabbits were labeled with BrdU, CFSE and GFP respectively. The efficiency of the three markers was quantified. Cell cycle and apoptosis ratio of labeled BMSCs were revealed, the growth curves were delineated, so as to identify whether the markers cast an influence on the cell growth characteristics in vitro. Furthermore, the traumatic brain injury model was introduced. And the labeled BMSCs were transplanted by means of lumbal puncture. 4 weeks later, the animals were sacrificed and the pathological sections underwent fluoroscopy.
Results: The efficiency of BrdU labeling was 73.3%, that of GFP was 81.6%, and that of CFSE was 99.1%. The three markers had no effects on the growth characteristics of BMSCs of rabbits cultured in vitro. The transplanted labeled cells were detected at the site of the injured area. And CFSE leakage was encountered.
Conclusions: BrdU, CFSE and GFP can serve as a label of BMSCs of rabbits. Each of BrdU and CFSE has its limitations.
Part three: Autologous transplantation of bone marrow stromal cells for treatment of traumatic brain injury in rabbits
Objective: To establish a stable TBI model in rabbits. To identify the therapeutics of treatment of TBI with autologous BMSCs transplantation through different pathways in rabbits. To explore a minimally invasive method for stem cell transplantation. And to clarify whether the optimizing pathway of stem cell transplantation does exist or not.
Methods: 45 New Zealand rabbits were randomly divided into five groups equally. BMSCs were injected into right ear-edge vein, subarachnoid cavity through lumbar puncture in one of the two treatment groups after TBI respectively. PBS was injected into subarachnoid cavity through lumbar puncture in another group. And BMSCs were injected into subarachnoid cavity through lumbar puncture in the last group without TBI. The last two groups together with the group receiving no engraftment after TBI served as control group. 72h after marked with Ad-GFP, 2×106BMSCs cultured for 10d in vitro, were harvested and suspended in 60μl sterile PBS. Moderate TBI models of rabbits were established. The cells were autologously implanted 24h after TBI. The motor dysfunction was assessed at 24h, 1w, 2w and 4w after cells transplantation respectively. And 2 models in each group were sacrificed at 1w, 2w and 4w after cells transplantation through cardio-perfusion with 4% paraformaldehyde (PFA). Then the brain specimen underwent frozen section. And the labeled cells were counted under fluorescence microscope.
Results: The recover of motor dysfunction in treated groups were better than that in the control groups. And the rehabilitation of motor function in the group of BMSCs transplantation by lumbar puncture was more outstanding. The transplanted cells found at the injured sites were not the same. And from many to few, they are by turns: the group of transplantation by mtraparenchymal injection, by lumbar puncture, by intraventricle injection, by intravenous injection.
Conclusions: A stable moderate TBI model of rabbits was established. BMSCs transplantation through various pathways can improve the motor dysfunction rehabilitation after TBI in rabbits. Among the several commonly used methods, the therapeutics of BMSCs injection into subarachnoid cavity by lumbar puncture is optimized. An effective and minimally invasive pathway for BMSCs transplantation to treat TBI has been explored.
引文
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