摘要
研究目的
本实验采用体外分离、培养并不同浓度的超顺磁性氧化铁(superparamagnetic iron oxid, SPIO)标记骨髓基质细胞(bone marrow stem cells, BMSCs),旨在明确不同浓度SPIO对BMSCs的活性影响；观察磁标记BMSCs在体MR成像时各序列特征,以期求得活体监测SPIO标记细胞的优选序列；动态观察创伤性脑损伤(traumatic brain injury, TBI)大鼠损伤对侧细胞移植后神经功能恢复及MR活体成像特点及规律,为临床干细胞移植治疗创伤性脑损伤的疗效观察与新方法开创提供依据。
实验方法
1. BMSCs的培养及鉴定：采用全骨髓贴壁法对BMSCs进行常规培养、纯化,观察细胞光镜及透射电镜下的形态；对所获得的细胞进行表面抗原(CD34、CD44、CD45、CD105)检测；采用DMSO+BHA诱导进行神经样细胞诱导分化；并用免疫组化的方法检测诱导后细胞神经元特异性烯醇化酶(neuron specific enolase, NSE)、神经胶质原纤维酸性蛋白(glial fibrillary acidiacprotein, GFAP)及神经上皮干细胞蛋白(Nestin)的表达。
2.标记细胞的鉴定及活性分析：不同浓度SPIO (Resovist)标记BMSCs,普鲁士蓝染色及电镜下观察细胞内铁,并通过MTT法分析不同浓度Resovist对细胞活性的影响。
3.标记细胞的脑内立体定位注射及MR活体示踪：采用Feeney's自由落体方法制备大鼠TBI模型,实验动物共分4组,A组：TBI后损伤对侧移植SPIO标记BMSCs; B组：TBI后移植未标记BMSCs; C组：TBI后移植纯培养液；D组(假手术组)：仅切开头皮不进行损伤和移植。移植后不同时间点(1天、1周、2周及4周)进行MR的TSE:T1WI、T2WI,GRE:T2*WI及磁敏感加权(susceptibility weighted imaging, SWI)序列动态观察其分布及迁徙。并经神经功能评分(参照平衡木试验的评分方法)对大鼠进行功能测试,评价细胞移植的疗效,最后经组织学检查验证标记细胞在脑内的存活及迁徙。
结果
1.全骨髓贴壁法培养至P3代后,光镜下细胞表现为均匀分布的梭形成纤维样细胞；透射电镜下表现为两种不同的结构类型；免疫细胞化学染色检测结果显示CD34、CD45表达阴性,CD44及CD105表达阳性；神经方向诱导6h后,倒置相差显微镜下细胞呈神经元样细胞形态,免疫细胞化学染色NSE、GAFP、Nestin呈阳性表达。
2. Resovist标记BMSCs后,光镜下可见胞浆内散在分布的棕黄色颗粒；普鲁士蓝染色胞质内可见阳性蓝色颗粒；电镜观察可见标记BMSCs胞质内含有许多包裹铁颗粒的囊泡。MTT比色试验结果显示14μg～112μg Fe/ml培养液对细胞活性没有影响。
3. BMSCs移植后1天、1周、2周及4周的神经功能评分显示A组和C组、B组和C组评分差异有统计学意义,而A组和B组评分差异无统计学意义。MR检查结果显示A组实验动物注射区在TSE:T1WI、T2WI, GRE:T2*WI及SWI四个序列均可显示低信号,以T2*WI及SWI信号丢失明显,其中SWI最敏感。且在2周及4周时的SWI序列上更清楚的显示线样低信号沿胼胝体逐渐向损伤区迁移,组织普鲁士蓝染色亦证实标记BMSCs沿胼胝体向对侧的迁移。
结论
1.无需转染介质,Resovist能够高效的进入BMSCs胞浆内,并且在14μgFe/ml～112μgFe/ml范围的铁浓度下对细胞的生长活性没有明显影响。
2.损伤区对侧脑皮质内移植细胞可以通过胼胝体向对侧迁移,并改善损伤鼠的神经功能；且MR活体示踪SPIO标记干细胞时,磁敏感加权成像序列最敏感。
Objective
In this study, bone marrow stem cells (BMSCs) were isolated, cultured and labled with different concentration of superparamagnetic iron oxid (SPIO) in vitro, Aimed at clarifying the activity of the labled cells with different concentration of SPIO; To observe the characteristics of the labled cells in different MR sequences in vivo, aim to obtain the preferred sequence for monitoring the SPIO labled cells; Identify the SPIO labled cells dynamics in vivo when transplanted in contralateral hemisphere of TBI model. We hope to find a easy and feasible technique and methods for future clinical trials of cellular therapy in TBI.
Methods
1. Culture and identification of BMSCs:BMSCs were derived from bone marrow aspirates of healthy adult rats by adherent culture method. Cells were identified by light microscope and transmission electron microscope; And detected the cell surface antigens (CD34, CD44, CD45, CD105); Induced the capability of neural differentiation of BMSCs by DMSO+BHA, and detected the expression of neuronspecific enolase (NSE), Glial Fibrillary Acidic Portein (GFAP) and nestin by immunocytochemistry technique.
2. The labeled cells' identification and activity analysis:Check the intracellular iron by Prussian blue staining and electron microscopy of different concentrations of SPIO (Resovist) labled BMSCs, and analysis the cell activity by MTT growth curves.
3. SPIO-labeled BMSCs were grafted stereotactically in the brain and tracked by MR in vivo:The brain injury model were developed as described by Feeney et al, and all of the rats were randomly and equally divided into 4 groups.Group A: TBI rats with BMSCs labeled by SPIO; Group B:TBI rats with BMSCs unlabeled by SPIO; Group C: negative control with only medium; Group D(sham group):only cut of the epicranium of rats without injury or transplatation. MR imaging were obtained on a 3.0T MR scanner to demonstrate the location, distribution and migration of the labled BMSCs at 1 day,1,2,4 weeks after injection by measuring Turbo Spin Echo (T1、T2), T2*-weighted GRE and susceptibility weighted imaging(SWI) sequence. Neurologic examinations (balance beam test) were performed at some time-points, such as 1 day,1 week, 2 weeks, and 4 weeks after the onset of TBI until sacrifice, aim to evaluate the effect of grafted cells. Finally after histological examination verified labeled cells in the brain's survival and migration.
Results
1. P3 generation of rat BMSCs demonstrated uniform distribution of the spindle or fiber-like cells; And showed the morphology of two different cell types under transmission electron microscope; Immunocytochemical staining results of BMSCs' CD molecules exhibited positive expression of CD44 and CD105 and negative expression of CD34 and CD45; The BMSCs were culture in nerve-induced liquid for 6 hours, the cells showed neuron-like cell under inverted phase contrast microscope, and express NSE, GAFP and Nestin when given immunocytochemical staining.
2. The BMSCs were labled by the Resovist,, scattered brown granules can be seen in the cytoplasm of the cells under inverted phase contrast microscope; Positive express on Prussian Blue staining; many vesicles of iron particles can be seen in the cytoplasm under transmission electron microscope. The MTT test show that 14μg～112μg Fe/ml has no damage on cell activity.
3. Neurological function score showed that A group vs. C, B group vs. C group had statistically significant between each other 1 day,1 week,2 weeks and 4 weeks after transplantation,but A group vs. B had no statistically significant between each other at any time-point. The injected site of the experimental animals MR results showed low signal intensity on T1WI, T2WI, T2*WI and SWI sequences, the signal loss significantly on T2*WI and SWI, and the SWI is the most sensitive sequence. The SPIO labled cells can be shown more clearly to cross from the contralateral hemisphere via a transcallosal path toward the lesion site by SWI at 2w and 4w time-point, and the tissue Prussian blue staining also confirmed the labled BMSCs migrated to the opposite side via a transcallosal path.
Conclusions
1. Resovist can access the cytoplasm of BMSCs efficiently without the help of transfection media,and it has no significant impact on cell growth and activity in the range of 14μg/ml～112μg/ml iron concentration.
2. SPIO labled cells have been shown to cross from the contralateral hemisphere via a transcallosal path toward the lesion site by MRI, especially the susceptibility-weighted imaging sequence, besides, the motor function of the models were improved.
引文
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