【作者】 徐锋；

【导师】 朱剑虹； 周良辅；

【作者基本信息】 复旦大学 ， 神经外科学， 2007， 博士

【摘要】 目的:(1)探讨骨髓间充质干细胞(MSCs)靶向胶质瘤迁移的能力。(2)明确超顺磁性氧化铁纳米粒子(SPIO)体外标记MSCs的适当浓度和不同标记浓度对细胞的生物学活性影响。(3)应用1.5T MRI探讨MSCs靶向颅内胶质瘤迁移的分布模式。(4)探讨单核细胞趋化蛋白-1(MCP-1)和基质细胞衍生因子-1a(SDF-1a)在MSCs靶向胶质瘤迁移过程中的作用。方法:(1)分离培养大鼠MSCs,应用增强型绿色荧光蛋白(EGFP)标记MSCs,观察MSCs在体外向胶质瘤细胞迁移的模式,Transwell系统定量分析MSCs的迁移能力。将LacZ标记的MSCs经静脉注入胶质瘤苛瘤大鼠体内,2 w后取下脑、心脏、肺脏、肝脏和肾脏进行X-gal染色观察MSCs在脑胶质瘤苛瘤大鼠的全身分布。(2)体外不同浓度SPIO标记MSCs,观察不同标记浓度细胞活力的改变及标记率,测量并绘制未标记细胞和标记细胞的MTI生长曲线,选取适当浓度标记量。(3)运用SPIO和EGFP双重标记MSCs,静脉移植双标细胞后应用MRI观察MSCs在颅内胶质瘤的时空分布。普鲁士蓝和免疫荧光染色检测MSCs靶向颅内胶质瘤迁移的分布模式。(4)通过RT-PCR及流式细胞仪检测第二代MSCs表达趋化因子受体CCR2和CXCR4的情况:利用Transwell系统探讨趋化因子MCP-1和SDF-1a对MSCs的体外趋化作用;抗体阻断后观察其对MSCs的胶质瘤趋向性的影响。结果:(1)将转染AAV-EGFP的MSCs与F98胶质瘤细胞共同培养后,绿色的MSCs聚集于F98克隆中,而在其它部位均不见绿色MSCs的分布。在不同时间点观察到MSCs在体外向胶质瘤细胞迁移的现象,随着观察时间的延长,更多的绿色MSCs向F98克隆聚集。体外研究显示正常脑组织溶解物或生理盐水不能刺激MSCs和成纤维细胞向其迁移,F98胶质瘤溶解物和F98胶质瘤细胞能显著诱导MSCs向其迁移。单因素方差分析表明不同类型细胞迁移能力存在显著不同(F=22.34;P＜0.001),不同刺激因素诱导MSCs的迁移能力也存在显著的不同(F=7.65;P=0.0002)。MSCs体外迁移能力明显强于成纤维细胞;F98胶质瘤细胞和F98胶质瘤溶解物较其它刺激因素具有更强的趋化作用。MSCs经静脉移植14d后,X-gal染色观察蓝色细胞很少分布于检测器官,而在脑肿瘤中则有大量蓝色的MSCs分布于其中。脑肿瘤内的蓝色MSCs(97.52±16.13/section)显著高于对侧正常脑组织(7.64±2.31/section)和其它器官(心脏:0/section;肺脏:3.76±1.74/section;旰脏:9.64±1.55/section;肾脏:19.78±2.63/section;脾脏:5.74±1.98/section)P<0.001)。(2)体外标记的MSCs普鲁士蓝染色见细胞浆内有许多蓝染的铁颗粒。25μg/ml标记组孵育24h和72h后,台盼蓝染色计数死细胞数约为12%-15%,与未标记细胞(约15%)相比无明显统计学差异。在100μg/ml和250μg/ml标记组孵育24h和72h后,台盼蓝染色计数活细胞数(约57%-78%)明显低于未标记细胞组(约85%)(P＜0.001)。MTT法检测发现SPIO标记MSCs的增殖活力与未标记MSCs相比无改变。1d,3d,1w和2w时测得的OD值比较,两组之间比较无统计学差异。(3)移植7d后,SPIO/EGFP双标细胞侵入肿瘤中,散布于肿瘤内部。14d后,双标细胞不再分布于肿瘤内部,大多数位于肿瘤和正常脑组织之间沿肿瘤边界分布。双标MSCs不仅分布于侵入边缘或肿瘤内部,并能够追踪浸润正常脑组织的肿瘤细胞。1.5 TMRI扫描发现,双标细胞经静脉移植7d后,肿瘤内部呈现一小片界限清晰的低信号暗区,而在移植未标记细胞的对照组则没有信号改变。14d后,在肿瘤的边缘呈现为不连续的弧形低信号。MRI显像所示低信号区与组织切片普鲁士蓝染色相符合。(4)MSCs表达趋化因子受体CCR2和CXCR4;当MCP-1浓度为4、20 ng/ml时,MSCs的迁移数量较对照组有显著性差异(P＜0.01),100、500ng/ml组较对照组无显著性差异。SDF-1a诱导MSCs的趋化作用呈剂量依赖关系,在100 ng/ml浓度时达高峰;抗MCP-1抗体或抗CXCR4抗体显著抑制胶质瘤细胞条件培养基对MSCs的趋化作用。结论:(1)MSCs具有高度特异性地向胶质瘤迁移的能力。(2)SPIO可以简便标记MSCs,并且在适当浓度下对MSCs的生物学活性没有影响。(3)MRI可以实时观察磁性标记MSCs在胶质瘤中的时空分布模式。(4)趋化因子MCP-1和SDF-1a介导MSCs的胶质瘤趋向性。更多还原

【Abstract】 Objective (1) To investigate the tropism capacity of rat mesenchymal stem cells (MSCs) for gliomas. (2) To in vitro evaluate the labeling efficiency of (MSCs) with different labeling concentrations of superparamagnetic iron oxide (SPIO) nanoparticels. (3) To delineate the pattern of MSCs distribution in glioma, and to track the migration and incorporation of magnetically labeled MSCs with clinical 1.5T MRI. (4) To investigate whether macrophage chemoattractant protein-1 (MCP-1) and stromal cell-derived factor-1a (SDF-1a) could play important roles in the migration of MSCs toward gliomas.Method (1) MSCs were isolated from Fischer 344 rats, cultured and labeled with enhanced green fluorescence protein (EGFP); The procedure of MSCs tropism to a F98 clone was captured by sequential photograph at 12 hours interval and the tropism capacity of MSCs was quantitatively assayed by Transwell system in vitro. For assessing the distribution of MSCs throughout the brain glioma burdened rats, their brain, heart, lungs, liver, kidneys and spleen examined histologically at 14 days after MSCs transplantation. (2) Various concentrations of SPIO nanoparticles were used to magnetically label cells. Cell viability was evaluated by trypan blue dye exclusion assay in relation to the concentration of SPIO and incubation times. To assess the effects of the particles on cell proliferation, MTT growth curves of each group were obtained at different time points. (3) Fisher344 rats MSCs were co-labeled with superparamagnetic iron oxide nanoparticles (SPIO) and enhanced green fluorescence protein (EGFP). To in vivo track the migration of MSCs, magnetic resonance imaging (MRI) was performed at 7 days and 14 days after systematic administration of labeled MSCs. After scanned, the distribution patterns of MSCs in glioma burdened rats were examined by Prussian blue and fluorescence staining. (4) RT-PCR and flow cytometry analysis were used to detect whether MSCs, in vitro, expressed CCR2 and CXCR4. The effect of MCP-1 and SDF-1a on MSCs migration was studied with migration assay. For neutralization studies, glioma cell conditioned medium was incubated with rabbit anti-rat MCP-1 or rat MSCs were incubated with anti-CXCR4 polyclonal antibody.Result (1) To visualize the migratory properties of MSCs, in vitro studies first assessed the relative migratory capacity of MSCs when mixed with glioma cells. 48 hours later, most of GFP labeled MSCs aggregated around the clone of glioma cells, while merely plated at any other place. To further quantitatively evaluate the migratory pattern of MSCs towards glioma cells, we assay the migration rate through calculating EGFP labeled green MSCs in the lower chamber of Transwell system by fluorescent microscope. Normal brain tissue lysate or saline induced only minimal migration of MSCs and fibrablast cells in vitro. By contrast, lysate from F98 gliomas and cultured F98 cells induced migration of MSCs. Using one-way ANOVA, we found a highly significant difference in the cell migration pattern between the different populations of cells (F=22.34; P<0.001). And there were significant differences between the different stimuli (F=7.65; P=0.002). Importantly, MSCs possessed significantly greater migratory capacity than fibrablast cells (P<0.001), and lysate of F98 glioma / cultured F98 cells showed more capacity to induce migration of cells than other stimuli (P<0.05). For assessing the distribution of MSCs throughout the brain glioma burdened rats, their brain, heart, lungs, liver, kidneys and spleen examined histologically at 14 days after MSCs transplantation. X-gal stained cells were found scarcely distributed in the organs examined, except for brain tumor mass, where blue MSCs were highly concentrated. The number of blue MSCs was significantly higher (MSCs in brain tumor versus MSCs in other tissues respectively, P<0.001) in the brain tumors (97.52±16.13/section) compared to contralateral side of nontumor-bearing brain (7.64±2.31/section) and other organs (heart: 0/section; lungs: 3.76±1.74/section; livers:9.64±1.55/section; kidneys: 19.78±2.63/section; and spleen: 5.74±1.98/section). (2) Prussian blue staining showed numerous blue stained particles in the cytoplasm of the labeled cells. When the iron concentration was 25μg/ml, no significant difference (i.e., labeled cells [12%-15%] vs. unlabeled cells [approximately 15%]) was detected in the percentage of dead cells compared with the unlabeled control MSCs at 24 and 72 hr. However, at the two highest concentrations of iron per milliliter evaluated (i.e., 100 and 250μg/ml), the percentage of viable cells (57%-78%) was significantly (P<0.001) decreased compared with the percentage of viable unlabeled control cells of 85% at 24 and 72hr. There is no difference of cell proliferation viability between SPIO-labeled and unlabeled MSCs by MTT detecting method. (3) At the 7 days after MSCs transplantation, SPIO/EGFP co-labeled MSCs infiltrated the tumor and distributed throughout the tumor. While, at the 14 days, the co-labeled cells no longer scattered through the tumor, but mostly found at the border between tumor and normal parenchyma. This result revealed that incorporation of systemically transplanted MSCs to brain tumors varied with the development of tumor. Moreover, SPIO labeled MSCs could not only be seen to distribute themselves to its invading edge or throughout the tumor, but could be seen to "trail" individual aggressive, dark red, elongated infiltrating tumor cells that migrate away from the main tumor mass. Additionally, EGFP-labeled MSCs were found to incorporate into the vessels at the edge of the tumor, and some labeled MSCs penetrated the vessels and streamed as a chain pattern toward glioma. In vivo MRI did demonstrate a hypointense region with small and well-defined dark features in the tumor at day 7 post transplantation in animals receiving the intravenous injection of SPIO labeled cells, as compared to animals that received unlabeled cells. At 14 days after transplantation, the hypointense areas developed as discontinued amorphous dark curves at the margin of tumor. For this method, we matched the MR images with histological sections as much as possible. The hypointense signals on MR images in panel are correspondent with multiple Prussian blue stained cells in the histological sections. (4) RT-PCR and FACS showed that MSCs express CCR2 and CXCR4, the respective receptors for MCP-1 and SDF-1a. In vitro analysis revealed that MCP-1 and SDF-1a induce the migration of MSCs. Futhermore, addition of the anti-MCP-1 neutralizing antibody or anti-CXCR4-blocking antidoby significantly attenuated the migration of MSCs toward glioma cell conditioned medium.Conclusion (1) MSCs have the ability to migrating toward gliomas. (2) MSCs can be easily and efficiently labeled by SPIO without interference on the cell viability and proliferation. (3) Systemically transplanted MSCs ’home’ to glioma with high specificity with a temporal-spatial pattern, which can be tracking by MRI. (4) MCP-1 and SDF-1a mediate the migration of MSCs toward gliomas in vitro.更多还原