【作者】 朱洁；

【导师】 郑健；

【作者基本信息】 第三军医大学 ， 神经病学， 2009， 博士

【摘要】 缺血性脑卒中是严重威胁中老年人健康和生活质量的重要疾病,干细胞移植是促进脑损伤修复和改善神经功能的具有实际应用前景的治疗手段。作为较理想的供体细胞,骨髓间充质干细胞(bone marrow stromal cells,BMSCs)移植在实验性脑缺血的研究中已被证明有明确的保护作用,但缺血局部微环境如何促进BMSCs趋向病灶迁移的确切机制仍不明确。趋化因子是一类结构和功能相关的多肽超家族,基本功能是诱导表达有相应受体的细胞的定向迁移。有研究提示趋化因子与其受体的作用可能参与促进移植的BMSCs在体内的定向迁移。基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)和fractalkine是为数不多组成性表达于中枢神经系统的趋化因子。当中枢神经系统发生炎症、缺血和缺氧等损伤后,病灶周围SDF-1和fractalkine表达上调,分别作用于其特异性受体CXCR4或CX3CR1,可促进小胶质细胞向病灶的迁移和活化,募集循环系统中的单核细胞、自然杀伤细胞和T淋巴细胞等,发挥清除坏死组织和促进损伤修复的作用。另有研究发现BMSCs也表达CXCR4和CX3CR1。由此提示SDF-1/CXCR4和fractalkine/ CX3CR1可能参与诱导移植的BMSCs向损伤脑组织的定向迁移,但目前仍缺乏在体研究报道。本研究旨在观察SDF-1与其受体CXCR4以及fractalkine与其受体CX3CR1的作用是否参与诱导移植的人骨髓间充质干细胞(bone marrow stromal cells,hMSCs)向缺血脑组织的定向迁移,以进一步解释移植BMSCs向脑损伤局部迁移的可能机制。方法1.人骨髓间充质干细胞经静脉注射移植后向大鼠缺血性脑损伤区的迁移以密度梯度离心贴壁筛选法分离纯化hMSCs,流式细胞仪检测细胞表面标记物进行细胞鉴定;采用线栓法制作大鼠大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)2h缺血/再灌注模型,于再灌注后24h经鼠尾静脉注射移植2×106 hMSCs;于移植后1d、3d和7d,取组织切片行小鼠抗人细胞核抗体mAb1281免疫组织化学染色,观察hMSCs在大鼠体内分布情况。2.大鼠脑缺血后SDF-1和fractalkine表达的变化线拴法制作大鼠大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)2h缺血/再灌注模型,采用real time PCR和免疫组织化学技术,观察脑缺血/再灌注后2d、4d和8d脑组织SDF-1和fractalkine mRNA和蛋白表达的变化。3.人骨髓间充质干细胞趋化因子受体CXCR4和CX3CR1的表达分离、纯化和扩增hMSCs,以real time PCR和western blotting检测hMSCs趋化因子受体CXCR4和CX3CR1表达情况;并模拟损伤微环境部分方面(低氧)培养hMSCs,观察趋化因子受体CXCR4和CX3CR1表达的变化。4.干扰CXCR4和CX3CR1基因表达对移植人骨髓间充质干细胞向缺血性脑损伤区迁移的影响采用可表达CXCR4或CX3CR1基因特异性siRNA的慢病毒载体病毒颗粒,以MOI=20的载体病毒量感染hMSCs;流式细胞仪分析测定载体病毒对hMSCs的感染效率;real time PCR和western blotting检测CXCR4和CX3CR1表达情况了解载体病毒感染后表达siRNA的干扰效率;将MCAO模型大鼠分为CX3CR1-RNAi-LV感染hMSCs移植组、CXCR4-RNAi-LV感染hMSCs移植组、阴性病毒感染hMSCs移植对照组和载体溶液注射对照组,每组按照移植后1d、3d和7d不同的时间点分为3个亚组,各组大鼠于规定时间点行全身灌注固定,观察hMSCs(GFP阳性细胞)在脑组织的分布情况。结果1.人骨髓间充质干细胞经静脉注射移植后向大鼠缺血性脑损伤区的迁移hMSCs呈长梭形、漩涡状生长,用流式细胞仪检测细胞表面标志物CD29和CD105阳性,CD14和CD45阴性;线拴法制作MCAO大鼠,术后Zea Longa五分制评分2~3分,TTC染色可见白色梗死区;hMSCs静脉移植后,在缺血/再灌注损伤侧脑组织中可观察到大量mAb1281阳性细胞,主要分布于缺血病灶周围区;在心、肝、脾、肺和肾等脏器以及损伤对侧脑组织中仅可观察到少量mAb1281阳性细胞散在分布。2.大鼠脑缺血后SDF-1和fractalkine表达的变化脑缺血/再灌注损伤2d、4d和8d组大鼠缺血侧大脑脑组织SDF-1 mRNA表达分别为正常对照组的2.285倍、2.543倍和1.710倍;fractalkine mRNA表达分别为正常对照组的1.154倍、2.453倍和1.341倍。免疫组织化学染色发现正常组大鼠脑组织中有SDF-1和fractalkine阳性表达,阳性细胞散在分布于皮质、纹状体等部位;脑缺血/再灌注损伤组大鼠脑组织SDF-1和fractalkine阳性细胞明显增多,损伤后2d、4d和8d组大鼠脑组织梗死灶周围区聚集大量SDF-1和fractalkine阳性细胞:SDF-1表达平均光密度由正常对照组的6.02±0.37,分别增高为37.8±1.30(2d组)、44.8±2.21(4d组)和30.6±1.08(8d组);fractalkine平均光密度由正常对照组的5.77±0.57,分别增高为31.3±1.80(2d组)、43.1±7.63(4d组)和30.2±4.18(8d组)。3.人骨髓间充质干细胞趋化因子受体CXCR4和CX3CR1的表达CXCR4和CX3CR1免疫细胞化学染色阳性表达分布于hMSCs的细胞膜和细胞浆;hMSCs在常规培养条件下有CXCR4和CX3CR1 mRNA表达,低氧条件下CXCR4和CX3CR1表达增高,分别为常规培养条件下的2.162倍(P<0.01)和2.524倍(P<0.05);western blotting检测发现hMSCs在正常培养条件下有CXCR4和CX3CR1表达,低氧条件下CXCR4和CX3CR1表达显著增高:CXCR4表达由正常组的0.535±0.061增高为0.901±0.029(P<0.01),CX3CR1表达由正常组的0.360±0.079增高为0.716±0.102(P<0.05)。4.干扰CXCR4和CX3CR1基因表达对移植人骨髓间充质干细胞向缺血性脑损伤区迁移的影响流式细胞分析慢病毒载体对hMSCs感染效率均高于90%。阴性病毒感染组hMSCs CXCR4和CX3CR1 mRNA表达与正常未感染组hMSCs比较无显著差异(P>0.05);CXCR4-RNAi-LV感染组hMSCs CXCR4 mRNA表达与正常未感染组hMSCs比较下降82.6%(P<0.01);CX3CR1-RNAi-LV感染组hMSCs CX3CR1 mRNA表达与正常未感染组hMSCs比较下降了74.4%(P<0.01)。western blotting检测发现阴性病毒感染组和正常未感染组CXCR4和CX3CR1表达无显著差异;CXCR4-RNAi-LV感染组较正常未感染组CXCR4表达下降80.0%(P<0.05),CX3CR1-RNAi-LV感染组较正常未感染组CX3CR1表达下降71.7%(P<0.05)。各组大鼠脑组织中均可观察到GFP阳性细胞,主要分布于缺血侧大脑半球病灶周围区;CXCR4-RNAi-LV感染hMSCs移植组和CX3CR1-RNAi-LV感染hMSCs移植组与阴性病毒感染hMSCs移植对照组相比,移植后1d、3d和7d,脑组织中GFP阳性细胞数量均显著减少(P<0.01)。结论1.静脉注射移植的骨髓间充质干细胞向缺血性脑损伤区定向迁移;2.缺血性脑损伤区趋化因子SDF-1和fractalkine表达增高;3.骨髓间充质干细胞表达趋化因子SDF-1受体CXCR4和fractalkine受体CX3CR1;4.干扰SDF-1受体CXCR4或fractalkine受体CX3CR1的基因表达后,移植的骨髓间充质干细胞向缺血性脑损伤区的迁移减少;5.趋化因子SDF-1、fractalkine与其特异性受体CXCR4、CX3CR1的相互作用参与诱导移植的骨髓间充质干细胞向缺血性脑损伤区的迁移。更多还原

【Abstract】 Ischemic stroke has become one of major diseases to harm the health and life quality of the aged. Stem cell transplantation can promote the repair of brain damage and improve neurological function. It has good prospects in clinical applications. As ideal donor cells, transplanted bone marrow stromal cells (BMSCs) have been demonstrated to have protective effects by many experimental studies of cerebral ischemia. The specific mechanisms involved in their migration to lesions are still to be fully elucidated. Chemokines form a superfamily that shares a common structure and function-related peptide. The basic function of a chemokine is to induce the directional chemotaxis of cells with corresponding receptors. It has been reported that chemokines and their receptors may be involved in the promotion of directional migration of transplanted BMSCs. Stromal cell-derived factor-1 (SDF-1) and fractalkine are the only chemokines constitutively expressed in the central nervous system (CNS). Studies have shown up-regulated expression of SDF-1 and fractalkine in select CNS lesions, such as those caused by inflammation, ischemia and hypoxia. Through interaction with their receptors, CXCR4 and CX3CR1 respectively, they promots the activation of microglial cells and their migration to the lesions, elevates the number of mononuclear cells, natural killer cells and T lymphocytes in the blood, plays a critical role in the clearance of necrotic tissue and promotes neurofunction repair. In other situations, it may aggravate the injury. Other studies found that BMSCs expressed CXCR4 and CX3CR1. These results indicate that SDF-1/CXCR4 and fractalkine/CX3CR1 may be involved in the induction of directional migration of transplanted BMSCs to injured brain regions. But there is still a lack of in vivo studies have reported. This study aimed to explore whether SDF-1/CXCR4 and fractalkine/CX3CR1 play an important role in the induction of directional migration of transplanted hMSCs to ischemic brain tissue. Furthermore, we aimed to explore the possible mechanisms involved in BMSCs migration.Methods1.The migration of intravenously grafted human bone marrow stromal cells toward ischemic brain lesionshMSCs were isolated from human bone marrow by combination of gradient centrifugation and different adherent time method. Cell surface markers were tested by flow cytometer. The transient middle cerebral artery occlusion (MCAO) was induced using a method of intraluminal vascular occlusion. At 24 hours after the onset of cerebral ischemia, model animals received 2×106 hMSCs transplantation. At 1, 3, and 7 days after cell transplantation, the directional migration of transplanted hMSCs to the damaged region was observed through detection of mAb1281 positive cells.2.SDF-1 and fractalkine expression in the infarcted brain after transient middle cerebral artery occlusionA rat model of MCAO was established. At 2, 4 and 8 days after cerebral ischemia, the model animals were sacrificed, the tissues were processed, and the expression of SDF-1 and fractalkine in the ischemic brain was determined by real time PCR and immunohistochemistry.3.CXCR4 and CX3CR1 expression in human bone marrow stromal cells hMSCs were isolated, purifIed and amplified. The CXCR4 and CX3CR1 expression were detected by real-time PCR , western blotting and immunocytochemistry. Then, after short-term exposure of hMSCs to 3% oxygen, the changes of CXCR4 and CX3CR1 expression were detected.4.The impact of down-regulation of CXCR4 or CX3CR1 expression to the migration of transplanted human bone marrow stromal cells to the damaged brainhMSCs were transduced with CXCR4 or CX3CR1 shRNA construct by lentivirus-mediated gene transfer at a multiplicity of infection (MOI) of 20. Transduction efficiency was measured by determining the frequency of green ?uorescent protein (GFP)-positive cells using flow cytometry. Cells were harvested 5-7 days following transduction, CXCR4 or CX3CR1 expression was determined by real-time PCR and western blotting. The MCAO rats were divided into 4 groups: rats in Group 1 (n=18) received 2×106 CXCR4 shRNA transduced hMSCs; rats in Group 2 (n=18) received CX3CR1 shRNA transduced hMSCs; rats in Group 3 (n=18) received 2×106 control-transduced hMSCs; rats in Group 4 (n=18) received PBS as a control. Rats were sacrificed at 1, 3, and 7 days after transplantation, and hMSCs distribution were analyzed by immunohistochemistry.Results1.The migration of intravenously grafted human bone marrow stromal cells toward ischemic brain lesionsAll of the BMSCs had a fibroblast-like morphology in culture and were uniformly positive for CD105 (99.38%) and CD29 (99.13%) and negative for CD34 (0.78 %) and CD45 (0.40 %), as determined by flow cytometry. MCAO was induced using a method of intraluminal vascular occlusion. Once the animals awoke, behavioral changes were evaluated according to the standard described by Longa et al. Rats with scores of 2-3 were included in the next experiments. The brains were stained with TTC 24 hours after MCAO. Normal brain (gray matter) tissue typically stains with TTC, but infarcted lesions show no or reduced staining. At 1, 3, and 7 days after cell transplantation, the transplanted hMSCs were mainly distributed in the ischemic hemisphere. Only a few of mAb1281 positive cells scattered in the heart, liver, spleen, lung and kidney.2.SDF-1 and fractalkine expression in the infarcted brain after transient middle cerebral artery occlusionSDF-1 mRNA expression was found to be up-regulated in the injured hemisphere on days 2, 4 and 8, in comparison with the normal control tissues (2.285, 2.543, and 1.710 times respectively). fractalkine mRNA expression was 1.154, 2.453, and 1.341 times higher at 2, 4, and 8 days respectively (P<0.05). Similarly, SDF-1 and fractalkine expression was dramatically up-regulated in the injured hemisphere at 2, 4, and 8 days after ischemia, as determined by immunofluorescence analysis. Quantification revealed that the density of SDF-1 or fractalkine-immunoreactive cells in the ischemic hemisphere was significantly (p<0.01) increased at 2, 4, and 8 days after ischemia.3.CXCR4 and CX3CR1 expression in human bone marrow stromal cellsThe result of immunocytochemistry revealed the localization of CXCR4 and CX3CR1 expression on the membranes and in the cytoplasm. In vitro study using real-time PCR and western blotting revealed that CXCR4 and CX3CR1 were expressed in normal cultured hMSCs. Exposure of hMSCs to 3% oxygen increased expression of the CXCR4 and CX3CR1, both as mRNA and as protein.4.The impact of down-regulation of CXCR4 or CX3CR1 expression to the migration of transplanted human bone marrow stromal cells to the damaged brainTransduction efficiencies were more than 90%, as indicated by the frequency of GFP-positive cells by ?ow cytometry. Real-time PCR analysis revealed that, the CXCR4 mRNA level in cells expressing the CXCR4 shRNA construct were about 80-fold lower (P<0.01) than those of non-transduced and control-transduced cells, and the CX3CR1 mRNA level in cells expressing the CX3CR1 shRNA construct were about 70-fold lower (P<0.01) than those of non-transduced and control-transduced cells. Also, in the hMSCs transduced with CXCR4 or CX3CR1 siRNA, a reduced level of CXCR4 or CX3CR1 protein was detected by western blotting. GFP-labeled cells were more abundant (P<0.01) in the ischemic hemisphere of rats injected with control-transduced hMSCs, than in that of rats injected with CXCR4 knock-down or CX3CR1 knock-down hMSCs at 1, 3 and 7 days after cell transplantation.Conclusion1.Intravenously grafted bone marrow stromal cells can directionally migrate to ischemic brain lesion.2.SDF-1 and fractalkine expression was up-regulated in the ischemic brain.3.Bone marrow stromal cells expressed CXCR4 the receptor of SDF-1and CX3CR1 the receptor of fractalkine.4.CXCR4-knockdown or CX3CR1-knockdown dramatically decreased the migration of transplanted human bone marrow stromal cells to the ischemic brain.5.The interactions of SDF-1 with its specific receptor CXCR4 and fractalkine with its receptor CX3CR1 were involved in the directional migration of transplanted bone marrow stromal cells to the ischemic damaged brain region.更多还原