【作者】 陈东平；

【导师】 张志坚；

【作者基本信息】 福建医科大学 ， 神经病学， 2009， 博士

【摘要】 缺血性脑卒中是中老年人致死致残的主要疾病之一,目前尚无有效药物能够阻止脑缺血和再灌注损伤引起的多重神经损害。许多动物实验表明,骨髓间质干细胞(MSCs)经全身或局部移植后能促进卒中后神经功能的修复。在大鼠脑卒中和创伤模型中,经静脉或大脑内直接注射MSCs后,MSCs可穿过血脑屏障向损伤脑组织聚集,进而修复神经功能。其中,损伤组织似乎可特异吸引MSCs向损伤区迁移,然而,目前对调节MSCs迁移进而向损伤脑组织聚集的机制还不清楚。已知基质细胞衍生因子-1α(SDF-1α)和其唯一特异受体CXCR4与干细胞定向迁移和损伤修复密切相关。在大鼠心肌梗死模型中,SDF-1在促使表达CXCR4的MSCs向缺血心肌的定向迁移、聚集中起很重要作用。而SDF-1α/CXCR4在低氧条件下表达增加,如急性肾衰、缺血性心脏病、脑缺血。为此,推测SDF-1α/CXCR4在MSCs向缺血脑组织的定向迁移中可能也起很重要作用。RNA干扰(RNAi)技术可稳定沉默哺乳动物细胞内的靶向基因,是研究基因功能的强大工具。为探讨脑缺血损伤中MSCs的迁移和调节机制,我们首先构建CXCR4 RNAi慢病毒载体,进而建立CXCR4基因稳定沉默的大鼠MSCs(rMSCs)。随后通过体内、外实验研究CXCR4基因沉默和SDF-1α对rMSCs迁移的影响。本课题分三部分。第一部分:CXCR4基因RNAi慢病毒载体的构建及鉴定1、材料和方法1)根据大鼠CXCR4 mRNA序列,选择三个21nts的靶序列,设计并合成包含各正、反义靶序列的互补DNA链;2)退火后插入pSUPER载体的H1 RNA启动子后面,产生pRiCXCR4a, b, c;3)酶切电泳和测序鉴定;4)质粒鉴定正确后,将其中的CXCR4 shRNA表达结构酶切,插入到慢病毒载体质粒pNL中,产生pNL-RiCXCR4a, b, c。2、结果1)酶切鉴定和测序都证实pRiCXCR4质粒构建正确。2)将pRiCXCR4中的CXCR4 shRNA表达结构插入pNL质粒,产生能同时表达增强型绿色荧光蛋白(EGFP)和转录产生CXCR4 shRNA的慢病毒载体质粒,测序鉴定正确。3、结论1)成功构建大鼠CXCR4 shRNA表达载体pRiCXCR4。2)成功构建大鼠CXCR4基因RNAi慢病毒载体pNL-RiCXCR4。3)为通过阻断大鼠CXCR4基因表达进而研究CXCR4功能奠定了基础。第二部分:CXCR4基因沉默的骨髓间质干细胞的构建、培养与鉴定1、材料和方法1)将三质粒pNL-RiCXCR4、pHELPER和pVSVG共转染293T细胞,包装生产慢病毒;2)经超速离心收集病毒颗粒,转导293T细胞,用流式细胞术测定EGFP蛋白表达率来测定慢病毒功能滴度;3)用相同滴度的慢病毒转导rMSCs ,建立CXCR4基因稳定沉默细胞系rMSCs/pNL-RiCXCR4(MSC-CXCR4-);4)用实时定量RT-PCR、Western Blot和流式细胞术检测CXCR4 mRNA、蛋白质和细胞表面表达水平,筛选有效的CXCR4基因沉默的细胞系MSC-CXCR4-;5)绘制细胞生长曲线,计算细胞倍增时间,比较CXCR4基因沉默的rMSCs与亲本细胞的生长变化。2、结果1)三质粒pNL-RiCXCR4、pHELPER和pVSVG共转染293T细胞,包装产生慢病毒。2)未浓缩含病毒细胞培养上清和浓缩病毒悬液的功能滴度分别为6.4×104TU/mL和6.9×106TU/mL。3) CXCR4基因稳定沉默细胞MSC-CXCR4-强烈表达EGFP。4)实时定量RT-PCR、Western Blot和流式细胞术一致证实pNL-RiCXCR4b组的CXCR4抑制作用最显著。5)亲本细胞、非特异细胞和CXCR4基因沉默细胞的生长曲线和倍增时间无明显差异。3、结论1)包装产生高滴度慢病毒,并能高效转导rMSCs。2)建立CXCR4基因稳定沉默细胞系MSC-CXCR4-。3)实时定量RT-PCR分析CXCR4 mRNA、Western Blot检测CXCR4蛋白和流式细胞术检测rMSCs表面CXCR4抗原,均证实所构建的慢病毒载体能使rMSCs的CXCR4基因表达显著下调。4)细胞生长曲线测定实验证明,CXCR4基因沉默rMSCs的生长未受到明显影响。第三部分:CXCR4基因沉默及SDF-1α对骨髓间质干细胞迁移作用的体内、外实验研究1、材料和方法1)体外实验在内置8μm孔径聚碳酸酯膜的48孔微迁移板中进行,趋化因子SDF-1α以不同浓度加入到迁移板下室,细胞rMSCs或CXCR4基因沉默的rMSCs加入到上室,观察细胞迁移情况,计算细胞迁移指数。2)取健康成年雄性SD大鼠制作短暂大脑中动脉阻塞(MCAO)模型。在MCAO前24h或MCAO后24h将表达EGFP(EGFP+)的rMSCs经股静脉移植入大鼠体内。其中,EGFP+的CXCR4基因沉默的rMSCs仅移植入MCAO后24h的大鼠体内。3)将EGFP阳性(EGFP+)的rMSCs经股静脉移植入正常大鼠体内后,立即将SDF-1α直接注射入左侧大脑皮质。作为对照,对侧相应部位只注射PBS。4)取大鼠骨髓制作涂片,同时取脑、心、肺、肝、脾、肾、骨骼肌、小肠组织标本制作冰冻切片,在荧光显微镜下观察EGFP+细胞的分布情况。5)为明确脑组织中SDF-1α表达和EGFP+细胞迁移之间的定位关系,对脑组织冰冻切片进行SDF-1α的免疫荧光染色。2、结果1)体外微迁移板实验表明,加入200ng/mL SDF-1α后可明显促进rMSCs的跨膜迁移,而rMSCs中CXCR4基因沉默后,rMSCs的跨膜迁移明显减弱。2)对MCAO前大鼠或对未进行MCAO的正常大鼠移植EGFP+细胞后,可在其肺、骨髓、脾、小肠发现EGFP+细胞,而在其他组织未发现。3)对MCAO后大鼠移植EGFP+细胞后,发现大量EGFP+细胞聚集于大脑半球缺血半暗带区,在肺中也可见EGFP+细胞分布,在缺血对侧大脑半球及其他组织中未发现。而rMSCs中CXCR4基因沉默后,这种EGFP+细胞在大脑半球缺血区的聚集作用被明显抑制。4)免疫荧光染色显示,在脑组织切片中SDF-1α主要由缺血半暗带区神经元和星形胶质细胞分泌,EGFP+细胞的数量和定位与SDF-1α的表达量大体一致。3、结论1)移植前的脑组织急性损伤对移植的rMSCs向脑局部缺血损伤区定向迁移起决定性作用,否则移植细胞将在骨髓和脾脏较长时间停留且不易再次迁移。2)损伤脑组织局部产生的SDF-1α和表达于rMSCs表面的CXCR4在介导rMSCs的损伤组织定向迁移中起很重要作用,在MSCs移植治疗脑梗死中,我们可能可通过调节SDF-1α/CXCR4表达以达到增进治疗作用的目的。更多还原

【Abstract】 Stroke is a leading cause of death and no pharmacological treatment is presently available to protect brain tissue from the multiple neurochemical cascades that are triggered by ischemia and reperfusion. Mounting experimental data obtained from animal models suggest that bone marrow mesenchymal stem cells (MSCs), administered locally or systemically, can improve functional outcome after stroke. In rat models for stroke and trauma, intravenously and transcerebrally injected MSCs have been demonstrated to go through the blood-brain barrier and accumulate in the parenchyma of the injured brain, and thereby induce neurological and functional recovery. It therefore seems that the injured tissues can specifically attract MSCs and mediate their migration behavior. However, the mechanisms regulating MSCs migration and accumulation in the injured brain remain to be revealed.Stromal cell-derived factor-1 (SDF-1α) and its cellular receptor CXCR4 have been demonstrated to direct the migration of stem cells associated with injury repair in many species and tissue types. In rat myocardial infarction model, SDF-1 plays an essential role in promoting the homing in of cells toward the ischemic myocardium by recruiting MSCs that express CXCR4. Expression of both SDF-1α/CXCR4 is predominantly promoted in hypoxic conditions, such as acute renal failure, ischemic cardiomyopathy, and ischemic brain. This raises the possibility that the SDF-1α/CXCR4 axis also plays essential roles in directing MSCs migration in ischemic brain.RNA interference (RNAi) technology, silencing targeted genes in mammalian cells, has become a powerful tool for studying gene function. In an effort to investigate the migration of rat MSCs (rMSCs) within the injured brain and the regulating mechanisms, we first constructed CXCR4 RNAi lentiviral vector and established CXCR4 gene silencing rMSCs. Then we focused on characterizing the effect of CXCR4 gene silencing and SDF-1αon the migration of rMSCs in vitro and vivo. This study consisted of three parts. Part1:Construction and identification of the lentiviral RNAi vector of CXCR4 gene Materials and Methods1) Three specific target sequences were selected according to rat CXCR4 mRNA. The complementary DNA which contained both sense and antisense oligonucleotides were synthesized.2) After phosphorylation and annealing, these double strands DNA were cloned to BglⅡand HindⅢsites of pSUPER.3) Then the product pRiCXCR4a, b, c was confirmed by electrophoresis and sequencing.4) CXCR4 shRNA was cloned to XbaI site of pNL, a transfer vector of lentivirus. Then the product pNL-RiCXCR4a, b, c were obtained.Results and Conclusions1) It is confirmed by restriction enzyme digestion and sequencing that CXCR4 shRNA expression structure is correctly cloned to pSUPER and pNL respectively.2) RNAi lentivirus vector of rat CXCR4 gene has been constructed, which is the essential building block required for the CXCR4 functional research. Part2:Establishment, culture and identification of CXCR4 gene silenced MSCsMaterials and Methods1) pNL-RiCXCR4 was cotransfected along with pHELPER and pVSVG into 293T to package lentivirus particles.2) According to the enhanced green fluorescent protein (EGFP) expression,the functional titer was determined by flow cytometry (FCM) after transduction into 293T cells.3) The rMSCs were transduced with lentiviral vectors at 2MOI and selected for stable integrants by using EGFP reporter gene to establish stable silenced cell line.4) Real-time RT-PCR analysis of CXCR4 mRNA, Western Blot analysis of CXCR4 protein and FCM analysis of CXCR4 on the cell membrane were conducted to compare the RNAi effect.5) The growth curve was obtained and the population doubling time was calculated to compare the growth of CXCR4-downregulated rMSCs and their parental cells.Results and Conclusions1) After cotransfection, lentiviral vector can be packaged in 293T cells. 2) The functional titer of unconcentrated virus and concentrated virus are 6.4×104TU/mL and 6.9×106TU/mL respectively.3) CXCR4 silenced cell line MSC-CXCR4- was successfully established.4) Down-regulation of CXCR4 expression in rMSCs was confirmed by Real-time RT-PCR, Western blot and FCM.Part3:Effect of CXCR4 gene silencing and SDF-1αon the migration of rMSCs in vitro and vivoMaterials and Methods1) In vitro migration of rMSCs in response to SDF-1αwas assessed in a modified 48-well microchemotaxis chamber using polycarbonate membranes with 8μm pore size. The cells were added to the upper chambers. SDF-1αwas added to the lower wells in different concentrations. The migrated cells were counted and the migration index was calculated to show the difference of migration.2) The healthy adult male SD rats were subjected to transient middle cerebral artery occlusion (MCAO).The rMSCs expressed EGFP (EGFP+) were infused into the femoral vein of rats at 24 hours before or after MCAO. The rMSCs with CXCR4 gene downregulation were also transplanted at 24 hours after MCAO.3) Immediately following femoral vein injection of EGFP-labeled rMSCs, the SD rats received an intracerebral injection of SDF-1αinto the left cerebral cortex. As control, the opposite side was injected with PBS alone.4) The tissue samples of brain, heart, lung, liver, spleen, kidney, skeletal muscle, small intestine and bone marrow of rats were collected. Then the frozen sections and bone marrow smear were prepared. The distribution of EGFP+ cells was observed under the fluorescent microscope.5) To clarify the relation and location of SDF-1αand EGFP-labeled rMSCs in the brain, SDF-1αimmunofluorescence staining was performed in the brain frozen sections.Results and Conclusions1) In vitro chemotaxis assay, the number of migrating cells significantly increased in the concentration of 200ng/mL SDF-1α, and significantly decreased while CXCR4 was knockdown with siRNA.2) In the groups of rats transplanted before or without MCAO, EGFP+ cells were found in bone marrow sample, lung, spleen and intestine samples, but not in the other tissues. 3) In the group of rats transplanted after MCAO, EGFP+ cells were localized in the ischemic penumbra, few marked cells were observed in the contralateral cerebral hemisphere or other tissues except a few in lung. CXCR4 downregulation in rMSCs exhibit strong inhibitory effects on rMSCs migration to the ischemic region of brain.4) Immunofluorescence staining showed that the source of SDF-1αis mainly from neurons and astrocytes in the ischemic penumbra, and the location and density of EGFP+ cells appears to be roughly associated with the level of SDF-1αimmunoreactivity in the brain frozen sections.5) The cerebral injury before transplantation is very important and necessary for intravenously grafted rMSCs selectively migrating to ischemic focus, otherwise, the grafted rMSCs will settle down in bone marrow, spleen, and never migrate to other tissues again.6) The interaction of locally produced SDF-1αand CXCR4 expressed on the rMSCs surface plays an important role in the migration of transplanted cells, suggesting that it might be a potential approach to modulate the expression of the two molecules in order to further facilitate the therapeutic effects using MSCs.更多还原