【作者】 俞晓岚；

【导师】 张志坚；

【作者基本信息】 福建医科大学 ， 神经病学， 2010， 博士

【摘要】 脑梗死是中老年人致残的主要疾病之一,目前对脑梗死的治疗尚缺乏理想的方法。在脑缺血损伤模型中,通过动、静脉注射的骨髓间质干细胞(MSCs)都能够迁移、聚集到损伤部位表明局部缺血微环境可能表达某些信号以促使循环中的MSCs迁移到该区域。目前,大多数学者倾向于用基质细胞衍生因子-1( SDF-1)及其唯一特异性受体CXCR4的相互作用来解释这一现象。SDF-1/CXCR4在骨髓干细胞的动员和归巢、内皮细胞的迁移、血管的发生、粘附分子表达、移植细胞增殖和存活方面起到重要作用。研究表明组织损伤后,损伤部位分泌的SDF-1与血循环中MSCs表达的CXCR4相互作用对MSCs产生向损伤组织的趋化聚集,在移植细胞向损伤部位迁移中发挥重要作用。由于CXCR4在MSCs的表达率比较低,可能影响其向损伤部位迁移。因此我们设想通过转基因技术将外源性CXCR4基因导入MSCs,并推测提高CXCR4在MSCs的表达率能增强MSCs向脑梗死区组织的趋化聚集。因此本实验构建表达CXCR4的慢病毒载体,并转导rMSCs,采用体内、体外实验观察过表达CXCR4基因的rMSCs是否具有较高的细胞迁移率,及过表达CXCR4基因的rMSCs移植治疗脑梗死是否有更好效果。本课题分四部分。第一部分:CXCR4重组慢病毒载体(pNL-CXCR4-IRES2-EGFP)的构建及鉴定1、材料和方法1)根据Genbank大鼠CXCR4编码区序列全长及两端酶切位点需要设计上下游引物;2)用RT-PCR方法从大鼠肝脏获取CXCR4的cDNA;3)双酶切将pNL- IRES2-EGFP慢病毒载体的线性化与含有CXCR4基因片段连接,转化,小量抽提质粒DNA,进行双酶切、PCR及测序鉴定。2、结果1)酶切鉴定和测序都证实pNL-CXCR4-IRES2-EGFP质粒构建正确。3、结论1)成功构建大鼠CXCR4基因重组慢病毒载体(pNL-CXCR4-IRES2- EGFP)。第二部分:建立过表达CXCR4骨髓间充质干细胞系(CXCR4-rMSCs)1、材料和方法1)将三质粒pNL-CXCR4-IRES2- EGFP、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)用相同滴度的转CXCR4基因慢病毒、空载体慢病毒、CXCR4基因沉默慢病毒(由本课题组的前期研究所构建)转导rMSCs,建立稳定过表达CXCR4基因细胞系CXCR4-rMSCs;4)用RT-PCR、Western Blot、细胞免疫荧光组织化学和流式细胞术检测转CXCR4基因rMSCs组(CXCR4-rMSCs组)、转空载体rMSCs组(null-rMSCs组)及CXCR4基因沉默rMSCs组(siRNA-rMSCs组中CXCR4表达情况。2、结果1)三质粒pNL-CXCR4-IRES2-EGFP、pHELPER和pVSVG共转染293T细胞,包装产生慢病毒。2)转CXCR4基因、空载体及CXCR4基因沉默rMSCs强烈表达EGFP。3) RT-PCR、Western Blot、细胞免疫荧光组织化学和流式细胞术一致证实CXCR4-rMSCs组强烈表达CXCR4,siRNA-rMSCs组的CXCR4表达明显被抑制。3、结论1)成功建立稳定过表达CXCR4骨髓间充质细胞系(CXCR4-rMSCs)。第三部分:SDF-1α对rMSCs迁移作用的体外研究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,CXCR4-rMSCs、null-rMSCs及siRNA-rMSCs加入到上室,观察细胞迁移情况,计算细胞迁移指数;2)抗CXCR4多抗阻断后各组rMSCs,观察细胞迁移情况,计算细胞迁移指数。2、结果1) SDF-1α可明显促进rMSCs的跨膜迁移,而且在一定浓度范围随着SDF-1α浓度梯度增高而增强。2)相同浓度SDF-1α作用下CXCR4-rMSCs跨膜细胞迁移指数明显高于null-rMSCs及siRNA-rMSCs。3) CXCR4-rMSCs及null-rMSCs抗体阻断后迁移指数明显下降,与抗体阻断前差别有显著性意义;siRNA-rMSCs抗体阻断前与抗体阻断后迁移指数无显著改变。3、结论1)体外微迁移板实验表明,表达于rMSCs表面的CXCR4在SDF-1α介导rMSCs跨膜迁移中起很重要作用。第四部分:rMSCs静脉移植后的迁移、分化及对脑梗死保护作用的研究1、材料和方法1)取健康成年雄性SD大鼠分为四组①CXCR4-rMSCs移植组;②null-rMSCs移植组;③siRNA-rMSCs移植组;④PBS组,在MCAO后24h将各组rMSCs或PBS经股静脉注入大鼠体内,在MCAO后7天观察以下各指标;2)体视荧光显微镜下观察EGFP+细胞的分布情况;3) TTC染色测定脑梗死体积;4)检测各组大鼠神经功能(mNSS)评分的变化;5)免疫荧光组化观察CXCR4表达、神经元、神经胶质细胞及内皮细胞表面标志观察;6)激光扫描共聚焦显微镜观察梗死灶周边区脑血流灌注量。2、结果1)移植EGFP+ rMSC主要存在于大鼠的脑梗死半球大脑皮质、皮质下和海马等处,对侧半球只可见少量EGFP+细胞。CXCR4-rMSCs移植组脑梗死半球特别在缺血半暗带区EGFP+ rMSC明显多于其他各组,siRNA-rMSCs移植组脑梗死半球缺血区EGFP+ rMSC明显少于null-rMSCs移植组,PBS组大鼠大脑两侧未发现EGFP+细胞聚集。2) MCAO后7天CXCR4-rMSCs和null-rMSCs两移植组脑梗死体积均低于PBS组及siRNA-rMSCs移植组,差异有显著性意义,且CXCR4-rMSCs移植组脑梗死体积明显低于null-rMSCs移植组,两移植组间差别亦有显著性意义。3) MCAO后7天CXCR4-rMSCs和null-rMSCs移植组mNSS评分均低于PBS组及siRNA-rMSCs移植组,差异有显著性意义,且CXCR4-rMSCs和null-rMSCs两移植组间mNSS评分差别亦有显著性意义,CXCR4-rMSCs移植组功能恢复明显优于null-rMSCs移植组。4)大部分移植EGFP阳性细胞表达CXCR4蛋白,小部分移植EGFP阳性细胞表达神经元标记物NSE、星形胶质细胞标记物GFAP或血管内皮细胞标记物vWF。5) MCAO后7天CXCR4-rMSCs和null-rMSCs两移植组梗死灶周边区脑血流灌注量均高于PBS组及siRNA-rMSCs移植组,差异有显著性意义,CXCR4-rMSCs移植组梗死灶周边区脑血流灌注量明显高于null-rMSCs移植组,两移植组间差别亦有显著性意义。3、结论1)脑梗死后脑组织局部产生的SDF-1α和表达于rMSCs表面的CXCR4在介导rMSCs向损伤组织迁移中起很重要作用。2)过表达CXCR4基因rMSCs能明显增强rMSCs向损伤组织迁移,增加梗死灶周边区脑血流灌注量,从而增进脑梗死后神经功能的恢复。更多还原

【Abstract】 Ischemic stroke remains a leading cause of adult disability and no pharmaco- logical treatment is presently available to protect brain tissue from the injury that are arosed by ischemia and reperfusion. Accumulated studies suggest that bone marrow mesenchymal stem cells (MSCs) participate in neuroprotection following Stroke. Increasing evidences suggest that systemically transplanted MSCs can survive, migrate toward injured tissue, and promote recovery of neurological function following cerebral infarction. It seems that the injured tissues can 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-1) and its unique receptor, CXCR4 play an important role in stem cell migration, chemotaxis, expression of adhesion molecules, engraftment, proliferation, and survival. Recent studies have shown that SDF-1 is expressed in the ischemic boundary zone of the brain and plays an important role in the migration of the transplanted cells. Due to low native levels of CXCR4 expression in MSCs, they migrate sluggishly toward injured tissue. We hypothesized that CXCR4 gene-modified MSCs will promote stem cell recruitment and injured brain tissue regeneration.In this study, we constructed the lentiviral vectors (LV)carrying the CXCR4 and genetically engineered rMSCs overexpressing CXCR4, and focused on that over-expression of CXCR4 in rMSCs will enhance their engraftment and protect brain subjected to MCAO. This study consisted of four parts.Part1:Construction and identification of the lentiviral vector carrying CXCR4Materials and Methods1) The total RNA was isolated from SD rat’s liver with Trizol. 2) The CXCR4 gene was amplified by RT-PCR.3) CXCR4 was inserted into the transfer vector of lentivirus after being digested with restriction endonuclease.4) Then the product pNL-CXCR4-IRES2-EGFP was confirmed by sequencing and being digested with restriction endonuclease.Results1) The result of restriction enzyme digestion and sequencing showed that the full-length fragment of CXCR4 gene was successfully cloned into the transfer vector of lentivirus.Conclusions1) The lentiviral vectors carrying the CXCR4 gene were successfully constructed.Part2:Construction of rMSCs over-expressing CXCR4 Materials and Methods1) pNL-CXCR4-IRES2-EGFP 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 after transduction into 293T cells.3) Lentiviral transduction was carried out to over-express either CXCR4/EGFP (CXCR4-rMSCs group), siRNA targeting CXCR4/EGFP (siRNA-rMSCs group) or EGFP alone (null-rMSCs group) in rMSCs. The rMSCs were selected for stable integrants by using EGFP reporter gene.4) The expressing of CXCR4 gene in rMSCs was evaluated with RT-PCR, Western blotting, cellular immunofluorescence and flow cytometry.Results1) Lentiviral vector can be packaged in 293T cells by cotransfection.2) The rMSCs from CXCR4-rMSCs group, siRNA-rMSCs group or null-rMSCs group strongly expressed EGFP.3) The result of FCM showed that CXCR4 expression was significantly higher in CXCR4-rMSCs group as compared with that of null-rMSCs group, and was lowest in siRNA-rMSCs group. The result was confirmed by RT-PCR, Western blotting and cellular immunofluorescence.Conclusions1) The rMSCs overexpressing the CXCR4 gene were successfully constructed. Part3:In vitro migration assay of rMCSs induced by SDF-1Materials and Methods1) In vitro migration of rMSCs in response to SDF-1αwas assessed in a 48-well microchemotaxis chamber using polycarbonate membranes with 8μm pore size. The rMSCs 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 rMSCs were incubated by anti CXCR4 monoclonal antibody then the chemotaxis assay was performed..Results1) The result showed that exposure of rMSCs to SDF-1αcaused a robust cell migration in a concentration dependent manner.2) The number of migrated rMSCs was significantly higher in CXCR4 -rMSCs group than that in null-rMSCs group. However, the migration of the rMSCs from the siRNA-rMSCs group in response to SDF-1αwas blocked.3) The migration of the rMSCs incubated by anti CXCR4 monoclonal antibody in response to SDF-1αwas blockedConclusions1) Over expression of CXCR4 enhances the ability of rMSCs to respond to SDF-1 induced chemotaxis.Part4:Migration, differentiation, neuroprotection and angiogenesis of donor rMSCs in stroke ratsMaterials and Methods1) The healthy adult male SD rats were divided into four groups: CXCR4-rMSCs group, siRNA-rMSCs group, null-rMSCs group and PBS group.Rats were received rMSCs or PBS transplantation via femoral vein injection 24 hours after the left middle cerebral artery occlusion (MCAO).2) 7 days after MCAO, the distribution of EGFP+ cells was observed under the fluorescent microscope.3) The total infarct volumes were calculated by 2,3,5- triph-enyltetrazolium chloride (TTC) stain.4) Behavioral tests ( modified Neurological Severity Score [mNSS]) was performed 1 and 7 days after MCAO.5) The expressing of CXCR4, neuronal marker NSE, astrocytic marker GFAP, and vascular phenotypes (vWF) in EGFP+ rMSCs was evaluated with immunohistochemical fluorescence.6) The volumes of the microvessels were analysed with a laser scanning confocal imaging system .Results1) The EGFP-positive cells were found in multiple areas of the ipsilateral hemisphere including cortex, striatum, and few cells were observed in the contralateral hemisphere. The number of migrating rMSCs to damaged brain area especially in ischemic boundary zone was significantly higher in CXCR4-rMSCs group as compared with null-rMSCs group. However, the number of EGFP-positive cells was significantly reduced in siRNA-rMSCs group as compared with null-rMSCs group.2) The average infarct volumes were significantly reduced in the CXCR4-rMSCs and null-rMSCs groups compared with those of the PBS and siRNA -rMSCs groups. The infarct volume was significantly decreased in the CXCR4 -rMSCs group as compared with the null-rMSCs group. There was no significant difference found between the infarct volumes of the siRNA -rMSCs and the PBS group..3) On 7th day, the mNSS scores of the null-rMSCs and CXCR4-rMSCs groups were significantly decreased compared with those of the siRNA-rMSCs and PBS groups. In addition, the score of the CXCR4-rMSCs group was significant decrease compared with that of the null-rMSCs group. There was no significant difference between the sores of the siRNA-rMSCs and PBS group. 4) Double-label fluorescence immunohistochemistry of brain sections of CXCR4-rMSCs group revealed that a number of EGFP-positive cells in the cerebral cortex, were neuronal marker NSE and astrocytic marker GFAP positive, and several EGFP positive cells showed vascular phenotypes (vWF) positive. Most of EGFP positive cells were CXCR4 positive.5) The result of three-dimensional image acquisition of brain slices indicated that the capillary vascular volume ratios were significantly higher in both null-rMSCs and CXCR4-rMSCs groups as compared with those in the PBS and siRNA-rMSCs groups, and the ratio in the CXCR4-rMSCs group was significantly higher than that in the null-rMSCs group. There was no significant difference found between the siRNA-rMSCs and the PBS group.Conclusions1)The interaction of locally produced SDF-1αand CXCR4 expressing on the rMSCs surface plays an important role in the migration of transplanted cells to infarcted brain.2)Overexpressing of CXCR4 in rMSCs was extremely effective in their engraftment in the infarcted brain for post-infarction recovery of neural function.更多还原