【作者】 张勇；

【导师】 程敬亮；

【作者基本信息】 郑州大学 ， 影像医学与核医学， 2010， 博士

【摘要】 背景和目的：缺血性脑血管病(ischemic cerebrovascular disease, ICVD)是一种致死率和致残率均很高的脑血管疾病,随着我国老龄化人口的增多,其发病率呈逐年升高趋势。ICVD的治疗手段有限,这也是其预后不良的主要原因。随着干细胞生物学特性研究的深入,冲破了成年生物体内神经细胞不可再生的传统观念,干细胞移植治疗ICVD已经成为研究的热点。尤其是活体状态下监测干细胞移植后在宿主脑组织中的分布、迁移及转归,对于评估干细胞移植后的疗效至关重要。本研究包括以下三部分内容：①体外分离、培养、纯化及鉴定大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs);②超顺磁性氧化铁颗粒(superparamagnetic iron oxide, SPIO)标记大鼠BMSCs,并探讨磁标记对干细胞生物学活性及向神经样细胞(neurocyte-like cells)分化的影响；③干细胞移植入大脑中动脉阻塞(middle cerebral artery occlusion, MCAO)大鼠脑内后,使用磁共振成像(magnetic resonace imaging, MRI)活体监测移植干细胞的存活及迁移。材料和方法：1.大鼠BMSCs的培养和鉴定：全骨髓贴壁培养筛选法分离和培养大鼠BMSCs,通过不断换液及传代纯化培养的细胞。倒置显微镜及透射电镜观察培养的BMSCs形态学特征,免疫组织细胞化学染色法检测BMSCs表面分子,流式细胞仪分析细胞周期以及酶标仪测定生长曲线的方法鉴定培养的细胞。最后对培养的BMSCs进行成骨、成脂及向神经样细胞分化的横向分化能力进行鉴定。2. SPIO标记大鼠BMSCs及磁共振细胞成像实验研究：首先通过使用扫描电镜观察Resovist和Feridex的分子表面形态及激光纳米粒度仪测定两种标记物的粒径和Zeta电位,对照两种标记物的不同。随后使用14μg/ml、28μg/ml、56μg/ml和112μg/ml铁浓度的Resovist标记大鼠BMSCs,并通过使用细胞爬片普鲁氏蓝染色及透射电镜观察细胞内铁颗粒,酶标仪及流式细胞仪分别测定Resovist标记对大鼠BMSCs生长曲线及细胞凋亡的影响；RT-PCR和Western blot对照研究Resovist未标记组及标记组向神经样细胞分化后,神经上皮干细胞蛋白(Nestin)、神经元特异性烯醇化酶(neuron specific enolase, NSE)和神经胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的mRNA及蛋白表达。最后对不同浓度Resovist标记的大鼠BMSCs进行离体试管及移植入正常大鼠脑内的磁敏感加权成像(Susceptibility weighted imaging, SWI)观察。3.Resovist标记大鼠BMSCs移植治疗缺血性脑梗死的磁共振成像监测：线栓法制作MCAO模型,实验动物共分5组：A组(假手术组)：仅分离结扎颈外动脉分支,不插入栓线；B组(MCAO组)：MCAO模型成功后不进行移植；C组(MCAO后假移植组)：MCAO后对侧顶叶皮层移植PBS液；D组(MCAO后实验1组)：MCAO后移植未经Resovist标记的BMSCs;E组(MCAO后实验2组)：MCAO后移植Resovist标记的BMSCs.移植成功后分别在1天、1周、2周和4周参照改良神经功能缺损评分法(modifiedNeurological Severity Scores, mNSS)进行神经功能缺损评分,SIEMENS Trio Tim 3.0 T超导高场磁共振扫描仪动态观察移植细胞的存活及迁移,将所得到的影像学检查结果与脑组织切片的HE染色及普鲁士蓝染色进行对照研究。结果：1.大鼠BMSCs传至P3代后,形态为均匀分布的梭形成纤维样细胞,透射电镜下可表现为为两种不同的细胞形态结构类型。免疫细胞化学染色法检测大鼠CD分子表达结果为CD44及CD105的阳性表达及CD34及CD45阴性表达,流式细胞周期分析显示G0/G1期细胞占90.18%。生长曲线显示大鼠BMSCs依次经历停滞期、指数递增期和平台期。横向分化能力检测结果显示：成骨诱导后14天ALP活性显著增高,成脂诱导后21天胞浆内可见大小不等的油滴状结构,神经方向诱导后NSE、Nestin及GFAP染色均为阳性。2.Resovist和Feridex的扫描电镜观察发现两者形态极为不同；粒径和Zeta电位分析显示,前者的粒径明显大于后者,而后者的Zeta电位绝对值大于前者。细胞普鲁氏蓝染色及透射电镜分别显示细胞内蓝染颗粒或高电子密度铁颗粒。MTT及流式细胞仪测定结果显示对照组与不同浓度标记细胞组相比,吸光度值差异及细胞凋亡率无统计学意义(P>0.05)>RT-PCR结果显示各组Nestin、NSE和GFAP的mRNA均呈高表达,Western blot显示未标记组及不同浓度标记组在240 Kd处均出现特异性蛋白阳性条带。离体试管SWI序列显示随标记浓度的增高,标记细胞的EP管信号强度在SWI序列图像上依次降低。标记细胞移植入正常大鼠脑内后7周仍可在SWI上显示为局部低信号。3. MCAO模型建立后HE染色显示神经元细胞数量减少,TTC染色显示缺血侧额颞顶皮层及基底节区为白色。BMSCs移植后1天、1周、2周和4周的神经功能评分显示A组和B组、A组和C组、C组和D组及C组和E组评分差异具有统计学意义(P<0.05)。磁共振检查结果显示移植后1天呈边界清晰的团块状类圆形低信号,1周后的低信号边界略显模糊,2周后移植位点形态变为“彗星状”,彗星尾指向对侧缺血病灶,4周后彗星尾向缺血侧延伸,甚至跨越中线迁移至对侧缺血异常高信号区域边缘。结论：1.全骨髓贴壁培养筛选法操作简便,需要的骨髓量少,原代培养时更符合细胞生长的微环境,是培养大鼠BMSCs的理想方案。2.不同浓度梯度Resovist标记大鼠BMSCs未对细胞细胞增殖造成影响,且未对向特定神经样细胞分化造成影响。3.不同浓度Resovist标记大鼠BMSCs在SWI上随标记浓度的增高,标记细胞信号逐渐降低,且移植入正常大鼠脑内的标记细胞7周后仍可显示。4. Resovist标记大鼠BMSCs移植入缺血性脑梗死大鼠脑内后,磁共振成像在长达4周的活体监测过程中,均可明确显示移植干细胞的部位及迁移。更多还原

【Abstract】 Background and Purpose:Ischemic cerebrovascular disease (ICVD) is a kind of significant high rate of deformity and mortality cerebral vascular disease, and its morbidity has the increasing tendency year by year, with the progressing of population aging and rising living standards in China. As we all known, ICVD has the limited method of treatment, which is the main reason for its poor prognosis. With the continuous researching of the biological characteristics of stem cell, traditional concepts of adult nerve cells do not have the ability of renewable had been broken down. In recent years, stem cell transplantation for treatment of ICVD becomes the research focus, especially researching the distribution, migration and turnover of transplanted stem cell in the host brain tissue in vivo after the transplantation of stem cell, which is essential for assessing the treatment efficacy of stem cell.Our research was divided into three parts to assessing the magnetic resonance monitoring after stem cell transplantation:1.Isolation, culture, purification and identification of rat bone marrow mesenchymal stem cells (BMSCs),2. Superparamagnetic iron oxide (SPIO) labeling of rat BMSCs and exploring the biological activity and the capability of differencing into neural-like cells after labeling,3.Magnetic resonance in vivo monitor the survival and migration of stem cells transplanted into the rat brain of middle cerebral artery occlusion (MCAO).Materials and Methods:1.Culture and identification of rat BMSCs:adherent culture and isolation method of whole bone marrow was employed to acquire BMSCs, and the cultured BMSCs were purified by continuous change of medium and pass on from generation to generation. Characteristic morphology of cultured BMSCs was observed by inverted phase contrast microscope and transmission electron microscopy, and the surface molecules of BMSCs was detected by immunocytochemistry staining, as well as the cell cycle and cell growth curve was analyzed by flow cytometry and microplate reader, respectively. Finally, the horizontal differentiation ability of BMSCs, including the osteogenic, adipogenic and neural-like differentiation,were identified. 2.SPIO labeling of rat bone marrow mesenchymal stem cells and magnetic resonance imaging of labeled cells:First, molecular surface morphology, as well as the size of particles and Zeta electric potential, between Resovist and Feridex, were measured by using scanning electron microscopy and laser particle size analyzer, respectively. Then, rat BMSCs were labeled by different concentration (14μg/ml,28μg/ml,56μg/ml and 112μg/ml) of Resovist, and intracellular iron particles were identified by using Prune’s blue staining and transmission electron microscopy. The influence of Resovist labeling on rat BMSCs cell growth curve were measured by microplate reader. After the neural-like differentiation of Resovist labeled and non-labeled BMSCs, the mRNA and protein expression of neuroepithelial stem cell protein (Nestin), neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were analyzed by RT-PCR and Western blot technique. Finally, rat BMSCs labeled with different concentrations of Resovist were measured in vitro, and transplanted into normal rat brain were observed by susceptibility weighted imaging.3.MR monitoring of SPIO labeled rat BMSCs transplanting into cerebral ischemic rats:left middle cerebral artery (MCA) was occluded with the suture, and all of the rats were randomly and equally divided into 5 groups. In A group (sham group), only separated branches of the external carotid artery (ECA), and tip of the suture was not inserted within the left common carotid artery (CCA).For B group (MCAO group):only the left common carotid artery was occluded, and did not performed transplantation. As to C group (sham transplantation after MCAO), contralateral parietal cortex transplantation of PBS solution was applied after MCAO.For D group (experimental group 1 after MCAO), rat BMSCs without Resovist labeled were transplanted into MCAO model. As well as E group (experimental group 2 after MCAO), rat BMSCs labeled with Resovist were transplanted. After the success of transplantation, modified Neurological Severity Scores (mNSS) methods were performed to assess the neurological deficit at 1 day,1 week,2 weeks and 4 weeks after transplantation. The survival and migration of transplanted BMSCs were dynamically observed by Siemens Trio Tim 3.0 T superconducting high-field MRI scanner. The imaging results and brain tissue sections of HE staining together with Prussian blue staining were researched contradistinctively.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 rat CD molecules exhibited positive expression of CD44 and CD105 and negative expression of CD34 and CD45.Flow cytometry cell cycle analysis showed G0/G1 stage of rat BMSCs accounting more than 90 percent. Growth curve showed rat BMSCs had undergone halt stage, index increase stage, and plateau phase. Horizontal differentiation results revealed:ALP activity was significantly increased 14 days after osteogenic induction, and different size of droplets-like structure were detected 21 days after adipogenic induction, as well as NSE, Nestin and GFAP staining was positive after neural induction.2.Scanning electron microscope manifested that there were extremely different between Resovist and Feridex, besides, particle size and Zeta potential analysis exhibited that the diameter of the former is significantly greater than the latter, whereas the absolute value of Zeta potential of the latter is greater than former. Cell Prune’s blue staining and transmission electron microscopy displayed blue iron particles or granules of high electron density, respectively. The control group and different concentrations of labeled cells group had no significant differences in optical density measured by MTT method. The mRNA of Nestin, NSE and GFAP showed high expression after neural induction detected by RT-PCR. Besides, specific positive bands were manifested in the 240 Kd position on Western blot. Susceptibility weighted imaging of in vitro EP tube revealed that the more increased concentration of labeling, the lower signal intensity were detected. Finally, lower signal density still could be demonstrated on susceptibility weighted imaging 7 weeks after transplanted into normal rat brain.3.After middle cerebral artery occlusion, HE staining showed decline of neurons number, besides, TTC staining manifest that the ischemic side of the frontal-temporal cortex and basal ganglia area had none staining. Neurological function score showed that A group vs. B and C group, C group vs. D and E group had statistically significant between each other 1 day,1 week,2 weeks and 4 weeks after transplantation. MRI results showed block oval low signal low signal with clear boundary 1 day after transplantation, along with blurred boundary 1 week later, as well as transformed to "comet-like" with a tail pointed contralateral ischemic lesions 2 weeks after transplantation, finally, tail extended to the ischemic side, and even migrate across the midline to the opposite edge of ischemic high intensity signal region 4 weeks after transplantation.Conclusion:1.The whole bone marrow adherent culture method, which had the advantage of simple, required less bone marrow, and accorded with the micro-environment of cell growth, is the ideal solution for rat BMSCs culture.2.Different concentrations labeling by using Resovist had none influence on cell proliferation of rat BMSCs, and none impact of specific neural induction.3.The more increased concentration of labeling, the lower signal intensity were detected on susceptibility weighted imaging, and lower signal density still could be demonstrated on susceptibility weighted imaging 7 weeks after transplanted into normal rat brain.4.The location and migration of Resovist labeled rat BMSCs could be detected by in vivo magnetic resonance imaging after transplanted into the ischemic rat brain as long as 4 weeks.更多还原