【作者】 李晶；

【导师】 牛勃；

【作者基本信息】 山西医科大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 研究背景间充质干细胞可自骨髓等多种组织分离得到,近年来,间充质干细胞移植已经成为心血管疾病,神经系统疾病等多种疾病治疗的种子细胞。研究目的本实验采用磁共振成像(MRI)进行标记细胞的体外示踪及移植细胞的糖尿病体内示踪,探讨不同浓度及时间超顺磁性氧化铁颗粒(SPIO)标记兔骨髓间充质干细胞(MSCs)的标记效率、标记后活力以及标记后体外成像情况,为临床体内MRI寻找最佳标记“浓度——时间”。尝试在常规1.5T磁共振(MR)的成像条件下,对较小动物兔的胰腺、肾脏等器官进行解剖成像和移植细胞的示踪;初步动态观察移植细胞在糖尿病兔体内的活体成像特点及规律,为临床干细胞移植治疗糖尿病疗效评估开创新方法。研究方法(1)MSCs的培养及鉴定。选择健康家兔,采用密度梯度离心法和贴壁筛选法分离、培养、纯化骨髓MSCs,观察细胞在光镜下的生长特征并记录其生长曲线,Von Kassa方法检测成骨诱导骨髓MSCs,对所获得的细胞进行表面抗原(CD44、CD45和CD71)的检测,确定所获细胞绝大多数为骨髓MSCs。(2)标记细胞的体外成像。配制不同浓度含SPIO的培养基(铁终浓度分别为25μg/ml、50μg/ml、75μg/ml、100μg/ml、150μg/ml),加入骨髓MSCs孵育标记。标记后1天、3天、1周、2周、3周、4周行普鲁士蓝染色、细胞活力检测及标记细胞体外MRI,并经MRI确定合适的标记时间和标记浓度。(3)移植细胞糖尿病兔MRI活体示踪。制备兔糖尿病模型,将SPIO标记的骨髓MSCs和未标记的骨髓MSCs经静脉移植入糖尿病兔体内,用测定血糖的方法评价细胞移植前、后对糖尿病高血糖的治疗效果。同时经MR动态观察移植细胞的定位、存活及迁徙。研究结果(1)骨髓MSCs培养、鉴定:骨髓MSCs呈梭形、漩涡状生长。骨髓MSCs经成骨诱导后表达骨细胞特征。流式细胞仪表面标志鉴定CD45呈阴性,CD44、CD71呈阳性。(2)SPIO可有效标记MSCs,细胞活力检测显示时间为1天和3天时,剂量0μg/ml组与25μg/ml组,0μg/ml组与50μg/ml组,25μg/ml组与50μg/ml组的细胞拒感率没有差别(P＞0.05)。体外MRI示标记3天时,标记浓度为50μg/ml组,T2WI及T2*WI信号降低最明显。(3)建立了兔糖尿病模型,其血糖稳定＞16.8mmol/L。血糖检测显示MSCs移植可有效降低糖尿病血糖升高,MR T2WI、T2*WI序列均可示踪糖尿病兔体内移植SPIO标记骨髓MSCs。MRI可对标记细胞示踪至注射后10天。研究结论联合采用密度梯度离心和贴壁筛选的方法易于分离、纯化获得纯度较高的MSCs。50μg/ml浓度,标记3天的组合不仅标记效率高,对细胞活力无影响而且细胞体外MRI信号强度降低最明显,为最适标记“浓度——时间”组合。磁标记与未标记的骨髓MSCs均能有效治疗糖尿病高血糖。临床型1.5TMR可对糖尿病兔体内标记细胞进行无创性活体示踪,并较长时间地动态观测细胞的存活与迁徙。更多还原

【Abstract】 Background Mesenchymal stem cells(MSCs) can be isolated from bone marrow and so on and can be used cell therapy which have been used cardiovascular diseases,nervous system diseases and so on.Objective The purpose of this study was labeled cells of vitro MRI test and to determine whether SPIO labeled bone marrow MSCs could be detected and tracked noninvasively by MRI in diabetic rabbit models.To explore the labeled efficiency,cellular viability of rabbit mesenchymal stem cells and vitro imaging of the labeled bone marrow MSCs with different concentration and time of superparamagnetic iron oxide(SPIO),and find the best labled concentration and labeled time for clinical vivo magnetic resonance imaging(MRI).We attempt to determine the feasibility of tracing rabbit transplanted cells by using a conventional clinical 1.5T MR scanner.The imaging characteristics and rules of cells also need dynamic observation preliminarily.We hope to find a kind of easy and feasible techniques and methods for future clinical bone marrow MSCs transplant therapy in diabetes.Methods Bone marrow MSCs were derived from bone marrow aspirates of healthy rabbits.Mononuclear cells(MNCs) were isolated using density gradient centrifugation,and cultured and expanded based on plastic adherence.Cellular growth characteristics were observed and recorded under light microscopy.Committed differentiation of bone marrow MSCs into osteoblast by von kossa stain.To identify most cells were bone marrow MSCs,detection and analysis of surface antigen of CD44、CD45 and CD7(?) was performed by flow cytometry.The culture media contained different concentration SPIO paticles were added to the bone marrow MSCs,the ultimate concentration of SPIO was 25μg/ml、50μg/ml、75μg/ml、100μg/ml、150μg/ml respectively.Prussian blue staining,trypan blue exclusion test and vitro magnetic resonance imaging of labeled cells were performed at 24h,72h,1w,2w,3w,4w respectively after labeled, and make sure rational labeled concentration and time.Prepared diabetic rabbit models,transplanted the SPIO labeled bone marrow MSCs and not labeled bone marrow MSCs into diabetic rabbits.Then evaluated the efficience of transplantation by determination of blood sugar and observed the location,distribution and migration.Results(1) the adherenced cells showed spindle shapes and vortex condition growth.Osteogenic induction showed the characters of bone cells.Flow cytometry analysis of the adherenced bone marrow MSCs indicated CD45(-)and CD44(+),CD7(?)(+)demonstrated the cells we get were bone marrow MSCs.(2) The labeled efficiency of all the groups reached to 95%-100%.trypan blue exclusion test showed the viability of the labeled cells with SPIO of 0μg/ml and 25μg/ml,0μg/ml and 50μg/ml,25μg/ml and 50μg/ml was not significantly statistical difference(P>0.05).The viability of the cells labeled with SPIO of labeled cells of ld and 3d was not significantly statistiscal difference(P>0.05).Vitro MRI test showed when labeled time is 72h and labeled concentration is 50μg/ml,signal intensity change is most remarkable.(3)The blood sugar of diabetic rabbits models are all more than 16.8mmol/L.Determination of blood sugar showed bone marrow MSCs transplantation can decrease the level of diabetic rabbits’ blood sugar. SPIO labeled cells could be identified on T2WI and T2~*WI sequence after injection into both normal and diabetic rabbits by using 1.5T MR.Bone marrow MSCs were observable and could be seen for at least 10days.Conclusion Bone marrow MSCs is easy to be isolated and purified by combined using density gradient centrifugation and plastic adherence.The SPIO concentration of 50μg/ml and the labeled time of 72h not only have high labeled efficiency and no effect on the viability of bone marrow MSCs but also signal intensity change is most remarkable with MRI in vitro,can be considered optimal labeled concentration and time with SPIO.Labeled bone marrow MSCs and non-labeled bone marrow MSCs can cure the hyperglycemia of the diabetes.Visualization of SPIO-labeled bone marrow MSCs in diabetic rabbit models by using a conventional 1.5T MR is feasible and the ability may be used to future clinical studies.更多还原