【作者】 李冬艳；

【导师】 宇丽；

【作者基本信息】 暨南大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 目的:1.从供体年龄、取材部位、提取方法三方面来探索稳定的脂肪间充质干细胞(ADMSCs)分离提取方法。2.在三维培养体系中探索ADMSCs向软骨细胞定向诱导分化的适宜条件。方法:一.ADMSCs分离提取方法的研究采用酶消化法分离培养原代兔ADMSCs,单层传代培养扩增。1.细胞爬片免疫荧光检测干细胞标志分子CD44、CD105的表达,进行表型鉴定。2.相差显微镜动态观察细胞形态;台盼蓝染色观察细胞活性;细胞计数法检测细胞生长动力学参数;流式细胞仪检测细胞周期。3.比较不同的供体年龄、取材部位、提取方法所获取的细胞产量和活性的差别。二.ADMSCs在三维培养体系中向软骨细胞定向诱导分化的研究分别以离心管成团培养和藻酸盐凝胶悬浮培养两种三维体系体外培养细胞,分别加入诱导培养液和普通培养液,培养第7、14、21天收集样本。1.HE染色,观察新生软骨组织形态。2.免疫荧光检测Ⅱ型胶原表达。3.羟脯氨酸法与阿新蓝(Alcian Blue)法测定细胞外基质中胶原与蛋白聚糖的含量。4.实时定量PCR从基因水平检测新生组织中软骨细胞特征性蛋白Ⅱ型胶原与蛋白聚糖的表达水平。结果:一.分离、鉴定结果1.多次消化收集法提取AMDSCs,产量高、活性良好、效果稳定。不同年龄的供体以及同一供体不同取材部位的脂肪组织中所提取的ADMSCs的生物学性质无差别,但是选择壮年兔以及脂肪组织丰富、成分单一的肌间隙、腹股沟部位取材,可以提高提取率。2.免疫荧光显示,绝大多数的细胞都表现为CD44和CD105阳性。证实了分离细胞纯度较好。生长曲线显示随代次的增加细胞增殖速度未见减慢。二.体外三维培养诱导检测结果1.两种体系中均形成了具有一定硬度的白色块状组织,HE染色显示,具有明显层次和分化区带的软骨组织特有形态。2.免疫荧光结果显示实验组第14、21天样本中有Ⅱ型胶原表达,实验组第7天样本和对照组全部样本呈阴性表达。羟脯氨酸法、阿新兰法结果显示,Ⅱ型胶原与蛋白聚糖在实验组的表达量第7、14、21天样本的表达量均呈上升趋势,对照组趋势不明显。实时定量PCR结果显示,随着时间延长,两种培养体系实验组各样本的Ⅱ型胶原与蛋白聚糖基因表达水平均呈上升趋势,并且在各时间点成团培养体系中基因的表达水平高于凝胶悬浮培养体系。结论:1.从成年个体、脂肪组织丰富、成分单一的部位取材,并采用多次消化收集法提取,可以提高AMDSCs提取效率,并保持良好的细胞活性。2.两种三维培养体系中培养的ADMSCs,在诱导剂的作用下均能向软骨细胞分化,表达软骨细胞特征性蛋白。3.与细胞密度相对较低的藻酸盐培养体系相比,高密度的成团培养体系可以提高诱导后细胞的特征性蛋白Ⅱ型胶原与蛋白聚糖的表达水平,即促进ADMSCs向软骨细胞的定向分化。更多还原

【Abstract】 Objective:1.In order to establish a stable extraction method for adipose tissue derived mesenchymal stem cells(ADMSCs),the research was carried out on three respects:donor age,location of fat obtained and extraction way.2.To Study the suitable conditions of the induced differentiation of adipose tissue derived mesenchymal stem cells into chondrocytes in three dimensional culture systems.Methods:1.Study on the ADMSCs’ extraction:①ADMSCs were extracted with the enzyme-digestion-separate method,cultured in monolayer and passenged,identified the phenotype by detected marker molecule of stem cells:CD44、CD105 with immunofluorescence.②Cells morphous was observed by phase-contrast microscope; Cytoactive was observed by stained with trypan blue;Cells growth was detected by cytometry;Cell life cycle was detected by flow cytometry.③The yield of extracted ADMSCs and the biological activity of cultured cells were compared among the groups with different extraction methods,from different locations and ages.2.Study on the induced differentiation of adipose tissue derived mesenchymal stem cells into chondrocytes in three dimensional culture systems:①The third passenged cells were incubated in pellet culture and in suspension culture variously.Induced or common medium was added into the experiment group or the control group,collect samples on the 7、14、21 days.②The histological structure of neocartilage was observed by species stained with H&E;③Immunofluorescence analysis was performed to detect the expression of collagenⅡ;④The production of matrix was estimated from the determination of hydroxyproline content and Alcian Blue method.;⑤The collagenⅡand GAGs DNA expression level of neocartilage was measured by real time PCR.Results:1.The eugonic ADMSCs could be stably extracted from adipose tissue with the multi-digestion-separate method,and the yield is high,the cells activity was good.The proliferation activity of extracted ADMSCs was similar among the rabbits from different ages and locations,but to select adult individuality,to draw the materials among muscles, and in groin where the adipose tissue is abundant and unitary could raise the yield significantly.2.Immunofluorescence results show that most cells display masculine of CD44、CD105,it confirmed a good purity;growth curve exhibited cells proliferation pace doesn’t step down with generation increased.3.ADMSCs formed cartilage-like tissue with distinct differentiated zones and regions in three dimensional culture systems in vitro.4.In three dimentional culture system and induction conditions,immunofluorescence results show that the 14、21 days samples of experiment group display masculine of collagenⅡ,the 7 days samples of experiment group and all the samples of control group display negative;The determination of hydroxyproline content and Alcian Blue method displayed in the experiment group the collagenⅡand GAGs content increased along the time,while the control group didn’t show a tendency;Real time PCR.results display the expression of collagenⅡand GAGs rose along with culture in experiment group,and the pellet culture was super than the suspension culture.Conclusion:1.To select adult individuality,draw the materials from where the adipose tissue is abundant and unitary,extract with the multi-digestion-separate method could raise the yield significantly,and keep cells in good activity.2.ADMSCs can differentiated into the chondrocytes under the action of derivative in these two three dimensional cuture,and secret the chondrocyte charatered protein.3.Pellet culture or high-density culture could be helpful to enhanced cartilage character matrix production compare with the Suspension,culture or low-density culture,it can stimulate the induction effect.更多还原