脂肪间充质干细胞在体外三维培养体系中向软骨细胞定向诱导分化的研究

发布时间：2018-04-25 23:35

本文选题：脂肪间充质干细胞 + 三维培养　；参考：《暨南大学》2008年硕士论文

【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。
[Abstract]:Objective: 1. to explore the stable.2. separation and extraction method of ADMSCs from the donor age, the location of the material and the extraction method, and to explore the suitable conditions for the directional differentiation of ADMSCs into the chondrocytes in the three-dimensional culture system.
Method:
Study on the separation and extraction method of a.ADMSCs
The primary rabbit ADMSCs was isolated by enzyme digestion. The monolayer was passaged and amplified.
1. cell crawling immunofluorescence was used to detect the expression of stem cell marker CD44 and CD105, and phenotypic identification was performed.
Cell morphology was observed dynamically by 2. phase contrast microscope, cell activity was observed by trypan blue staining, cell growth kinetics parameters were detected by cell count method, and cell cycle was detected by flow cytometry.
3. compare the different donor age, location, and the difference in cell yield and activity obtained by the extraction method.
Differentiation of two.ADMSCs into chondrocytes in three dimensional culture system
Two three dimensional systems were cultured in vitro by centrifuge tube formation and alginate gel suspension culture. The cultured cells were added to the induction culture medium and ordinary medium respectively, and the samples were collected on day 7,14,21.
1.HE staining was used to observe the tissue morphology of newborn cartilage.
2. the expression of type II collagen was detected by immunofluorescence.
The content of collagen and proteoglycan in extracellular matrix was determined by 3. hydroxyproline method and Alcian Blue method.
4. real-time quantitative PCR was used to detect the expression level of characteristic protein type II collagen and proteoglycan in chondrocytes from the gene level.
Result:
1. Separation and identification results
1. multiple digestion and collection methods were used to extract AMDSCs, with high yield, good activity and stable effect. There was no difference in the biological properties of ADMSCs extracted from the donor and the same donor site of the same donor and the same donor site. Extraction rate.
2. immunofluorescence showed that most of the cells were CD44 and CD105 positive. The purity of the isolated cells was better. The growth curve showed that the proliferation rate of cells did not slow down with the increase of generation.
Two. The results of in vitro three-dimensional culture induction
1. in two systems, white patches with a certain degree of hardness were formed. HE staining showed the unique morphology of cartilage tissue with obvious stratified and differentiated zones.
2. immunofluorescence results showed that type 2 collagen was expressed in the samples on day 14,21 of the experimental group, while negative expression was found in all the samples of the seventh day and the control group.
The results showed that the expression of type II collagen and proteoglycan in the experimental group was 7,14.
The expression level of the 21 day samples showed an upward trend, and the trend of the control group was not obvious.
The results of real time quantitative PCR showed that the expression level of type II collagen and proteoglycan gene in each sample of the two culture systems increased with time, and the expression level of the gene in the system was higher than that in the gel suspension culture system at all time points.
Conclusion: 1. from adult individuals, rich in fat tissue, single component parts and multiple digestion and collection can improve the efficiency of AMDSCs extraction and maintain good cell activity in the two three dimensional culture system of ADMSCs, which can differentiate into chondrocytes under the action of inducer and express the characteristic egg of cartilage cells. Compared with the low cell density of the alginate culture system, white.3. can increase the expression level of the characteristic protein type II collagen and proteoglycan in the induced cells, that is, to promote the directional differentiation of ADMSCs to cartilage cells.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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