微孔水凝胶共培养体系的构建及其诱导干细胞软骨分化的研究

发布时间：2018-03-27 19:40

本文选题：软骨细胞　切入点：水凝胶　出处：《广州医科大学》2017年硕士论文

【摘要】：研究目的构建微孔水凝胶,令小鼠胚胎瘤细胞(ATDC5)与原代猪软骨细胞在微孔水凝胶中直接共培养,探索该共培养体系中最适合的细胞环境,以达到ATDC5最佳的软骨诱导效果,从而获得更符合人体的透明软骨特性的组织工程软骨,为临床软骨修复提供一种理想的方法。方法:一、共培养的细胞采集以及培养共培养所需的原代猪软骨细胞从鲜猪膝关节软骨中提取,并使用软骨培养液于175cm2培养瓶进行培养。ATDC5从美国圣地亚哥公司(San Diego,USA)购买,用ATDC5培养液于175cm2培养瓶进行培养。二、明胶微球的制备及微孔水凝胶猪软骨细胞培养的研究所需的80-120微米大小的明胶微球通过双乳液法进行制备,然后用罗丹明染色液标记后包埋在水凝胶,观察明胶微球降解情况。再用此方法获得的微球制备微孔海藻酸钠水凝胶培养原代软骨细胞,通过荧光显微镜观察活/死细胞检测结果,并使用CCK8方法测定细胞活力值。三、微孔水凝胶共培养体系的构建以1:3、1:1、3:1的软骨细胞与ATDC5细胞比例(分别为1C3A、AC、3C1A组)混合,对照组分别为单纯ATDC5组(A组)和软骨细胞组(C组)。该5组细胞分别混合到含有适当比例的明胶微球的海藻酸钠溶液后,制作成细胞/微孔水凝胶复合物,并用软骨诱导液进行培养。四、共培养细胞的活力以及软骨特征性表达的检测整个培养周期中,在对应的时间点收集相应的样本,通过细胞增殖活力(Cell Counting Kit-8,CCK-8)、软骨核酸的表达(Quantitative real-time PCR,qRt-PCR)、组织学染色、免疫组织化学染色(Immunohistochemistry,IHC)和细胞代谢相关蛋白表达(Western blot,WB)的检测结果,分析共培养细胞软骨特征性基因或蛋白的表达水平。结果:一、成功从新鲜猪软骨中提取原代软骨细胞和将P7ATDC5复苏。成功在体外培养增殖并了解其生长形态、生长活力和绘制其细胞活力曲线。二、通过不同的搅拌速度,获得不同直径的明胶微球。实验表明,搅拌速度越快,产生的微球粒径越小。在显微镜下观察到,明胶微球在37℃的培养环境下能够自然降解,水凝胶内部产生微孔。活/死细胞染色结果观察到原代软骨细胞能够在材料中均匀地分布和生长,在微孔边缘生长的软骨细胞甚至能够突破边缘,将空腔位置占据生长。细胞活力检测数据表明微孔水凝胶组中的软骨细胞的增殖能力比对照组中的细胞显著提高。三、成功构建出原代软骨细胞和ATDC5细胞的微孔水凝胶共培养体系。从共培养细胞的细胞活力,软骨特异性核酸表达,软骨相关蛋白表达以及组织化学染色方面综合来看,在微孔水凝胶共培养体系中,软骨细胞和ATDC5以1:3的比例最为理想,并且发现丝裂原活化蛋白激酶(the mitogen-activated protein kinase,MAPK)中P44/42与该共培养体系中的细胞增殖相关。结论:该微孔水凝胶共培养体系能够为ATDC5提供理想的定向软骨分化环境,特别在软骨与ATDC5以1:3的比例下,ATDC5的定向软骨分化效果最理想。本研究为软骨组织工程的进一步应用提供扎实的实验基础,为日后临床治疗软骨损伤提供理想的对策。
[Abstract]:Study objective to construct porous hydrogel, the mouse embryonic cells (ATDC5) and primary porcine chondrocytes in microporous hydrogel in direct co culture, exploring the most suitable environment for cell co culture system, in order to achieve the best effect of ATDC5 induced cartilage, resulting in cartilage tissue engineering with hyaline cartilage properties of the human body, provide an ideal method for clinical cartilage repair. Methods: a cell co culture and co culture collection of primary cultured porcine cartilage cells to extract from fresh porcine articular cartilage, cartilage and use medium in 175cm2 flask and cultured.ATDC5 from the Santiago company (San Diego USA) to buy. Using ATDC5 medium in 175cm2 flask were cultured. Two, gelatin microspheres 80-120 micron size of gelatin microspheres and porous hydrogel cartilage cell culture required by double emulsion method The preparation, then use the Luo Danming dye labeled entrapped in the hydrogel, observe the gelatin microspheres degradation of microspheres obtained by this method. The preparation of microporous sodium alginate hydrogel in primary cultured chondrocytes, live / dead cell results detected by fluorescence microscopy, and the determination of cell viability by CCK8 method. Three, system to construct cartilage cells and ATDC5 cell ratio of 1:3,1:1,3:1 co cultured with microporous hydrogel (1C3A, AC, 3C1A group) mixed control group respectively ATDC5 alone group (A group) and chondrocyte group (C group). The 5 groups of cells were mixed with sodium alginate solution gelatin microspheres containing the appropriate proportion of the, made into cell / porous hydrogel composite, and were cultured in chondrogenic fluid. Four, co cultured cell viability and expression of cartilage characteristic throughout the culture period, at the corresponding time points from the corresponding The sample, through the cell proliferation (Cell Counting, Kit-8, CCK-8), the expression of cartilage (Quantitative real-time nucleic acid PCR, qRt-PCR), histological staining, immunohistochemical staining (Immunohistochemistry, IHC) expression and cell metabolism related protein (Western, blot, WB) of the test results, the expression level of cell specific genes or cartilage protein co culture analysis. Results: a successful, extracted from fresh pig cartilage chondrocytes and P7ATDC5 recovery. Success in vitro proliferation and to understand the growth morphology, growth vigor and the cell viability curve. Two, through different stirring speeds, obtain gelatin microspheres with different diameters. Experiments show that the stirring speed faster, the particle size is smaller. Observed under the microscope, gelatin microspheres can be naturally degraded in the environment under 37 DEG C, hydrogel generated inside the hole. Live / dead cell staining The observed primary chondrocytes can be distributed uniformly and the growth in the material, the chondrocytes in microporous edge growth can even break edge, will occupy the cavity position growth. Cell viability test data showed that the chondrocytes in microporous hydrogel group proliferation significantly increased than in the control group. Cell three, successfully constructed the porous hydrogel primary cartilage cells and ATDC5 cells in co culture system. The co cultured cell viability, expression of cartilage specific expression of cartilage associated protein and nucleic acid staining comprehensive to see, in the microporous hydrogel coculture system, cartilage cells and ATDC5 with the ratio of 1:3 is the most ideal, and found mitogen activated protein kinase (the mitogen-activated protein kinase, MAPK) in P44/42 associated with the co culture system in cell proliferation. Conclusion: the micro pore water gel Co cultured cartilage differentiation system can provide ideal environment for ATDC5, especially in cartilage and ATDC5 with the ratio of 1:3, the most ideal cartilage differentiation effect of ATDC5. This study provides a solid foundation for further experimental application of cartilage tissue engineering, countermeasures provide an ideal clinical treatment of cartilage injury after.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68

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