Ⅰ型胶原形态对细胞软骨分化的影响及其机理研究

发布时间：2018-06-14 20:05

本文选题：软骨分化 + 胶原　；参考：《华南理工大学》2014年博士论文

【摘要】：组织工程与再生医学为解决软骨有限的修复能力提供了全新的方法，间充质干细胞（MSCs）可克服软骨细胞的来源限制而在这一修复方法中广泛应用。如何更安全高效地将干细胞诱导为软骨细胞是目前的瓶颈之一。细胞所处的微环境及与细胞相互接触的胞外基质都对细胞的分化有重要影响。因此针对软骨缺损修复材料的发展，研究材料介导细胞分化的功能，评价材料对细胞分化的影响具有重要意义。 I型胶原因其优异的生物相容性与生物活性而广泛应用于组织工程与再生医学，但其是否具有MSCs软骨分化的诱导性，不同的研究结果不尽相同。导致这一差异性结果的原因主要有细胞来源、细胞代数、培养条件等所导致的细胞状态与分化性能的差异，以及所采用的胶原的来源、纯度、材料形态等的区别。ATDC5等细胞株可以避免MSCs上述不稳定因素带来的影响，本论文以ATDC5建立了材料对细胞软骨分化影响的评价体系，比较了不同形态的I型胶原对细胞软骨分化的作用，并研究了胶原对FAK和MAPK信号通路的影响，以期为软骨修复材料的设计和研发提供指导。通过geNorm算法分析16个常用内参基因在ATDC5软骨分化过程中的稳定性，结合内参基因的表达水平分析，筛选出Ppia和Hprt作为软骨分化过程中qPCR实验的理想内参基因。以小鼠MSCs证实了Ppia和Hprt在MSCs软骨分化过程中同样稳定。随后优化了ATDC5在评价材料对细胞软骨分化影响实验中的各种参数。通过细胞增殖、基因表达、染色及定量等方法证实了含TGF-β3的诱导液可缩短ATDC5的增殖期并迅速进入软骨分化进程，表达软骨相关表型，比ATDC5软骨分化中常用的胰岛素诱导液更适于评价材料对细胞的软骨分化。筛选出TGF-β3的最优添加浓度为10ng/ml，在此浓度下ATDC5细胞软骨表型基因表达最高，且内骨化表型基因表达最低。本研究制备了三种不同形态的I型胶原。通过冷冻干燥制备了非重组纤维结构的海绵支架，孔间为连通结构，，孔径约为300μm。采用NaOH中和法制备了网状重组纤维结构凝胶，纤维直径为100-200nm。通过I型胶原体外重组与原位除盐制备了重组纤维膜包被的细胞培养器皿，天狼猩红染色及三维立体显微镜检测结果显示在细胞培养器皿表面包被的胶原膜均一、平整；SEM、TEM、AFM等显示纤维的直径为100-200nm，周期性螺纹结构D-band约68nm，螺纹gap深度约3.46nm，同体内胶原纤维的天然形态基本一致。在评价体系建立的基础上，本论文研究了不同形态I型胶原对ATDC5细胞软骨分化早期的影响。通过分析细胞的生长形态及相关基因的表达水平，发现在软骨诱导液下胶原对细胞软骨分化的具体作用依赖于胶原的形态。网状重组纤维结构凝胶使细胞形态转为利于软骨分化的圆形，比阻碍细胞自发凝聚及形态转变的重组纤维膜与非重组纤维结构海绵支架更利于ATDC5的软骨分化。同无胶原微团形式培养的ATDC5相比，胶原的存在提供了胞外基质信号，促进了细胞的软骨分化。其次，研究了不同形态的I型胶原对ATDC5细胞软骨分化后期肥大与内骨化的作用。含β-磷酸甘油、维生素C和地塞米松的OM诱导液对ATDC5内骨化有诱导作用，且能保证细胞不过度增殖，使所有细胞都可与材料相互接触。细胞形态、基因表达、染色与定量等检测分析均显示单层培养形式下胶原重组纤维膜对ATDC5内骨化无促进与诱导作用，而非重组纤维结构的海绵胶原支架对ATDC5细胞内骨化不仅有促进，而且有诱导的作用。最后，采用hBMSCs检测0.25mg/ml及2.5mg/ml的I型胶原凝胶对细胞软骨分化的影响，证实了ATDC5细胞所得到的结果。基因表达、组织染色与免疫组化等检测手段均显示在TGF-β3的诱导下，I型胶原可促进hBMSCs的软骨分化，0.25mg/ml的胶原凝胶比2.5mg/ml的促进效果好。通过对MAPK家族相关信号蛋白磷酸化水平的分析及FAK表达水平的检测，发现在TGF-β3的诱导下，I型胶原的存在维持了FAK表达水平的相对稳定并激活了细胞内的MAPK信号通路。
[Abstract]:Tissue engineering and regenerative medicine provide a new method to solve the limited repair ability of cartilage. Mesenchymal stem cells (MSCs) can overcome the limitation of the origin of cartilage cells and are widely used in this repair method. It is one of the bottlenecks in how to induce the stem cells into chondrocytes more safely and efficiently. The extracellular matrix contact with each other has an important effect on cell differentiation. Therefore, it is of great significance to study the function of materials to mediate the differentiation of cells and to evaluate the effect of materials on cell differentiation in the development of cartilage defect repair materials.
I gum is widely used in tissue engineering and regenerative medicine because of its excellent biocompatibility and bioactivity. But whether it has the inducibility of MSCs cartilage differentiation, different results are different. The main reasons for this difference are cell status and differentiation caused by cell sources, cell generations, culture conditions and so on. The difference in chemical properties, as well as the difference between the collagen sources, purity and material morphology,.ATDC5 and other cell lines can avoid the effects of the above unstable factors of MSCs. In this paper, the evaluation system of the effect of the material on the differentiation of cell cartilage was established by ATDC5, and the effect of different forms of I collagen on the differentiation of cartilage was compared. The effects of collagen on FAK and MAPK signaling pathways were studied in order to provide guidance for the design and development of cartilage repair materials.
The geNorm algorithm was used to analyze the stability of 16 common internal reference genes in the ATDC5 cartilage differentiation process. Combined with the analysis of the expression level of the internal reference genes, Ppia and Hprt were selected as the ideal internal reference genes for the qPCR experiment during the cartilage differentiation process. The mice MSCs confirmed that Ppia and Hprt were also stable in the process of MSCs soft bone differentiation. Then the AT was optimized for AT. DC5 was used to evaluate the various parameters in the experiment of cell cartilage differentiation. Through cell proliferation, gene expression, dyeing and quantitative methods, it was proved that the inducer containing TGF- beta 3 could shorten the proliferation period of ATDC5 and quickly enter the cartilage differentiation process and express the cartilage related phenotype, which is more suitable than the insulin inducer commonly used in the differentiation of ATDC5 cartilage. The optimal concentration of TGF- beta 3 was selected as 10ng/ml, and the highest expression of phenotypic gene expression in ATDC5 cells was found at this concentration, and the expression of internal ossification phenotype was the lowest.
Three different forms of I collagen were prepared in this study. The non recombinant fibrous scaffold was prepared by freeze-drying. The interconnected structure was connected, and the pore size was about 300 m.. The reticulated fibrous structure gel was prepared by NaOH neutralization. The fiber diameter was 100-200nm. by recombinant I collagen in vitro and in situ desalination. The results of Sirius red staining and three-dimensional microscopic examination showed that the collagen membrane of the cell culture utensils was uniform and smooth; SEM, TEM, AFM, etc. showed the diameter of the fiber was 100-200nm, the periodic thread structure was D-band 68nm, the thread gap depth was about 3.46nm, and the natural form of the inner collagen fibers. The state of the state is basically the same.
On the basis of the establishment of the evaluation system, this paper studied the effect of different forms of I collagen on the early differentiation of cartilage in ATDC5 cells. By analyzing the cell growth morphology and the expression level of related genes, it was found that the specific effect of collagen on the differentiation of cartilage under cartilage induced solution depends on the morphology of collagen. The gel makes the cell morphology beneficial to the circle of cartilage differentiation, which is more conducive to the differentiation of ATDC5 cartilage than the recombinant fibrous membrane and non recombinant fibrous scaffold, which hinders the spontaneous agglomerate and morphologic transformation of the cells. The existence of collagen provides the extracellular matrix signal and promotes the chondrodifferentiation of the cells compared with the ATDC5 without collagen micro mass.
Secondly, the effects of different forms of collagen type I on the hypertrophy and ossification of ATDC5 cells in the late stage of differentiation were studied. The OM inducer of beta phosphoric acid, vitamin C and dexamethasone can induce the ossification of ATDC5, and it can ensure that the cells do not proliferate so that all the cells can contact each other. Both the color and the quantitative analysis showed that the collagen membrane of the monolayer culture did not promote and induce the ossification in ATDC5, but the sponge collagen scaffold that was not the structure of the recombinant fibrous structure not only promoted the ossification of ATDC5 cells, but also had the induced effect.
Finally, the effect of I collagen gel on cell cartilage differentiation of 0.25mg/ml and 2.5mg/ml was detected by hBMSCs. The results of ATDC5 cells, gene expression, tissue staining and immunohistochemistry showed that TGF- beta 3 induced cartilage differentiation in hBMSCs, and 0.25mg/ml collagen gel was more than 2.5mg/ml. Through the analysis of the phosphorylation level of MAPK family related signal protein and the detection of FAK expression level, it was found that under the induction of TGF- beta 3, the existence of type I collagen maintains the relative stability of FAK expression level and activates the MAPK signaling pathway in the cell.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08

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