發(fā)布時間：2018-06-14 20:05

  本文選題：軟骨分化 + 膠原　； 參考：《華南理工大學》2014年博士論文

【摘要】：組織工程與再生醫(yī)學為解決軟骨有限的修復能力提供了全新的方法，間充質(zhì)干細胞（MSCs）可克服軟骨細胞的來源限制而在這一修復方法中廣泛應用。如何更安全高效地將干細胞誘導為軟骨細胞是目前的瓶頸之一。細胞所處的微環(huán)境及與細胞相互接觸的胞外基質(zhì)都對細胞的分化有重要影響。因此針對軟骨缺損修復材料的發(fā)展，研究材料介導細胞分化的功能，評價材料對細胞分化的影響具有重要意義。 I型膠原因其優(yōu)異的生物相容性與生物活性而廣泛應用于組織工程與再生醫(yī)學，但其是否具有MSCs軟骨分化的誘導性，不同的研究結(jié)果不盡相同。導致這一差異性結(jié)果的原因主要有細胞來源、細胞代數(shù)、培養(yǎng)條件等所導致的細胞狀態(tài)與分化性能的差異，以及所采用的膠原的來源、純度、材料形態(tài)等的區(qū)別。ATDC5等細胞株可以避免MSCs上述不穩(wěn)定因素帶來的影響，本論文以ATDC5建立了材料對細胞軟骨分化影響的評價體系，比較了不同形態(tài)的I型膠原對細胞軟骨分化的作用，并研究了膠原對FAK和MAPK信號通路的影響，以期為軟骨修復材料的設計和研發(fā)提供指導。 通過geNorm算法分析16個常用內(nèi)參基因在ATDC5軟骨分化過程中的穩(wěn)定性，結(jié)合內(nèi)參基因的表達水平分析，篩選出Ppia和Hprt作為軟骨分化過程中qPCR實驗的理想內(nèi)參基因。以小鼠MSCs證實了Ppia和Hprt在MSCs軟骨分化過程中同樣穩(wěn)定。隨后優(yōu)化了ATDC5在評價材料對細胞軟骨分化影響實驗中的各種參數(shù)。通過細胞增殖、基因表達、染色及定量等方法證實了含TGF-β3的誘導液可縮短ATDC5的增殖期并迅速進入軟骨分化進程，表達軟骨相關表型，比ATDC5軟骨分化中常用的胰島素誘導液更適于評價材料對細胞的軟骨分化。篩選出TGF-β3的最優(yōu)添加濃度為10ng/ml，在此濃度下ATDC5細胞軟骨表型基因表達最高，且內(nèi)骨化表型基因表達最低。 本研究制備了三種不同形態(tài)的I型膠原。通過冷凍干燥制備了非重組纖維結(jié)構(gòu)的海綿支架，孔間為連通結(jié)構(gòu)，，孔徑約為300μm。采用NaOH中和法制備了網(wǎng)狀重組纖維結(jié)構(gòu)凝膠，纖維直徑為100-200nm。通過I型膠原體外重組與原位除鹽制備了重組纖維膜包被的細胞培養(yǎng)器皿，天狼猩紅染色及三維立體顯微鏡檢測結(jié)果顯示在細胞培養(yǎng)器皿表面包被的膠原膜均一、平整；SEM、TEM、AFM等顯示纖維的直徑為100-200nm，周期性螺紋結(jié)構(gòu)D-band約68nm，螺紋gap深度約3.46nm，同體內(nèi)膠原纖維的天然形態(tài)基本一致。 在評價體系建立的基礎上，本論文研究了不同形態(tài)I型膠原對ATDC5細胞軟骨分化早期的影響。通過分析細胞的生長形態(tài)及相關基因的表達水平，發(fā)現(xiàn)在軟骨誘導液下膠原對細胞軟骨分化的具體作用依賴于膠原的形態(tài)。網(wǎng)狀重組纖維結(jié)構(gòu)凝膠使細胞形態(tài)轉(zhuǎn)為利于軟骨分化的圓形，比阻礙細胞自發(fā)凝聚及形態(tài)轉(zhuǎn)變的重組纖維膜與非重組纖維結(jié)構(gòu)海綿支架更利于ATDC5的軟骨分化。同無膠原微團形式培養(yǎng)的ATDC5相比，膠原的存在提供了胞外基質(zhì)信號，促進了細胞的軟骨分化。 其次，研究了不同形態(tài)的I型膠原對ATDC5細胞軟骨分化后期肥大與內(nèi)骨化的作用。含β-磷酸甘油、維生素C和地塞米松的OM誘導液對ATDC5內(nèi)骨化有誘導作用，且能保證細胞不過度增殖，使所有細胞都可與材料相互接觸。細胞形態(tài)、基因表達、染色與定量等檢測分析均顯示單層培養(yǎng)形式下膠原重組纖維膜對ATDC5內(nèi)骨化無促進與誘導作用，而非重組纖維結(jié)構(gòu)的海綿膠原支架對ATDC5細胞內(nèi)骨化不僅有促進，而且有誘導的作用。 最后，采用hBMSCs檢測0.25mg/ml及2.5mg/ml的I型膠原凝膠對細胞軟骨分化的影響，證實了ATDC5細胞所得到的結(jié)果�；虮磉_、組織染色與免疫組化等檢測手段均顯示在TGF-β3的誘導下，I型膠原可促進hBMSCs的軟骨分化，0.25mg/ml的膠原凝膠比2.5mg/ml的促進效果好。通過對MAPK家族相關信號蛋白磷酸化水平的分析及FAK表達水平的檢測，發(fā)現(xiàn)在TGF-β3的誘導下，I型膠原的存在維持了FAK表達水平的相對穩(wěn)定并激活了細胞內(nèi)的MAPK信號通路。
[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.
【學位授予單位】：華南理工大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R318.08

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