本文關(guān)鍵詞：人臍帶間充質(zhì)干細(xì)胞復(fù)合海藻酸鈣水凝膠支架材料構(gòu)建組織工程軟骨的實(shí)驗(yàn)研究　出處：《暨南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 臍帶 間充質(zhì)干細(xì)胞 軟骨 海藻酸鈣 組織工程

【摘要】：目的：體外分離培養(yǎng)人臍帶間充質(zhì)干細(xì)胞,并對其進(jìn)行生物學(xué)鑒定及軟骨方向誘導(dǎo)。構(gòu)建海藻酸鈣水凝膠,同時觀察其生物相容性。檢測人臍帶間充質(zhì)干細(xì)胞與海藻酸鈣水凝膠構(gòu)建組織工程軟骨修復(fù)軟骨缺損的效果。 方法：用酶消化法分離培養(yǎng)人臍帶間充質(zhì)干細(xì)胞,通過傳代培養(yǎng),擴(kuò)增。倒置光學(xué)顯微鏡及原子力顯微鏡觀察細(xì)胞形態(tài)。MTT檢測細(xì)胞增殖曲線,流式細(xì)胞儀檢測細(xì)胞周期及免疫表型。在低密度、高密度、微團(tuán)的環(huán)境下對hUCMSCs向軟骨方向誘導(dǎo)。誘導(dǎo)成軟骨過程中應(yīng)用倒置光學(xué)顯微鏡觀察細(xì)胞結(jié)構(gòu)的變化,并對不同組別進(jìn)行甲苯胺藍(lán)雜色、免疫組化檢測。制備海藻酸鈣水凝膠支架材料。將1UCMSCs與支架材料復(fù)合,體外成軟骨誘導(dǎo)培養(yǎng)3周,掃描電鏡觀察細(xì)胞黏附情況,MTT法分析細(xì)胞增殖情況來評價其組織相容性。并通過原位植入兔股骨髁上軟骨缺損,通過組織學(xué)等手段檢測其軟骨修復(fù)效果。 結(jié)果：采用酶消化法能有效分離純化人臍帶間充質(zhì)干細(xì)胞。細(xì)胞增值能力強(qiáng)。流式細(xì)胞儀分析第3代細(xì)胞均強(qiáng)烈表達(dá)CD29、CD44、CD105,不表達(dá)CD34、CD45和HLA-DR。經(jīng)成軟骨誘導(dǎo)分化后,低密度誘導(dǎo)組及各對照組甲苯胺藍(lán)雜色、Ⅱ型膠原免疫組化染色弱陽性；高密度誘導(dǎo)組甲苯胺藍(lán)雜色、Ⅱ型膠原免疫組化染色陽性,微團(tuán)誘導(dǎo)組染色均表現(xiàn)出強(qiáng)陽性。掃描電鏡示細(xì)胞海藻酸鈣水凝膠支架上吸附,生長良好。MTT示hUCMSCs在材料上增殖能力良好。hUCMSCs構(gòu)建的組織工程軟骨能夠修復(fù)軟骨缺損。 結(jié)論：本實(shí)驗(yàn)建立了一套體外穩(wěn)定培養(yǎng)擴(kuò)增人臍帶間充質(zhì)干細(xì)胞的方法,所培養(yǎng)的細(xì)胞成分單一,擴(kuò)增迅速,生物學(xué)形狀穩(wěn)定；并能使其在體外采用不同環(huán)境下誘導(dǎo)向成軟骨細(xì)胞分化。海藻酸鈣水凝膠支架材料是一種具有良好生物相容性的支架材料。hUCMSCs與之構(gòu)建組織工程軟骨可修復(fù)軟骨缺損。
[Abstract]:Objective: to isolate and culture human umbilical cord mesenchymal stem cells in vitro and to construct calcium alginate hydrogel. At the same time, the biocompatibility of human umbilical cord mesenchymal stem cells and calcium alginate hydrogel was observed. Methods: human umbilical cord mesenchymal stem cells were isolated and cultured by enzyme digestion. The proliferation curves were observed by inverted optical microscope and atomic force microscope. Flow cytometry was used to detect cell cycle and immunophenotype. HUCMSCs was induced to cartilage in the environment of microclusters. The changes of cell structure were observed by inverted optical microscope during the process of cartilage formation, and toluidine blue hybrids were performed on different groups. Immunohistochemical examination. Calcium alginate hydrogel scaffold materials were prepared. 1UCMSCs were combined with scaffold materials and cultured in vitro for 3 weeks. The adhesion of cells was observed by scanning electron microscope (SEM). MTT method was used to evaluate the histocompatibility of the cells, and the cartilage repair effect was detected by in situ implantation of rabbit supracondylar cartilage defect. Results: human umbilical cord mesenchymal stem cells could be effectively isolated and purified by enzyme digestion. CD34, CD45 and HLA-DR were not expressed. After chondrogenic differentiation, toluidine blue heterochromatic staining and type 鈪，

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