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

  更多相關(guān)文章： 臍帶 間充質(zhì)干細胞 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)干細胞,并對其進行生物學鑒定及軟骨方向誘導。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)干細胞與海藻酸鈣水凝膠構(gòu)建組織工程軟骨修復軟骨缺損的效果。 方法：用酶消化法分離培養(yǎng)人臍帶間充質(zhì)干細胞,通過傳代培養(yǎng),擴增。倒置光學顯微鏡及原子力顯微鏡觀察細胞形態(tài)。MTT檢測細胞增殖曲線,流式細胞儀檢測細胞周期及免疫表型。在低密度、高密度、微團的環(huán)境下對hUCMSCs向軟骨方向誘導。誘導成軟骨過程中應用倒置光學顯微鏡觀察細胞結(jié)構(gòu)的變化,并對不同組別進行甲苯胺藍雜色、免疫組化檢測。制備海藻酸鈣水凝膠支架材料。將1UCMSCs與支架材料復合,體外成軟骨誘導培養(yǎng)3周,掃描電鏡觀察細胞黏附情況,MTT法分析細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,通過組織學等手段檢測其軟骨修復效果。 結(jié)果：采用酶消化法能有效分離純化人臍帶間充質(zhì)干細胞。細胞增值能力強。流式細胞儀分析第3代細胞均強烈表達CD29、CD44、CD105,不表達CD34、CD45和HLA-DR。經(jīng)成軟骨誘導分化后,低密度誘導組及各對照組甲苯胺藍雜色、Ⅱ型膠原免疫組化染色弱陽性；高密度誘導組甲苯胺藍雜色、Ⅱ型膠原免疫組化染色陽性,微團誘導組染色均表現(xiàn)出強陽性。掃描電鏡示細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軟骨缺損。 結(jié)論：本實驗建立了一套體外穩(wěn)定培養(yǎng)擴增人臍帶間充質(zhì)干細胞的方法,所培養(yǎng)的細胞成分單一,擴增迅速,生物學形狀穩(wěn)定；并能使其在體外采用不同環(huán)境下誘導向成軟骨細胞分化。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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