【摘要】：目的:探索兔脂肪組織中分離、培養(yǎng)及鑒定干細(xì)胞的方法;觀察體外長(zhǎng)期培養(yǎng)對(duì)新西蘭大白兔脂肪間充質(zhì)干細(xì)胞(Adipose-Derived Stem Cells,ADSCs)的基本生物學(xué)性狀及定向改良誘導(dǎo)分化為軟骨細(xì)胞的能力。方法:在無(wú)菌條件下從雄性大白兔頸部區(qū)取皮下脂肪組織約5～10g,I型膠原酶消化后,1500r/min離心10min,待細(xì)胞長(zhǎng)滿(mǎn)瓶底80%時(shí)用2.5g/L胰蛋白酶消化,按1:3比例傳代培養(yǎng)、鑒定,至第3代時(shí)加入軟骨誘導(dǎo)培養(yǎng)基14d后通過(guò)免疫熒光和RT-PCR對(duì)其相關(guān)表型進(jìn)行檢測(cè),阿新藍(lán)染色及II型膠原免疫組織化學(xué)檢測(cè)脂肪間充質(zhì)干細(xì)胞向軟骨細(xì)胞的誘導(dǎo)分化。結(jié)果:兔脂肪間充質(zhì)干細(xì)胞形態(tài):原代及傳代的兔脂肪干細(xì)胞均呈長(zhǎng)梭形或多邊形貼壁生長(zhǎng),生長(zhǎng)分化活躍。兔脂肪間充質(zhì)干細(xì)胞的生長(zhǎng)曲線(xiàn):傳代的兔脂肪間充質(zhì)干細(xì)胞生長(zhǎng)曲線(xiàn)呈“S”形。新西蘭大白兔脂肪組織分離培養(yǎng)出的細(xì)胞經(jīng)流式細(xì)胞儀檢測(cè)CD44、CD49d表達(dá)陽(yáng)性,CD45表達(dá)陰性。加入軟骨誘導(dǎo)培養(yǎng)基定向誘導(dǎo)72h后,細(xì)胞生長(zhǎng)緩慢,由長(zhǎng)梭形逐漸變成圓形,并漸漸有軟骨結(jié)節(jié)形成,胞漿中出現(xiàn)棕色顆粒,細(xì)胞表面有較多偽足,具備正常細(xì)胞器結(jié)構(gòu)。誘導(dǎo)分化2周后,熒光免疫檢測(cè)有特異軟骨基質(zhì)Ⅱ型膠原和蛋白聚糖的表達(dá), RT- PCR檢測(cè)顯示有Ⅱ型膠原、Aggrecan和Sox- 9 mRNA的表達(dá),阿新藍(lán)染色及II型膠原免疫組織化學(xué)檢測(cè)陽(yáng)性。結(jié)論:新西蘭大白兔脂肪組織能夠分離培養(yǎng)出脂肪干細(xì)胞,兔源性脂肪干細(xì)胞體外以成纖維細(xì)胞樣形態(tài)貼壁生長(zhǎng),在體外具有生長(zhǎng)穩(wěn)定,增殖較快的特點(diǎn),生物學(xué)特性與骨髓間充質(zhì)干細(xì)胞相似,能向軟骨細(xì)胞分化,在體外長(zhǎng)期培養(yǎng)后,其一般生物學(xué)性狀可保持穩(wěn)定。
[Abstract]:Objective: to explore the method of isolation, culture and identification of stem cells in rabbit adipose tissue. To observe the basic biological characteristics of adipose mesenchymal stem cells (Adipose-Derived Stem Cells,ADSCs) of New Zealand white rabbits and their ability to differentiate into chondrocytes in vitro. Methods: the subcutaneous adipose tissue of male white rabbits was digested with subcutaneous adipose tissue of about 5U 10g / L collagenase under aseptic condition and centrifuged by 1500r/min for 10 mins. The cells were digested with 2.5g/L trypsin when the cells grew to the bottom of the bottle and cultured at 1:3. The phenotypes were detected by immunofluorescence and RT-PCR, and the differentiation of adipose mesenchymal stem cells into chondrocytes was detected by Arxin blue staining and II collagen immunohistochemistry. Results: the morphology of rabbit adipose mesenchymal stem cells: primary and passage of rabbit adipose stem cells were long fusiform or polygonal adherent growth and active growth and differentiation. The growth curve of rabbit adipose mesenchymal stem cells: the growth curve of rabbit adipose mesenchymal stem cells was "S" shape. The positive expression of CD44,CD49d and negative expression of CD45 were detected by flow cytometry in adipose tissue isolated from New Zealand white rabbit. After 72 hours of induction in cartilage induction medium, the cells grew slowly, gradually became round from long spindle shape, and gradually formed cartilage nodules, brown granules appeared in the cytoplasm, and the cell surface had more pseudopodia, with normal organelle structure. After 2 weeks of differentiation, the expression of type 鈪，

本文編號(hào)：2281697