發(fā)布時(shí)間：2018-01-03 21:13

  本文關(guān)鍵詞：三種亞型軟骨組織來(lái)源干細(xì)胞的分離培養(yǎng)及鑒定　出處：《中國(guó)修復(fù)重建外科雜志》2015年04期 　論文類(lèi)型：期刊論文

  更多相關(guān)文章： 軟骨組織工程 軟骨干細(xì)胞 軟骨亞型 纖維連接蛋白

【摘要】：目的分離培養(yǎng)豬不同亞型軟骨組織(彈性軟骨、透明軟骨以及纖維軟骨)來(lái)源干細(xì)胞并鑒定,為軟骨組織工程提供理想種子細(xì)胞。方法利用纖維連接蛋白黏附法分別從豬耳軟骨、關(guān)節(jié)軟骨以及椎間盤(pán)軟骨中分離培養(yǎng)干細(xì)胞,并進(jìn)行傳代。倒置相差顯微鏡下觀察細(xì)胞形態(tài)變化,流式細(xì)胞術(shù)鑒定細(xì)胞表面抗原表達(dá)水平(陽(yáng)性標(biāo)志物CD29、CD90及陰性標(biāo)志物CD34、CD45),單克隆形成實(shí)驗(yàn)鑒定軟骨干細(xì)胞單克隆形成能力。三向誘導(dǎo)分化鑒定軟骨來(lái)源干細(xì)胞的成軟骨、成骨及成脂多向分化潛能。RT-PCR檢測(cè)成骨(Ⅰ型膠原、Ⅹ型膠原)、成軟骨[蛋白聚糖(Aggrecan)、II型膠原]、成脂[脂聯(lián)素(Adiponectin)、脂肪酸合成酶(fatty acid synthase,FAS)]相關(guān)基因表達(dá),并以豬BMSCs作為對(duì)照。結(jié)果通過(guò)纖維連接蛋白黏附法分別從耳軟骨(彈性軟骨)、關(guān)節(jié)軟骨(透明軟骨)、椎間盤(pán)軟骨(纖維軟骨)分選出一群細(xì)胞,細(xì)胞高表達(dá)干細(xì)胞表面陽(yáng)性標(biāo)志物CD29、CD90,幾乎不表達(dá)干細(xì)胞表面陰性標(biāo)志物CD34、CD45。經(jīng)過(guò)體外2周培養(yǎng),單個(gè)細(xì)胞均能形成細(xì)胞克隆。三向誘導(dǎo)分化顯示軟骨來(lái)源的干細(xì)胞具備成軟骨、成骨和成脂分化能力。RT-PCR結(jié)果顯示,成骨誘導(dǎo)后關(guān)節(jié)和椎間盤(pán)來(lái)源軟骨干細(xì)胞的Ⅰ、Ⅹ型膠原基因相對(duì)表達(dá)量明顯高于BMSCs(P0.05),耳軟骨來(lái)源干細(xì)胞與BMSCs比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);成軟骨誘導(dǎo)后,3種亞型軟骨組織來(lái)源干細(xì)胞Aggrecan、Ⅱ型膠原基因相對(duì)表達(dá)量均高于BMSCs(P0.05);成脂誘導(dǎo)后,3種來(lái)源軟骨干細(xì)胞Adiponectin及FAS基因相對(duì)表達(dá)量均低于BMSCs,但比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論不同亞型的豬軟骨組織中均存在軟骨干細(xì)胞,具有干細(xì)胞的典型特征。
[Abstract]:Objective to isolate and identify the stem cells derived from different subtypes of cartilage (elastic cartilage, hyaline cartilage and fibrocartilage) of pigs. Methods Stem cells were isolated from porcine ear cartilage, articular cartilage and intervertebral disc cartilage by fibronectin adhesion method. The changes of cell morphology were observed under inverted phase contrast microscope and the expression level of cell surface antigen was identified by flow cytometry (positive marker CD29 CD90 and negative marker CD34). CD45, monoclonal formation assay was used to identify the ability of forming chondrocytes. Three ways of inducing differentiation were used to identify the chondrogenic cartilage of chondrogenic stem cells. Detection of osteogenesis (type I collagen, type X collagen, cartilage formation) by reverse transcription-polymerase chain reaction (RT-PCR). [Proteoglycan type II collagen. [Adiponectin (Adiponectin), fatty acid synthase (FAS)). Results A group of cells were isolated from ear cartilage (elastic cartilage), articular cartilage (hyaline cartilage) and intervertebral disc cartilage (fibrous cartilage) by fibronectin adhesion method. The cells expressed CD29 + CD90 on the surface of stem cells and almost no CD34 + CD45 on the surface of stem cells. The cells were cultured for 2 weeks in vitro. Three-way differentiation showed that the chondrogenic stem cells had the ability of chondrogenesis, osteogenesis and adipogenic differentiation. RT-PCR results showed that. After osteogenic induction, the relative expression of type 鈪，

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