【摘要】：目的：比較兔脂肪來源和骨髓來源的兩種間充質(zhì)干細(xì)胞的成軟骨能力。 方法：選取六月齡新西蘭大白兔,取腹部脂肪,分離培養(yǎng)ADSCs并進(jìn)行傳代。取兔雙側(cè)股骨,采用全骨髓培養(yǎng)法分離培養(yǎng)BMSCs并進(jìn)行傳代。使用倒置顯微鏡觀察兩種間充質(zhì)干細(xì)胞的形態(tài)及生長增殖情況。比較兩種細(xì)胞的原代及2、3代的傳代時(shí)間。繪制3代ADSCs和BMSCs的生長曲線,測定兩種細(xì)胞的倍增時(shí)間并進(jìn)行比較。選取兩種間充質(zhì)干細(xì)胞的3代細(xì)胞進(jìn)行成軟骨誘導(dǎo),實(shí)驗(yàn)分為四組：ADSCs成軟骨誘導(dǎo)組(A1),ADSCs一般DMEM培養(yǎng)基培養(yǎng)組(A2),BMSCs成軟骨誘導(dǎo)組(B1), BMSCs一般DMEM培養(yǎng)基培養(yǎng)組(B2)。其中A1、B1為實(shí)驗(yàn)組,A2、B2為對照組。倒置顯微鏡下觀察各組細(xì)胞形態(tài)及生長增殖情況。培養(yǎng)14天后,實(shí)驗(yàn)組行甲苯胺藍(lán)染色和Ⅱ型膠原免疫組化染色,對照組行Ⅱ型膠原免疫組化染色。Ⅱ型膠原免疫細(xì)胞化學(xué)染色灰度值評分法比較實(shí)驗(yàn)組中A1、B1的成軟骨能力。 結(jié)果：BMSCs原代細(xì)胞呈聚集樣生長,而ADSCs原代細(xì)胞呈單個(gè)、散在生長。自第二代開始,BMSCs的生長方式與ADSCs一致,以單個(gè)、散在方式生長。單層培養(yǎng)時(shí),這兩種間充質(zhì)干細(xì)胞增殖迅速且穩(wěn)定,形態(tài)上均為成纖維細(xì)胞樣,呈梭形,沒有觀察到明顯的形態(tài)學(xué)差異。原代ADSCs傳代時(shí)間為6天,第2代、第3代的ADSCs傳代時(shí)間為3天。原代BMSCs傳代時(shí)間為10天,第2代、第3代的BMSCs傳代時(shí)間為5天。原代ADSCs傳代時(shí)間較原代BMSCs快4天,而第2代、第3代的ADSCs傳代時(shí)間較同代BMSCs快2天。生長曲線示ADSCs增殖速度要快于BMSCs, ADSCs倍增時(shí)間為26h,BMSCs為36h。實(shí)驗(yàn)組加入成軟骨誘導(dǎo)液后,細(xì)胞的增殖緩慢,細(xì)胞的形態(tài)由長梭形逐漸變成了多角形,或者圓形。而對照組細(xì)胞的形態(tài)無變化,皆為梭形。成軟骨誘導(dǎo)14天后,實(shí)驗(yàn)組A1和B1甲苯胺藍(lán)染色及Ⅱ型膠原免疫組化染色皆為陽性表達(dá)。而對照組Ⅱ型膠原免疫組化染色陰性表達(dá)。Ⅱ型膠原免疫細(xì)胞化學(xué)染色灰度值評分示,B1組表達(dá)Ⅱ型膠原水平高于A1組(p0.05)。 結(jié)論：單層培養(yǎng)時(shí),ADSCs與BMSCs形態(tài)上均為成纖維細(xì)胞樣,呈梭形,兩者之間沒有明顯的形態(tài)學(xué)差異。ADSCs與BMSCs增殖皆迅速且穩(wěn)定,但是ADSCs的生長增殖速度更快。單層培養(yǎng)時(shí),特定條件下,ADSCs與BMSCs均能向軟骨細(xì)胞轉(zhuǎn)化。兩者成軟骨分化時(shí),BMSCs比ADSCs有更高的潛能。
[Abstract]:Objective: to compare the cartilage formation ability of rabbit adipose stem cells and bone marrow derived mesenchymal stem cells. Methods: six months old New Zealand white rabbits were selected, abdominal fat was taken, ADSCs was isolated and cultured and subcultured. The bilateral femurs of rabbits were isolated and cultured by whole bone marrow culture and subcultured. The morphology, growth and proliferation of two kinds of mesenchymal stem cells were observed by inverted microscope. The primary passage time and the passage time of 2 and 3 generations of the two kinds of cells were compared. The growth curves of ADSCs and BMSCs were drawn, and the doubling time of the two cells was measured and compared. Three generations of cells from two kinds of mesenchymal stem cells were selected for cartilage induction. The experiment was divided into four groups: ADSCs cartilage induction group (A1), ADSCs general DMEM medium culture group (A2), BMSCs cartilage induction group B1). BMSCs general DMEM medium culture group (B2). Among them, A1, B1 were the experimental group, A2 and B2 were the control groups. The morphology, growth and proliferation of cells in each group were observed under inverted microscope. After 14 days of culture, the experimental group was stained with toluene blue and type 鈪，

本文編號：2484129