軟骨細(xì)胞和骨髓間充質(zhì)干細(xì)胞混合培養(yǎng)構(gòu)建組織工程軟骨的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2019-01-12 09:10
【摘要】： 目的: 探討軟骨細(xì)胞和骨髓間充質(zhì)干細(xì)胞(BMSCs)混合培養(yǎng)對(duì)構(gòu)建組織工程軟骨的影響,并確定兩者的最佳比例。 方法: 取一月齡新西蘭兔六只,雌雄不限。體外分離培養(yǎng)擴(kuò)增兔關(guān)節(jié)軟骨細(xì)胞和BMSCs。收獲第一代末的軟骨細(xì)胞和BMSCs,按不同比例(軟骨細(xì)胞/BMSCs:4/1,2/1,1/1,1/2,1/4,單純軟骨細(xì)胞)混合培養(yǎng)一代。以4×107/ml的細(xì)胞終濃度接種40μl于聚乳酸聚羥基乙酸聚合物支架( PLGA:4mm×4mm×2mm)上靜態(tài)培養(yǎng)2天,移入周期性壓力場(chǎng)下培養(yǎng)(壓力:0～200kPa ,頻率:0.1 Hz,時(shí)間:8h/d)三周。三周后觀察各組組織工程軟骨的大小形態(tài)質(zhì)地光澤和組織彈性;HE染色法觀察組織工程軟骨細(xì)胞增殖及分布;甲苯胺藍(lán)染色法觀察氨基糖胺聚糖(GAG)的分泌及分布狀況,并用1,9二甲基亞甲藍(lán)法定量檢測(cè)GAG的含量;采用Ⅱ型膠原免疫組織化學(xué)法觀察Ⅱ型膠原的分泌及分布,并用image-proplus6.0圖象分析系統(tǒng)對(duì)Ⅱ型膠原染色面積行半定量分析;比色法測(cè)定組織工程軟骨的DNA含量觀察組織工程軟骨的細(xì)胞增殖情況。 結(jié)果: 軟骨細(xì)胞和BMSCs混合培養(yǎng)組與單純軟骨細(xì)胞組相比,體積較大,表面光滑,有彈性,有光澤。組織學(xué)顯示混合培養(yǎng)組結(jié)構(gòu)致密,細(xì)胞外基質(zhì)分布更均勻,其中2/1組可見(jiàn)軟骨陷窩�；旌吓囵B(yǎng)組的Ⅱ型膠原染色面積、GAG含量、DNA含量高于單純軟骨細(xì)胞組,其中2/1組含量最高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論: 軟骨細(xì)胞和BMSCs混合培養(yǎng)能顯著提高組織工程軟骨的質(zhì)量,其中軟骨細(xì)胞和BMSCs的濃度比為2/1時(shí)效果最好。
[Abstract]:Aim: to investigate the effect of co-culture of chondrocytes and bone marrow mesenchymal stem cells (BMSCs) on the construction of tissue engineered cartilage and determine the optimal ratio of the two. Methods: six New Zealand rabbits (male and female) of one month old were selected. Isolation and amplification of rabbit articular chondrocytes and BMSCs. in vitro The first generation of chondrocytes and BMSCs, were harvested and cultured in different proportion (chondrocytes / BMSCs:4/1,2/1,1/1,1/2,1/4, pure chondrocytes). The cells were inoculated with 40 渭 l of 4 脳 107/ml cells in PLGA:4mm 脳 4mm 脳 2mm for 2 days, then cultured under cyclic pressure (pressure: 0~200kPa, frequency: 0. 1 Hz, time: 8h/d) for three weeks. The size of tissue engineered cartilage in each group was observed three weeks later. Form? Texture? HE staining was used to observe the proliferation and distribution of tissue engineered chondrocytes, toluidine blue staining was used to observe the secretion and distribution of aminoglycosaminoglycan (GAG), and the content of GAG was detected by 1- (9) dimethylmethylene blue method. The secretion and distribution of type 鈪，
本文編號(hào)：2407604
[Abstract]:Aim: to investigate the effect of co-culture of chondrocytes and bone marrow mesenchymal stem cells (BMSCs) on the construction of tissue engineered cartilage and determine the optimal ratio of the two. Methods: six New Zealand rabbits (male and female) of one month old were selected. Isolation and amplification of rabbit articular chondrocytes and BMSCs. in vitro The first generation of chondrocytes and BMSCs, were harvested and cultured in different proportion (chondrocytes / BMSCs:4/1,2/1,1/1,1/2,1/4, pure chondrocytes). The cells were inoculated with 40 渭 l of 4 脳 107/ml cells in PLGA:4mm 脳 4mm 脳 2mm for 2 days, then cultured under cyclic pressure (pressure: 0~200kPa, frequency: 0. 1 Hz, time: 8h/d) for three weeks. The size of tissue engineered cartilage in each group was observed three weeks later. Form? Texture? HE staining was used to observe the proliferation and distribution of tissue engineered chondrocytes, toluidine blue staining was used to observe the secretion and distribution of aminoglycosaminoglycan (GAG), and the content of GAG was detected by 1- (9) dimethylmethylene blue method. The secretion and distribution of type 鈪，
本文編號(hào)：2407604
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