本文選題：間充質(zhì)干細(xì)胞 + Buffy　； 參考：《延邊大學(xué)》2017年碩士論文

【摘要】：目的:比較分析兔骨髓間充質(zhì)干細(xì)胞(Mesenchymal Stem Cells,MSC)與含有單核細(xì)胞、生長(zhǎng)因子和細(xì)胞因子等的Buffy coat在體外軟骨分化,為Buffy coat治療軟骨損傷提供實(shí)驗(yàn)依據(jù)。方法:動(dòng)物選取新西蘭系大白兔12只(16周齡,體重3±0.5kg),隨機(jī)分為:MSC組(陰性對(duì)照組,n=3),采集骨髓的間充質(zhì)干細(xì)胞(MSC),不添加轉(zhuǎn)化生長(zhǎng)因子-β(Transforing growth factor-β,TGF-β)的誘導(dǎo)下培養(yǎng)。Buffy coat組(實(shí)驗(yàn)組,n=6),采用Buffy coat不經(jīng)過(guò)體外的傳代培養(yǎng),在軟骨誘導(dǎo)液下培養(yǎng)。MSC+TGF組(陽(yáng)性對(duì)照組,n=3),采集骨髓的間充質(zhì)干細(xì)胞,添加TGF-β的誘導(dǎo)下培養(yǎng)。通過(guò)大體觀察、組織學(xué)觀察、糖胺多糖(Glycosaminoglycan,GAG)檢測(cè),和體外軟骨BERN的評(píng)分比較三組在體外分化的能力。結(jié)果:1)大體觀察:MSC組培養(yǎng)的軟骨部分已經(jīng)溶解,邊緣模糊;MSC+TGF組與Buffy coat組基本沒(méi)有溶解現(xiàn)象,邊緣圓潤(rùn)、清晰。2)番紅O染色鏡下觀察:MSC組軟骨細(xì)胞生長(zhǎng)緩慢,分布不均勻,數(shù)量極少,有極少量GAG的分泌現(xiàn)象出現(xiàn),分泌不規(guī)則;有少數(shù)細(xì)胞可以找到軟骨細(xì)胞特有的Cartilage lacuna結(jié)構(gòu)。Buffy coat組與MSC+TGF組軟骨細(xì)胞生長(zhǎng)速度較快,細(xì)胞分布較均勻,細(xì)胞數(shù)量較多,表面較平整、周圍較圓潤(rùn),有較多GAG分泌現(xiàn)象出現(xiàn),基本分布在軟骨細(xì)胞周圍;有較多細(xì)胞可以找到Cartilage lacuna結(jié)構(gòu)。3)糖胺多糖(GAG)含量測(cè)定:MSC 組:7.78± 1.79μg,Buffy coat 組:22.83±3.11μg,MSC+TGF 組:24.05±3.35μg。三組間存有顯著差異,MSC 組與 MSC+TGF 組、MSC 與 Buffy coat 組,組間存在顯著差異(P0.05);Buffy coat組與MSC+TGF組,組間無(wú)顯著差異(P0.05)。4)體外軟骨 BERN 評(píng)分:MSC 組:2.83± 1.16,Buffy coat 組:6.16±1.16,MSC+TGF組:6.68±0.81。三組間存在顯著差異,MSC組與MSC+TGF組、MSC與Buffy coat組,組間存在顯著差異(P0.05),Buffy coat組與MSC+TGF組,組間無(wú)顯著差異(P0.05)。結(jié)論:Buffy coat具有良好的體外軟骨分化能力,可以解決目前體外分化軟骨必須添加生長(zhǎng)因子這個(gè)難題,為Buffy coat在臨床應(yīng)用提供了實(shí)驗(yàn)依據(jù)。
[Abstract]:Aim: to compare and analyze the cartilage differentiation of rabbit bone marrow mesenchymal Stem cells (MSCs) and Buffy coat containing monocytes, growth factors and cytokines in vitro, so as to provide experimental evidence for the treatment of cartilage injury by Buffy coat. Methods: twelve New Zealand white rabbits aged 16 weeks were selected. Body weight was 3 鹵0.5 kg 路kg ~ (-1), and was randomly divided into two groups: 1 / growth group (negative control group). MSCs were collected from bone marrow and cultured without TGF- 尾-尾 -Transforing growth factor- 尾 (TGF- 尾). Buffy coat group (experimental group) was cultured without Buffy coat in vitro. Mesenchymal stem cells (MSCs) were collected from bone marrow and cultured with TGF- 尾. The ability of differentiation of the three groups in vitro was compared by gross observation, histological observation, glycosaminoglycan glycosaminoglycan gag test and cartilage BERN score in vitro. Results (1) the chondrocytes cultured in the TGF group were dissolved, but there was no dissolution in the TGF group and the Buffy coat group. The edges were smooth and clear. Under the microscope, the chondrocytes in the Buffy coat group grew slowly and distributed unevenly. A few cells could find the specific Cartilage lacuna structure of chondrocytes. Buffy coat group and MSC TGF group had a faster growth rate, more evenly distributed cells and more cells. The surface was smooth, the surrounding was smooth, and there were more GAG secretion phenomena, which were mainly distributed around the chondrocytes, and the Cartilage lacuna structure. 3) glycosaminoglycan (Gagg) content could be found in the Cartilage lacuna group. The content of Cartilage was 7. 78 鹵1. 79 渭 g / m Buffy coat group and 20. 83 鹵3. 11 渭 g / m GAG TGF group. There was significant difference among the three groups. There was significant difference between the two groups (P0.05 Buffy coat group and MSC TGF group). There was no significant difference among the three groups (P0.05. 4) the in vitro cartilage BERN score was 2.83 鹵1.16 Buffy coat group, 6.16 鹵1.16 MSC coat group, 6.68 鹵0.81MSC TGF group. There was significant difference among the three groups. There was no significant difference between the two groups (P0.05Buffy coat group and MSC TGF group). Conclusion coat has a good ability of cartilage differentiation in vitro, which can solve the problem that growth factor must be added to the differentiation cartilage in vitro, and provide experimental basis for the clinical application of Buffy coat.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R687;R318.08

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