【摘要】：目的采用正交實(shí)驗(yàn)研究滑膜間充質(zhì)干細(xì)胞(synovial derived MSCs,SMSCs)成纖維軟骨分化的條件。方法 5只成年新西蘭白兔,活檢取滑膜組織。貼壁法獲取SMSCs后采用流式細(xì)胞儀及成脂、成骨、成軟骨誘導(dǎo)分化鑒定。根據(jù)預(yù)實(shí)驗(yàn)與文獻(xiàn)綜述尋找與SMSCs成纖維軟骨分化可能相關(guān)的條件,采用缺失實(shí)驗(yàn)初篩必要條件后,TGF-β1、BMP-2、地塞米松、脯氨酸、檸檬酸(ascorbic acid,ASA)、丙酮酸、胰島素+轉(zhuǎn)鐵蛋白+亞硒酸預(yù)混液、牛血清白蛋白、b FGF、間斷靜水壓、BMP-7、IGF納入正交實(shí)驗(yàn),采用SPSS18.0統(tǒng)計(jì)軟件設(shè)計(jì)L60(212)正交實(shí)驗(yàn)及表頭,定義2水平條件,在SMSCs-三維小腸黏膜下層(small intestinal submucosa,SIS)支架上誘導(dǎo)成纖維軟骨分化。使用流式細(xì)胞儀計(jì)數(shù),檢測CD151+/CD44+細(xì)胞并記錄SMSCs向纖維軟骨分化的轉(zhuǎn)換率,采用免疫組織化學(xué)染色,結(jié)合細(xì)胞形態(tài)、甲苯胺藍(lán)染色、半定量RT-PCR檢測SOX9、聚集蛋白聚糖、Ⅰ型膠原(collagen typeⅠ,ColⅠ)、ColⅡ、ColⅨ基因表達(dá),進(jìn)一步驗(yàn)證結(jié)果。檢驗(yàn)指標(biāo)rate是CD151+/CD44+細(xì)胞與高表達(dá)ColⅠ細(xì)胞的比例乘積。同時采用Pico Green Assay測量細(xì)胞總DNA量,以反映細(xì)胞擴(kuò)增情況。正交實(shí)驗(yàn)結(jié)果采用直觀觀察和主體間方差分析方法,考慮部分因子間的1階交互作用,組間差異采用LSD和q檢驗(yàn)驗(yàn)證,使用Ⅲ型平方和校正模型,檢驗(yàn)水準(zhǔn)α=0.05。結(jié)果實(shí)驗(yàn)獲取的細(xì)胞為SMSCs,細(xì)胞倍增時間為28 h。成纖維軟骨分化過程中SMSCs-三維SIS支架體積5 d倍增,14 d后得到甲苯胺藍(lán)染色陽性的細(xì)胞-支架復(fù)合物。正交實(shí)驗(yàn)結(jié)果直觀分析示,TGF-β1對成纖維軟骨分化轉(zhuǎn)化率的作用最明顯,BMP-7采用水平2有利于得到更高的轉(zhuǎn)化率,但與BMP-2存在交互作用;其余第1區(qū)組水平值加和高于22.5的因子有DEX、ASA、ITS、轉(zhuǎn)鐵蛋白、b FGF,模型的相關(guān)性好。方差分析校正模型P=0.000,能夠滿足實(shí)驗(yàn)設(shè)計(jì);截距P=0.000,說明各因子對因變量影響差異不完全相同。TGF-β1、ASA、b FGF、IGF對因變量調(diào)控作用較其他因子顯著,差異有統(tǒng)計(jì)學(xué)意義,與直觀觀察結(jié)果相似。結(jié)論 TGF-β1、ASA、b FGF、IGF顯著影響SMSCs成纖維軟骨分化,通過合理調(diào)整上述因子濃度,可顯著提高SMSCs成纖維軟骨分化轉(zhuǎn)化率;精確的調(diào)控條件和調(diào)控機(jī)制有待進(jìn)一步探索。
[Abstract]:Objective to study the condition of fibrocartilage differentiation of synovial mesenchymal stem cells (synovial derived MSCs,SMSCs) by orthogonal experiment. Methods 5 adult New Zealand white rabbits were biopsied for synovial tissue. SMSCs obtained by adherent method was identified by flow cytometry, adipogenesis, osteogenesis and cartilage-induced differentiation. According to the pre-experiment and literature review, the possible conditions related to the differentiation of SMSCs fibroblast cartilage were found. After screening the necessary conditions for TGF- 尾 _ 1 BMP-2, dexamethasone, proline, citric acid (ascorbic acid,ASA), pyruvate. Insulin transferrin selenite premixed solution, bovine serum albumin (BSA), b FGF, intermittent hydrostatic pressure, BMP-7,IGF was included in orthogonal experiment, L60 (212) orthogonal experiment and its head were designed by SPSS18.0 software, and 2 level conditions were defined. Fibrocartilage differentiation was induced on SMSCs- 3 D small intestinal submucosal (small intestinal submucosa,SIS scaffold. Flow cytometry was used to detect CD151 / CD44 cells and to record the conversion rate of SMSCs into fibrochondrocytes. Immunohistochemical staining, cell morphology, toluidine blue staining and semi-quantitative RT-PCR were used to detect SOX9, aggregation proteoglycan. The expression of (collagen type 鈪，

本文編號：2300002