本文選題：椎間盤退行性變　切入點(diǎn)：髓核間充質(zhì)干細(xì)胞　出處：《南方醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：實(shí)驗(yàn)?zāi)康?研究髓核間充質(zhì)干細(xì)胞和髓核細(xì)胞修復(fù)退變椎間盤能力的差異,為髓核間充質(zhì)干細(xì)胞治療退變椎間盤的進(jìn)一步應(yīng)用提供實(shí)驗(yàn)根據(jù)。實(shí)驗(yàn)方法:1.髓核間充質(zhì)干細(xì)胞的分離培養(yǎng)及生物活性檢測:髓核間充質(zhì)干細(xì)胞是通過流式細(xì)胞分選術(shù)獲得并培養(yǎng),并進(jìn)行成骨、成軟骨、成脂細(xì)胞誘導(dǎo)分化實(shí)驗(yàn)。成骨分化檢測包括堿性磷酸酶染色和和茜素紅染色。成軟骨分化與成脂肪檢測分別是通過甲苯胺藍(lán)染色鑒定與油紅O染液染色完成。2.毮核細(xì)胞的分離培養(yǎng)及生物活性檢測:髓核細(xì)胞是通過細(xì)胞貼壁法獲得并培養(yǎng),進(jìn)行增殖能力的檢測、Ⅱ型膠原染色和甲苯胺藍(lán)染色。3.實(shí)驗(yàn)分組:將32只兔子進(jìn)行退變椎間盤模型建立,造模后隨機(jī)分為A、B、C、D組,分別為空白對照組,髓核間充質(zhì)干細(xì)胞移植組,髓核細(xì)胞移植組,單純培養(yǎng)液對照組。4.影像學(xué)檢查:在細(xì)胞移植1d前及細(xì)胞移植12w后,行X線檢查以評估椎間盤高度指數(shù)(DHI%),行MRI檢查以計(jì)算髓核區(qū)域相對信號強(qiáng)度(RSI)。5.PCR檢測:提取總RNA,把內(nèi)參引物序列GAPDH的表達(dá)量除膠原蛋白Ⅱ、蛋白聚糖的表達(dá)量,得到膠原蛋白Ⅱ、蛋白聚糖的相對表達(dá)量。6.蛋白聚糖及Ⅱ型膠原蛋白定量檢測。7.統(tǒng)計(jì)學(xué)分析:選用spss20.0軟件進(jìn)行數(shù)據(jù)統(tǒng)計(jì)學(xué)分析。計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,組間進(jìn)行單因素方差分析,以P0.05表示統(tǒng)計(jì)學(xué)差異顯著,P0.05表示無明顯統(tǒng)計(jì)學(xué)差異。主要結(jié)果:1.髓核間充質(zhì)干細(xì)胞在成骨誘導(dǎo)分化及檢測中,堿性磷酸酶染色和和茜素紅染色均顯示陽性顯色。在成軟骨和成脂誘導(dǎo)分化及檢測中見甲苯胺藍(lán)染色見細(xì)胞呈藍(lán)色,并有大小不等的脂滴形成。2.兔原代髓核細(xì)胞呈圓形,形態(tài)大小均一。細(xì)胞增殖活力在早期較低,而在5d-13d時(shí)明顯增強(qiáng)。甲苯胺藍(lán)染色與Ⅱ型膠原蛋白免疫組化染色見陽性。3.在移植12w后,B組的高度指數(shù)(DHI%)與其他組相比明顯增高,有顯著性差異(P0.05)。B組的RSI值比C組明顯增強(qiáng),顯著性差異(P0.05)。4.在PCR檢測和蛋白聚糖及Ⅱ型膠原蛋白定量檢測可知,B組在蛋白聚糖及Ⅱ型膠原蛋白的基因表達(dá)水平高于其他組,有統(tǒng)計(jì)學(xué)意義。B組比C組蛋白聚糖的蛋白表達(dá)水平高,有顯著性差異,但四組在Ⅱ型膠原蛋白表達(dá)水平上差異無統(tǒng)計(jì)學(xué)意義(P0.05)。實(shí)驗(yàn)結(jié)論:髓核間充質(zhì)干細(xì)胞與髓核細(xì)胞均能提高退變椎間盤內(nèi)蛋白聚糖和Ⅱ型膠原蛋白基因水平的表達(dá),促進(jìn)退變椎間盤的修復(fù),但髓核間充質(zhì)干細(xì)胞的修復(fù)效果比髓核細(xì)胞更好。髓核間充質(zhì)干細(xì)胞或許能進(jìn)一步應(yīng)用于治療退變椎間盤方面。
[Abstract]:Objective: to study the difference of the ability of mesenchymal stem cells and nucleus pulposus cells to repair degenerative intervertebral disc. To provide experimental basis for further application of nucleus pulposus mesenchymal stem cells in the treatment of degenerative intervertebral disc. Experimental method: 1. Isolation, culture and bioassay of mesenchymal stem cells of nucleus pulposus: mesenchymal stem cells of nucleus pulposus are derived from flow cytometry. Sorting is obtained and cultivated, And osteogenesis, cartilage formation, Osteogenic differentiation was detected by alkaline phosphatase staining and alizarin red staining. Cartilage differentiation and adipogenesis were detected by toluidine blue staining and oil red O staining. Isolation, culture and bioactivity of nuclear cells: nucleus pulposus cells were obtained and cultured by cell adherence. The proliferative ability was tested, type 鈪，

本文編號：1610280