發(fā)布時間：2018-04-02 10:54

  本文選題：軟骨分化　切入點(diǎn)：海藻酸鈉　出處：《山西醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:觀察周期性動態(tài)壓縮應(yīng)力對骨髓間充質(zhì)干細(xì)胞軟骨分化的作用,同時對比分析力學(xué)因素對敲減SOX9基因骨髓間充質(zhì)干細(xì)胞軟骨分化過程中的作用,探討力學(xué)因素與SOX9在軟骨分化過程中的重要作用。方法:取4周齡昆明小鼠骨髓間充質(zhì)干細(xì)胞(C),體外培養(yǎng)至第三代,流式細(xì)胞儀鑒定細(xì)胞表型確定為干細(xì)胞,將構(gòu)建的小鼠SOX9基因干擾的慢病毒載體體外轉(zhuǎn)染小鼠骨髓間充質(zhì)干細(xì)胞,構(gòu)建SOX9敲減的骨髓間充質(zhì)干細(xì)胞(si C)。分別將C細(xì)胞和si C細(xì)胞以1x107/ml的密度與2%海藻酸鈉凝膠混勻,制成圓柱形載體。根據(jù)實驗過程中對載體的處理因素的不同將試驗分為8組:各組在試驗的前7天均給予軟骨誘導(dǎo)液處理。A組:C細(xì)胞加力未誘導(dǎo)組:在軟骨誘導(dǎo)液誘導(dǎo)7天后再給予7天的加力未誘導(dǎo)處理;B組:C細(xì)胞加力誘導(dǎo)組:在軟骨誘導(dǎo)液誘導(dǎo)7天后再給予7天的加力誘導(dǎo)處理;C組:誘導(dǎo)不加力組:在軟骨誘導(dǎo)液誘導(dǎo)7天后再給予7天的誘導(dǎo)不加力處理;D組:si C細(xì)胞加力未誘導(dǎo)組;E組:si C細(xì)胞加力誘導(dǎo)組;F組:si C細(xì)胞誘導(dǎo)不加力組;D、E、F組的處理分別同A、B、C組。G、H組為分別為C細(xì)胞、si C細(xì)胞未加力、未誘導(dǎo)的對照組:誘導(dǎo)7天后予以普通培養(yǎng)液培養(yǎng);實驗采用Flexcell-5000基底加載系統(tǒng)對細(xì)胞盤施加正弦波、0.5HZ、20Kpa、2h/d壓縮應(yīng)力。在相關(guān)處理后采用RT-PCR分析SOX9、ROCK1及COL-Ⅱ的表達(dá)。應(yīng)用免疫熒光技術(shù)對各組處理前后的COL-Ⅱ表達(dá)進(jìn)行監(jiān)測。結(jié)果:RT-PCR檢測:SOX9及COL-Ⅱm RNA表達(dá):B組高于C組及A組,C組較G組表達(dá)高,E組高于F組及D組,D組高于H組(P0.05)。A組與G組表達(dá)相當(dāng),B組及E組表達(dá)無顯著差異(P0.05)、ROCK1m RNA:E組高于B組(P0.05)。免疫熒光檢測顯示B組及E組均有二型膠原免疫熒光表達(dá)。結(jié)論:周期性動態(tài)壓縮應(yīng)力可以促進(jìn)間充質(zhì)干細(xì)胞的軟骨分化。周期性動態(tài)力學(xué)加載可以增加敲減SOX9基因骨髓間充質(zhì)干細(xì)胞的SOX9、COL-Ⅱ的表達(dá),降低ROCK1的表達(dá),周期性動態(tài)壓縮應(yīng)力可能通過SOX9相關(guān)通路促進(jìn)間充質(zhì)干細(xì)胞的軟骨分化。
[Abstract]:Aim: to observe the effect of cyclic dynamic compression stress on the differentiation of bone marrow mesenchymal stem cells (MSCs), and to compare the effects of mechanical factors on the differentiation of bone marrow mesenchymal stem cells (BMSCs) from SOX9 gene. Methods: bone marrow mesenchymal stem cells (BMSCs) of 4-week-old Kunming mice were cultured to the third generation in vitro and identified as stem cells by flow cytometry. Murine SOX9 gene interfering lentivirus vector was transfected into mouse bone marrow mesenchymal stem cells in vitro, and bone marrow mesenchymal stem cells (BMSCs) knocked out by SOX9 were constructed. C cells and si C cells were mixed with 2% sodium alginate gel and 1x107/ml density, respectively. The experiment was divided into 8 groups according to the different factors of treatment of the carrier during the experiment. Each group was treated with cartilage inducer on the first 7 days of the experiment. Group B: after 7 days of cartilage induction, 7 days after 7 days of fluid induction, group C: group C: group B: induced by chondrocytes: group C: group B: induced by chondrocytes: group B: group B: group B: group B: group B: induced by chondrocytes: group B: group B: after 7 days of induction of cartilage: group C: group C: induced by chondrocytes. After 7 days of induction, 7 days after induction, no additional treatment was given. Group D, group D, group D, group E, group E, group E, group E, group E, group E, group E, group E, group E, group C, group C, group C, group C, group D, group D, group F, treatment, respectively. Si C cells were not stimulated. Uninduced control group: after 7 days of induction, the medium was cultured in normal medium. The Flexcell-5000 substrate loading system was used to apply 2h / d compression stress to the cell disk. After the correlation treatment, the expressions of SOX9 roCK1 and COL- 鈪，

本文編號：1700068