本文選題：機械牽拉　切入點：肌腱干細(xì)胞　出處：《中國修復(fù)重建外科雜志》2017年04期

【摘要】：目的探討不同機械牽伸條件對肌腱干細(xì)胞(tendon stem cells,TSCs)分化的影響,尋求TSCs成肌腱分化、成骨分化以及成脂肪分化的最佳單軸循環(huán)牽伸載荷。方法取8周齡雄性SD大鼠跟腱,采用酶消化法分離培養(yǎng)TSCs。取第3代TSCs,隨機分為不同牽拉條件組(實驗組A~D組)及靜態(tài)培養(yǎng)組(對照組E組),其中A組牽拉強度4%、頻率1 Hz,B組牽拉強度4%、頻率2 Hz,C組牽拉強度8%、頻率1 Hz,D組牽拉強度8%、頻率2 Hz。利用課題組自行研發(fā)的體外細(xì)胞單軸循環(huán)牽拉設(shè)備,沿培養(yǎng)皿長軸對A~D組細(xì)胞進(jìn)行單軸循環(huán)機械牽伸,E組細(xì)胞行靜態(tài)培養(yǎng)。分別處理12、24、48 h后收集各組細(xì)胞,采用實時熒光定量PCR檢測成腱分化相關(guān)基因Scleraxis(SCX)、抗細(xì)胞黏合素C(Tenascin C,TNC),成脂肪分化相關(guān)基因CCAAT/增強子結(jié)合蛋白-α(CCAAT-enhancer-binding protein-α,CEBPα)、脂蛋白脂肪酶(lipoprteinlipase,LPL)及成骨分化相關(guān)基因RUNX2、遠(yuǎn)端缺失基因5(distal-less homeobox 5,DLX5)的表達(dá);Western blot檢測TNC、CEBPα及RUNX2蛋白表達(dá)。結(jié)果實時熒光定量PCR檢測示:SCX、TNC m RNA相對表達(dá)量在B組牽拉24 h時顯著高于其余各組,差異有統(tǒng)計學(xué)意義(P0.05);CEBPα、LPL m RNA相對表達(dá)量在D組牽拉48 h時顯著高于其余各組,差異有統(tǒng)計學(xué)意義(P0.05);RUNX2、DLX5 m RNA相對表達(dá)量在C組牽拉24 h時顯著高于其余各組,差異有統(tǒng)計學(xué)意義(P0.05)。Western blot檢測示:B組牽拉各時間點TNC蛋白表達(dá)均高于E組(P0.05),同時牽拉24 h與E組相比CEBPα表達(dá)有顯著抑制作用(P0.05);C組牽拉24 h RUNX2蛋白表達(dá)顯著高于E組(P0.05),同時牽拉24、48 h TNC蛋白表達(dá)顯著低于E組(P0.05);D組牽拉48 h CEBPα蛋白表達(dá)顯著高于E組(P0.05),TNC蛋白表達(dá)顯著低于E組(P0.05),RUNX2蛋白表達(dá)與E組比較差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論機械力學(xué)刺激可以促進(jìn)TSCs發(fā)生分化,而且不同條件的牽拉載荷會引起不同方向分化。4%、2 Hz牽拉24 h為成腱分化最佳條件,8%、1 Hz牽拉24 h為成骨分化最佳條件,8%、2 Hz牽拉48 h為成脂肪分化最佳條件。
[Abstract]:Objective to investigate the effects of different mechanical drafting conditions on the differentiation of tendon stem cells (TSCs) from tendon stem cells, and to find out the best uniaxial cyclic drafting load for TSCs tendon differentiation, osteogenic differentiation and adipogenic differentiation. Methods the Achilles tendon of 8-week-old male Sprague-Dawley rats was obtained. TSCs were isolated and cultured by enzyme digestion. TSCs of the third generation were randomly divided into two groups: group A (group A) and group A (group A) and group E (control group). The pulling strength of group 1 was 8 and that of group D was 8 and that of group D was 2 Hz.Using the uniaxial retraction equipment of extracorporeal cells developed by the research group, The cells in group E were cultured statically along the long axis of culture dish for uniaxial circulatory mechanical drafting, and the cells in each group were collected after 48 hours of treatment. Real-time fluorescence quantitative PCR was used to detect the genes associated with tendon differentiation, such as Scleraxis Scleraxis, C(Tenascin, CCAAT/ enhancer, CCAAT-enhancer-binding protein 偽, Lipoprteinlipase LPL2, and RUNX2, which are associated with osteogenic differentiation, and the CCAAT/ enhancer of adipogenic differentiation related gene, CCAAT-enhancer-binding protein 偽, Lipoprteinlipase LPL2, and RUNX2, which are associated with osteogenic differentiation, are detected by real-time fluorescence quantitative PCR. The expression of 5(distal-less homeobox 5 (DLX5) was detected by Western blot. Results Real-time quantitative PCR analysis showed that the relative expression of RNA in group B was significantly higher than that in other groups at 24 h. The relative expression of CEBP 偽 -LPLM RNA in group D was significantly higher than that in other groups at 48 h, and the relative expression of RUNX2 + DLX5 m RNA in group C was significantly higher than that in other groups at 24 h. The expression of TNC protein in group B was higher than that in group E at all time points, and the expression of CEBP 偽 was significantly inhibited at 24 h compared with that in group E. The expression of RUNX2 protein in group C was significantly higher than that in group E at 24 h. At the same time, the expression of TNC protein in group E was significantly lower than that in group E at 48 h. The expression of CEBP 偽 protein in group D was significantly higher than that in group E (P 0.05). There was no significant difference in the expression of RUNX2 protein between group E and group E. Conclusion Mechanical force has no significant difference between group E and group E. Conclusion the expression of CEBP 偽 protein in group E is significantly lower than that in group E (P 0.05). Conclusion the expression of RUNX2 protein in group E is significantly lower than that in group E (P 0.05). Stimuli can promote the differentiation of TSCs. Moreover, the optimal condition of tendon differentiation was that the pulling load of different conditions would cause differentiation in different directions. 4Hz for 24 h was the best condition for tendon differentiation. The best condition for osteogenic differentiation was the best condition for osteogenic differentiation, the best condition for osteogenic differentiation was the best condition for osteogenic differentiation, and the best condition for adipogenic differentiation was 82Hz retraction for 48 h.
【作者單位】： 第三軍醫(yī)大學(xué)西南醫(yī)院骨科全軍矯形外科中心;第三軍醫(yī)大學(xué)神經(jīng)生物教研室;
【基金】：國家自然科學(xué)基金重點資助項目(81230040) 全軍醫(yī)學(xué)科研基金創(chuàng)新專項重點資助項目(13CXZ008)~~
【分類號】：R686.1

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