本文選題：肌腱干細(xì)胞 + 循環(huán)牽拉　； 參考：《中國(guó)修復(fù)重建外科雜志》2017年01期

【摘要】：目的探討機(jī)械刺激對(duì)大鼠跟腱來(lái)源肌腱干細(xì)胞(tendon stem cells,TSCs)立早基因c-fos表達(dá)的影響。方法取8周齡雄性SD大鼠跟腱組織,用酶消化法分離、培養(yǎng)TSCs,取第3代細(xì)胞用于實(shí)驗(yàn)。利用單拉循環(huán)牽伸載荷模型在1 Hz條件下,分別用4%和8%牽拉強(qiáng)度牽拉細(xì)胞,并在牽拉0、5、15、30、60、120 min后收集細(xì)胞進(jìn)行實(shí)時(shí)熒光定量PCR檢測(cè),觀察c-fos m RNA相對(duì)表達(dá)量隨牽拉時(shí)間的變化,并找到表達(dá)最高時(shí)間點(diǎn)Tmax。然后分別用2%、4%、6%、8%和12%的牽拉強(qiáng)度,在牽拉Tmax時(shí)收集細(xì)胞,觀察c-fos m RNA相對(duì)表達(dá)量隨牽拉強(qiáng)度的變化。接著分別用2%、4%、6%、8%和12%的牽拉強(qiáng)度,對(duì)TSCs經(jīng)0、5、15 min短時(shí)間牽拉后靜置至Tmax,觀察c-fos m RNA相對(duì)表達(dá)量經(jīng)短時(shí)刺激后的變化情況。最后分別用4%和8%牽拉強(qiáng)度牽拉細(xì)胞0、Tmax、120 min,檢測(cè)成肌腱分化標(biāo)志基因Ⅰ型膠原、腱調(diào)蛋白(tenomodulin,TNMD),成骨分化標(biāo)志基因Runx2、遠(yuǎn)端缺失基因5(distal-less homeobox 5,Dlx5),成脂肪分化標(biāo)志基因脂肪酸結(jié)合蛋白4(fatty acid binding protein 4,FABP4)的表達(dá)情況。結(jié)果與0 min相比,在4%和8%牽拉強(qiáng)度下,c-fos m RNA相對(duì)表達(dá)量在牽拉15 min時(shí)顯著升高(P0.05),30 min時(shí)出現(xiàn)峰值(P0.05),至60 min時(shí)表達(dá)量恢復(fù)至起始水平(P0.05);故Tmax為30 min。牽拉30 min時(shí),2%的牽拉強(qiáng)度即可使c-fos m RNA相對(duì)表達(dá)量升高,且6%、8%和12%牽拉強(qiáng)度較2%、4%牽拉強(qiáng)度進(jìn)一步升高,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。經(jīng)過(guò)5 min的短時(shí)間牽拉,并靜置至30 min時(shí)即可使c-fos m RNA相對(duì)表達(dá)量升高(P0.05)。4%牽拉強(qiáng)度下,在牽拉30 min和120 min時(shí),各分化標(biāo)志基因相對(duì)表達(dá)量與0 min比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);而8%牽拉強(qiáng)度下,在牽拉30 min時(shí),Runx2基因相對(duì)表達(dá)量顯著升高,在牽拉120 min時(shí),Ⅰ型膠原、TNMD、Dlx5、Runx2等基因相對(duì)表達(dá)量均升高,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論循環(huán)牽拉能夠影響大鼠跟腱來(lái)源TSCs立早基因c-fos的m RNA表達(dá),并且呈時(shí)間和強(qiáng)度依賴(lài)性;提示機(jī)械刺激的時(shí)間和強(qiáng)度可能在作用早期即對(duì)TSCs的分化產(chǎn)生影響,對(duì)進(jìn)一步研究TSCs機(jī)械-細(xì)胞內(nèi)信號(hào)耦聯(lián)機(jī)制具有重要意義。
[Abstract]:Objective to investigate the effect of mechanical stimulation on c-fos expression of tendon stem cells (TSCs) in rat tendon stem cells derived from Achilles tendon. Methods the Achilles tendon of 8-week-old male SD rats was isolated and cultured by enzyme digestion. The third passage cells were used in the experiment. In this paper, we used the single pull cycle drawing load model at 1 Hz, and used 4% and 8% stretch strength to pull the cells, and collected the cells after pulling 0 ~ (5 ~ 5) ~ 1530 ~ (30) ~ 60120 min for real-time fluorescence quantitative PCR detection, and observed the relative expression of c-fos _ m RNA with the pulling time. And find the highest expression time point Tmax. Then the cells were collected with 8% and 12% tensile strength of 2% and 4%, respectively, and the relative expression of c-fos m RNA was observed. Then, with 8% and 12% tensile strength of 2% and 4%, respectively, the relative expression of c-fos m RNA was observed when the relative expression of c-fos m RNA was stimulated for a short period of time. Finally, the type I collagen, a marker gene of tendon differentiation, was detected with 4% and 8% stretch strength of Tmax1 for 120 min, respectively. The expression of tenomodulinus tenomonas tenomodulinus TNMDN, osteoblast differentiation marker gene Runx2, distal deletion gene 5(distal-less homeobox 5, fatty acid binding protein 4(fatty acid binding protein 4 and FABP4) were detected. Results compared with 0 min, the relative expression of c-fos m RNA at 4% and 8% tensile strength was significantly increased at 15 min, the peak value of P0.05 was found at 30 min, and returned to the initial level at 60 min, so Tmax was 30 min. At 30 min, the relative expression of c-fos m RNA was increased by 2% and 6% and 12% respectively, and the tensile strength of 6% and 12% was higher than that of 2% and 4%, the difference was statistically significant (P 0.05). The relative expression of c-fos m RNA was increased at 30 min and 120 min after a short stretch of 5 min, and the relative expression of c-fos m RNA was increased at 30 min and 120 min. There was no significant difference in the relative expression of all differentiation marker genes between 0 min and 0 min, but the relative expression of Runx2 gene increased significantly at 30 min of traction and 120 min after pulling at 8% retraction intensity. The relative expression of Dlx5, Runx2 and other genes in type I collagen was increased, and the difference was statistically significant (P 0.05). Conclusion cyclic retraction can affect the expression of m RNA in c-fos of TSCs gene derived from Achilles tendon in a time-and intensity-dependent manner, suggesting that the time and intensity of mechanical stimulation may have an effect on the differentiation of TSCs in the early stage of action. It is of great significance to further study the mechanism of mechanical-cellular signal coupling in TSCs.
【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(guó)家自然科學(xué)基金重點(diǎn)項(xiàng)目(81230040)~~
【分類(lèi)號(hào)】：R686

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