發(fā)布時(shí)間：2019-05-10 00:36

【摘要】：目的：聯(lián)合應(yīng)用堿性成纖維細(xì)胞生長(zhǎng)因子（basic fibroblast growth factor，bFGF）與胰島素樣生長(zhǎng)因子IGF-1作為誘導(dǎo)劑探討向肌腱細(xì)胞方向轉(zhuǎn)化的誘導(dǎo)技術(shù)，以探索一種新的肌腱組織工程種子細(xì)胞的獲取方法。 方法： 1.分離、培養(yǎng)大鼠BMSCs并進(jìn)行分子生物學(xué)鑒定：密度梯度離心法體外分離BMSCs，并對(duì)培養(yǎng)的BMSCs進(jìn)行干細(xì)胞表型鑒定與成骨、成脂肪及成軟骨多向誘導(dǎo)確定所獲取的BMSCs是具有多向分化能力的BMSCs。 2.觀察bFGF和IGF-1聯(lián)合應(yīng)用對(duì)BMSCs向肌腱細(xì)胞分化的作用：將BMSCs隨機(jī)分為4組，即實(shí)驗(yàn)組A(IGF-1+bFGF)、實(shí)驗(yàn)組B(IGF-1)、實(shí)驗(yàn)組C(bFGF)及空白對(duì)照組D于37℃、5%CO2孵箱中培養(yǎng)。用相差顯微鏡觀察細(xì)胞形態(tài)的變化；RT-PCR方法檢測(cè)各組細(xì)胞內(nèi)I型膠原、III型膠原以及scleraxis mRNA在3天、7天、14天和21天時(shí)的表達(dá)水平；免疫熒光染色檢測(cè)各組細(xì)胞內(nèi)I型膠原、III型膠原14天的表達(dá)水平。 結(jié)果： 1.分離培養(yǎng)的BMSCs在形態(tài)學(xué)上表現(xiàn)為長(zhǎng)梭形、呈漩渦狀生長(zhǎng)。流式細(xì)胞儀檢測(cè)結(jié)果顯示細(xì)胞表面CD29和CD90表達(dá)陽(yáng)性，分別為99.24%、99.06%；CD45表達(dá)陰性，為98.71%。BMSCs經(jīng)誘導(dǎo)后茜素紅、油紅O、甲苯胺藍(lán)及II型膠原免疫細(xì)胞化學(xué)染色均為陽(yáng)性。 2.誘導(dǎo)后的BMSCs A組、B組、C組、與空白對(duì)照組相比其I、III、SCX在基因及蛋白水平上具有顯著差異，從3天開始不斷遞增，14天后轉(zhuǎn)而下降，以7天至14天的變化最明顯與空白對(duì)照組差異最大。而在A、B、C三組中，以B組I、III、SCX基因的表達(dá)最明顯，免疫熒光染色檢測(cè)中B組細(xì)胞內(nèi)I型膠原、Ⅲ型膠原的表達(dá)水平最高。 結(jié)論： 大鼠BMSCs體外誘導(dǎo)各組所獲得的細(xì)胞與陰性對(duì)照組細(xì)胞在形態(tài)與功能上存在顯著差別，，其形態(tài)特征及胞內(nèi)基質(zhì)表達(dá)近似肌腱細(xì)胞；尤其是IGF-1組。bFGF與IGF-1兩種細(xì)胞因子聯(lián)用沒有明顯明顯協(xié)同效應(yīng)。
[Abstract]:Objective: to explore the induction technique of transformation to tendon cells by using basic fibroblast growth factor (basic fibroblast growth factor,bFGF and insulin-like growth factor IGF-1 as inducers. In order to explore a new method for obtaining seed cells from tendon tissue engineering. Method: 1. Rat BMSCs was isolated, cultured and identified by molecular biology: BMSCs, was isolated in vitro by density gradient centrifugation and stem cell phenotype and osteogenesis of cultured BMSCs were identified. Multidirectional induction of adipogenesis and chondrogenesis determines that the obtained BMSCs is a BMSCs. with multidirectional differentiation ability. 2. To observe the effect of bFGF and IGF-1 on the differentiation of BMSCs into tendon cells: BMSCs was randomly divided into four groups: experimental group A (IGF-1 bFGF), experimental group B (IGF-1), experimental group C (bFGF) and blank control group D at 37 鈩

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