發(fā)布時(shí)間：2019-01-04 12:47

【摘要】：目的探討周期性機(jī)械應(yīng)力條件下上調(diào)整合素表達(dá)量對(duì)大鼠軟骨細(xì)胞的影響。方法1.體外分離培養(yǎng)6-8d的SD大鼠軟骨細(xì)胞,取第3代接種于無(wú)菌6孔板中,隨機(jī)分為三組,分別為:(1)整合素上調(diào)組:將含有整合素過(guò)表達(dá)質(zhì)粒的慢病毒轉(zhuǎn)染至大鼠軟骨細(xì)胞中(2)整合素下調(diào)組:將含有整合素低表達(dá)質(zhì)粒的慢病毒轉(zhuǎn)染至大鼠軟骨細(xì)胞中(3)對(duì)照組:將含有整合素空白質(zhì)粒的慢病毒轉(zhuǎn)染至大鼠軟骨細(xì)胞中。三組細(xì)胞在37℃,5%CO2條件下培養(yǎng)3d后,用熒光顯微鏡觀察細(xì)胞的轉(zhuǎn)染情況,Western blot法測(cè)定三組細(xì)胞整合素表達(dá)量。2.將三組細(xì)胞分別置于有、無(wú)周期性機(jī)械應(yīng)力作用的培養(yǎng)基中培養(yǎng),周期性機(jī)械應(yīng)力場(chǎng)條件為0-200k Pa、0.1Hz。培養(yǎng)1h后,用Western blot法檢測(cè)蛋白激酶(ERK1/2)的磷酸化水平;培養(yǎng)8h后,采用實(shí)時(shí)定量熒光PCR(Real-time PCR)法檢測(cè)蛋白聚糖(aggrecan)、二型膠原(collagen II)基因表達(dá)的水平;加壓8h/d,連續(xù)培養(yǎng)3d后,用CCK-8法檢測(cè)細(xì)胞增殖。3.將轉(zhuǎn)染過(guò)慢病毒的三組細(xì)胞分別接種于PLGA支架上,在周期性機(jī)械應(yīng)力條件下(0-200k Pa、0.1Hz)培養(yǎng)6周(每天加壓8小時(shí)),獲取軟骨塊。觀察軟骨塊大體形態(tài),對(duì)軟骨塊濕重和GAG含量測(cè)定,HE和番紅染色,二型膠原免疫組化染色。結(jié)果1.將三組轉(zhuǎn)染慢病毒的軟骨細(xì)胞置于熒光顯微鏡下觀察發(fā)現(xiàn),超過(guò)80%的軟骨細(xì)胞已被慢病毒轉(zhuǎn)染,表明三組軟骨細(xì)胞具有良好的轉(zhuǎn)染效率。Western blot法檢測(cè)各組整合素β1蛋白的表達(dá)水平結(jié)果顯示整合素過(guò)表達(dá)組中整合素β1蛋白表達(dá)量較對(duì)照組明顯提高(p0.05),而整合素低表達(dá)組中整合素β1蛋白表達(dá)量較對(duì)照組明顯降低(p0.05) 2.整合素過(guò)表達(dá)后,周期性機(jī)械應(yīng)力場(chǎng)中培養(yǎng)的軟骨細(xì)胞ERK1/2的磷酸化水平升高(P0.05),蛋白聚糖、二型膠原基因表達(dá)的水平升高(P0.05),CCK-8法檢測(cè)軟骨細(xì)胞增殖增加(P0.05)。而在沒(méi)有周期性機(jī)械應(yīng)力刺激條件下培養(yǎng)的三組軟骨細(xì)胞之間ERK1/2的磷酸化水平?jīng)]有變化(P0.05),蛋白聚糖、二型膠原基因表達(dá)的水平?jīng)]有明顯差異(P0.05),CCK-8法檢測(cè)軟骨細(xì)胞增殖無(wú)明顯改變(P0.05)。3.整合素過(guò)表達(dá)組軟骨塊最大,濕重較其他兩組顯著升高(P0.05),GAG含量增多(P0.05)、二型膠原免疫組化染色陽(yáng)性面積較其他兩組顯著升高(P0.05)。結(jié)論在周期性機(jī)械應(yīng)力作用下,整合素過(guò)表達(dá)能促進(jìn)大鼠軟骨細(xì)胞功能,提高組織工程軟骨質(zhì)量。
[Abstract]:Objective to investigate the effect of upregulation of integrin expression on rat chondrocytes under cyclic mechanical stress. Method 1. The chondrocytes of SD rats were isolated and cultured for 6-8 days in vitro. The third passage was inoculated in the sterile 6-hole plate and randomly divided into three groups. The results were as follows: (1) integrin up-regulation group: lentivirus containing integrin overexpression plasmid was transfected into rat chondrocytes (2) integrin down-regulation group: lentivirus containing integrin low expression plasmid was transfected into rat soft tissue. (3) control group: lentivirus containing integrin blank plasmid was transfected into rat chondrocytes. After three groups of cells were cultured at 37 鈩，

本文編號(hào)：2400314