發(fā)布時間：2019-01-27 20:32

【摘要】：目的探討軟骨源性形態(tài)發(fā)生蛋白1(cartilage-derived morphogenetic protein 1,CDMP1)轉(zhuǎn)基因細(xì)胞片的構(gòu)建及其活性鑒定。方法取1月齡新西蘭白兔骨髓分離培養(yǎng)BMSCs,取第3~6代細(xì)胞進(jìn)行實(shí)驗(yàn)。實(shí)驗(yàn)分為3組,A組:腺病毒(adenovirus,Ad)-巨細(xì)胞病毒(cytomegalovirus,CMV)-人CDMP1(human CDMP1,hCDMP1)-內(nèi)部核糖體進(jìn)入位點(diǎn)(internal ribosome entry site,IRES)-增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)轉(zhuǎn)染BMSCs,B組:Ad-CMV-EGFP(空載體腺病毒)轉(zhuǎn)染BMSCs,C組:未轉(zhuǎn)染的BMSCs。倒置熒光顯微鏡觀察3組細(xì)胞熒光表達(dá)情況,MTT法檢測3組細(xì)胞增殖活力。將3組處于對數(shù)生長期的細(xì)胞接種于溫度敏感性6孔板獲得細(xì)胞片,行形態(tài)學(xué)及HE染色觀察,RT-PCR及Western blot檢測3組細(xì)胞片hCDMP1和Ⅱ型膠原mRNA及蛋白表達(dá),阿利新藍(lán)染色檢測糖胺聚糖(glycosaminoglycans,GAG)表達(dá)。結(jié)果倒置熒光顯微鏡下觀察示轉(zhuǎn)染細(xì)胞在72 h表達(dá)明亮熒光,轉(zhuǎn)染效率達(dá)90%;MTT法檢測示,3組細(xì)胞生長曲線基本呈S形,培養(yǎng)1~9 d吸光度(A)值比較差異均無統(tǒng)計學(xué)意義(P0.05)。通過溫度敏感性6孔板體外可成功收獲完整的三維立體細(xì)胞片結(jié)構(gòu),RT-PCR及Western blot檢測示,A組細(xì)胞片中hCDMP1和Ⅱ型膠原mRNA及蛋白均呈陽性表達(dá),B、C組均為陰性。HE染色和阿利新藍(lán)染色示,A組纖維組織豐富,細(xì)胞外基質(zhì)較多,藍(lán)色異染顆粒多;B、C組細(xì)胞外基質(zhì)相對較少,無明顯特異性藍(lán)染顆粒;A組GAG陽性染色面積明顯低于B、C組,灰度值明顯高于B、C組(P0.05)。結(jié)論 hCDMP1基因轉(zhuǎn)染的BMSCs細(xì)胞片能表達(dá)Ⅱ型膠原和GAG,具有成軟骨活性,其克服了傳統(tǒng)組織工程中細(xì)胞利用率低、支架異質(zhì)性等缺點(diǎn),有望構(gòu)建出致密的組織工程軟骨,為軟骨組織工程進(jìn)一步發(fā)展提供了新思路。
[Abstract]:Objective to investigate the construction and activity identification of chondrogenic morphogenetic protein 1 (cartilage-derived morphogenetic protein 1 CDMP1) transgenic cells. Methods 1 month old New Zealand white rabbits were isolated from bone marrow and cultured with BMSCs, for 3 ~ (th) passage. The experiment was divided into three groups: group A: adenovirus (adenovirus,Ad)-cytomegalovirus (cytomegalovirus,CMV)-human CDMP1 (human CDMP1,hCDMP1)-internal ribosomal entry site (internal ribosome entry site,IRES)-enhanced green fluorescent protein (enhanced green fluorescent protein, EGFP) transfected BMSCs,B group: Ad-CMV-EGFP (empty vector adenovirus) transfected BMSCs,C group: untransfected BMSCs. The fluorescence expression of the three groups was observed by inverted fluorescence microscope, and the proliferative activity of the three groups was detected by MTT assay. Three groups of cells in logarithmic growth phase were inoculated into the temperature-sensitive 6-well plate to obtain the cell pieces. Morphological and HE staining were performed. RT-PCR and Western blot were used to detect the expression of hCDMP1 and type 鈪，

本文編號：2416672