软骨源性形态发生蛋白1转基因细胞片的初步探讨
发布时间：2019-01-27 20:32
【摘要】：目的探讨软骨源性形态发生蛋白1(cartilage-derived morphogenetic protein 1,CDMP1)转基因细胞片的构建及其活性鉴定。方法取1月龄新西兰白兔骨髓分离培养BMSCs,取第3~6代细胞进行实验。实验分为3组,A组:腺病毒(adenovirus,Ad)-巨细胞病毒(cytomegalovirus,CMV)-人CDMP1(human CDMP1,hCDMP1)-内部核糖体进入位点(internal ribosome entry site,IRES)-增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转染BMSCs,B组:Ad-CMV-EGFP(空载体腺病毒)转染BMSCs,C组:未转染的BMSCs。倒置荧光显微镜观察3组细胞荧光表达情况,MTT法检测3组细胞增殖活力。将3组处于对数生长期的细胞接种于温度敏感性6孔板获得细胞片,行形态学及HE染色观察,RT-PCR及Western blot检测3组细胞片hCDMP1和Ⅱ型胶原mRNA及蛋白表达,阿利新蓝染色检测糖胺聚糖(glycosaminoglycans,GAG)表达。结果倒置荧光显微镜下观察示转染细胞在72 h表达明亮荧光,转染效率达90%;MTT法检测示,3组细胞生长曲线基本呈S形,培养1~9 d吸光度(A)值比较差异均无统计学意义(P0.05)。通过温度敏感性6孔板体外可成功收获完整的三维立体细胞片结构,RT-PCR及Western blot检测示,A组细胞片中hCDMP1和Ⅱ型胶原mRNA及蛋白均呈阳性表达,B、C组均为阴性。HE染色和阿利新蓝染色示,A组纤维组织丰富,细胞外基质较多,蓝色异染颗粒多;B、C组细胞外基质相对较少,无明显特异性蓝染颗粒;A组GAG阳性染色面积明显低于B、C组,灰度值明显高于B、C组(P0.05)。结论 hCDMP1基因转染的BMSCs细胞片能表达Ⅱ型胶原和GAG,具有成软骨活性,其克服了传统组织工程中细胞利用率低、支架异质性等缺点,有望构建出致密的组织工程软骨,为软骨组织工程进一步发展提供了新思路。
[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，
本文编号：2416672
[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，
本文编号：2416672
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