应用转BMP7基因软骨细胞构建组织工程软骨  

Reconstruction of tissue engineered cartilage with BMP7 gene transfected chondrocytes

作　　者：曲福军[1] 侯宜[1] 刘丽玲 周余来[1] 侯立中[1] 

机构地区：[1]吉林大学再生医学科学研究所 [2]哈尔滨兽医研究所,黑龙江省哈尔滨市150001

出　　处：《中国临床康复》2006年第13期59-61,i0004,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金(30271319);国家高技术研究发展计划(863计划)资助(2004AA205020)~~

摘　　要：目的:利用转BMP7基因软骨细胞构建组织工程软骨,测定BMP7基因在构建的组织工程软骨中的表达,探讨与未转BMP7基因软骨细胞构建的组织工程软骨的生物学差异。方法:实验于2004-06/2005-01在哈尔滨医科大学附属第二医院临床药学药物研究所完成。应用转染重组pcDNA3.1-BMP7真核表达载体质粒并用G418筛选14d的阳性克隆软骨细胞,以胶原-纤维蛋白凝胶为支架,构建转BMP7基因组织工程软骨。以未转BMP7基因组织工程软骨作为对照,软骨细胞的接种密度为5×109L-1。用甲苯胺蓝、苏木精-伊红染色对组织工程软骨培养物组织学分析;原位杂交法检测Ⅱ型胶原mRNA的表达;透射电镜观察组织工程软骨培养物的超微结构;原位杂交和免疫组化法测定体外培养物BMP7mRNA和蛋白的表达。结果:①BMP7基因转染软骨细胞情况:G418筛选7d时大量细胞死亡,约30%的细胞存活,且贴壁生长。G418连续筛选14,28d均有阳性克隆细胞生成,贴壁生长率100%,存活的阳性克隆细胞为转染成功的软骨细胞。②组织工程软骨培养物大体形态及显微镜观察结果:构建的组织工程软骨培养物呈浅粉红色胶冻样圆盘状,透明,体积约为16mm×16mm×3mm。显微镜下只能观察到呈圆形的浅层细胞。③组织工程软骨培养物组织学分析结果:组织工程软骨培养物体外培养7,14,28d,细胞周围有丰富的细胞外基质,转BMP7基因组织工程软骨培养物细胞基质较多,体外培养14d软骨细胞合成和分泌最为活跃。④组织工程软骨培养物Ⅱ型胶原表达mRNA原位杂交检测结果:软骨细胞核周围呈棕黄色,有mRNA表达。⑤不同培养时间下组织工程软骨培养物DNA含量的测定情况:DNA含量随着培养时间的推移逐渐增加,21d时达到高峰,转BMP7基因比未转染BMP7基因的组织工程软骨培养物DNA含量高(P<0.05)。⑥不同培养时间下组织工程软骨培养物糖胺多糖含量的测定情况:转BMPAIM： To construct tissue engineering cartilage transfected with BMP7 gene, determine the expression of BMP7 gene in the tissue engineering cartilage and probe into the biological difference between transfected and non-transfected with BMP7 gene in biology. METHODS： This experiment was conducted at the Institute of Clinical Pharmacy and Pharmacolog, Second Hospital Affiliated to Harbin Medical University from June 2004 to January 2005. We used the recombinant pcDNA3,1-BMP7 eukaryotic expression vector plasmid and screened positive clones with G418 for 14 days,Collagen-fibrin gel composite was used to construct tissue engineering cartilage transfected with BMP7 gene, Tissue engineering cartilage non-transfected with BMP7 gene was used as control, The inoculated density of chondrocytes was 5×10^9 L^-1. The culture of tissue engineering cartilage was given histological analysis with Toluidine blue, hematoxylin-eosin staining; The expression of type Ⅱ collagen mRNA was detected with in situ hybridization; The microstructure of the culture of tissue engineering cartilage was observed under the transmission electron microscope; The expression of BMP7 mRNA and protein cultured in vitro were measured with in situ hybridization and immunohistochemical staining. RESULTS： ① Chondrocytes transfected with BMP7 gene： After screened with G418, a lot of cells died and about 30% cells survived and greW, Positive clone cells appeared after 14- and 28-day consecutive screening with G418 , and the rate of adhesion grow was 100%. The survived positive clone cells were chondrocytes transfected successfully. ② Morphology of tissue engineering cartilage culture and observation results under the microscope： The culture of constructed tissue engineering cartilage presented light pink gel-like, round plate in shape, transparent, and 16 mm×16 mm×3 mm in volume. Only a round thin cell layer was observed under the microscope.③ Histological analysis of the culture of tissue engineering cartilage： After the culture of ti

关 键 词：生物医学工程 骨形态发生蛋白质类/分析 转基因 软骨细胞 

分 类 号：R318[医药卫生—生物医学工程]