重组hTGF-β1基因转染的BMSCs在体内成软骨能力的初步研究  

Chondrogenesis potential of bone marrow stromal cells transfected with adeno-hTGF-β1 in pig

作　　者：夏万尧[1] 王毅敏[1] 刘伟[1] 丁文龙[2] 钟梅芳 周广东[1] 崔磊[1] 曹谊林[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院整形外科上海市组织工程重点实验室,上海200011 [2]上海交通大学医学院人体解剖学教研室,上海200011

出　　处：《解剖科学进展》2010年第4期297-301,306,共6页Progress of Anatomical Sciences

基　　金：国家重点基础研究发展规划资助项目("973"计划项目)(2005CB522702);上海市重点实验室基金(06DZ22029)

摘　　要：目的重组hTGF-β1腺病毒(adeno-hTGF-β1)转染的BMSCs在体内成软骨能力的初步研究,探讨其作为组织工程化软骨的种子细胞的可行性。方法重组adeno-hTGF-β1转染猪BMSCs,酶联免疫吸附定量检测(ELISA法)重组腺病毒转染hTGF-β1蛋白的表达。然后消化收集重组腺病毒转染后的BMSCs,均匀接种于圆盘状PGA支架上,对照组转染adeno-LacZ,然后植入裸鼠背部皮下,在植入后第3周取材,分别行大体观察、组织学、II型胶原免疫组化和蛋白聚糖含量检测对形成组织进行评价。结果 ELISA结果显示adeno-hTGF-β1转染的BMSCs,其hTGF-β1表达量是转染adeno-lacZ 的BMSCs 2.65倍( P<0.05)。植入裸鼠体内后3周取材,实验组HE染色观察可见有软骨形成,但较不均匀,并被纤维组织分割,形成的软骨组织区域可见软骨细胞包埋在软骨陷窝内;对照组可见仅有少量软骨形成,被大量的纤维组织和未降解的PGA支架包裹,实验组和对照组形成软骨占总组织百分比,分别为(41.83±4.64)%和(17.50±2.85)%,P<0.05。Safranin’O染色显示,实验组形成的软骨组织区域都有被染成桔红色蛋白多糖类基质分泌,着色比对照组更深。实验组形成的软骨组织区域有大量棕黄色的II型胶原颗粒,而对照组仅有少量的棕黄色的II型胶原颗粒,实验组形成的软骨组织中的蛋白聚糖含量多于正常猪耳软骨。结论重组hTGF-β1腺病毒转染BMSCs作为种子细胞,在裸鼠体内能促使软骨组织形成,从而为hTGF-β1基因转染的BMSCs在软骨组织工程应用中奠定了基础。Objective To investigate the feasibility of chondrogenesis differentiation of bone marrow stromal cells （BMSCs） transfected with an adenoviral vector containing human transforming growth factorβ1（hTGF-β1） in pig. Methods A replication-deficient adenovirus expression vector carrying hTGF-β1 gene was applied to infect BMSCs. ELISA was adopted to quantify the amount of hTGF-β1 protein in the cultural medium released from transfected BMSCs. The disk-shaped polyglycolic acid （PGA） scaffolds were co-cultured with transduced BMSCs, which were implanted into the subcutaneous tissue of mice at dorsal site. As a control, each BMSCs was transduced with adeno-LacZ vector. The BMSCs-PGA complex constructs were harvested and assessed by gross observation, histological examination, immunohistochemical localization of type II collagen and glycosaminoglycan （GAG） content after 3 weeks of implantation. Results By ELISA analysis, hTGF-β1 gene transfection resulted in about 2.65-fold increases in hTGF-β1 protein secretion comparing to the control group of vector transfection （p0.05）. After 3 weeks of implantation, all harvested specimens exhibited neocartilage formation in the experimental group. Tissues made from cells transduced with AdhTGF-β1 exhibited neocartilage formation, although the tissue was not homogeneous. The tissues comprised of nests of cartilage separated by strips of fibrous tissue, mature chondrocytes were embedded in lacunae in the regions of cartilage. However, tissues made from cells transduced with Ad-LacZ showed only a small quantity of neocartilage surrounded by abundant fibrous tissue and remnants of undegraded PGA scaffolds. The neocartilage volume, expressed as a percent of the total tissue volume, for the experimental group and the control group were （41.83±4.64）% and （17.50±2.85）% respectively, according to the Image-Pro Plus analysis. Furthermore, Safranin’O staining confirmed the presence of a great deal of sulfated proteoglycans in the ECM of the regions of cart

关 键 词：骨髓间充质干细胞 基因转染 腺病毒 转化生长因子Β1 猪 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学]