华西口腔医学杂志

• 基础研究 •    下一篇

腺病毒介导的骨形态发生蛋白-2基因转染脂肪间充质干细胞的实验研究

郑培惠1,魏奉才2,晋国营2,单秀丽3,孙树阳3   

  1. 1.山东大学山东省立医院  口腔颌面外科,山东  济南 250021;2.山东大学齐鲁医院  口腔颌面外科;3.山东大学口腔医院  中心实验室,山东  济南 250012
  • 收稿日期:2006-06-25 修回日期:2006-06-25 出版日期:2006-06-20 发布日期:2006-06-20
  • 通讯作者: 魏奉才,Tel:0531-82169002
  • 作者简介:郑培惠(1969-),男,山东人,副主任医师,博士研究生
  • 基金资助:

    国家自然科学基金资助项目(30471796)

Experimental Studies of Human Adipose Tissue-derived Stromal Cells Transfected with Ad-hBMP-2 Gene

ZHENG Pei-hui1, WEI Feng-cai2, JIN Guo-ying2, SHAN Xiu-li3, SUN Shu-yang3   

  1.  1. Dept. of Oral and Maxillofacial Surgery, Shandong Provincial Hospital, Shandong University, Jinan 250021, China; 2. Dept. of Oral and Maxillofacial Surgery, Qilu Hospital, Shandong University, Jinan 250012, China; 3. Central Laboratory,Hospital of Stomatology, Shandong University, Jinan 250012, China
  • Received:2006-06-25 Revised:2006-06-25 Online:2006-06-20 Published:2006-06-20

摘要:

目的  探讨腺病毒介导的人骨形态发生蛋白-2(Ad-hBMP-2)基因转染大鼠脂肪间充质干细胞(ADSCs)的可行性及其成骨特性。方法  取4周龄SD大鼠腹股沟的脂肪组织,经体外分离培养的方法获得ADSCs,分别转染以腺病毒为载体的绿色荧光蛋白(Ad-GFP)基因和Ad-hBMP-2基因。用荧光显微镜每隔12 h观察转染Ad-GFP的细胞,并确定转染率,观察其形态变化,绘制生长曲线;利用免疫细胞化学法对转染Ad-hBMP-2的细胞作碱性磷酸酶(ALP)染色,用免疫细胞化学法作骨钙素(OC)检测确定成骨活性,Western blot法检测hBMP-2的表达。结果  12 h后荧光显微镜观察转染Ad-GFP的细胞,有52%的阳性表达细胞,48 h后转染率达95%;转染Ad-hBMP-2后倍增时间延长,ALP、OC检测为阳性表达,hBMP-2蛋白转染后48 h为阳性表达,并持续至第5代。结论  ADSCs可以作为腺病毒转染的载体,转染Ad-hBMP-2基因后有明显的成骨作用,可以作为骨组织工程的种子细胞。

关键词: 脂肪间充质干细胞, 骨形态发生蛋白-2, 腺病毒, 基因转染

Abstract:

Objective  To investigate the possibility of adipose-derived stromal cells(ADSCs) transfeced by ade-novirus containing human bone morphogenetic protein-2(Ad-hBMP-2) gene and their osteogenic potential. Methods ADSCs were obtained from inguinal fat tissue of 4 weeks old SD rats. After exposure to adenovirus containing green fluorescent protein(Ad-GFP), fluorescent microscope was used to observe gene transfection effect once 12 hours. After transfected with Ad-hBMP-2,cytochemistry, immucytochemistry and Western blot were used to examine the ex- pression of  alkaline phosphatase(ALP), osteocalcin(OC) and hBMP-2. Results  After exposed to Ad-GFP 12 hours, 52% ADSCs were observed being transfected and 48 hours later reached 95%. The double number time belonged after transfecting with Ad-hBMP-2, and cytochemistry, immucytochemistry and Western blot examines indicated positive results of ALP, OC, hBMP-2 after 48 hours. Conclusion  Adipose tissue contains abundant ADSCs which could be transfected as gene vectors by adenovirus, ADSCs transfected with Ad-hBMP-2 can convert to ostoeblasts, and can act as a kind of seed cells for osteo-tissue engineering. 

Key words: adipose tissue-derived stromal cells, bone morphogenetic protein-2, adenovirus, gene transfection