CONSTRUCTING AAV-TGFβ_1 AND COMPARISING ITS BIOLOGICAL EFFECTS ON PROTEOGLYCAN SYNTHESIS OF NUCLEUS PULPOUS CELLS WITH AV-TGFβ_1

作     者：赛佳明 胡有谷 王德春 SAI Jia-ming, HU You-gu, WANG De-chun Department of Orthopaedic Surgery, Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, China

作者机构：Department of Orthopaedic Surgery Affiliated Hospital of Medical College Qingdao University Orthopaedic LaboratoryOrthopaedics and Traumatology Hospital Qingdao266003 China. 

出 版 物：《Journal of Shanghai Second Medical University(Foreign Language Edition)》 (上海第二医科大学学报（英文版）)

年 卷 期：2007年第19卷第2期

页      面：90-94页

学科分类：1002[医学-临床医学] 100210[医学-外科学(含：普外、骨外、泌尿外、胸心外、神外、整形、烧伤、野战外)] 10[医学] 

基　　金：the Natural Science Foundation of China (30271318) 

主　　题：adeno-associated virus adenovirus transforming growth factor β1 nucleus pulpous proteoglycan 

摘      要：Objective To construct adeno-associated virus express system for TGFβ1 (AAV-TGFβ1) and compare its biological effects on proteoglycan synthesis of the rabbit lumbar disc nucleus pulpous (NP) cells with adenovirus (Ad) express system for TGFβ1 (AV-TGFβ1). Methods TGFβ1 gene was obtained by polymerase chain reactions (PCR). The upstream of TGFβ1 contained restriction enzyme site of EcoR Ⅰ, and the restriction enzyme site of Sal Ⅰ was at the downstream of TGFβ1. Using the multiple cloning sites (MCS) in plasmid AAV and the corresponding contained restriction enzyme site in PCR product of TGFβ1, TGFβ1 gene was subcloned into AAV. The recombinant plasmid AAV-TGFβ1 was detected by restriction enzyme digestion and DNA sequencing. Then, AAV-TGFβ1 virus was packaged and TGFβ1 expression mediated by AAV was detected by immunofluence analysis in H293 cells. AAV transfection rate to NP cells was evaluated with AAV-PEGF. After NP cells were respectively transfected by AAV-TGFβ1 virus and AV-TGFβ1 virus, proteoglycan synthesis was detected and compared by using Antonopulos methods. Results DNA sequencing revealed that the PCR-amplified TGFβ1 gene was consistent with NCBI Gene Bank. The recombinant plasmid was proved to be constructed successfully by restriction enzyme digestion. AAV could be transfected into NP cells and mediate an efficient expression of TGFβ1 protein. AV-TGFβ1 virus could quickly enhance the proteoglycan synthesis of the NP cells, but its biological effect was transient. AAV-TGFβ1 virus could enhance stably proteoglycan synthesis. Conclusion AAV-TGFβ1 virus was successful constructed and enhanced stably proteoglycan synthesis of NP cells.