CAJ | 학술논문

背景:能否通过病毒或者非病毒载体将骨形态发生蛋白2转导到骨髓基质细胞发挥成骨作用?目的:观察真核表达载体pcDNA3-hBMP2转染兔骨髓基质细胞后体外表达,同时将MSC转染后但未筛选兔骨髓基质细胞自体回植入肌组织,利用X线观察其成骨情况.设计、时间及地点:观察对比实验.实验于2004-11/2005-04在辽宁医学院骨科研究室完成.材料:6只成年新西兰大白兔,雌雄不拘,体质量2.0～3.0 kg.BMP2抗体为美国Sanaka公司产品,pcDNA3-hBMP2由解放军第四军医大学生化教研室蒲勤教授提供,限制性内切酶购于大连宝生物工程有限公司.方法:从大肠杆菌提取超纯质粒pcDNA3-hBMP2,从成年兔股骨抽取骨髓,密度梯度分离法分离培养骨髓基质干细胞,将细胞分成4组:A组:pcDNA3-hBMP2转染进行G418筛选;B组:pcDNA3-hBMP2转染未用G418筛选;C组:给予pcDNA3空载体转染;D组:仅加入脂质体转染试剂Fugene 6.主要观察指标:①应用免疫组织化学法检测转染后瞬时表达.②应用免疫组织化学法检测细胞骨钙素表达,分别应用原位杂交法检测细胞Ⅰ型胶原表达.③转染2周后将B组细胞自体回植入兔后腿肌组织中,移植4周后应用X射线观察成骨情况. 结果:①pcDNA3-hBMP2成功转染入骨髓基质干细胞内并100%瞬间表达BMP2.②基因转染4周后,A组细胞骨钙素及Ⅰ型胶原表达高于C组及D组.③B组细胞回植肌组织4周后,X射线可显示新骨形成.结论:pcDNA3-hBMP2能安全有效转染兔骨髓基质干细胞,通过其分泌物BMP2来作用诱导细胞加速分化为成骨细胞.
배경:능부통과병독혹자비병독재체장골형태발생단백2전도도골수기질세포발휘성골작용?목적:관찰진핵표체재체pcDNA3-hBMP2전염토골수기질세포후체외표체,동시장MSC전염후단미사선토골수기질세포자체회식입기조직,이용X선관찰기성골정황.설계、시간급지점:관찰대비실험.실험우2004-11/2005-04재료녕의학원골과연구실완성.재료:6지성년신서란대백토,자웅불구,체질량2.0～3.0 kg.BMP2항체위미국Sanaka공사산품,pcDNA3-hBMP2유해방군제사군의대학생화교연실포근교수제공,한제성내절매구우대련보생물공정유한공사.방법:종대장간균제취초순질립pcDNA3-hBMP2,종성년토고골추취골수,밀도제도분리법분리배양골수기질간세포,장세포분성4조:A조:pcDNA3-hBMP2전염진행G418사선;B조:pcDNA3-hBMP2전염미용G418사선;C조:급여pcDNA3공재체전염;D조:부가입지질체전염시제Fugene 6.주요관찰지표:①응용면역조직화학법검측전염후순시표체.②응용면역조직화학법검측세포골개소표체,분별응용원위잡교법검측세포Ⅰ형효원표체.③전염2주후장B조세포자체회식입토후퇴기조직중,이식4주후응용X사선관찰성골정황. 결과:①pcDNA3-hBMP2성공전염입골수기질간세포내병100%순간표체BMP2.②기인전염4주후,A조세포골개소급Ⅰ형효원표체고우C조급D조.③B조세포회식기조직4주후,X사선가현시신골형성.결론:pcDNA3-hBMP2능안전유효전염토골수기질간세포,통과기분비물BMP2래작용유도세포가속분화위성골세포.
BACKGROUND: Whether bone morphogenetic protein 2 (BMP-2) can be transduced into marrow stromal cells (MSCs) and produce osteogenic effects by viral or non-viral vector remains unclear? OBJECTIVE: To observe the expression of cultured rabbit MSCs transfected with pcDNA3-hBMP2 in vitro. Simultaneously, the MSCs were transfected but not screened and then transplanted into autologous muscle to investigate the osteogenic capability by X-ray. DESIGN, TIME AND SETTING: A controlled observation experiment was performed at the Department of Orthopedics, Liaoning Medical University between November 2004 and April 2005. MATERIALS: Six adult New Zealand rabbits, of either gender, weighing 2.0-3.0 kg, were included for this study. BMP2 antibody was the product of Sanaka Company, USA. pcDNA3-hBMP2 was provided by Professor Pu Qin from the Department of Biochemistry, Fourth Military Medical University of Chinese PLA). Restriction enzyme was purchased from Takara biotechnology (Dalian) CO., LTD., China. METHODS: Super-purified plasmid pcDNA3-hBMP2 was extracted from E. coli. Bone marrow was taken from the adult rabbit femur for harvesting MSCs by density gradient separation. The MSCs were divided into the following 4 groups: Group A, cells were transfected and screened by G418; Group B, cells were transfected by pcDNA3-hBMP2; Group C, cells were transfected by empty vector pcDNA3; Group D, only transfection reagent Fugene 6 was added.MAIN OUTCOME MEASURES: Transient BMP2 expression was analyzed by immunohistochemistry. Expression of osteocalcin and collagen I was examined by immunohistochemistry and in situ hybridization, respectively. Two weeks after transfection, MSCs from the group B were autologously transplanted into the muscle. Four weeks later, X-ray assay was used to observe bone formation.RESULTS: pcDNA3-hBMP2 was successfully transduced into MSCs and transiently expressed BMP2 100%. Four weeks after gene transfection, expression levels of osteocalcin and collagen I were significantly higher in the group A than in the groups C and D. X-ray results demonstrated new bone formation four weeks after MSCs transplanted into the muscle.CONCLUSION: pcDNA3-hBMP2 can safely and efficiently transfect MSCs and induce them to differentiate towards osteoblasts by secreting BMP2.