含RGD肽基因导入系统介导TGF-β1基因转染对兔骨髓基质干细胞增殖和体外诱导成骨的影响

Effects on Proliferation and Osteoblastic Phenotype of Rabbit Bone Marrow Stromal Cells Transfected with Transforming Growth Factor-beta 1 Gene Mediated with RGD-containing Peptide Gene Delivery System in vitro

目的探讨一种新型靶向性非病毒载体—含RGD肽K16GRGDSPC(记作K16-RGD)介导外源基因转染骨髓基质干细胞(BMSCs)的可行性,观察转化生长因子β1(TGF-β1)基因转染对BMSCs的增殖和体外诱导成骨的影响。方法固相合成法合成K16-RGD,以其为载体将转化生长因子β1质粒pcDNA3-TGF-β1导入兔BMSCs,以导入空载体pcDNA3作为对照,G418筛选得到阳性细胞集落(分别命名为BMSCs-TGF-β1和BMSCs-pcDNA3),逆转录聚合酶链反应(RT-PCR)检测TGF-β1基因的表达;采用四甲基偶氮唑蓝微量酶反应比色法(MTT)和流式细胞仪(FCM)检测细胞体外增殖活性,并进行成骨诱导扩增,检测转基因细胞经成骨诱导后碱性磷酸酶(ALP)活性、骨钙蛋白(OCN)含量、核心结合因子a1(Cbfa1)的表达、矿化(钙结节形成)情况。以经诱导的BMSCs-pcDNA3和未转染BMSCs作为对照。结果RT-PCR显示转基因细胞能高表达TGF-β1,BMSCs-TGF-β1的体外增殖活性明显高于对照组(P<0.01);BMSCs-TGF-β1经成骨诱导后表达的成骨特征性表型(ALP、OCN、Cbfa1的表达和矿化)与对照组有显著的差异(P<0.01)。结论含RGD肽K16-RGD可以作为一种较理想的新型基因转移载体介导外源基因转染BMSCs;TGF-β1可以作为骨组织工程种子细胞BMSCs向成骨细胞定向分化的理想调控因子之一。

Objective To evaluate the feasibility of an novel targeted non-viral gene delivery system—RGD containing peptide K16GRGDSPC （Abbr. K16-RGD） to mediate exogenous gene into bone marrow stromal cells （rBMSCs） and study the effects on proliferation and osteoblastic phenotype of rabbit BMSCs transfected with transforming growth factor-beta 1 （TGF-β1） gene mediated with K16-RGD in vitro. Methods K16-RGD was synthesized by solid-phase batch peptide synthesizer and characterized. Using it as vector the pcDNA3-TGF-β1 plasmid was transfected into rBMSCs and empty vector pcDNA3 was transfected as control. The positve colonies named BMSCs-TGF-β1 and BMSCs-pcDNA3 respectively were selected by G418 and induced to differentiate into osteoblasts with ossific medium in vitro. Their proliferation capacity, alkaline phosphate activity, osteocalcin synthesis, core binding factor al （Cbfa1） expression and mineralization capacity were examined and compared with those of untransfected cells. Results TGF-β1 gene strengthened the cells＇ proliferation capacity and enhanced the ALP activity, osteocalcin synthesis, Cbfal expression and mineralization capacity significantly. Conclusion K16-RGD could be used as a new targeted non-viral vector to mediate exogenous gene into BMSCs and TGF-β1 could be an ideal factor to regulate BMSCs to differentate into osteoblasts orientationally.