摘要
目的探讨载基因仿生基质材料能否调控骨髓基质干细胞(BMSCs)向成骨细胞定向分化。方法将非病毒载体K16GRGDSPC共价接枝于新型骨基质材料聚丙交酯/乙交酯/天冬氨酸/聚乙二醇(PLGA-[ASP-PEG])构建非病毒基因转染体系。该体系与转化生长因子(TGF)-β1基因复合制备成载基因仿生基质材料pcDNA3-TGF-β1/K16GRGDSPC/PLGA-[ASP-PEG],并将兔BMSCs与其复合培养,以pcDNA3/K16GRGDSPC/PLGA-[ASP-PEG]作为对照。通过逆转录-聚合酶链反应(RT-FCR)、Western blot和酶联免疫吸附测定(ELISA)检测TGF-β1基因的表达;应用碱性磷酸酶(ALP)试剂盒检测BMSCs的ALP活性,并通过RT-PCR检测细胞ALP、骨钙素(OCN)、骨桥蛋白(OPN)和Ⅰ型胶原的表达,通过免疫荧光染色检测核心结合因子a1(Cbfa1)表达,观察BMSCs向成骨细胞方向分化的情况。结果XPS图谱证实K16GRGDSPC被成功地接枝到PLGA-[ASP- PEG]表面。载基因仿生基质材料复合BMSCs培养结果表明,TGF-β1基因被成功地导入细胞内并表达,而且其成骨标志物(ALP、OCN、OPN、Ⅰ型胶原和Cfba1)的表达均显著高于对照组(P<0.05)。结论这种载基因基质材料既可以作为骨组织工程支架材料,又可介导外源TGF-β1基因转染BMSCs并定向调控BMSCs成骨分化。
Objective To explore whether gene-activated biomimetic matrix scaffold materials were able to regulate the directional osteogenic differentiation of rabbit bone marrow stromal cells （ BM- SCs） in vitro. Methods PLGA-[ ASP-PEG] scaffold materials were linked with a nonviral vector K16GRGDSPC through cross linker to construct a novel nonviral gene transfer system and then detected by XPS. Eukaryotic expressing vector encoding transforming growth factor beta 1 （pcDNA3-TGFβ1） was mixed and encapsulated by the system. The BMSCs obtained from rabbit were cultured on PLGA [ ASP- PEG] modified with K16GRGDSPC and TGF-β1 gene, while those modified with K16GRGDSPC only taken as controls. The gene expression of TGF-β1 in the cells was detected by reverse transcription-polymerase chain reaction （RT-PCR）, Western blot and enzyme-linked immune absorbent assay （ELISA）. The alkaline phosphatase （ALP） activity of the cells was measured by ALP assay kit and the mRNA levels of ALP,osteocalcin （OCN） ,osteopontin （OPN） and collagen I were assessed by RT-PCR. Immunofluorescence stain was also used to detect the expression of core binding factor a1 （ Cbfa1 ） which was an osteogenic maker as well. Results XPS patterns confirmed that K16GRGDSPC was successfully linked to the surface of PLGA [ ASP-PEG] by the cross-linker. TGF-β1 was expressed in the BMSCs which were cocultured on TGF-β1 gene-activated PLGA-[ ASP-PEG] materials. The expression of osteogenic makers of the cells such as ALP, OCN, OPN, type Ⅰ collagen and Cfbal was significantly higher than that in control group （P 〈 0.05）. Conclusion TGF-β1 gene-activated biomimetic materials can not only be used as a scaffold in bone tissue engineering,but also to mediate exogenous TGF-β1 gene into BMSCs and regulate their directional osteogenic differentiation in vitro.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2007年第9期1105-1107,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(30200063
30470483)
关键词
骨
分化
组织工程
多肽
Bone
Differentiation
Tissue engineering
Peptide
