hBMP-2基因转染的MSC与藻酸钙凝胶的生物相容性研究

Biocompatibility studies on MSC-transfected hBMP-2 gene combined with calcium alginate hydrogel

目的探讨人类骨形态发生蛋白-2(hum an bone morphogenetic prote in-2,hBMP-2)基因转染的兔骨髓基质细胞(bone m arrow strom al cells,MSC)与藻酸钙凝胶的生物相容性,为骨组织工程中生物材料的选择提供依据。方法应用脂质体介导的方法将真核表达载体pCDNA3介导的质粒pCDNA3-hBMP-2导入体外培养的兔MSC中,免疫组织化学和RT-PCR检测转染后hBMP-2基因的表达;将G418筛选获得的阳性克隆继续扩增培养,与藻酸钙凝胶复合作为实验组,单纯转染的细胞为对照组,在不同的时间行扫描电镜、四甲基偶氮唑蓝(MTT)比色法、碱性磷酸酶(ALP)、台盼蓝染色检查。结果免疫组织化学和RT-PCR均证实基因转染成功,4周时仍有表达;转染的MSC与藻酸钙复合良好,并能在其中保持活性,活力未受影响。结论hBMP-2基因转染的MSC能够获得瞬时及稳定表达,是一种新型的种子细胞;其与藻酸钙凝胶复合用于骨缺损的修复具有较强的可行性。

Objective To study the biocompatibility of bone marrow stromal cells transfected human bone morphogenetic protein-2 （hBMP-2） gene combined with calcium alginate hydrogel in bone tissue engineering. Methods By means of lipofectamine, the pCDNA3 was transferred into the MSC. The expression of hBMP-2 gene was determined by immunohistochemical analysis and RT-PCR. Subsequently, the positive cells screened out by G418 were harvested and expanded, then cultured with or without calcium alginate hydrogel, at last observed with scan electron microscopy, MTT assay, the activity of alkaline phosphatase and trypan blue stain. Results The cultured MSC were proved to be transfected successfully. The expression of hBMP-2 was still observed after four weeks. Transfected MSC were well compounded with calcium alginate hydrogel and keep the activity with the growth not affected. Conclusions MSC transfected gene, a new kind of seed, can express hBMP-2 transiently and perminenfly. Calcium alginate hydrogel-transfected MSC composite may succeed in reconstructing bone defects.