壳聚糖对转染hBMP2基因的NIH3T3细胞相容性研究

Cellular biocompatibility of chitosan with NIH3T3 cells transfected with human bone morphogenetic protein 2 gene

目的:研究壳聚糖对转染人骨形成蛋白2(BMP2)基因的NIH3T3细胞生物学行为的影响,评价该多聚糖对转基因细胞的相容性。方法:用阳离子聚合物试剂将pcDNA3.1-BMP2真核表达质粒转染NIH3T3细胞,G418筛选克隆,收集扩大培养后接种于2%壳聚糖膜表面,在倒置光学显微镜、扫描电镜的辅助下,观察细胞的黏附和生长情况。用MTT法检测增殖情况,并行碱性磷酸酶活性检测。结果:壳聚糖能够促进转基因细胞在其表面的黏附和形态保持,增殖指数增高(P<0.05),碱性磷酸酶活性两组无显著差异(P>0.05)。结论:壳聚糖具有良好的细胞相容性,能够促进转基因细胞的增殖,不影响细胞的成骨分化,可作为牙周组织工程的载体材料。

Objective： To study the effects of chitosan membrane on biological behavior of NIH3T3 cells transfected with hBMP2 gene in vitro,and to evaluate the celluar biocompatibility of chitosan with the transfected cells. Methods： pcDNA3.1-BMP2 was transfect into NIH3T3 cells by using SofastTM, a positive compound transfection agent. The transfected ceils were selected by GAI8 and the positive clone was collected and expanded. The expanded cells were then divided into chitosan membrane group （in which cells were cultured with chitosan membrane ） and control group （in which cells were cultured with glass cover slip without chitosan ）. The cells were observed under inverted phase contrast microscope and scanning electric microscope. Proliferation of transfected cells was evaluated by MTT assay . Alkaline phosphatese （ALP） activity was also estimated. Results：The transfected cells could adhere to chitosan membrane well and keep cellular morphology, and proliferation index of the ceils on the membrane was higher compared with control group（P 〈 0.05）. ALP activity Was not inhibited. Conclusion. Chitosan membrane can promote proliferation of the transfected cells, and does not inhibit osteogenic differentiation. It might potentially find application for peridontal tissue engineering.