聚乙烯亚胺-蛋白纳米复合体转染蛋白进入骨髓间充质干细胞的应用研究

目的:探索纳米材料聚乙烯亚胺(PEI)携载不同分子量的蛋白质转染进人骨髓间充质干细胞(h BMSCs)的作用及优化其最佳配比条件。方法:利用物理化学作用构建6组PEI-DNase I(DNA酶I)复合体,运用激光散射技术初步摸索其最佳摩尔比,用透射电镜直观复原纳米-蛋白复合体外貌;同时,原代分离培养健康人的骨髓间充质干细胞并进行体外扩增,通过构建成功的4组PEI-GFP(绿色荧光蛋白)纳米蛋白复合体对h BMSCs进行转染,共聚焦荧光显微镜下观察荧光表达情况,再通过MTT法检测该复合体对细胞增殖的毒性影响,并用β-半乳糖苷酶实验验证其转入蛋白的活性表现。结果:当PEI与蛋白质的摩尔比为4∶1时,形成的复合体转染效率最高,β-半乳糖苷酶实验细胞染色变蓝。结论:PEI能与蛋白质形成纳米复合体,并能转染多种蛋白质进入人骨髓间充质干细胞,所转染的蛋白质分子仍具有活性,为细胞重编程技术提供了新途径。

Proteins are transfected into bone marrow mesenchymal stem cells by polyethyleneimine-protein nano-complexes

AIM: To investigate the role of encapsulated protein transfected into human bone marrow mesenchymal stem cells (hBMSCs) by polyethyleneimine (PEI), and to optimize the best mole ratio of PEI-proteins. METHODS: 6 groups of DNase I-PEI complexes were constructed and the best mole ratio was explored by laser scattering analysis. The appearance of complexes was presented under transmission electron microscope. Meanwhile, 4 groups of constructed GFP-PEI complexes were utilized to transfect into the hBMSCs, which were isolated and expand in vitro. The fluorescence intensity of transfected cells was observed under confocal microscope. In addition, the cytotoxicity of the complexes on the cell proliferation was detected by MTT assay. The activity of the intracellular proteins was testified by a β-galactosidase staining experiment. RESULTS: When the mole ratio of PEI and protein was adjusted to 4:1, the complex transfection efficiency was the best, and β-galactosidase color test turned blue. CONCLUSION: PEI has the character of encapsulating various proteins to nano-complexes. The proteins transfected into bone marrow mesenchymal stem cells are confirmed to have functional activity. As a protein carrier, PEI is of high efficiency and low toxicity, thus providing a new way for stem cell reprogramming.