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目的研究新型非病毒载体磷酸钙纳米颗粒的生物学特性,评估其在肿瘤基因治疗中应用的前景。方法化学方法制备,氯化钙修饰纳米颗粒,用琼脂糖凝胶电泳分析磷酸钙纳米颗粒与DNA的结合效率及对DNA的保护作用,用绿色荧光蛋白基因作报告基因,将磷酸钙纳米颗粒为基因载体转染鼻咽癌(CNE-2)细胞和在动物体内转染肿瘤细胞;以及将磷酸钙纳米与自杀基因yCDglyTK结合,并在体外对鼻咽癌进行基因治疗。结果电镜观察证实磷酸钙纳米颗粒进入细胞内,磷酸钙纳米颗粒与DNA结合后,能对DNA起保护作用;磷酸钙纳米颗粒作为基因转染的载体,将绿色荧光蛋白基因导入CNE-2细胞,用荧光显微镜观察到高水平的绿色荧光蛋白表达;体内转染结果显示,纳米介导的基因在肿瘤组织中有很好的表达;以磷酸钙纳米为载体介导自杀基因yCDglyTK在体外治疗鼻咽癌的实验中显示,在加入5-FC后大量的CNE-2细胞出现死亡。结论磷酸钙纳米颗粒具有优良的生物学特性,是有应用前景的基因转染和基因治疗载体之一。
OBJECTIVE: To study the biological characteristics of novel non-viral vector calcium phosphate nanoparticles and evaluate their potential application in tumor gene therapy. Methods: The calcium chloride modified nanoparticles were prepared by chemical method. The binding efficiency of calcium phosphate nanoparticle to DNA and the DNA protective effect were analyzed by agarose gel electrophoresis. The green fluorescent protein gene was used as the reporter gene. The calcium phosphate nanoparticles were Gene vector transfected nasopharyngeal carcinoma (CNE-2) cells and in vivo transfected tumor cells; and calcium phosphate nano and suicide gene yCDglyTK binding, and in vitro gene therapy of nasopharyngeal carcinoma. Results Electron microscopy confirmed that calcium phosphate nanoparticles enter the cells, and calcium phosphate nanoparticles can protect DNA after binding to DNA. As a vector for gene transfection, calcium phosphate nanoparticles are introduced into CNE-2 cells, High-level expression of green fluorescent protein (GFP) was observed by fluorescence microscopy. The results of in vivo transfection showed that Nano-mediated gene expression was well expressed in tumor tissues. In vitro treatment of nasopharyngeal carcinoma with yCDglyTK mediated by calcium phosphate nanoparticles Cancer experiments showed that a large number of CNE-2 cells died after the addition of 5-FC. Conclusion Calcium phosphate nanoparticles have excellent biological properties and are potential carriers of gene transfection and gene therapy.