Apoptin基因/壳聚糖纳米粒的制备及其对U937细胞凋亡的诱导作用

Construction and Effect of Apoptin Gene Delivery System on Inducing Apoptosis of U937 Cells

目的以羧甲基壳聚糖为基质材料,制备apoptin基因缓释微球,探讨其对U937细胞凋亡的作用。方法采用复凝聚法制备apoptin/壳聚糖微球,分别用光镜观察微球形态、内切酶研究其稳定性、DNA电泳阻滞分析apoptin/壳聚糖最佳比例、PCR测定apoptin基因作为复制摸板能力、用MTT法检测其抗肿瘤活性。结果壳聚糖与apoptin基因可形成稳定的微球,其直径在200～300之间,成球性较好,apoptin/壳聚糖微球P/N最佳质量比为5.5:1。微球能够有效防止DNA酶的降解作用,apoptin/微球载体中的基因仍具有DNA复制摸板功能,并能有效地转染U937细胞,转染48h可诱导U937细胞发生凋亡,从而抑制瘤细胞生长。结论apoptin基因与羧甲基壳聚糖可形成稳定的缓释微球,并能有效地转染肿瘤细胞,诱导肿瘤细胞发生凋亡。

Objective To construct the delivery nanoparticles system of apoptin gene with O-carboxymethylated chitosan（CMC） and study its inducing apoptosis effect on human melanoma cell U937 in vitro. Methods CMC nanoparticles containing apoptin gene were prepared by an ultrasonic method and cbaracterized witb restriction enzymes, DNA gel retardation and PCR to identify apoptin gene stability and decide the best N/P ratio as well as determine the model effect in the progress of replication. The human melanoma cell U937 was transiently transformed by nanoparticles containing apoptin gene and apoptosis rates were measured by MTT assay at various time period. Results Morphology studies revealed that the particles were spherical in shape with smooth surface and the diameter ranged 200~30Ohm. The ratio of the chitosanm to apoptin DNA （N/Pratio） was 5.5：1. The apoptin gene in chitosan/apoptin nanoparticles could be protected from DNase degradation and used as the model in the process of replication.The apoptin gene nanoparticles could induce apoptosis of U937 cells in vitro at 48h after transformation, the apoptosis rates of U937 cells were associated positively with dosage of apoptin gene after transformation. Conclusion A safe gene delivery nanoparticles system for the chitosan vector and apoptin gene could be formed and be able to transfect U937 cells and induce apoptosis in human melanoma cell U937.