Apoptin基因／壳聚糖缓释微球的制备及其对A375细胞的凋亡诱导作用

Construction of apoptin gene delivery system and its effect on apoptosis of A375 cells

目的:以羧甲基壳聚糖为基质材料,制备apoptin基因缓释微球。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200～300nm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复制模板功能,并能有效地转染A375细胞,诱导A375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。

AIM： To construct the delivery nanoparticles system of apoptin gene with O-carboxymethylated chitosan （CMC） and study its effect on inducing apoptosis of human melanoma cells A375 in vitro. METHODS： CMC nanopartides containing apoptin gene were prepared by an ultrasonic method. Restriction enzymes, DNA gel retardation assay and PCR were used to identify apoptin gene stability and to decide the best N/P ratio as well as the model effect in the progress of replication. Human melanoma cells A375 are transiently transformed by nanoparticles containing apoptin gene and apoptosis was measured by MTT assay at various time period. RESULTS： Morphology studies revealed that the particles were spherical in shape with smooth surface. The mean particle diameter ranged from 200 -300 nm. The ratio of the chitosan to apoptin DNA （N/P ratio） was 5.5： 1. The apoptin gene in chitosan/apoptin nanoparticles could be protected from DNase degradation and could be used as the model in the process of replication. The nanoparUcles with apoptin gene could induce apoptosis of A375 cells in dose- dependent manner in vitro at 48 h after transformation. CONCLUSION： The chitosan vector and apoptin gene could be combined to be a safe gene delivery nanoparticles system, which could induce apoptosis in human melanoma cells A375.