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

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 nanoparticles 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 nanoparticles 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.