Experimental Study Of Nano Gene Carrier Induced Survivin RNAi Gene-Nano Complex In Treatment Of Colon Cancer

Objective:To prepare the PEGylated chitosan (CS-PEG) nano-gene carrier with high transfection rate and low cytotoxicity by a novel preparing method; construct the CS-PEG induced Survivin shRNA gene nano complex, which can transfect to HT-29 cells, to investigate its effect on the expression of survivin mRNA.To investigate its effect on the propagation and apoptosis of colon carcinoma cell lines HT-29, and to discuss its treatment effect on colon carcinomaMethods:CS-PEG nanoparticles with small particle size were prepared by an optimized graft copolymerization method. Malvem Zetasizer 3000E was used to measure the particle size and zeta potential; and atomic force microscope was used to observe the morphology.Construct the survivin shRNA recombinant plasmid targeted at human survivin gene (NM₀01168).The accuracy of the sequence was tested by gene sequencing analysis and the best survivin shRNA recombinant plasmid was selected. Synthesized the CS-PEG induced survivin RNAi gene nano complex and transfected to HT-29 (lipsome that carried the same shRNA was used as positive control and nonsense shRNA was used as negative control).RT-PCR and Western blot were used to check the change of survivin mRNA expression, respectively; and MTT was performed to see the apoptosis.Results:Malvem Zetasizer 3000E was used to test the particle size and distribution, the results showed that the CS-PEG particles size was 61nm. Atomic force microscope testing results showed that the shape of the particles was spherical like, with smooth surface and less adsorption. The nano particles had low cytotoxicity and high transfection rate. Gene sequencing analysis showed that the survivin shRNA recombinant plasmid was accurately constructed. The transfection rate of the gene nano complex was much higher than the controls (P<0.01);the expression of survivin mRNA in gene nano complex group was lower than the lipsome group (P<0.01);besides, the death rate and apoptosis (P<0.01).Conclusion:In this study the CS-PEG nanoparticles with a particle size of 61nm, and well distributed are prepared;its cytotoxicity is low and transfection rate is high. The gene nano complex can effectively transfect to colon carcinoma cell lines HT-29 and interfere survivin. Compared with that of the control groups, the gene nano complex group shows higher effect in inducing the apoptosis and suppressing the propagation. It can effectively silence the expression of survivin in HT-29 at a comparatively long time.