Research Of Damage Efficiency By Livin/Survivin RNA Interference On Colorectal Cancer Cell Mediated By MPEGylated-chitosan Nanoparticles

Object:to prepare mPEG-CS nanoparticles and to explore the feasibility of mPEG-CS as gene carrier, and to study the gene transfection efficiency to colorectal cancer cell HT-29 mediated by mPEG-CS nanoparticles.Methods:Preparing mPEG-CS nanoparticles by ionic cross-linking method and preparing mPEGylated-chitosan-livin shRNA nanoparticles by Static adsorption.Detecting the pattern, size and Zeta potential of blank nanoparticles and mPEGylated-chitosan-livin shRNA nanoparticles by Zeta-size analyzer and transmission electron microscope (TEM). Measuring encapsulation efficiency of gene-nano complex.Validating the protection of nanoparticles for gene through Gel electrophoresis block experiment and DNase I enzyme digestion experiment. Comparing the transfection efficiency of livin shRNA to CRC cell HT-29 mediated by mPEG-CS nanoparticles or not.Results:Successfully prepared out mPEG-CS nanoparticles of the size about 60nm. When nanoparticles/gene ratio was 3:1, size of gene-nano complex is 100nm and encapsulation efficiency is 94.32±0.35%. The gel electrophoresis blocking test showed that nano particles could effectively combine with the plasmid, and Dnase I test proved that the nanoparticles could protect the plasmid. Transfection efficiency mediated by mPEG-CS is higher than naked gene and working a longer time.Conclusion:as gene carrier, mPEG-CS nanoparticles can protective gene well, and it is able transfect livin shRNA recombinant plasmid into colorectal cancer cells and express in a Long time. The nano-gene vector overcomes the shortage of relatively short time of RNA interference in gene therapy for tumor. Part two:Research of damage efficiency by livin/survivin RNA interference on colorectal cancer cell mediated by nanoparticlesObjective:to study livin/survivin RNA interference mediated by mPEG-CS nano carrier and to compare the CRC HT-29 proliferation and apoptosis through livin and survivin double gene silence with only livin or survivin gene silence mediated by mPEG-CS nanoparticles, so as to explore the feasibility of double gene interference for colorectal cancer.Methods:using mPEGylated-chitosan-livin shRNA and mPEGylated-chitosan-survivin shRNA nanoparticles to transfect CRC cell HT-29 jointly or respectively in the best gene-nano ratio. Using MTT method to Detect RNA interference inhibition effects on CRC cell proliferation mediated by nano carrier. Using RT-PCR and western blot to detection livin and survivin gene expression respectively. Using Hoechst dyeing to detect gene silencing for cell apoptosis, and observing cell shape on fluorescence microscope.Results:MTT experiment and Hoechst apoptosis dyeing experiments results showed that livin and survivin double gene silence can reduce tumor cell proliferation rate and increase cell apoptosis rate. The function of inhibiting cell growth and promoting cell apoptosis are better than single livin or survivin gene interference. Livin mRNA and protein expression inhibition rate by livin and survivin double gene interference is 58.62% and 59.41%, and survivin mRNA and protein expression inhibition rate by livin and survivin double gene interference is 60.2% and 61.39%.Conclusion:livin and survivin double gene silence mediated by mPEG-CS nanoparticles can reduce Livin and Survivin gene expression in CRC cells. It also can Inhibit CRC cell proliferation and Induce cell apoptosis, and the function are stronger than separately livin or survivin gene interference. Livin and survivin double gene silence has cooperative enhancement.