Study Of RNA Interference Plasmid Carrided By Tf-PEG-PEI Targeting Nucleostemin Of PC-3 Cell In Vitro

Objective: We synthesize multifunctional cationic polymers Tf-PEG-PEI, to establish a high biocompatibility, targeted and efficient RNAi delivery systems,and successfully silencing nucleostemin of prostate cancer PC-3 cell line by RNA interference.Methods: Firstly,we identify and extract RNA interference plasmid nucleostemin factor(NS-shRNA), while synthesis Tf-PEG-PEI and correct identification of its structure. NS-shRNA plasmid complexes were combine with two composite electrolyte, and the case of nucleases present in serum, PEG of the complexes after binding ratio, particle size and the cationic polymer PEI and Tf-PEG-PEI formed size and zeta potential, the carrier is able to protect DNA from nuclease degradation, and whether the vector Tf-PEG-PEI for prostate cancer PC-3 cells targeting.Then we cultured prostate cancer cell PC-3 lines, through MTT assay test vector Tf-PEG-PEI on the toxicity of NIH3T3 cells by fluorescence inverted microscope and flow cytometry transfection efficiency, while Tf-PEG-PEI as a carrier-mediated nuclear factor NS-shRNA interference plasmid transfected PC-3 cells.we extract total cellular RNA and protein,test the effect of gene silencing by RT-PCR and Western blot Nucleostemin(NS).Results: The plasmid amplification and sequencing of sh-RNA sequence is successfully inserted on the carrier GV248, sequencing patterns in line with expectations. Through the carrier protein gel electrophoresis elution peak, proven successful synthesis of non-viral vector Tf-PEG-PEI. After hydration PEG-modified,Tf-PEG-PEI / NS-shRNA complex in the electrolyte, the presence of the protein,reducing the particle size, zeta potential of 10 mV, to maintain a weak positive,helping improve the transfection efficiency. Tf-PEG-PEI / NS-shRNA complex in N /P ratio of 7.5 can be completely complex; when Tf-PEG-PEI: NS-shRNA of N / P ratio is 20, the maximum transfection efficiency in fluorescence microscopy and flow cytometry showed that the transfection efficiency was maximal; MTT results showed that when the concentration of Tf-PEG-PEI should be 40 μ g / mL, NIH3T3 cells survival rates was above 80%. Westemblot by Realtime-PCR and detection, NSmRNA and protein expression after Tf-PEG-PEI / NS-shRNA-transfected with non-transfected group and empty vector group, mRNA and protein expression was significantly lower NS, has statistically significant(P <0.05).Conclusion: Multifunction carrier Tf-PEG-PEI and NS-shRNA complex can protect DNA Free endogenous nuclease degradation, PEG hydration layer can prevent aggregation between the polymer, reducing plasma protein aggregation and immune phagocytic organs removed, but the access PEG segment will affect the ability to escape and transport within the material of the cells to a certain extent. Through prostate cancer PC-3 transferrin receptor on the cell surface can be achieved targeted effect on the tumor. Multifunction carrier Tf-PEG-PEI NS-shRNA can mediate effectively silencing expression in prostate cancer PC-3 NS gene, Tf-PEG-PEI is a good biocompatibility, targeted, efficient gene delivery system. NS has potential significance in the clinical treatment. In gene therapy program, RNAi technology is a great hope of treatment strategies.