Synthesis Of A New Cationic Liposome And Study On Transfection And Expression Of Human Beta Defensin-2 In Eukaryotic Cells

As a gene transfection vector, cationic liposome possesses many advantages. Even though, researchers keep on exploring and studying a new cationic liposome with high stability and effectiveness because its present transfection has not well enough results in comparison with viral vector and is easily influenced by many factors. Human β-defensin-2 (hBD-2) is a small molecular anti-bacterial peptide with broad spectrum of activity which was found in the secretion of some cells from human beings.It plays an important role in protecting the epithelium and lung mucosa. The aim of this study is firstly to prepare a new cationic liposome with high transfection rate by organic chemical synthesis, and then screen and evaluate the optimal condition and patterns for in vitro cell culture and in vivo transfection in order to establish a novel approach of gene transfection by inhaling through respiratory route. What's more, the fragment of hBD-2 gene is artificially produced and constructed into a recombinant eukaryotic expressing vector followed by transfected into cultured cells and in vivo cells respectively with prepared cationic liposome. The expressions of hBD-2 in the cultured cells and the in vivo cells are studied and evaluated so as to explore a feasible approach for anti-infection therapy.Methods (1)A new compound, cytofectin N4-spermine cholesteryl carbamate, was synthesized with organic chemical methods to serve as gene delivery vehicle in gene therapy of lung diseases. The structure of the obtained compound was identified. (2)The cationic liposome was prepared with the obtained compound as positive component by selective coupling and deprotecting, isolating and purify with column chromatography. Electron microscopy and gel electrophoresis were employed to define the condition for transfection. The prepared cationic liposome was used in the transfection of a fluorescence plasmid to lung cancer cell line SPC and the rate of the transfection was evaluated.(3) According to the gene sequence of hBD-2 in Genebank, 3 primers were designed and synthesized. hBD-2 gene was produced using the successive extension PCR technique. The synthesized gene was digested and inserted into a plasmid pLXSN to construct an eukaryoctic expressing vector pLXSN-hBD2. (4) With the aid of prepared cationic liposome, the transfection of recombined pLXSN-hBD2 to cultured rat airway epithelial cells was conducted. PCR and Western blotting were used to study the integration and expression of hBD-2.(5) This transfection was also studied when the aerosolized recombinant pLXSN-hBD2 was administered torat lung by inhaling.Results (1) The objective compound was synthesized , and its structure was confirmed by mass spectrography, infrared spectrography and nuclear magnetic resonancespectroscopy. (2) Electron microscopy showed that the prepared cationic liposome was global microcapsule in size of 0.1-1μm. The highest transfection rate of 60.5% was attained when the liposome was at 0.25 mg/ml and in a ratio of 6-8: 1 with plasmid. (3) Both gel electrophoresis and sequence analysis indicated that the sequence of PCR-prepared hBD-2 gene was identical to that in Genebank , and it was successfully inserted into the plasmid pLXSN. (4)PCR and Western blotting confirmed that the recombined pLXSN-hBD2 was successfully integrated with the genome of rat airway epithelial cells and could be expressed. (5) hBD-2 was also expressed in the airway epithelial cells of rats receiving the inhalation of aerosolized pLXSN-hBD-2 mediated with self-prepared cationic liposome. Western blotting indicates that hBD-2 is successively expressed on the 2nd, 6th and 15th day after transfection, and the expression is reduced gradually till the 21th day. Conclusion (1) Our objective compound, cytofectin N4-spermine cholesteryl carbamate, of a high purity is obtained .(2)A new cationic liposome is prepared with cytofectin N4-spermine cholesteryl carbamate as its positive component, and sound transfection activity is observed under a certain circumstance.(3...