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With the completion of the Human Genome Project, it had a deeper understanding of the genetics of the disease the material basis of gene therapy has become a promising treatment. The primary problem is that of the gene therapy gene into the target cells, due to the target gene (e.g., DNA, RNA, and antisense nucleic acids, etc.) is a highly hydrophilic macromolecular substances, the surface with a strong negative charge, and therefore, it is difficult to wear through the cell membrane, and susceptible to intracellular nuclease degradation, the biological half-life is short, how to build a safe and effective intracellular gene delivery system efficient and targeted gene expression regulation is one of the tasks of the pharmacy workers. The delivery systems currently used for the introduced gene viral vectors and non-viral vectors, former transduction efficiency is high, and the majority of cells having targeting, but there is a serious safety and immunogenicity problems. Non-viral-based carrier is a liposome, nanoparticle, polymeric micelle, wherein the cationic liposomes (cationic liposomes, CL) as the representative of the particle delivery system (nanoparticles DELIVERY SYSTEMS) currently used in gene therapy research. a wide range of non-viral vectors one. CL The main advantage is the preparation is relatively simple, in vivo biodegradable, and can be repeated transfection, but compared with the non-virus-containing carrier, the transfection efficiency was significantly lower. Combine the advantages of the virus and non-virus-class carrier, people would come up with a low toxicity of the new gene into the carrier - fusogenic liposomes (the fusogenic liposomes FL) upcoming liposomes with specific UV off live virus fusion, it is necessary to maintain the characteristics of the non-virus-like carrier, and use of the nature of the virus itself, and the cell fusion, which can further improve the efficiency of transfection. Telomerase (telomerase) is a special reverse transcriptase ribonucleoprotein composed of Zhejiang University, a master's degree thesis, cationic membrane fusion liposome gene delivery system by the two parts of the RNA and protein to own RNA template RT telomeric repeat sequences, so as to maintain the ends of chromosomes has stabilized, the cells acquire the ability to proliferate indefinitely. The study concluded that: increased telomerase activity and malignant tumor development are closely related. Telomerase has anti-apoptotic effects, inhibit telomerase activity can inhibit tumor cell growth and induce apoptosis. Application of telomerase the antisense oligonucleotide past acid (antisensesoligonucleotides, AsON), inhibition of telomerase activity of the telomere is lost within the tumor cells, thereby promoting tumor cell apoptosis to treat cancer gene therapy is a very attractive new target . The research Szoka reverse evaporation Preparation of cationic liposomes (/ a) charge different than telomerase ASON complex with Sendal virus fusion obtained cationic fusogenic liposomes (eationie fusogenie liposomes , CFL) with AsON of the complex. display form a round, uniform size the eL and eFL in the transmission electron microscope. The after Zetasizer300oHS laser diffraction particle size analyzer was the CFL good particle size distribution, average particle diameter were 2552lun gross potential was 37.5 mV. The particle size of the drug-loaded CFL and gross potential complexes (/ a) charge ratio. Ultrafiltration measured the CFL drug loading rate as the charge ratio increases when the charge ratio of 4:1 the phospholipid / ASON uniform eighteen), CFL drug loading was 89.1%. 20% denaturing polyacrylamide gel electrophoresis (PAGE) analysis of the degradation of ASON ASON free within 10 min. Quickly DNasel degradation CFL / ASON complexes in 4h hardly degradable. When the detergent pre destruction CFL phospholipid bilayer degradation with free the ASON consistent. It is speculated the ASON not simple but wrapped in which adsorbed on the surface of the CFL. The stability investigation showed that the blank CFL at 4 ℃ storage a month later, the its stuffy value, particle size not change significantly, but three months later, particle size, and pH values ??have changed over the The drug loading CFL vitro release slowly takes 5 release more than 90%. MTT assay CFL cytotoxicity experiments found that the size of the cell toxicity and amount of carrier and the duration of action of the cells. Under the same conditions, CFL the cytotoxicity is less than CL. Uptake with flow cytometry the fluorescently labeled ASON amount, and the experimental results show that: factors affecting the cellular uptake of the CFL's prescription, the formation of the complex (/ a) charge ratio, transfection conditions. When prescriptions De a Chol content of 33.3%, the total intracellular fluorescence intensity (total fluoreseence intensity, TFI) 133.3-fold increase compared with the control group, and higher than the other two prescriptions: With CFL / ASON complexes with cells role of time and the increase of the charge ratio, TFI corresponding enhanced significantly increased, longer duration of action than sh, enter the amount of Zhejiang University, a master's degree thesis, intracellular cation membrane fusion liposome gene delivery system ASON: When the charge ratio is less than 4:1, the CFL the ASON-cell mediated uptake is higher than under the same conditions Cl; serum can affect the CFL and CL mediated cellular uptake, but the greater impact of the CL mediated, when the culture medium containing 10% serum, the CL group intracellular opened I, 76% lower than serum-free, but only a 18.6% reduction in group CFL. CFL endocytosis mechanism: in the training system in the low-energy -4 ° C and containing lysosomal enzyme inhibitors - the chlorine cha conditions, the intracellular scared I did not change significantly, it is speculated that the CFL-mediated cell transfection The dyeing mechanism is fusion rather than endocytic pathway. In accordance with the results of the cellular uptake experiments to optimize transfection conditions to coding knife the name acz report gene model CFL transfection efficiency (2.3 ± 13%), CL transfection efficiency (3.9 ± 2.1)% . CFL mediated ASON complementary to the telomerase RNA inhibit the proliferation of 7404 cells was the role of time and dose dependent. Immunohistochemical results showed that: the CFL / ASON complex set of p53 protein and the expression of human telomerase reverse transcriptase (h ~ telomerasereverse transcriptase, hll, Rl,) was significantly lower than the control group. Solid tumor in tumor-bearing mice after local injection CFL / A SON, tumor growth was inhibited, began four days after the tumor volume became smaller and slowly shrinking, while the control group, the tumor volume is increasing.
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