| Telomere is natural structure located at the ends of linear eukaryotic chromosomes, which is essential for protecting chromosomes from degradation and fusion. The length of telomeres is shortened with the division of normal cells , eventually the cells will senescence, even death. The synthesis of telomere DNA repeats in most eukaryotes is performed by a special enzyme, telomerase, which is a ribonucleoprotein that can compensates for the loss of telomeres through synthesizing (TTAGGG) hexametric repeats onto chromosomal ends using an RNA component as a template. The length of telomeres is generally maintained by the expression of telomerase in most tumor cells, and the expression may be necessary and a critical step in cell immortalization and oncogenesis. The researches showed that telomerase is positive in 80%~85% lung cancer tissue, and it is negative in normal tissue and tissue near the cancer, it indicats that the activation of telomerase act as an allimportant role during the occuring and development course of lung cancer.hTERT is a catalytic subunit of telomerase and it is the rate-limiting factor of human telomerase activity, and it has been shown that the expression of hTERT is closely associated with the activity of telomerase .Therefore, hTERT is a perfect target for anticancer strategy.The antisense oligodeoxynucleotides (ASODN) that are complementary to hTERT mRNA, to inhibit hTERT gene expression and telomerase activity ,to induce cells apoptosis for the treatment of cancer were synthesized. In order to over come the defect that ASODN is easy to be degraded, a method of phosphorothioate oligonucleotide ( PS-ODN ) was adapted, but this kind of method is very expensive and difficult to apply in clinic. With the development of nanoparticulate systems, nanoparticles are used to the delivery system of oligonucleotides (ODNs), ODNs associated to nanoparticles are shown to be protected against degradation, to penetrate more easily into different types of cells,and to enhance the ODNs stability in biological medium and their weak intracellular penetration,The study is to prepare polybutylcyanoacrylate nanoparticles ( PBCA-NPs), to investigate the cytotoxicity of PBCA-NPs and the intracellular penetration of ASODN mediated by PBCA-NPs,to observe the influence of hTERT ASODN on the proliferation, the cell cycles and the expression of hTERT of A549 cells and to explore the mechanism that ASODN inhibit A549 cells. Methods:A549 cells were maintained in RPMI-1640 medium supplemented with 10% FCS and cultured in humidified atmosphere with 5% CO2 at 37℃.1. The cationic PBCA-NPs were prepared by an emulsion polymerization process in the presence of DEAE-Dextran.2. The cytotoxicity of PBCA-NPs on A549 cells was detected by MTT assay.3. Intracellular fluorescence intensity after transfecting the 5'-FITC-labeled ASODN (FASODN) was determined by Flow Cytometry (FCM).4. Three ASODNs complementary to different sites of hTERT mRNA, ASODN1, ASODN2, ASODN3,and one sense oligodeoxynucleotides( SODN) were synthesized. Each was transfected into A549 cells by PBCA-NPs.5. Cells were cultured continually, the modality and proliferation of A549 cells were observed by cell count and MTT assay; cell cycles was determined by FCM; the expression of hTERT mRNA was determined by RT-PCR. The data were expressed asmean±S.D., and analyzed by statistical software SPSS 10.0. P-value of <0.05 was considered to be statistically significant. Results:1. Under the condition of pH=3, the total concentration of DEAE-Dextran and Dextran-70 was 1.2 %, the volume proportion of them was 2: 3, the concentration of BCA was 0.8%, prepare ideal blank nanoparticles, their average diameter were 90.2nm,zeta potential were+40.1mv;The concentration of PBCA-NPs no more than 2.0 g · L-1 had no cytotoxicity obviously; The intracellular fluorescence in FASODN-NP group was stronger obviously than in FASODN group (PBCA-NPs free) after transfection for 24 h (P<0.01).2. The inhibitory effect of ASODN3 , ASODN2, ASODNl on cells proliferation were time depedent and concentration dependent and had the increasing tendency in turn. There was significant difference between the three ASODNs and PBCA-NPs, SODN or control group (P < 0.01), but there were no significant different between the three ASODNs (P > 0.05).3. After transfecting for 72 h, the cell number of ASODN group in G0/G1 phase increased obviously and the cell number in S phase decreased obviously, and the cell cycles of had no difference compared with PBCA-NPs , SODN and control group(P<0.01).4. Compared with PBCA-NPs, SODN or control group, the hTERT mRNA expression of ASODN group decreased observably (P<0.01).Conclusion:1. The cationic PBCA-NPs modified by DEAE-Dextran ,the diameter ,zeta potential are suitable for adsorping ASODN, the concentration of PBCA-NPs under 2.0 g· L-1 had no cytotoxicity. As the carrier of ASODN, PBCA-NPs can enhance the cells permeability of ASODN which is a perfect vector for gene transfer.2. ASODN can inhibit the proliferation of A549 cells , induce A549 cells apoptosis , A549 cells are blocked in G0/G1 phase ,and the cells number in S phase are decreased obviously, ASODN can also down-regulate the expression of hTERT mRNA. It reveals that ASODN can exert inhibitory effect on A549 cells.Theinhibitory effect of the three ASODNs on A549 cells are efficient , ASODN1 is the most effective segment to the inhibition on A549 cells. |
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