Study Of Building Up Nano Gene Vehicle PRNA And Its Ability To Silence Asthma STAT5b Gene

Objective To build up nano gene vehicle pRNA that contains siRNA and RNA aptamer, and to study its ability to silence STAT5b gene of spleen lymphocytes in asthmatic mice.Methods Build up pRNA/siRNA monomer that can silence STAT5b gene by method of PCR and transcription in vitro, and build up pRNA/aptamer(8) monomer and pRNA/aptamer(14) monomer that can bind to CD4 specially by the same way. Then to build up the pRNA/siRNA-pRNA/aptamer(8) dimer and pRNA/siRNA-pRNA/aptamer(14) dimer by assembling the previous monomer. Establish a mouse model of asthma by sensitizing with ovalbumin and challenging with repeated exposure to aerosolized ovalbumin.Use lymphocyte separation medium to isolate spleen lymphocytes. The lymphocytes were intervened by the previous pRNA monomer and pRNA dimer with or without transfection reagent. The effect of intervention was tested by RT-PCR. The cytotoxicity of pRNA/siRNA monomer and pRNA/aptamer(8) monomer was tested by CCK-8.Results (1) Clinical manifestation and the results of lung pathology and white blood cells counting of bronchoalveolar lavage fluid indicated that mouse model of asthma was successfully built. (2) The sequence and secondary structure forecasting of pRNA showed that pRNA/siRNA monomer,pRNA/aptamer(8) monomer and pRNA/aptamer(14) monomer were all built up successfully. But non-denaturing PAGE gel electrophoresis used in this experiment can't identify pRNA/siRNA-pRNA/aptamer(8) dimer and pRNA/siRNA-pRNA/aptamer(14) dimer. (3) The RT-PCR result indicated that if intervening with pRNA/siRNA monomer or pRNA/aptamer(8) monomer, there was no significant effect on mRNA expression of STAT5b gene, but if intervening with pRNA/aptamer(14) monomer,pRNA/siRNA-pRNA/aptamer(8) dimer or pRNA/siRNA-pRNA/aptamer(14) dimer, mRNA expression of STAT5b gene was reduced. (4) The CCK-8 result indicated that the cytotoxicity of pRNA/siRNA monomer and pRNA/aptamer(8) monomer was very low.Conclusion1 pRNA monomer can be successfully built up and gained by method of PCR and transcription in vitro. 2 There is no significant effect on mRNA expression of STAT5b gene in asthmatic mice lymphocyte by intervening with pRNA/siRNA monomer without transfective reagent.3 pRNA/aptamer(8) monomer and pRNA/aptamer(14) monomer could fullfill ligand function of aptamer.4 pRNA/siRNA-pRNA/aptamer(8) dimer and pRNA/siRNA-pRNA/aptamer(14) dimer can exist in the serum environment, and have the ability to reduce mRNA expression of STAT5b gene in asthmatic mice lymphocyte. And the mechanism may be is the expected features: reducing mRNA expression of STAT5b gene in targeted cells CD4+T lymphocyte.5 Nano gene vehicle pRNA is worthy of further study, and is hopeful to be used in the gene therapy of asthma.