| Summary: | 碩士 === 國立中央大學 === 化學工程與材料工程學系 === 101 === Small interfering RNA (siRNA) can specifically inhibit certain gene expression, but siRNA cannot approach to negatively-charged cell membrane and not even be functionalized into cytoplasm. To overcome the above problems, a suitable carrier to cover siRNA from degradation and to deliver it into cell is required.
In this study, we selected Indolicidin(IL), its derivative ILF89, PEI(M.W=25k da)-ILC、PEI(M.W=25k da)-CIL、PEI(M.W=750k da)-ILC以及PEI(M.W=750k da)-CIL as the gene carriers. Indolicidin may assemble into a complex with siRNA by electrostatic interaction at different amine over phosphate molar ratio (N/P ratio). First, we covered siRNA with IL and ILF89, and found that the sizes of siRNA-peptide complex are 800nm to 1000nm and 300nm to 600nm, respectively. On the other hand, the zeta potential of siRNA-IL complex at N/P ratio less than 20 is neutral and negatively charged while that of N/P ratio higher than 30 is above 18mV. In contrast to siRNA-IL complex, zeta potential of siRNA-ILF89 complex is completely negatively charged. We applied the above method to the inhibitory examination on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of HEK293T cell line. From the real-time polymerase chain reaction (QPCR) analysis, we found that the inhibitory efficiencies of siRNA-IL and siRNA-ILF89 complex are 30% to 40% of mRNA and 0％ of mRNA, respectively, which is relatively lower than the expected result.
To another gene, enhanced green fluorescent protein (EGFP), expressed from HT1080 cell line, there is little decrease in the fluorescence intensity from the green fluorescent protein expression of both siRNA-IL and siRNA-ILF89 complex, which was analyzed by QPCR. We attributed the above results to the long half-life of EGFP (26 hour) that makes the protein stable resulting in low inhibitory efficiency.
From results of PEI-IL, we found that the inhibitory efficiencies of PEI-IL are higher than PEI and the inhibitory efficiencies of PEI(M.W=25kda)-IL are higher than PEI(M.W=750kda)-IL. Thus, IL shows enhanced transfection ability.
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