靶向sirna体外抑制大鼠肾间质成纤维细胞mas 基因表达的研究-内科学专业论文.docxVIP

PAGE PAGE 10 关键词：小分子干扰 RNA；基因沉默；MAS Research on screening of siRNA targetting to silence expression of MAS of NRK Abstract ： Objective: Through direct transfecting small interfering RNA(siRNA) of the targeted to NRK-49F MAS gene in vitro, to observe mRNA express inhibition and protein level of the MAS gene in the NRK-49F, so as to select the effective siRNA，for further researching the MAS gene mechanism of action in kidney disease provide the experimental basis. Methodes :（1）Design and synthesis of siRNA ： three pairs of siRNA and a pair of negative-controled siRNA were synthesized according to siRNA order design philosophy by retrieveing MAS gene sequence in NCBI. The three of them are respectively siRNA-1（5-CCUGACCAGAGCUUUCAAATT -3，5-UUUGAAAGC UCUGGUCAGGTT-3），siRNA-2(5- GACCAAUCAAAUAUGACAU TT-3,5-AUGUCAUAUUUGAUUGGUCTT-3) and siRNA-3(5-GCC AUUACUACACAAUCGUTT- 3, 5- ACGAUUGUGUAGUAAUGGCT T -3)，at the same times，negative control was siRNA (siRNA-neg)（5- UUCUCCGAACGUGUCACGUTT - 3, 5- ACGUGACACGUUCGGA GAATT -3）. （2）Cell culture: the NRK-49F cell placed in culture medium of DMEM/F12 which contained 10% of fetal calf serum， 100U/ml of penicillin and 100U/ml of streptomycin at 37℃, 5% of carbon dioxide sterile incubator，1×105/ml of cell suspension were made and inoculated in six orifice plate for experiment after cells in suspension culture showing logarithmic growth. (3) Groups: five groups were divided: group One was MAS siRNA-1 which mixed transfection complexes contained the siRNA sequence one , group two and three espectively contained the siRNA sequence two and three, group four was negative control which mixed the transfection complexes of the siRNA sequence negative, the last group just put into hiperfect transfection reagent, each group had the three replicate wells. (4) Transfection: after 12ul of hiperfect transfection reagent mixed nutrient solution without serum and double antibody into 100ul of transfection complexes, added 10n