A Study About AP-1Interacting With NF-κB In Human Pregnant Myometrium Cell By NMBR

Objective:To study whether Nuclear Factor kappa B (NF-κB) and Activator protein1(AP-1) have interactions by the effect of Neuromedin B receptor activator Neuromedin B in late pregnancy on human uterine myometrial smooth muscle.Methods:1. Using tissue culture method obtain the primitive culture in late pregnancy on human uterine myometrial smooth muscle and to identify by immune cell chemical and detect NMBR expression on the cell.2. Inducting cells with10-6M、10-8M、10-10M concentrations of NMB after48hours, testing AP-1and NF-κB in all three groups by the mean of Western blotting. The use of co-immunoprecipitation(co-IP) to each group with anti-Jun antibody or anti-NF-κBp65antibody. After that, Western blotting verifies NF-κB or AP-1existing in conjugations which have been enriched by co-IP.3. Inducting cells for12hours,24hours and48hours with the best concentration of NMB. Testing AP-1and NF-κB protein through WB. Application of co-IP and WB methods at positive and negative to testify whether there is interaction between AP-1and NF-κB.Results:1. Primitive culture and identifying successfully in late pregnancy on human uterine myometrial smooth muscle, at least five generations, the a-SMA identified as high purity uterine smooth muscle cells.And NMBR express positively in the cell.2. Inducting cells with10-6M、10-8M、10-10M concentrations of NMB after48hours, The use of co-immunoprecipitat-ion(co-IP) with anti-NF-KB p65antibody, precipitation complexes with anti-c-Jun antibody as a line of WB identification, molecular weight36KD appear banding. By the same way, the reverse validation by the anti-c-Jun antibody concentraton of immune coprecipitation compound, the use of anti-NF-KB p65antibody as a line of WB identification, molecular weight of65KD appear banding. Inducting cells with10-10M concentrations of NMB, the corresponding objective stripe grey value is higher than other induced concentration stripe grey value.3. Inducting cells for12hours,24hours and48hours with10"10M concentration of NMB, and validation of anti-NF-KB p65/anti-c-Jun antibody positive and negative to testify the concentration of immune coprecipitation compounds. WBtesting36KD/65KD molecular weight bands appeared. AND induction time for24h, the corresponding objective stripe grey value is higher than other induction time stripe grey value.Conclusion:AP-1and NF-κB exist specific interaction in late pregnancy on human uterine myometrial smooth muscle under the NMB-NMBR. By10-10MNMB induction24h, the interaction become strongest.