Intracellular Translocation Of NF-κB And AP-1in The Onset Of Labor And NMB Induction

Objective:1.To verify the expression and correlation between NF-κB and AP-1via NMB in the onset of labor.2.To study the intracellular translocation of NF-κB and AP-1via NMB in the onset of labor.Methods:1. Using the method of tissue adhesive in the primary culture to gain primary smooth muscle cells of gestation uterine,which is confirmed by immunocytochemistry.2. Using immunofluorescence to determine the expression of NF-κB (p65) and AP-1(c-Jun) in the smooth muscle cells of lower segment of gestation uterine, which were induced by NMB.Multiple immunofluorescence staining is used to verify the colocalization of NF-κB and AP-1.3.Organizations from lower segment of uterine around spontaneous labor are fixed and paraffin-embedded,and then use hematoxylin-eosin staining to test the slicing quality.4. Using immunohistochemical to determine the expression of NF-κB and AP-1in the smooth muscle cells from lower segment of uterine around spontaneous labor.5. Using Image-Pro Plus6.0software to measure the integrated optical density value of NF-κB and AP-1expression (IOD),and then use the SPSS19.0to analysis of differences of expression and correlation between NF-κB and AP-1.Results:1.Adherent tissue primary cultured uterine smooth muscle cell growth in good condition,immunocytochemistry α-SMA identification and NMBR detection show that cell purity is nearly100%, indicating that cultured cells are uterine smooth muscle cells.2. Tissue were fixed and paraffin-embedded, and then HE staining. Structure of smooth muscle cell from the lower segment of uterine is clear, indicating that the method is feasible for embedded tissue fixation, tissue sections are suitable for immunohistochemical staining.3. The p65, c-Jun localization in smooth muscle cells of lower segment of uterine around spontaneous labor:not labor(NL)group mainly in the cytoplasm, and the labor group mainly located nucleus. The expression in labor group is higher than NL group, the difference was significant, p <0.05.4. Pearson correlation analysis of p65and c-Jun expression in uterine smooth muscle cells around spontaneous labor:Both in NL Group and labor group p65had positive correlation with c-Jun, correlation coefficients were0.659,0.879, respectively.p<0.05.5.NMB induction on p65, c-Jun localization:control group they are in the cytoplasm; NMB4hour group major locate in the peripheral region of nuclear membrane; NMB8hour group major in the nucleus, cytoplasm expression was low.6. NMB induction on p65, c-Jun expression:the IOD value in NMB8hour group was significantly higher than NMB4hour group and control group, the difference was significant, P<0.05; differences between NMB4hour group and the control group had no statistically significant, p>0.05.7.The p65and c-Jun co-localization:the control group,NMB4hour group and NMB8hour group, multiple immunofluorescence staining showed they consistently express at the same sites in the cell, both mainly in the cytoplasm before NMB induction, after NMB induced8hours both in the nucleus. The major expression site of p65and c-Jun almost completely overlap.8. Pearson correlation analysis of NMB induced p65and c-Jun expression:the control group, NMB4hour group, NMB8hour group, all of them p65had positive correlation with c-Jun, correlation coefficients were0.587,0.653,0.874, respectively.p<0.05.Conclusion: 1.Before the onset of labor NF-κB and AP-1both with located in cytoplasm,and they migrate into the nucleus after the onset of labor, changes of the location and direction of migration were synchronous.2.NMB simultaneously can induce NF-κB and AP-1translocation,with the performance of both the migration,while increasing the amount of expression,suggesting that NF-κB and AP-1may have a synergistic effect.