| Objective1. To explore Shoutaiwan different concentration and different time effects on the expression of mRNA, protein of HLA-Gsubtypes.2. To explore Shoutaiwan different concentration and different time effects on the expression of mRNA, protein of ILT2, KIR2DL4.3. To explore the traditional prescription of Shoutaiwan by regulating signaling pathway on the maternal fetal interface, which HLA-G ligand and its receptor KIR2DL4, ILT2 mediated, play a role on the treatment of unexplained recurrent spontaneous abortion.4. To explore the effect of HLA-G subtypes on unexplained recurrent spontaneous abortion.Methods1. Culture JEG-3 cells which specific express HLA-G, and NK92 cells which express ILT2, KIR2DL4 receptor.2. JEG-3 cells and NK92 cells in the logarithmic growth phase, and with the good state were cultivated in 24 pore plate evenly, every 12 hours with 4 holes to count, until the cell abundance was 100%. Draw growth curve with culturing time as horizontal axis, the cell number as the vertical axis.3. Through the MTT cell proliferation and toxicity experiment, to determine the effective concentration range of the culture medium containing Shoutaiwan lyophilized powder respectively in JEG-3 cells, NK92 cells, making the gradient concentration.4. Different gradient concentration of culture medium containing Shoutaiwan lyophilized powder effect on JEG-3 cells, NK92 cells, RT-PCR detection of the mRNA expression of HLA-G subtypes, ILT2, KIR2DL4 gene per 12 hours.5. Different gradient concentration of culture medium containing Shoutaiwan lyophilized powder effect on JEG-3 cells, NK92 cells, cultured for 24hours, FlowCytoMetry detect the protein expression of HLA-G1 (membrane full length subtype), ILT2, KIR2DL4 gene per 12 hours.6. JEG-3 cells and NK92 cells in the logarithmic growth phase, and with the good state, according to the different ratio of the number, cocultured for 24 hours, the effect target ratio determined by the MTT cytotoxicity assay.7. Coculture JEG-3 cells and NK92 cells in effector target ratio, RT-PCR detect the expression of HLA-G subtypes, ILT2, KIR2DL4 mRNA in 24 hours,48 hours respectively.8. After arrangement of the data, analyse with SPSS13.0 through repeated measures analysis of variance, get the overall trend of eachgene, and the contribution of time factor, concentration factor, and interaction effect on the gene expression. Split the data, using repeated measurement for multiple comparisons of paired t test (Bonferroni method) to performance the two-two comparison of the same concentration at each time point; using the multifactor analysis of variance to performance the two-two groups with different drug concentrations at the same time.Results1. KouShiMethod calculate the IC50 concentration of Shoutaiwan medium for JEG-3 cells was 1.884mg/ml, for NK92 cells was 0.6088mg/ml.2. Some effects of different time, different concentration on the expression of HLA-G1 mRNA, and there is interaction (F=775.340,P=0.000) 3mg/ml concentration performed inhibition on the expression.2mg/ml and the concentrations below, to relieve promote on the expression of HLA-G1 after inhibition effect at 24h, and 0.5mg/ml concentration performed the most significant. Taking 36h as the boundary points, before 36h HLA-G1 expression is positively related to the concentration; the expression of HLA-G1 is negatively related to the concentration after 36h.3. Some effects of different time, different concentration on the expression of HLA-G2 mRNA, and there is interaction (F=102.692,P=0.000). The 0.5mg/ml group is positively related to the time, other concentration group expression level and time factors have no dependence, every concentration performance the highest expression at 60H.3mg/ml and the lower concentrations has promoting effect on the expression of HLA-G2 after transient inhibition at 24h, and 0.5mg/ml concentration performed the most significant.4. Some effects of different time, different concentration on the expression of HLA-G3 mRNA, and there is interaction (F=203.727, P=0.000). Every concentrations groups in 12h was the lowest, second troughs appeared 48h,60h highest expression, the expression and the factor of time without dependency. The expression of HLA-G3 at each time point and concentration has a positive relation. The highest expression at 60h is in 0.5mg/ml, and the expression level of 0.5mg/ml group at each time point is always located above the no dosing control group.5. Some effects of different time, different concentration on the expression of HLA-G4 mRNA, and there is interaction (F=197.812, P=0.000) Except the 0.5mg/ml group is positively related to the time, the expression level of other concentration and the time factors without dependency,60h at different concentration were the highest expression; the expression levels of 0.5mg/ml group after 24h were above other concentration group at the same time, and each time point expression is higher than the no dosing control group.6. Some effects of different time, different concentration on the expression of HLA-G5 mRNA, and there is interaction (F=472.895,P=0.000). The concentration of HLA-G5 mRNA expression after 24h are promotion and positively related to the time.7. Some effects of different time, different concentration on the expression of HLA-G6 mRNA, and there is interaction (F=295.869, P=0.000) 2mg/ml,3mg/ml concentration promoted the expression of HLA-G6, the highest level at 60h.1.5mg/ml,0.5mg/ml concentration play an inhibitory effect HLA-G6 expression.8. Some effects of different time, different concentration on the expression of HLA-G7 mRNA, and there is interaction (F=468.075, P=0.000). There was no obvious promotion in 3mg/ml group according to the time accumulation.2mg/ml and the lower concentration groups were promoted by time.9. Some effects of different time, different concentration on the expression of KIR2DL4 mRNA, and there is interaction (F=326.133,P=0.000) The expression of each concentration group and the factor of time without dependency,24h was the lowest,36h was the highest; 12h,48h,60h in different concentration groups had no difference in the expression; the expression at 24h,36h were negatively related to the concentration, the 0.5mg/ml group at different time points are not less than other concentration and no dosing control group.10. Some effects of different time, different concentration on the expression of ILT2 mRNA, and there is interaction (F=1487.783, P=0.000) Expression level and the factor of time without dependency.48h and 60h with different concentration groups expression levels are equal, the expression of the rest time point were negatively related to the concentration. Expression of 2mg/ml and 1.5mg/ml concentrations at each time point were not higher than no dosing control group, no obvious promotion effect;the 0.5mg/ml concentration at each time point expression are not less than no dose control group, has the effect of promotion.11. Different time influence the expression of HLA-G1 protein (F=53.102, P=0.000), in different concentration within 24 hours, not affected the expression of HLA-Gl protein (F=1.609, P=0.247). The expression level of each concentration was negatively related to the time factor, the level of 12h was higher than 24h, the expression of 12h was positively related to the concentration, the expression of 24h was negatively related to the concentration. In 24h drug had no obvious promotion on the expression of HLA-Gl protein,0.5mg/ml above concentration group play an inhibition role.12. Different time influence the expression of ILT2 protein (F=23.946, P=0.001), different concentration influence the expression of ILT2 protein(F=8.894, P=0.002), and there was no interaction (F=l.579, P=0.254). The expression level of each concentration was positively related to the time factor, the expression level at each time point were above the no dosing control groups; the expression level at each time point were negatively to the concentration, and above the no dose control group.13. Shoutaiwan had no effect on low expression of KIR2DL4 protein at NK92 cell surface within 24 hours (P≥0.423)14. NK92 cells as the effector cells, JEG-3 cells as the target cells, when ratio was 10:1 the killing rate is as high as 60.40%; killing rate below 50% when effector-target rate 5:1-1:1, and no significant difference (P≥0.05), the follow-up experiment choose 1:1 ratio.Conclusion1. Shoutaiwan can promote the expression of HLA-G gene 7 subtypes, and there is time effect, concentration effect and interaction.2. Shoutaiwan can promote the expression of KIR2DL4 and ILT2. There is time effect, concentration effect and interaction.3. Showtaiwan can promote the expression of HLA-G, KIR2DL4 and ILT2; promote the transduction of NK cell inhibitory signal, then induce the maternal fetal tolerance; Showtaiwan can play a role on the treatment of unexplained recurrent spontaneous abortion through the signaling pathway mediated by HLA-G. |
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