The Effect Of Bilirubin On Neonatal Cord Blood Mononuclear Cells MD-2 And TRAF6 MRNA Gene Expression

Objective: To observed the changes of myeloid differentiatial protein 2 (MD-2) and tumor necrosis factor receptor associated 6 (TRAF6) mRNA expressions in monocyte under the incubation of different concentrations of bilirubin. To explore the effect of bilirubin on the immune functions of neonatal cord blood monocyte. Methods: The cord blood of ten newborns of different gestational age was collected. The method of gelatin/plasma coated flasks was used to isolate mononuclear cells and to measure its purity with flow cytometry. There were 10 parallel experimental group, including blank control group (A group), LPS control group (B group), 1mg bilirubin group (C1 group), 2.5mg bilirubin group (C2 group), 1mg bilirubin + LPS group (D1 group), 2.5mg bilirubin + LPS group (D2 group), 6mg bilirubin + LPS group (D3 group), 9mg bilirubin + LPS group (D4 group), 12.9mg bilirubin + LPS group (D5 group), 18mg bilirubin + LPS group (D6 group). The level of gene expression of MD-2, TRAF6 mRNA was measured by RT-PCR, and correlation analysis between them was done. Results: 1. The effect of bilirubin on CBMC MD-2 mRNA gene expression:â‘ There was no statistical significance between LPS control group and blank control group (P> 0.05). There was no statistical significance between 1mg/dl, 2.5mg/dl bilirubin group and blank control group (P> 0.05). That is to say, there were no significant effect of LPS and low concentrations of bilirubin (1mg/dl, 2.5mg/dl) on CBMC MD-2 mRNA gene expression level.â‘¡There was no statistical significance when comparing different concentrations of bilirubin + LPS group with the LPS control group(P> 0.05); Either were there among different concentrations of bilirubin + LPS group when compared overrally or between any two of them (P > 0.05). MD-2 mRNA gene expression levels with bilirubin concentration were negatively correlated (r = -0.2947, P <0.05). That is to say, there was the trend of bilirubin inhibiting CBMC MD-2 mRNA gene expression. The higher, the more obvious. 2. The effect of bilirubin on CBMC TRAF6 mRNA gene expression:â‘ There was no statistical significance between LPS control group and blank control group (P> 0.05). There was no statistical significance between 1mg/dl, 2.5mg/dl bilirubin group and blank control group (P> 0.05). That is to say, there were no significant effect of LPS and low concentrations of bilirubin on CBMC TRAF6 mRNA gene expression level.â‘¡There were statistical significance when comparing the TRAF6 mRNA gene expression of 1mg/dl bilirubin + LPS group, 2.5mg/dl bilirubin + LPS group with the LPS control group, respectively(P <0.05); so were there between the TRAF6 mRNA gene expression of 6mg/dl bilirubin + LPS group, 18mg/dl bilirubin + LPS group and LPS control group, respectively (P <0.05); when comparing the overall levels TRAF6 mRNA gene expression of D1, D2, D3, D4, D5, D6, there was statistical significance (M = 774, P <0.05); Inter-group comparison showed that the difference between D1 and D2 was not significant (P> 0.05);when comparing D1 with D3, D4, D5, D6 respectively and comparing D2 with D3, D4, D5, D6 respectively, there were both statistically statistical significance (P <0.05);when comparing the D3,D4,D5,D6 each other, there were not statistical significance(P<0.05).That is, low concentrations of bilirubin + LPS stimulated CBMC TRAF6 mRNA gene expression, while high concentrations of bilirubin inhibited its expression. The higher, the more obvious. TRAF6 mRNA gene expression levels with bilirubin concentration were negatively correlated (r = -0.3948, P <0.01) 3. MD-2 and TRAF6 correlation analysis: with the effect of bilirubin, the MD-2 and TRAF6 mRNA expressions were positively correlated ( r = 0.3108, P <0.05). That is, under different concentrations of bilirubin, with the MD-2 gene expression declines, TRAF6 gene expression decreases accordingly.Conclusion: 1. Low concentrations of bilirubin do not affect the MD-2, TRAF6 mRNA gene expression of monocytes obviously or even tend to stimulate their expression, while higher concentrations of bilirubin are more likely to suppress the expression dose-dependently, which indicates that high concentrations of bilirubin have obvious immunotoxicity that lead to the decline of cell-mediated immune functional. 2. The levels of MD-2 mRNA gene expression and that of TRAF6 mRNA gene expression were positively correlated. That is, under different concentrations of bilirubin, with the MD-2 gene expression declines, TRAF6 gene expression decreases accordingly, which suggests both TRAF6 and MD-2 in are expressed in the signal transduction pathway of monocytes and involved in the immune mechanism of neonatal hyperbilirubinemia.