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目的观察脐血单核细胞(CBMC)经不同水平胆红素孵育后髓样分化蛋白2(MD-2)、TNF受体相关因子6(TRAF6)mRNA表达的变化。方法采集10例健康足月新生儿的脐血,经纤维蛋白结合法分离得CBMC,将其加入含不同水平胆红素(0μmol.L-1、17.1μmol.L-1、42.8μmol.L-1、102.6μmol.L-1、153.9μmol.L-1、220.6μmol.L-1和307.8μmol.L-1)的牛血清清蛋白溶液中孵育1 h,再加入脂多糖(LPS)刺激30 min。收集CBMC,采用反转录-PCR方法测定其MD-2、TRAF6 mRNA的表达水平。结果 1.LPS单独作用对CMBC MD-2 mRNA和TRAF6 mRNA表达水平无明显影响,低水平胆红素(17.1μmol.L-1、42.8μmol.L-1)单独作用对CBMC MD-2 mRNA和TRAF6 mRNA表达水平无明显影响。2.先经不同水平胆红素孵育,再接受LPS刺激,胆红素有抑制细胞MD-2 mRNA表达的趋势,随着胆红素水平的升高,抑制趋势越明显;先经不同水平胆红素孵育,再接受LPS刺激,低水平胆红素促进细胞TRAF6 mRNA的表达,当胆红素≥102.6μmol.L-1时,抑制TRAF6 mRNA表达,胆红素水平越高,抑制作用越明显。在相同胆红素水平作用下,随着MD-2 mRNA表达水平的降低,TRAF6 mRNA表达水平随之降低。结论低水平胆红素对CBMC MD-2 mRNA的表达无明显影响,对TRAF6 mRNA的表达有促进作用。高水平胆红素对二者的表达均有抑制作用,随着胆红素水平升高,抑制作用越来越明显。高胆红素血症患儿免疫功能低下可能与高浓度胆红素对免疫细胞的抑制作用有关。
Objective To observe the changes of mRNA expression of MD-2 and TNF receptor-related factor 6 (TRAF6) in cord blood mononuclear cells (CBMCs) incubated with different levels of bilirubin. Methods Cord blood was collected from 10 healthy full-term newborns and CBMCs were isolated by fibrin-binding method. CBMCs with different levels of bilirubin (0μmol.L-1, 17.1μmol.L-1, 42.8μmol.L- 1,102.6μmol.L-1, 153.9μmol.L-1,220.6μmol.L-1 and 307.8μmol.L-1) in bovine serum albumin solution for 1 h, then add lipopolysaccharide (LPS) to stimulate 30 min. CBMCs were collected and their MD-2, TRAF6 mRNA expression levels were determined by reverse transcription-PCR. Results 1.LPS alone had no significant effect on the expression of MD-2 mRNA and TRAF6 mRNA in CMBC. The effect of low level of bilirubin (17.1μmol.L-1.42.8μmol.L-1) TRAF6 mRNA expression level had no significant effect. Bilirubin first incubated with different levels, then LPS stimulation, bilirubin inhibited the expression of MD-2 mRNA in the trend, with the bilirubin levels increased, the more obvious inhibition trend; first by a different level of gall When incubated with erythrocytes and then with LPS, low level of bilirubin promoted the expression of TRAF6 mRNA. When bilirubin≥102.6μmol.L-1, TRAF6 mRNA expression was inhibited. The higher the level of bilirubin, the more obvious the inhibitory effect . Under the same bilirubin level, TRAF6 mRNA expression decreased with the decrease of MD-2 mRNA expression. Conclusion Low-level bilirubin has no effect on the expression of MD-2 mRNA in CBMC and promotes the expression of TRAF6 mRNA. High levels of bilirubin have inhibitory effect on both the expression, with the bilirubin levels increased, the inhibitory effect is more and more obvious. Immunocompromised children with hyperbilirubinemia may be related to the inhibitory effect of high concentration of bilirubin on immune cells.