胆红素对脐血T淋巴细胞功能的影响

Effects of bilirubin on the functions of cord blood T-lymphocytes

目的探讨胆红素对新生儿T淋巴细胞功能的影响。方法收集并分离21例正常足月新生儿脐血单个核细胞（cord blood mononuclear cell，CBMC），按培养液中胆红素的浓度相应分为A、B、C、D四组。A组：胆红素终浓度205．2μmol／L（12mg／dl）；B组：胆红素终浓度153．9μmol／L（9mg／d1）；C组：胆红素终浓度102．6μmol／L（6mg／d1）；D组：胆红素终浓度0μmol／L。培养后检测细胞凋亡率和CD25^＋细胞百分率，ELISA法检测培养上清液中可溶性Fas（soluble Fas，sFas）、可溶性Fas配体（soluble Fasligand，sFasL）、IFN-γ及IL-4水平，并分析它们之间的相关性。结果（1）A、B组细胞凋亡率分别为（61．49±8．93）％和（56．36±9．24）％，高于C、D组，分别为（32．44±8．72）％和（28．72±6．21）％，差异均有统计学意义（P〈0．01）；（2）A、B组CD25^＋细胞百分率分别为（19．89±6．72）％和（22．12±4．61）％，低于C、D组，分别为（61．28±4．87）％和（64．25±6．18）％，差异均有统计学意义（P〈0．01）；（3）A、B组sFas、sFasL水平低于C、D组，C组sFas、sFasL水平低于D组，差异均有统计学意义（P〈0．01）；（4）A、B组IFN-γ水平和IFN-γ／IL-4比值低于C、D组，差异有统计学意义（P〈0．01）C组IFN-γ水平和IFN-γ／IL-4比值低于D组，差异有统计学意义（P〈0．05）；四组间IL-4水平差异无统计学意义（q=0．47～3．30，P〉0．05）；（5）CD25^＋细胞百分率、培养上清液sFas、sFasL、IFN-γ水平及IFN-γ／IL-4比值均与凋亡细胞百分率呈负相关（r=0．79～-0．37，P〈0．01），而IL-4水平与细胞凋亡率无相关性（r=0．20，P〉0．05）。结论胆红素具有明显的免疫抑制活性，在生理性黄疸状态的较高水平[血清蛋红素≥153．9μmol／L（9mg／dl）]即能促进T淋巴细胞凋亡，进一步加重新生儿时期辅助性T细胞亚群失衡状态，导致细胞免疫功能下降。

Objective To study the effects of bilirubin on the functions of neonatal T- lymphocytes. Methods Cord blood mononuclear cells （CBMC） of 21 full-term neonates were in vitro preincubated with various levels of unconjugated bilirubin. Levels of unconjugated bilirubin in group A were 205.2μmol/L（12 mg/dl） , 153.9μmol/L（9 mg/dl） in group B, 102.6μmol/L in group C （6 mg/dl）and 0 μmol/L in group D. The apoptosis rate of lymphocytes in CBMC after cultivation was determined by morphological methods. The positive expression rate of CD25 was detected by indirect immuno-fluorescence procedure. The levels of soluble Fas（sFas）, soluble Fas ligand（sFasL）, interferon-γ（IFN-γ） and interleukin-4（IL-4） in the supernatants of CBMC were detected by ELISA. Results （1）The apoptosis rate of lymphocytes in group A and B （61. 49 ± 8. 93）% and （56. 36± 9.24）%]was higher than those in group C and D[（32. 44± 8. 72）% and （28. 72 ± 6. 21）%], respectively（P〈0. 01）. （2）The positive expression rate of CD25 in lymphocytes in group A and B [（19.89±6.72）% and （22.12±4.61）%] was lower than those in group C and D[（61.28±4.87）% and （64.25±6.18）%] ,respectively（P〈0.01）. （3）The levels of sFas,sFasL in group A and B were lower than those in group C and D,and which in group C were lower than group D（P〈0.01）. （4）The level of IFN-γ and the ratio of IFN-γ/IL-4 in group A and B were lower than those in group C and D（P〈0.01） ,and which in group C were lower than group D（P〈0.05）. No significant difference of IL-4 level in supernatants of CBMC was found among the four groups （q=0.47～3.30, P〉0.05）. （5）The positive expression rate of CD25, the levels of sFas, sFasL and IFN-γ in the supernatants had negative correlation with the apoptosis rate of lymphocytes （r= -0. 79-0. 37,P〉0.01）. However, the IL-4 level did not correlate with the apoptosis rate of lymphocytes （r = -0. 20, P 〉0. 05）. Conclusions Bilirubin has obvious immune inhibitory activity. It could promote apoptosis and inhibit activities of T-lymphocytes, and result in further imbalance of Th1/Th2 even in neonates with physical jaundice. The effects are more significant when the level of bilirubin is higher than 153.9μmol/L.