褪黑素对新生儿脐带血单个核细胞活化增殖的影响

Effect of melatonin on the activation and proliferation of neonatal cord blood mononuclear cell

目的探讨褪黑素（melatonin，MLT）对新生儿脐带血单个核细胞（cord blood mononuclear cells，CBMC）活化增殖的影响。方法运用相差显微镜及。H-TdR掺入法观察MLT对CBMC形态学的影响及增殖的调节，并与MLT对成人血单个核细胞（peripheral blood mononuclear cells，PBMC）的影响进行比较。结果CBMC的。H-TdR掺入率随培养基中MLT浓度（50pg／ml～50ng／m1）的增加而增加，在5ng／ml效果最佳。在加入MLT（5ng／m1）的培养基中，经72h培养后，高倍光镜观察下见细胞较培养前明显密集，为较多体积增大的单个核细胞。CBMC培养基中分别加入不同刺激物MLT（5ng／m1）、Interleukin-2（IL-2，50ng／m1）、MLT＋phytohemagglutinin（PHA，5μg/ml）、MLT＋IL-2，其^3H-TdR掺入率（cpm）分别为114327±52863、16087±9006、118360±59207和17682±7391。与细胞悬液组（14133±8688）比较，MLT组和MLT＋PHA组CBMC的。H-TdR掺入率显著增加（t=5．9143，P〈0.001；t=5．5078，P〈0．001），IL-2组、MLT＋IL-2组CBMC的。H-TdR掺入率无明显变化（t=0．4983，P〉0．05；t=0．9839，P〉0.05）；而MLT组或MLT＋PHA组CBMC的。H-TdR掺入率与培养基中仅加入PHA组（110397±48663）比较，差异无统计学意义（t=0．1730，P〉0．05；t=0．3286，P〉0．05）。各种刺激物加入培养基后，CBMC的^3 H-TdR掺入率均明显高于PBMC。结论MLT不但可诱导PBMC活化增殖，也可诱导CBMC的活化增殖，且其对CBMC的作用强于PBMC。

Objective A growing body of evidence suggests that the pineal hormone, melatonin （MLT）, has immunomodulatory properties; MLT can induce an increment of cell proliferation and an increase or a decrease of a number of cytokines in adults＇ peripheral blood mononuclear cells （ PBMC）. However, the influence of MLT on the modulation of neonatal cord blood mononuclear cells （CBMC） proliferation has not been reported. The present study aimed to investigat the possible regulatory effects of MLT on the proliferation of CBMC in vitro. Methods Ten cord blood preparations from placenta vena umbilicalis of 10 healthy full-term normally deliveed newborns and 10 healthy adult volunteers＇ peripheral blood preparations as controls were obtained. Cord/peripheral blood mononuclear cells suspension were prepared, 2 x 10^5 cells were added to 96-well plate and co-cultured with different stimulants （ in cell cultures containing 5 μg phytohemagglutinin （PHA）/ml, 50 ng MLT/ml, 5 ng MLT/ml, 500 pg MLT/ml, 50 pg MLT/ml, 50 ng IL-2/ml, 5 ng MLT/ml ＋5 μg PHA/ml or5 ng MLT/ml ＋50 ng IL-2/ml） for72 h, while the cell suspension （with no stimulant） was used as controls, also cultured for 72 h. With the methods of microscopic examination and ^3H-TdR incorporation test, the influence of melatonin on CBMC morphology and proliferation were investigated, and the effects of MLT on the proliferation of CBMC and PBMC were compared with LSD-t T test and independent samples T test. Results In CBMC, MLT（50 pg/ml -50 ng/ ml）increased the ^3H-TdR incorporation rates in a dose-dependent manner, the rate was the highest in the concentration of 5 ng/ml. After 72 h of cell culture, the number of cells in the MLT （5 ng/ml）-exposed group was higher than that recorded before incubation when observed under the high power microscope,including many big mononuclear cells. After adding different stimulans MLT （ 5 ng/ml ） , IL-2 （50 ng/ml） , MLT plus PHA （ 5μg/ml ） or MLT plus IL-2 into mediums, the 3 H-TdR incorporation rates of CBMC （cpm） was 114 327±52 863, 16 087±9006, 118 360±59 207 and 17 682±7391 respectively. In comparison with the controls （ 14 133±8688 ）, the incorporation rates of both MLT-exposed group and MLT ＋ PHA-exposed group increased significantly （ t = 5. 9143, P 〈 0. 001 ; t = 5. 5078, P 〈 0. 001 ） ; the rate of IL-2-treated group or MLT ＋ IL-2-treated group demonstrated no significant changes （ t = 0. 4938, P 〉 0. 05 ; t =0. 9839, P 〉0. 05） ; while the incorporation rates of MLT-exposed group or MLT ＋ PHA-exposed group had no significant difference compared with that of PHA-exposed group （ t = 0. 1730, P 〉 0.05 ; t = 0. 3286, P 〉 0. 05 ）. After adding different stimulants into the medium, the incorporation rates of CBMC were all higher than those of PBMC. Conclusions MLT can promote not only the proliferation of PBMC, but also the proliferation of CBMC, and the effects of MLT on CBMC were stronger than those on PBMC. This suggests that MLT could be involved in the regulation of the newborn immune system and suggests a new immunotherapeutic strategy in the treatment of certain diseases of neonates.