鼠尾草酸增加1,25(OH)2D3诱导HL-60细胞分化及其机制研究

目的：已证实1, 25(OH)2D3可以诱导HL-60细胞向成熟单核细胞分化，但其不足之处是高钙血症和形成耐药株。该实验应用鼠尾草酸（CA）联合1, 25(OH)2D3研究对HL-60细胞分化的影响及引起细胞内活性氧（ROS）水平和细胞内钙离子浓度的改变。方法：应用鼠尾草酸，1，25（OH）2D3单独和联合应用处理HL-60细胞，共分为5组：空白对照组；低浓度1，25（OH）2D3（1 nmol/L）组、鼠尾草酸组；联合处理组（10 μmol/L鼠尾草酸和1 nmol/L 1，25（OH）2D3）；高浓度1，25（OH）2D3（100 nmol/L）组。每24 h监测1次，通过四唑氮蓝(MTT)法观察细胞生长,共72 h；收集处理后72 h的HL-60细胞，通过光学显微镜观察细胞形态，用流式细胞仪（FCM）检测不同处理组细胞周期及单核细胞分化标记CD14的表达，ROS和细胞内钙离子浓度。结果：72 h后，联合处理组HL-60细胞与空白对照组相比，细胞增殖明显受抑制（Ab= 0.560±0.020; P<0.01），细胞形态具有成熟单核细胞特征，CD14的表达率升高（57.62%；P<0.01），G0/G1 期细胞显著增多, ROS水平下降［（52.67±10.76）%，P＜0.01］；联合处理组细胞内钙离子水平同空白对照组比较差异无显著性（115.64±17.74 nmol/L，P>0.05），但与高浓度1，25（OH）2D3组相比细胞内钙离子水平明显下降(P<0.01)。结论：鼠尾草酸可增强1，25（OH）2D3对HL-60细胞的诱导分化、增强抑制HL-60细胞增殖作用，细胞阻滞于G0/G1期，细胞内活性氧水平降低，这可能与细胞的诱导分化机制有关，且这一联合作用不增高细胞内钙离子水平。

Enhancement of differentiation induction of HL-60 cells by 1,25-dihydroxyvitamin D3 in combination with carnosic acid

OBJECTIVE: 1,25-dihydroxyvitamin D3 1,25(OH)2D3] is a potent inducer of differentiation in myeloid leukemia cells, but its clinical use is limited due to its hypercalcaemic effect and resistance. Carnosic acid is a plant-derived polyphenol food preservative with chemoprotective effects against carcinogens. Recent research has shown that carnosic acid potentiates the effects of 1,25(OH)2D3 on differentiation of human leukemia cells. This study examined the effects of 1,25(OH)2D3 in combination with carnosic acid on monocytic differentiation as well as intracellular reactive oxygen species (ROS) and Ca2+ levels in human leukemia HL-60 cells. METHODS: HL-60 cells were randomly treated with 1 nmol/L 1,25(OH)2D3, 100 nmol/L 1,25(OH)2D3, 10 micromol/L carnosic acid, a combination of 1 nmol/L 1,25(OH)2D3 and 10 micromol/L carnosic acid or placebo. Cell growth was observed by MTT assay for 72 hrs at an interval of 24 hrs. Cells were harvested after 72 hrs of culture. Morphologic features of the cells were observed by microscopy. Flow cytometry was used to detect cell cycle, monocytic differentiation marker CD14 expression, and intracellular ROS and Ca2+ levels. RESULTS: A combination of 1 nmol/L 1,25(OH)2D3 and 10 micromol/L carnosic acid resulted in greater proliferation inhibition (Ab: 0.56 0+/- 0.020 vs 1.482 +/- 0.327; P <0.01), mature monocytic features, G0 /G1 cell arrest, higher CD14 expression (57.62 +/- 0.817% vs 2.76 +/- 0.828%; P <0.01), lower intracellular ROS levels (52.67 +/- 10.76% vs 86.46 +/- 40.52%; P <0.01 and similar intracellular Ca2+ levels in HL-60 cells when compared with the placebo group. The ability of a combination of 1 nmol/L 1,25(OH)2D3 and 10 micromol/L carnosic acid to inhibit the proliferation and induce the differentiation of HL-60 cells was similar to that of 100 nmol/L 1,25(OH)2D3, while the intracellular Ca2+ level (115.64 +/- 17.74 nmol/L vs 185.75 +/- 27.38 nmol/L) was significantly lower than that in the 100 nmol/L 1,25(OH)2D3 group. CONCLUSIONS: Low concentration of 1,25(OH)2D3 combined with 10 micromol/L carnosic acid can produce enhanced differentiation, proliferation inhibition and antioxidant effects of HL-60 cells. The combination of the two inducers dose not increases intracellular Ca2+ levels.