滋肾调冲法对大鼠外周血内皮祖细胞影响的实验研究

Effect of Zishentiaochong therapy on peripheral blood endothelial progenitor cells in rats

[目的]观察滋肾调冲法代表方功血宁Ⅱ号方对骨髓单个核细胞移植大鼠外周血内皮祖细胞(EPCs)数量的影响,探讨该法对骨髓EPCs的动员作用。[方法]受鼠雌性大鼠经137Cs全身照射后,从尾静脉输注雄性大鼠标记4′,6-二脒基-2-苯基吲哚(DAPI)荧光的骨髓单个核细胞。受鼠随机分为移植对照组、功血宁Ⅱ号组和乌鸡白凤丸组,另取未照射大鼠尾静脉输注等量培养液为假孕对照组。骨髓移植24 h后各组给予相应药物或等容积的生理盐水。受鼠造血重建后,各组均制备假孕模型。在黄体早中晚期各组处理1/3数的大鼠,眼内眦取血,流式细胞术检测外周血EPCs的数量。血液涂片,荧光显微镜观察DAPI标记细胞的分布。[结果]荧光显微镜下显示,移植各组黄体各期血液涂片可见DAPI标记细胞,且早期数量为多,功血宁Ⅱ号组尤为明显。各组黄体早期外周血EPCs数目最多,中期略有下降,晚期明显减少(P<0.05或P<0.01)。功血宁Ⅱ号组、乌鸡白凤丸组黄体各期EPCs数目较假孕对照组显著增多(P<0.05或P<0.01);功血宁Ⅱ号组早期较移植对照组和乌鸡白凤丸组显著增多(P<0.05)。[结论]滋肾调冲法能够动员骨髓EPCs,提高外周血中EPCs数量,对EPCs进一步归巢至卵巢黄体,促进黄体血管新生具有重要作用。

[Objective] To study the effect of Gongxuening 11 prescription, a representative of Zishentiaochong therapy on the number of peripheral blood endothelial progenitor cells （EPCs） in rats transplanted with single marrow nuclear cells and explore the mobilization effect on EPCs. [Methods] Female Wistar rats with bone marrow cells transplantation were taken as experimental animals. Each of them was prepared by injecting bone marrow mononuclear cells suspension into the tail veins within 6 h after a general irradiation by ~37Cs. The bone marrow mononuclear cells were conventionally extracted and separated from the bilateral femoral marrows of adult male Wistar rats under aseptic conditions and fluorescence labeled by 4, 6- two acetyl-2-phenyl indole （DAPI）. The experimental female rats with the transplantation were randomly divided into three groups and some normal female Wistar rats with a infusion of equivalent blank medium via tail vein were taken as the fourth group. After 24 h of the bone marrow transplantation, the first group rats with the transplantation was intragastrically given physic liquor of Gongxuening // as Gongxuening 11 group, the second group rats was given isometric physic liquor of WujiBaifeng pills as WujiBaifeng Wan group, both the third and the fourth groups were given isometric physiological saline as transplantation control group and pseudopregnancy control group. After hematopoietic reconstitution in the rats with the transplantation, all the rats of the three groups and the fourth normal control group were prepared the pseudopregnancy model by using PMCG ＋ HCG. At the early, medium and advanced luteal stages 1/3 of each group were killed for getting intraocular canthal blood, making blood smears , observing the distribution of DAPI staining cells in the blood by fluorescence microscopy and detecting peripheral blood EPCs number by flow cytometry. [Results] It was revealed by fluorescence microscopy that in the early stage a larger number of DAPI staining cells can be found in blood smears made from transplanted rats, especially from the rats of Gongxuening II group. In all groups, the number of peripheral blood EPCs was maximum in early luteal stage and slightly reduced in medium luteal stage. The number of peripheral blood EPCs in advanced luteal stage was significantly reduced compared with the results in early or medium luteal stage （P〈0.05 or P〈0.OI ）. In every stage the numbers of peripheral blood EPCs from the both Gongxuening II group and WujiBaifeng Wan group were obviously higher than those from pseudopregnancy control group （P〈0.05 or P〈0.01）. In the early stage the number of EPCs from Gongxuening ]/group was significantly increased compared with the numbers of EPCs from WujiBaifeng Wan group and transplantation control group （P〈0.05）. [Conclusion] Zishentiaochong therapy can increase the number of circulating blood EPCs through mobilizing bone marrow EPCs into peripheral blood. It is important to home EPCs to the corpus luteum and promote luteal angiogenesis.