续断水煎液对小鼠胚胎肢芽细胞体外培养中增殖、分化、凋亡的影响

Effect of Radix Dipsaci on cell proliferation,differentiation and apoptosis of mouse embryo limb bud cells

目的探讨续断水煎液对小鼠胚胎肢芽细胞增殖,分化和凋亡的影响,以评价续断水煎液的胚胎发育毒性作用。方法取E13(embryos on day 13)后肢芽制成细胞悬液,接种到细胞培养板中,分别加入不同剂量(325、162.5、40.625)mg/ml续断水煎液和蒸馏水,作为高,中,低剂量组和空白对照组。体外培养72 h后,用MTT法检测肢芽细胞的增殖,甲苯胺蓝染色法检测分化,Hochest 33258染色法及流式细胞仪检测凋亡情况。结果各剂量组吸光度值均高于空白对照组,其中高和中剂量组与空白对照组比较具有显著性差异(P<0.05),且各剂量组软骨细胞集落数均多于空白组,与对照组比较均具有显著性差异(P<0.05);高和中剂量组细胞凋亡率明显低于空白组(P<0.05)。结论在本实验剂量范围内未发现续断水煎液对胚胎肢芽细胞的毒性作用,反而促进了肢芽细胞的增殖和分化,抑制了肢芽细胞的凋亡。

Objective： To explore the effect of Dipsaci Radix water decoction on proliferation, differentiation and apoptosis of embryonic limb budcells to evaluate its embryotoxicity in mice. Methods： The hindlimb buds were amputated from mice embryos on day 13, then were made into cell suspension.The cells were cultured by （325, 162.5, 40.625） mg/ml Dipsaci Radix water decoction and the distilled water respectively, which were divided into the high, middle and low doses and the blank control group for 72 h. The limb bud cells proliferation and differentiation were determined by MTT and cartilage cell colony numbers which were stained with toluidine blue. Cell apoptosis was determined by Hochest33258 staining and flow cytometry. Results： The absorbance value of each dose group was higher than that of the control group. Compared with the control group, the high and middle dose group had significant difference （P〈0.05） . The number of cartilage cells in each dose group was more than that in the blank group. Compared with the control group, each group had significant difference （P〈0.05） . The apoptosis rate of high and middle dose group was significant lower than that of control group （P〈0.05） . Conclusion： It is not found embryotoxicity effect of the decoction of Radix Dipsaci on mouse embryo limb bud cell.In contrast, the decoction of Radix Dipsaci can promote the proliferation and differentiation and inhibit apoptosis of limb bud cells.