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目的以原代培养的胚胎大鼠大脑皮质神经细胞进行缺氧缺糖再给氧处理,观察党参皂苷L1对神经细胞凋亡的抑制作用。方法胚胎大鼠大脑皮质神经细胞原代培养,含连二亚硫酸钠的无糖Earle s液进行缺氧缺糖处理造模,MTT法测定细胞存活率,Hoechst33342和PI原位双染法荧光显微镜观察细胞形态学变化和坏死、凋亡细胞百分率,APO BRDU法流式细胞术检测凋亡细胞百分率,Fluo3法流式细胞术检测细胞内Ca2+浓度,评价药效。结果(1)细胞存活率(%)与模型组(29.89±6.28)比较党参皂苷L11.0mg L组(46.05±8.92)、0.1mg L组(46.83±6.76)、0.01mg L组(41.05±9.18)均能显著提高细胞存活率(P<0.01和P<0.05);(2)细胞坏死率(%)与模型组(44.99±12.81)比较,党参皂苷L11.0mg L组(19.45±2.31)、0.1mg L组(18.78±4.95)和0.01mg L组(22.37±4.44)能显著降低细胞坏死率(P<0.001);(3)原位双染法细胞凋亡率(%)与模型组(17.44±4.82)比较,党参皂苷L11.0mg L组(8.75±1.88)、0.1mg L组(9.73±1.76)能显著降低细胞凋亡率(P<0.01和P<0.05);(4)流式细胞术检测细胞凋亡率(%)与模型组(8.55±3.84)比较,党参皂苷L11.0mg L组(4.21±2.31)、0.1mg L组(3.83±2.95)能显著降低细胞凋亡率(P<0.05);(5)细胞内Ca2+平均荧光值,与模型组(47.37±9.68)比较,?
Objective To observe the inhibitory effects of Codonopsis saponin L1 on apoptosis of neurons after primary cultured embryonic rat cerebral cortical neurons were subjected to hypoxia/glucose deprivation and then to oxygenation. Methods Embryonic rat cerebral cortical neurons were cultured in primary culture. Sugarless Earle s solution containing sodium dithionite was used to model hypoxia-glucose deprivation. Cell viability was measured by MTT assay. Hoechst 33342 and PI were used to observe the cells by fluorescence microscope in situ. Morphological changes and necrosis, percentage of apoptotic cells, percentage of apoptotic cells detected by APO BRDU flow cytometry, intracellular Ca2+ concentration detected by Fluo3 flow cytometry, and efficacy were evaluated. Results (1) The cell viability (%) was compared with that of the model group (29.89±6.28). The results showed that the Codonopsis saponin L11.0mg L group (46.05±8.92), 0.1mg L group (46.83±6.76), 0.01mg L group (41.05±9.18) Both of them significantly increased cell viability (P<0.01 and P<0.05); (2) compared cell necrosis rate (%) with model group (44.99±12.81), codonopsis saponin L11.0 mg L group (19.45±2.31), The 0.1 mg L group (18.78±4.95) and 0.01 mg L group (22.37±4.44) could significantly reduce the rate of cell necrosis (P<0.001); (3) In-vivo double staining method of apoptosis rate (%) and model group ( Compared with 17.44±4.82), codonopsis saponin L11.0 mg L (8.75±1.88) and 0.1 mg L group (9.73±1.76) could significantly reduce the apoptosis rate (P<0.01 and P<0.05); (4) flow pattern Compared with the model group (8.55±3.84), the cell apoptosis rate (%) was significantly lower in the codonopsis saponin L11.0 mg L group (4.21±2.31) and the 0.1 mg L group (3.83±2.95). P<0.05); (5) The average fluorescence of intracellular Ca2+, compared with the model group (47.37±9.68),