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利用粉纹夜蛾Trichoplusia ni Hbner卵细胞系(Hi-5细胞系)在细胞水平研究了印楝素(azadirachtin)A杀卵活性的毒性机理。以MTT法研究了印楝素A对粉纹夜蛾Hi-5细胞的生长抑制率,结果表明最初两天印楝素A对Hi-5细胞无较明显活性,但随后几天抑制率显著增加。用Giemsa染色法对细胞进行染色,观察细胞形态发生的变化,发现:1.25μg/mL印楝素A处理Hi-5细胞1d后,细胞已无法贴壁,形状变圆,接着细胞形态变得极不规则,有凋亡小体出现。用Ho33342染料对Hi-5细胞核DNA染色,通过荧光显微镜观察发现:经印楝素A处理后第1天,部分细胞核染色体发生异常凝聚,此后异常细胞核比例增多,核膜严重破损。以异硫氰酸荧光素(FITC)荧光染料研究了Hi-5细胞的蛋白质含量变化,发现1.25μg/mL印楝素A处理Hi-5细胞1d后,细胞蛋白质指数(DI)为1.070±0.018,至第3dDI值上升到1.912±0.019。分析了印楝素A处理后Hi-5的还原性谷胱甘肽(GSH)的相对含量变化,发现1.25μg/mL处理浓度下,各天处理组GSH抑制率有显著差异。结果显示印楝素A能够抑制Hi-5细胞增殖,影响细胞骨架正常功能,降低细胞活力。
The toxicity mechanism of azadirachtin A on ovicidal activity was studied at the cellular level using the Trichoplusia ni Hbner egg cell line (Hi-5 cell line). The inhibitory rate of azadirachtin A to Hi-5 cells was studied by MTT assay. The results showed that azadirachtin A had no obvious activity on Hi-5 cells in the first two days, but the inhibition rate was significantly increased in the next few days . The cells were stained with Giemsa staining and the morphological changes of cells were observed. It was found that after treated with 1.25μg / mL azadirachtin A for 1 day, the cells were unable to attach to the cells and became round in shape. Then the cell morphology became extremely Irregular, apoptotic bodies appear. The staining of the nuclear DNA of Hi-5 cells with Ho33342 dye was observed by fluorescence microscope. On the first day after treatment with azadirachtin A, part of the nuclear chromosomes were abnormally aggregated, and then the proportion of abnormal nuclei increased and the nuclear membrane severely damaged. The protein content of Hi-5 cells was studied by Fluorescein isothiocyanate (FITC) fluorescent dye. The results showed that the protein index (DI) of Hi-5 cells treated with 1.25μg / mL azadirachtin A for 1 day was 1.070 ± 0.018 , To the first 3dDI value rose to 1.912 ± 0.019. The change of relative content of reduced glutathione (GSH) in Hi-5 treated with azadirachtin A was analyzed. The results showed that there was a significant difference in the GSH inhibition rate of each treatment group at the concentration of 1.25μg / mL. The results showed that azadirachtin A can inhibit Hi-5 cell proliferation, affecting the normal function of cytoskeleton, reducing cell viability.