发酵桦褐孔菌对肝癌HepG-2细胞凋亡作用初探

发布时间：2018-04-21 18:21

本文选题：发酵桦褐孔菌 + HepG-2细胞　；参考：《延边大学》2017年硕士论文

【摘要】：目的初步探讨发酵桦褐孔菌对肝癌HepG-2细胞的凋亡作用。方法分别采用硫酸苯酚法、Folin-Ciocalteu法、比色法测定未发酵、发酵桦褐孔多糖、萜类和多酚的含量;以肝癌HepG-2细胞为实验对象,采用MTT法测定发酵桦褐孔菌对肝癌HepG-2细胞增殖的影响;HE染色法观察发酵桦褐孔菌对肝癌HepG-2细胞形态学的影响;流式细胞仪测定发酵桦褐孔菌对肝癌HepG-2细胞凋亡作用及细胞周期的影响;琼脂糖凝胶电泳法观察发酵桦褐孔菌对肝癌HepG-2细胞DNA裂解的影响。结果与未发酵桦褐孔菌比较,发酵桦褐孔菌多糖、多酚和萜类含量明显升高(P0.01);发酵桦褐孔菌显著抑制肝癌HepG-2细胞增殖,其抑制作用与给药时间和给药浓度呈正相关,且与未发酵桦褐孔菌相比,发酵桦褐孔菌浓度为100mg/L,处理时间为48h对肝癌细胞的抑制作用更明显(P0.05),且呈时间与剂量依赖性;与对照组比较,发酵桦褐孔菌组肝癌HepG-2细胞细胞间距增大、细胞核固缩、染色加深、细胞膜皱缩、形成凋亡小体,出现典型凋亡形态,凋亡程度随着剂量和时间的增加而增强,与未发酵组相比,肉眼观察无差异;发酵桦褐孔菌可显著诱导肝癌HepG-2细胞凋亡,并且随着浓度的增加其诱导凋亡作用增强,与未发酵桦褐孔菌相比,当发酵桦褐孔菌浓度为200mg/L、400mg/L,诱导肝癌细胞凋亡作用更显著(P0.01);与对照组相比,发酵桦褐孔菌组肝癌HepG-2细胞细胞周期分布受到影响,G0/G1期细胞比率显著升高(P0.05),S期和G2/M期比率显著下降(P0.05),与未发酵桦褐孔菌组相比,发酵桦褐孔菌浓度为200mg/L,作用时间为48h时G0/G1期细胞周期分布存在显著性差异(P0.05);与对照组相比,发酵桦褐孔菌作用48h肝癌HepG-2细胞DNA均发生裂解,呈现典型的梯形凋亡形态,与未发酵桦褐孔菌相比,发酵桦褐孔菌DNA裂解更明显。结论发酵桦褐孔菌可显著诱导肝癌HepG-2细胞凋亡,且与未发酵桦褐孔菌相比,发酵桦褐孔菌更显著诱导肝癌HepG-2细胞凋亡。
[Abstract]:Objective to investigate the apoptosis of HepG-2 cells by fermenting Betula obliquus. Methods Folin-Ciocalteu method and colorimetric method were used to determine the contents of unfermented and fermented birch brownhole polysaccharides, terpenoids and polyphenols respectively. The effect of Betula obliquus fermentation on the proliferation of HepG-2 cells was determined by MTT method. The morphological changes of HepG-2 cells were observed by HE staining. The apoptosis and cell cycle of HepG-2 cells were determined by flow cytometry, and the DNA cleavage of HepG-2 cells was observed by agarose gel electrophoresis. Results compared with unfermented Betula obliquus, the contents of polysaccharides, polyphenols and terpenes in fermented Betula obliquus increased significantly (P 0.01), and the inhibition of P0.01C was positively correlated with the time of administration and the concentration of the drug. Compared with unfermented Betula obliquus, the concentration of 100 mg / L and the treatment time of 48 h had more obvious inhibitory effect on hepatoma cells in a time and dose-dependent manner, compared with the control group. In the fermentative group, the HepG-2 cell spacing increased, the cell nuclear pyknosis and staining deepened, the cell membrane shrank and formed apoptotic bodies, and the apoptotic degree increased with the increase of dose and time. Compared with the unfermented group, there was no significant difference between the unfermented group and the unfermented group, and the apoptosis of the HepG-2 cells was significantly induced by the fermented Betula obliquus, and the apoptotic effect was enhanced with the increase of the concentration, compared with that of the unfermented Betula obliquus. When the concentration of fermenting Betula obliquus was 200 mg / L, 400 mg / L, the apoptotic effect of hepatoma cells was more significant than that of control group. The cell cycle distribution of HepG-2 cells in fermenting Betula obliquus group was significantly increased in G _ 0 / G _ 1 phase, the ratio of P0.05 / M phase and G _ 2 / M phase was significantly decreased compared with that of unfermented Betula platyphylla group. There was a significant difference in cell cycle distribution of HepG-2 cells in G0/G1 phase at the concentration of 200mg 路L ~ (-1) and the time of treatment for 48 h. Compared with the control group, the DNA of hepatoma HepG-2 cells were lysed at 48 h after treatment by the fermenting strains of Betula obliquus, which showed typical ladder apoptotic morphology. The DNA cleavage of fermented Betula obliquus was more obvious than that of unfermented Betula obliquus. Conclusion the apoptosis of hepatoma HepG-2 cells was significantly induced by fermenting Betula obliquus, and the apoptosis of HepG-2 cells was more obvious than that of unfermented Betula obliquus.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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