益气养阴方及其拆方通过线粒体途径诱导KG-1a细胞凋亡的研究

发布时间：2018-07-09 21:10

本文选题：急性髓系白血病 + 益气养阴方　；参考：《山东中医药大学》2016年硕士论文

【摘要】：目的:以NOD/SCID小鼠为研究平台,以KG-1a细胞株建立人急性髓系白血病模型,探讨益气养阴方及其拆方通过线粒体途径诱导急性髓系白血病KG-1a细胞凋亡的可能作用机理,为中医药通过线粒体途径诱导白血病细胞凋亡为靶点治疗急性髓系白血病寻求实验学支持,以期在此理论指导下研究新的抗白血病中药提供理论依据。方法:以NOD/SCID小鼠为实验对象,经60Co全身辐照,尾静脉注射KG-1a细胞株,建立急性髓系白血病小鼠模型。将造模成功后小鼠随机分为对照组、扶正组、祛邪组、全方组并以非造模小鼠为空白对照组作为阴性对照,观察小鼠的一般生存状态,濒死小鼠行骨髓系形态学及组织病理学检测,给药4周后脱颈处死小鼠,取脾细胞制成细胞悬液应用流式细胞术检测NOD/SCID小鼠细胞周期与细胞凋亡率;采用免疫组化法检测线粒体相关凋亡因子Cyt-C、Apaf-1、Smac/Diablo和AIF的表达。结果:药物治疗后,用药组和空白对照组小鼠生存时间较对照组明显延长(P0.01),全方组和空白对照组小鼠生存时间较扶正组与祛邪组长(P0.01),全方组小鼠较空白对照组比较无统计学意义(P0.05),祛邪组小鼠生存时间较扶正组延长(P0.01)。用药组细胞G1期停滞率和细胞凋亡率较对照组和空白对照组明显升高(P0.01),全方组细胞G1期停滞率和细胞凋亡率较扶正组与祛邪组升高(P0.01),祛邪组细胞G1期停滞率较扶正组略高(P0.05),祛邪组细胞凋亡率较扶正组升高(P0.01)对照组与空白对照组比较无统计学意义(P0.05)。小鼠骨髓中Cyt-C、Apaf-1、Smac/Diablo、AIF的表达均为用药组较对照组升高(P0.05),其中Cyt-C与Apaf-1表达显著升高(P0.01);全方组均高于扶正组与祛邪组(P0.01);祛邪组均较扶正组升高(P0.05),其中Cyt-C、Apaf-1和Smac/Diablo表达显著升高(P0.01)。其中对于各指标的影响全方组均优于扶正组与祛邪组,祛邪组均优于扶正组。结论:实验结果表明,益气养阴方能够使停滞于G1期的细胞增加,进入S期的细胞减少,从而了抑制白血病细胞增殖,并上调线粒体相关凋亡因子Cyt-C、Apaf-1、Smac/Diablo、AIF的表达,诱导白血病细胞凋亡,提高白血病细胞的凋亡率,因此延长了白血病小鼠生存期,揭示其对急性髓系白血病具有一定的治疗作用。
[Abstract]:Objective: to establish human acute myeloid leukemia model with KG-1a cell line and to explore the possible mechanism of inducing apoptosis of KG-1a cells by mitochondrial pathway. The aim of this study is to seek experimental support for the treatment of acute myeloid leukemia by inducing apoptosis of leukemic cells through mitochondrial pathway in order to provide theoretical basis for the study of new anti-leukemia Chinese medicine under the guidance of this theory. Methods: the mouse model of acute myeloid leukemia was established by injecting KG-1a cell line into caudal vein after 60Co irradiation. The mice were randomly divided into three groups: control group, Fuzheng group, dispelling evil group, the whole prescription group and the non-model mice as the negative control group. The general living state of the mice was observed. Bone marrow morphology and histopathological examination were performed in the dying mice. The mice were killed after 4 weeks of administration. The splenocytes were made into cell suspensions and the cell cycle and apoptosis rate of nod / SCID mice were detected by flow cytometry. The expression of mitochondrial related apoptosis factor Cyt-Capaf-1 Smac-Diablo and AIF was detected by immunohistochemical method. Results: after drug treatment, The survival time of mice in the medication group and blank control group was significantly longer than that in the control group (P0.01). The survival time of mice in the whole prescription group and blank control group was significantly longer than that in the Fuzheng group and in the blank control group (P0.01), but there was no statistical significance in the whole prescription group compared with the blank control group (P0.05). The survival time of mice in evil group was longer than that in Fuzheng group (P0.01). Compared with the control group and the blank control group, the G1 phase arrest rate and cell apoptosis rate of the medication group were significantly higher than those of the control group and the blank control group (P0.01). The G1 phase arrest rate and the apoptosis rate of the whole prescription group were higher than those of the Fuzheng group and the dispelling evil group (P0.01). The positive group was slightly higher (P0.05), and the apoptosis rate of the pathogenic group was higher than that of the Fuzheng group (P0.01). There was no significant difference between the control group and the blank control group (P0.05). The expressions of Cyt-C and Apaf-1 in bone marrow of mice were higher than those of control group (P0.05), the expression of Cyt-C and Apaf-1 were significantly higher in the whole prescription group than in the Fuzheng group and dispelling evil group (P0.01), and the expression of Cyt-Capaf-1 and Smac-Diablo in the pathogenic group were significantly higher than those in the Fuzheng group (P0.05), and the expression of Cyt-Capaf-1 and Smac-Diablo in the treatment group were significantly higher than that in the control group (P0.01). The effect on each index in the whole prescription group was better than that in the Fuzheng group and the dispelling evil group, and that in the dispelling evil group was better than that in the Fuzheng group. Conclusion: the experimental results showed that Yiqi Yangyin prescription could increase the number of cells in G1 phase and decrease the number of cells in S phase, thereby inhibiting the proliferation of leukemia cells and up-regulating the expression of Cyt-Cnapaf-1, a mitochondrial related apoptosis factor, in Smac-P / Diablosi-AIF. The apoptosis of leukemia cells was induced and the apoptosis rate of leukemia cells was increased. Therefore, the survival time of leukemia mice was prolonged, and it was revealed that it had a certain therapeutic effect on acute myeloid leukemia.
【学位授予单位】：山东中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R273

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