乳铁蛋白素对Jurkat细胞增殖、分化和凋亡机制的研究

发布时间：2018-04-25 12:11

  本文选题：牛乳铁蛋白素 + Jurkat细胞　； 参考：《东北农业大学》2013年硕士论文

【摘要】：牛乳铁蛋白素（lactoferricin B，LfcinB）是一种抗菌活性非常高的微生物活性肽，且来源广泛。在发挥抗肿瘤活性的浓度范围内，LfcinB对人体正常的细胞没有影响，但是它对人类许多肿瘤细胞系具有细胞毒作用，呈现出一定的针对性。本课题将人工合成的LfcinB作用于人急性T细胞白血病的T淋巴细胞（Jurkat cell），应用流式细胞术（Flow Cytometry，FCM）、蛋白质免疫印迹分析法（Western Blotting）研究LfcinB对Jurkat细胞增殖、分化和凋亡的影响，将细胞增殖、分化和凋亡的机制系统地结合起来进行分析和讨论。 本课题的主要研究内容包括四部分：（1）CCK-8法检测LfcinB对Jurkat细胞增值的影响。实验以浓度为100nmol/mL的雷帕霉素作为对照组，以作用浓度分别为0μg/mL、50μg/mL、100μg/mL、200μg/mL和400μg/mL的LfcinB作为实验组，分别测定Jurkat细胞的增值抑制率。结果显示：LfcinB对Jurkat细胞增殖的影响效果随着其作用浓度和时间的增加而增大，即呈现明显的浓度时间依赖性；（2）利用荧光倒置显微镜检测浓度分别为0μg/mL、50μg/mL、100μg/mL、200μg/mL和400μg/mL的LfcinB作用Jurkat细胞24h后的形态学变化。结果显示：实验组细胞的细胞核发生浓缩，呈浓密染色状态，而空白组细胞的细胞核则呈均匀染色状态，此结果表明实验组的细胞发生凋亡；（3）AnnexinⅤ-FITC/PI双标记流式细胞术分析不同浓度的LfcinB作用Jurkat细胞不同时间后，细胞发生早期凋亡的变化规律。实验以浓度为100nmol/mL的雷帕霉素作为对照组，以作用浓度分别为0μg/mL、50μg/mL、100μg/mL、200μg/mL和400μg/mL的LfcinB作为实验组，分别测定Jurkat细胞的早期凋亡率。结果显示：在相同的LfcinB作用时间内，Jurkat细胞的早期凋亡率随着LfcinB作用浓度的增大而增加；对于同一作用浓度的LfcinB，当作用时间到达72h时，其早期凋亡率有明显的下降趋势，而晚期凋亡或是坏死细胞的比例有所增加；（4）Western blot法检测并分析不同浓度的LfcinB作用Jurkat细胞不同时间后，Jurkat细胞内P21waf1、AKT、mTOR的磷酸化程度变化和Nocth1、C-MYC、Bcl-2、Bax、Cyclin D1的蛋白表达程度的变化，实验分为8组，分别为雷帕霉素组(100nmol/mL)、0μg/mL、50μg/mL、100μg/mL、200μg/mL、400μg/mL、雷帕霉素+PI3K抑制剂和LfcinB+PI3K抑制剂；结果显示：Jurkat细胞内的P21waf1、AKT、mTOR的磷酸化程度随着LfcinB浓度的升高和作用时间的延长而降低；Nocth1、C-MYC、Bcl-2、Cyclin D1的蛋白表达程度与LfcinB的作用时间和浓度呈负相关，而Bax的蛋白表达程度与LfcinB的浓度以及作用时间呈正相关。
[Abstract]:Lactoferricin (lactoferricin) LfcinB is a highly antimicrobial bioactive peptide, and has a wide range of sources. LfcinB has no effect on human normal cells in the concentration range of antitumor activity, but it has cytotoxic effect on many human tumor cell lines, showing some pertinence. In this study, synthetic LfcinB was used to study the effects of LfcinB on the proliferation, differentiation and apoptosis of human T cell leukemia (Jurkat) cells. Flow cytometry was used to study the effects of LfcinB on the proliferation, differentiation and apoptosis of Jurkat cells, and the flow cytometry was used to study the effects of LfcinB on the proliferation, differentiation and apoptosis of Jurkat cells. The mechanisms of differentiation and apoptosis are systematically analyzed and discussed. The main contents of this study include four parts: the effect of LfcinB on the proliferation of Jurkat cells was detected by CCK-8 method. The inhibitory rate of proliferation of Jurkat cells was measured by using rapamycin at the concentration of 100nmol/mL as control group, and with 50 渭 g / mL of 50 渭 g 路mL ~ (-1) or 100 渭 g / mL ~ (-1) LfcinB as experimental group, and with a concentration of 100 渭 g / mL ~ (-1) LfcinB of 400 渭 g/mL. The results showed that the effect of 1: LfcinB on the proliferation of Jurkat cells increased with the increase of its concentration and time. The morphological changes of Jurkat cells exposed to 100 渭 g 路mL ~ (-1) 100 渭 g / mL ~ (100 渭 g 路mL ~ (-1)) and 400 渭 g/mL LfcinB for 24 h were detected by fluorescence inverted microscope. The results showed that the nuclei of the experimental group were thickly stained and the nuclei of the cells in the blank group were uniformly stained. The results showed that the apoptosis of Jurkat cells in the experimental group was analyzed by flow cytometry with Annexin 鈪，

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