本文選題：牛乳鐵蛋白素 + Jurkat細(xì)胞�。� 參考：《東北農(nóng)業(yè)大學(xué)》2013年碩士論文

【摘要】：牛乳鐵蛋白素（lactoferricin B，LfcinB）是一種抗菌活性非常高的微生物活性肽，且來源廣泛。在發(fā)揮抗腫瘤活性的濃度范圍內(nèi)，LfcinB對人體正常的細(xì)胞沒有影響，但是它對人類許多腫瘤細(xì)胞系具有細(xì)胞毒作用，呈現(xiàn)出一定的針對性。本課題將人工合成的LfcinB作用于人急性T細(xì)胞白血病的T淋巴細(xì)胞（Jurkat cell），應(yīng)用流式細(xì)胞術(shù)（Flow Cytometry，F(xiàn)CM）、蛋白質(zhì)免疫印跡分析法（Western Blotting）研究LfcinB對Jurkat細(xì)胞增殖、分化和凋亡的影響，將細(xì)胞增殖、分化和凋亡的機制系統(tǒng)地結(jié)合起來進行分析和討論。 本課題的主要研究內(nèi)容包括四部分：（1）CCK-8法檢測LfcinB對Jurkat細(xì)胞增值的影響。實驗以濃度為100nmol/mL的雷帕霉素作為對照組，以作用濃度分別為0μg/mL、50μg/mL、100μg/mL、200μg/mL和400μg/mL的LfcinB作為實驗組，分別測定Jurkat細(xì)胞的增值抑制率。結(jié)果顯示：LfcinB對Jurkat細(xì)胞增殖的影響效果隨著其作用濃度和時間的增加而增大，即呈現(xiàn)明顯的濃度時間依賴性；（2）利用熒光倒置顯微鏡檢測濃度分別為0μg/mL、50μg/mL、100μg/mL、200μg/mL和400μg/mL的LfcinB作用Jurkat細(xì)胞24h后的形態(tài)學(xué)變化。結(jié)果顯示：實驗組細(xì)胞的細(xì)胞核發(fā)生濃縮，呈濃密染色狀態(tài)，而空白組細(xì)胞的細(xì)胞核則呈均勻染色狀態(tài)，此結(jié)果表明實驗組的細(xì)胞發(fā)生凋亡；（3）AnnexinⅤ-FITC/PI雙標(biāo)記流式細(xì)胞術(shù)分析不同濃度的LfcinB作用Jurkat細(xì)胞不同時間后，細(xì)胞發(fā)生早期凋亡的變化規(guī)律。實驗以濃度為100nmol/mL的雷帕霉素作為對照組，以作用濃度分別為0μg/mL、50μg/mL、100μg/mL、200μg/mL和400μg/mL的LfcinB作為實驗組，分別測定Jurkat細(xì)胞的早期凋亡率。結(jié)果顯示：在相同的LfcinB作用時間內(nèi)，Jurkat細(xì)胞的早期凋亡率隨著LfcinB作用濃度的增大而增加；對于同一作用濃度的LfcinB，當(dāng)作用時間到達72h時，其早期凋亡率有明顯的下降趨勢，而晚期凋亡或是壞死細(xì)胞的比例有所增加；（4）Western blot法檢測并分析不同濃度的LfcinB作用Jurkat細(xì)胞不同時間后，Jurkat細(xì)胞內(nèi)P21waf1、AKT、mTOR的磷酸化程度變化和Nocth1、C-MYC、Bcl-2、Bax、Cyclin D1的蛋白表達程度的變化，實驗分為8組，分別為雷帕霉素組(100nmol/mL)、0μg/mL、50μg/mL、100μg/mL、200μg/mL、400μg/mL、雷帕霉素+PI3K抑制劑和LfcinB+PI3K抑制劑；結(jié)果顯示：Jurkat細(xì)胞內(nèi)的P21waf1、AKT、mTOR的磷酸化程度隨著LfcinB濃度的升高和作用時間的延長而降低；Nocth1、C-MYC、Bcl-2、Cyclin D1的蛋白表達程度與LfcinB的作用時間和濃度呈負(fù)相關(guān)，而Bax的蛋白表達程度與LfcinB的濃度以及作用時間呈正相關(guān)。
[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，

本文編號：1801254