黑水虻抗菌肽分离纯化及活性成分对CNE2细胞影响的研究

发布时间：2018-02-01 03:51

本文关键词： 黑水虻抗菌肽鼻咽癌细胞增殖细胞凋亡端粒酶　出处：《遵义医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:从黑水虻幼虫血淋巴中分离纯化抗菌肽,筛选抗菌肽活性组分。探讨黑水虻抗菌肽活性组分对鼻咽癌(CNE2)细胞的影响及其机制,为挖掘黑水虻抗菌肽医药研发价值提供理论依据。方法:(1)利用金黄色葡萄球菌针刺诱导,提取抗菌肽粗提物,利用Tricine-SDSPAGE电泳检测诱导后黑水虻幼虫血淋巴中小分子抗菌肽的存在,药敏纸片法检测抗菌肽粗提物的抑菌效果;利用RP-HPLC法对抗菌肽粗提物进行分离纯化,并收集各纯化峰对应组分,测定各纯化峰对应组分的抑菌效果,筛选出具有抑菌活性的单一组分,并测定活性组分的MIC值。(2)采用CCK-8法检测各组分对CNE2细胞体外增殖的影响,筛选出抑癌活性组分,采用RP-HPLC法检测抑癌活性组分的纯度。(3)Hoechst33342染色法检测抑癌活性组分对CNE2细胞形态的影响;FCM法检测抑癌活性组分对CNE2细胞凋亡率的影响;细胞划痕实验检测抑癌活性组分对CNE2细胞迁移的影响。(4)RT-PCR法检测抑癌活性组分对CNE2细胞端粒酶逆转录酶h TERT基因表达的影响;Western blotting法检测抑癌活性组分对CNE2细胞端粒酶逆转录酶h TERT蛋白表达的影响。结果:(1)通过Tricine-SDS-PAGE电泳及RP-HPLC法分离纯化得到黑水虻抗菌肽3个组分(HI-1、HI-2、HI-3),药敏纸片法检测结果表明,仅组分HI-3(以下称抗菌肽HI-3)有抑菌活性,其对金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌和产气肠杆菌均有抑菌活性,MIC分别为80μg/ml、160μg/ml、80μg/ml、80μg/ml。(2)抗菌肽HI-3可有效抑制CNE2细胞体外增殖,抑制率达到40.56±6.80%,与HI-1(4.38±0.33%)和HI-2(4.16±0.14%)组相比较均具有统计学意义(P0.05),抗菌肽HI-3对HUV-C细胞抑制作用不明显,抑制率仅为4.65±1.45%,通过RP-HPLC分析抗菌肽HI-3的纯度为96.1%。(3)抗菌肽HI-3可增加CNE2细胞膜通透性,当浓度为160μg/ml时可观察到CNE2细胞有典型的凋亡现象,早期凋亡率可达27.59±1.14%;但抗菌肽HI-3未改变HUV-C细胞膜通透性,早期凋亡率与对照组相比无统计学意义。抗菌肽HI-3可降低CNE2细胞的迁移能力,HI-3处理组在12h、24h和48h的迁移率分别为24.43±0.47%、61.51±0.04%和80±1.46%,与相同时段的阴性对照组相比较,迁移率显著降低(P0.05)。(4)抗菌肽HI-3处理组端粒酶活性明显低于阴性对照组,h TERT基因表达量及蛋白表达量与对照组相比均显著降低(P0.05)。结论:(1)黑水虻幼虫可经金黄色葡萄球菌针刺诱导在其血淋巴中产生具有抑菌活性的小分子多肽物质(HI-3),其对革兰阴性菌和革兰阳性菌均有一定的抑制作用;(2)抗菌肽HI-3可有效抑制CNE2细胞的体外增殖,诱导其发生凋亡并降低其迁移力,但对正常细胞HUV-C无显著影响;(3)抗菌肽HI-3可抑制CNE2细胞内端粒酶逆转录酶h TERT的表达,下调端粒酶活性,发挥其抑制CNE2细胞增殖和诱导CNE2细胞凋亡的作用。
[Abstract]:Objective: to isolate and purify antimicrobial peptides from hemolymph of the larva of Tabanus nigra, and to screen the active components of antimicrobial peptides, and to investigate the effect and mechanism of the active components of antimicrobial peptides on nasopharyngeal carcinoma (NPC) CNE2 cells. In order to provide the theoretical basis for the research and development of antimicrobial peptides of the black water gadfly. Methods the crude extract of antimicrobial peptides was extracted from Staphylococcus aureus by the acupuncture of Staphylococcus aureus. Tricine-SDSPAGE electrophoresis was used to detect the presence of small molecular antimicrobial peptides in the hemolymph of the induced larvae of Tabanus hirsutum, and the antimicrobial effect of crude extracts of antimicrobial peptides was detected by the drug sensitive disk method. The antimicrobial peptide crude extract was separated and purified by RP-HPLC, and the corresponding components of each purification peak were collected, the antibacterial effect of the corresponding components of each purification peak was determined, and the single component with antibacterial activity was screened out. The effect of each component on the proliferation of CNE2 cells in vitro was detected by CCK-8 assay, and the tumor suppressor components were screened out. RP-HPLC assay was used to detect the purity of tumor suppressor components. Hoechst33342 staining method was used to detect the effect of tumor suppressor active components on the morphology of CNE2 cells. FCM assay was used to detect the effect of tumor suppressor components on the apoptosis of CNE2 cells. Effect of tumor suppressor components on migration of CNE2 cells detected by cell scratch assay. The expression of telomerase reverse transcriptase h TERT gene in CNE2 cells was detected by RT-PCR assay. Expression of telomerase reverse transcriptase h TERT protein in CNE2 cells was determined by Western blotting assay. Three antimicrobial peptides (HI-1) were isolated and purified by Tricine-SDS-PAGE electrophoresis and RP-HPLC. The results of HI-2HI-3, drug sensitive disk method showed that only HI-3 (hereinafter referred to as antimicrobial peptide HI-3) had antimicrobial activity against Staphylococcus aureus and Bacillus subtilis. The MIC of Escherichia coli and Enterobacter aerogenes were 80 渭 g / ml, 160 渭 g / ml and 80 渭 g / ml, respectively. The antibacterial peptide HI-3 (80 渭 g 路ml. 2) could effectively inhibit the proliferation of CNE2 cells in vitro, and the inhibition rate was 40.56 卤6.80%. Compared with HI-1(4.38 卤0.33) and HI-2(4.16 卤0.14) group, there was statistical significance (P 0.05). The inhibitory effect of antimicrobial peptide HI-3 on HUV-C cells was not obvious, and the inhibitory rate was only 4.65 卤1.45%. The purity of antimicrobial peptide HI-3 was 96. 1 and the purity of HI-3 was 96. 3) HI-3 could increase the permeability of CNE2 cell membrane. When the concentration was 160 渭 g / ml, typical apoptosis of CNE2 cells was observed, and the early apoptosis rate was 27.59 卤1.14. But the antibacterial peptide HI-3 did not change the permeability of HUV-C cell membrane and the rate of early apoptosis was not statistically significant compared with the control group. The antibacterial peptide HI-3 could reduce the migration ability of CNE2 cells. The mobility at 24 h and 48 h in HI-3 group was 24.43 卤0.47 and 61.51 卤0.04% and 80 卤1.46%, respectively. Compared with the negative control group at the same time, the telomerase activity in the HI-3 treated with antimicrobial peptide was significantly lower than that in the negative control group. H TERT gene expression and protein expression were significantly decreased compared with the control group (P 0.05). Black water gadfly larvae can be induced by staphylococcus aureus acupuncture in their hemolymph to produce a small polypeptide with bacteriostasis activity of HI-3). It can inhibit Gram-negative bacteria and Gram-positive bacteria to some extent. 2) Antimicrobial peptide HI-3 could effectively inhibit the proliferation of CNE2 cells in vitro, induce apoptosis and decrease its migration, but had no significant effect on HUV-C of normal cells. The antimicrobial peptide HI-3 could inhibit the expression of telomerase reverse transcriptase h TERT and down-regulate telomerase activity in CNE2 cells. It can inhibit the proliferation of CNE2 cells and induce apoptosis of CNE2 cells.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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