本文關(guān)鍵詞： 黑水虻 抗菌肽 鼻咽癌細(xì)胞 增殖 細(xì)胞凋亡 端粒酶　出處：《遵義醫(yī)學(xué)院》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:從黑水虻幼蟲(chóng)血淋巴中分離純化抗菌肽,篩選抗菌肽活性組分。探討黑水虻抗菌肽活性組分對(duì)鼻咽癌(CNE2)細(xì)胞的影響及其機(jī)制,為挖掘黑水虻抗菌肽醫(yī)藥研發(fā)價(jià)值提供理論依據(jù)。方法:(1)利用金黃色葡萄球菌針刺誘導(dǎo),提取抗菌肽粗提物,利用Tricine-SDSPAGE電泳檢測(cè)誘導(dǎo)后黑水虻幼蟲(chóng)血淋巴中小分子抗菌肽的存在,藥敏紙片法檢測(cè)抗菌肽粗提物的抑菌效果;利用RP-HPLC法對(duì)抗菌肽粗提物進(jìn)行分離純化,并收集各純化峰對(duì)應(yīng)組分,測(cè)定各純化峰對(duì)應(yīng)組分的抑菌效果,篩選出具有抑菌活性的單一組分,并測(cè)定活性組分的MIC值。(2)采用CCK-8法檢測(cè)各組分對(duì)CNE2細(xì)胞體外增殖的影響,篩選出抑癌活性組分,采用RP-HPLC法檢測(cè)抑癌活性組分的純度。(3)Hoechst33342染色法檢測(cè)抑癌活性組分對(duì)CNE2細(xì)胞形態(tài)的影響;FCM法檢測(cè)抑癌活性組分對(duì)CNE2細(xì)胞凋亡率的影響;細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)抑癌活性組分對(duì)CNE2細(xì)胞遷移的影響。(4)RT-PCR法檢測(cè)抑癌活性組分對(duì)CNE2細(xì)胞端粒酶逆轉(zhuǎn)錄酶h TERT基因表達(dá)的影響;Western blotting法檢測(cè)抑癌活性組分對(duì)CNE2細(xì)胞端粒酶逆轉(zhuǎn)錄酶h TERT蛋白表達(dá)的影響。結(jié)果:(1)通過(guò)Tricine-SDS-PAGE電泳及RP-HPLC法分離純化得到黑水虻抗菌肽3個(gè)組分(HI-1、HI-2、HI-3),藥敏紙片法檢測(cè)結(jié)果表明,僅組分HI-3(以下稱(chēng)抗菌肽HI-3)有抑菌活性,其對(duì)金黃色葡萄球菌、枯草芽孢桿菌、大腸桿菌和產(chǎn)氣腸桿菌均有抑菌活性,MIC分別為80μg/ml、160μg/ml、80μg/ml、80μg/ml。(2)抗菌肽HI-3可有效抑制CNE2細(xì)胞體外增殖,抑制率達(dá)到40.56±6.80%,與HI-1(4.38±0.33%)和HI-2(4.16±0.14%)組相比較均具有統(tǒng)計(jì)學(xué)意義(P0.05),抗菌肽HI-3對(duì)HUV-C細(xì)胞抑制作用不明顯,抑制率僅為4.65±1.45%,通過(guò)RP-HPLC分析抗菌肽HI-3的純度為96.1%。(3)抗菌肽HI-3可增加CNE2細(xì)胞膜通透性,當(dāng)濃度為160μg/ml時(shí)可觀(guān)察到CNE2細(xì)胞有典型的凋亡現(xiàn)象,早期凋亡率可達(dá)27.59±1.14%;但抗菌肽HI-3未改變HUV-C細(xì)胞膜通透性,早期凋亡率與對(duì)照組相比無(wú)統(tǒng)計(jì)學(xué)意義�？咕腍I-3可降低CNE2細(xì)胞的遷移能力,HI-3處理組在12h、24h和48h的遷移率分別為24.43±0.47%、61.51±0.04%和80±1.46%,與相同時(shí)段的陰性對(duì)照組相比較,遷移率顯著降低(P0.05)。(4)抗菌肽HI-3處理組端粒酶活性明顯低于陰性對(duì)照組,h TERT基因表達(dá)量及蛋白表達(dá)量與對(duì)照組相比均顯著降低(P0.05)。結(jié)論:(1)黑水虻幼蟲(chóng)可經(jīng)金黃色葡萄球菌針刺誘導(dǎo)在其血淋巴中產(chǎn)生具有抑菌活性的小分子多肽物質(zhì)(HI-3),其對(duì)革蘭陰性菌和革蘭陽(yáng)性菌均有一定的抑制作用;(2)抗菌肽HI-3可有效抑制CNE2細(xì)胞的體外增殖,誘導(dǎo)其發(fā)生凋亡并降低其遷移力,但對(duì)正常細(xì)胞HUV-C無(wú)顯著影響;(3)抗菌肽HI-3可抑制CNE2細(xì)胞內(nèi)端粒酶逆轉(zhuǎn)錄酶h TERT的表達(dá),下調(diào)端粒酶活性,發(fā)揮其抑制CNE2細(xì)胞增殖和誘導(dǎo)CNE2細(xì)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 鹵6.80%. Compared with HI-1(4.38 鹵0.33) and HI-2(4.16 鹵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 鹵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 鹵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 鹵0.47 and 61.51 鹵0.04% and 80 鹵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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R915

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