本文選題：化香樹果序　切入點：鼻咽癌　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景:鼻咽癌起源于人鼻咽部粘膜上皮和腺體的惡性腫瘤,發(fā)病人群主要分布于我國華南地區(qū),廣東省人數(shù)尤其較多,因此又稱為“廣東癌”。目前,鼻咽癌的臨床治療主要以放療、化療為主,但這兩種方法副作用較大且有約30%的患者即使運用這兩種方法進行綜合治療,5年生存率仍較低。近年來研究發(fā)現(xiàn),多種中藥具有抗腫瘤作用,且以放化療為主、中藥為輔的綜合治療方案能在一定程度上提高腫瘤患者的5年生存率。因此,中藥被認為是一種治療腫瘤的有效輔助藥物�；銟涔蚴且环N主要產(chǎn)于華南和西南各省的中藥材。既往研究表明,其水煎物對人鼻咽癌細胞CNE2具有細胞毒性作用,但具體藥理機制尚未闡明。Methuosis死亡的發(fā)生機制尚未完全闡明,目前部分研究認為methuosis死亡是macropinocytosis過程出現(xiàn)異常時發(fā)生的一種細胞死亡方式。在藥物和外界刺激下,macropinosomes會在細胞內(nèi)大量積累,彼此相互融合逐步形成更大的液泡,同時細胞核不會發(fā)生明顯改變,隨后細胞代謝活動會逐漸減少,細胞膜發(fā)生破裂,最終細胞死亡。Methuosis細胞死亡方式的發(fā)現(xiàn)可為對抗凋亡藥物耐藥的惡性腫瘤患者提供新的治療途徑。此前,我們課題組通過體內(nèi)外實驗證實,化香樹果序醇提物(Alcohol extract of Platycarya strobilacea sieb.et Zucc,PSZ)能抑制CNE1 和CNE2增殖并誘導(dǎo)其發(fā)生methuosis死亡,發(fā)揮抗腫瘤作用。目前,國內(nèi)外對methuosis死亡發(fā)生的具體機制仍未有明確的闡述。本研究旨在前期研究基礎(chǔ)上進一步探究化香樹果序醇提物誘導(dǎo)人鼻咽癌細胞發(fā)生methuosis死亡的相關(guān)信號通路,并初步篩選methuosis死亡發(fā)生過程中的關(guān)鍵靶點,為中藥治療惡性腫瘤提供實驗室依據(jù)。目的:研究化香樹果序醇提物誘導(dǎo)人鼻咽癌細胞發(fā)生methuosis細胞死亡的范圍及methuosis死亡發(fā)生的相關(guān)信號通路,同時初步篩選導(dǎo)致該死亡方式發(fā)生的關(guān)鍵靶點。方法:第1部分化香樹果序生藥材的提取工藝及其主要成分分析用乙醇回流方法提取化香樹果序生藥材,隨后濃縮、干燥藥物,利用高效液相色譜技術(shù)(High Performance Liquid Chromatography,HPLC)分析醇提物的主要成分。第2部分檢測化香樹果序醇提物對不同病理類型人鼻咽癌細胞的作用效果取對數(shù)生長期的SUNE1、CNE1、CNE2和5-8F細胞分別接種于6孔培養(yǎng)板(1.2×105cells/ml,2ml/孔),細胞培養(yǎng)箱孵育24h后分別進行相應(yīng)處理,倒置顯微鏡下觀察細胞在4h、8h、12h、24h、48h這5個時間點的形態(tài)學(xué)改變。第3部分化香樹果序醇提物誘導(dǎo)人鼻咽癌細胞發(fā)生methuosis死亡的機制研究3.1生物信息學(xué)分析在本課題組前期全基因組測序的基礎(chǔ)上,結(jié)合相關(guān)文獻,利用KEGG數(shù)據(jù)庫篩選與methuosis死亡相關(guān)的信號通路。3.2熒光實時定量PCR(Real-time qPCR)檢測各組相關(guān)基因的表達水平化香樹果序醇提物(1.0mg/m)干預(yù)24h后,分別提取各組RNA,逆轉(zhuǎn)錄,擴增,RT-qPCR比較H-Ras、Arf6、Rac1、MAPK1、MAPK3和FOS六個相關(guān)基因的表達差異。3.3 Western Blot檢測H-Ras和Racl蛋白的表達水平化香樹果序醇提物(1.0mg/m)干預(yù)24h后,提取各組細胞總蛋白,Bicinchoninic acid(BCA)法蛋白定量檢測,Western Blotting(WWB)測定細胞內(nèi)H-Ras和Rac1蛋白的表達水平。3.4 Rac1抑制劑EHT1864干預(yù)實驗將處于對數(shù)生長期的CNE1和CNE2細胞分別接種于6孔培養(yǎng)板(1.2×105 cells/ml,2ml/孔),細胞箱孵育24h后分別進行相關(guān)藥物處理,倒置顯微鏡下觀察細胞在4h、8h、12h、24h、48h各時間點的形態(tài)學(xué)改變。統(tǒng)計學(xué)分析:本課題組實驗數(shù)據(jù)均采用IBM SPSS Statistics 20.0軟件進行分析,實驗結(jié)果中的計量資料以均數(shù)±標(biāo)準(zhǔn)差(χ±s)表示,兩組比較時如果方差齊用t檢驗,方差不齊用秩和檢驗。P0.05認為差異有統(tǒng)計學(xué)意義。作圖由GraphPadPrism 6.0軟件提供。結(jié)果:1.本次實驗化香樹果序醇提物出膏率為4.1%�；銟涔虼继嵛锏闹饕煞趾S酮類物質(zhì),與課題組前期實驗結(jié)果相同。2.化香樹果序醇提物在1.0mg/ml濃度下可誘導(dǎo)人鼻咽癌細胞株SUNE1、CNE1和CNE2細胞內(nèi)出現(xiàn)大量空泡,空泡不斷相互融合、增大,細胞膜相互融合,最后細胞破裂,此期間細胞核無明顯改變。同時,5-8F細胞相同條件下加入1.0mg/ml濃度的化香樹果序醇提物未發(fā)生明顯的細胞形態(tài)學(xué)改變。3.根據(jù)KEGG數(shù)據(jù)庫找到可能與methuosis死亡方式的發(fā)生有關(guān)的H-Ras/Arf6/Rac1 信號通路。4.RT-qPCR結(jié)果顯示,化香樹果序醇提物干預(yù)后CNE1和CNE2細胞中H-Ras、Rac1和MAPK1基因表達量增加,Arf6、MAPK3和FOS基因表達量降低。5.Western Blotting結(jié)果表明經(jīng)化香樹果序醇提物處理后CNE1、CNE2細胞中H-Ras和Rac1蛋白表達量均增加。6.Rac1蛋白抑制劑EHT1864干預(yù)下,加了 1.Omg/ml化香樹果序醇提物的CNE1、CNE2細胞不再發(fā)生methuosis死亡。結(jié)論:1.乙醇回流法能夠得到成分穩(wěn)定的化香樹果序醇提物,該方法為一種潛在的可批量工業(yè)化生產(chǎn)香樹果序有效藥物成分的技術(shù)。2.化香樹果序醇提物在1.0mg/ml濃度下時可誘導(dǎo)人鼻咽癌SUNE1、CNE1和CNE2細胞株發(fā)生methuosis死亡,但不能誘導(dǎo)5-8F細胞株發(fā)生methuosis死亡。3.化香樹果序醇提物誘導(dǎo)人鼻咽癌細胞發(fā)生methuosis死亡的機制可能是通過調(diào)控RAS/Arf6/Rac1信號通路,進而影響MAPK信號通路的轉(zhuǎn)導(dǎo),最終誘導(dǎo)人鼻咽癌細胞發(fā)生methuosis死亡。4.Rac1可能是化香樹果序醇提物誘導(dǎo)人鼻咽癌細胞發(fā)生methuosis死亡的關(guān)鍵靶點。
[Abstract]:Background: human nasopharyngeal epithelium and gland malignant tumor incidence of nasopharyngeal carcinoma in origin, mainly distributed in China's Southern China region, Guangdong Province, the number of especially more, it is also known as the "Guangdong cancer". At present, the clinical treatment of nasopharyngeal carcinoma with radiotherapy, chemotherapy is the main, but the two methods of side-effects. About 30% of the patients and even use the two methods for comprehensive treatment, 5 year survival rate is still low. In recent years, a variety of traditional Chinese medicine has anti-tumor effect, and chemotherapy, combined therapy of traditional Chinese medicine supplement can improve the 5 year survival rate of cancer patients in a certain extent. Therefore, Chinese medicine is considered effective medicine for treating tumor. Dyetree fruit order is one of Chinese herbal medicine is mainly produced in Southern China and the southwest provinces. Previous studies showed that the decoction has cytotoxicity on human nasopharyngeal carcinoma cell CNE2 Use, but the specific pharmacological mechanism has not been elucidated the mechanism of.Methuosis's death has not been fully elucidated, some studies suggest that methuosis is a kind of abnormal cell death occurs when the death of macropinocytosis. In the process of drugs and external stimulation, macropinosomes accumulated in the cells, merging each other gradually formed larger vacuoles, and the nucleus do not change, then the cell metabolism will be gradually reduced, the cell membrane rupture, the final cell death of.Methuosis cell death that may provide a new therapeutic approach for patients with drug resistance against apoptosis of malignant tumor. Previously, our group through in vivo experiments confirmed that Platycarya strobilacea sieb.et Zucc extracts (Alcohol extract of Platycarya strobilacea sieb.et Zucc, PSZ CNE1 and CNE2) can inhibit the proliferation and induce the death of methuosis, hair The volatile anti tumor effect. At present, domestic and foreign to the specific mechanism of methuosis deaths has not yet clear in this paper. The purpose of this study is to further explore the preliminary study on the basis of Platycarya strobilacea sieb.et Zucc extracts induced methuosis signal pathway related death of human nasopharyngeal carcinoma cells, and preliminary screening of methuosis death key targets during the process. Provide laboratory evidence for traditional Chinese medicine in the treatment of malignant tumors. Objective: To study the dyetree infructescence ethanol extract on human nasopharyngeal carcinoma cell methuosis cell death and incidence of methuosis associated with death pathway occur at the same time, preliminary screening leads to key targets the death. Methods: first part of the extraction process of incense tree fruit Xusheng medicinal herbs and its main component analysis to extract dyetree fruit Xusheng herbs, ethanol refluxing method then concentrated, dried drugs by high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) principal component analysis extracts. The second part detection of Platycarya strobilacea sieb.et Zucc extracts the effect of different pathological types of human nasopharyngeal carcinoma cells in logarithmic growth phase of SUNE1, CNE1, CNE2 and 5-8F cells were inoculated in 6 well plates (1.2 x 105cells/ml, 2ml/ hole). Cell culture for processing box respectively after 24h incubation, cells were observed under inverted microscope at 4h, 8h, 12h, 24h, 48h and morphological changes of the 5 time points. The third part of the infructescence of Platycarya strobilacea ethanol extract on human nasopharyngeal carcinoma cells to study mechanism of methuosis's death 3.1 bioinformatics analysis based on this ourprevious whole genome sequencing, combined with relevant literature, using methuosis KEGG database screening and death signaling pathways related to.3.2 real-time PCR (Real-time qPCR) to detect the expression level of each gene of incense tree Infructescences alcohol extract (1.0mg/m) after 24h intervention, respectively, were extracted RNA, reverse transcription, amplification, RT-qPCR H-Ras, Arf6, Rac1, MAPK1, MAPK3 and FOS expression levels of six genes related to the differential expression of.3.3 Western Blot to detect H-Ras and Racl protein Platycarya strobilacea sieb.et Zucc extracts (1.0mg/m) intervention 24h, extracted the total cell protein, Bicinchoninic acid (BCA) method for quantitative detection of protein Western, Blotting (WWB) for determination of the expression level of.3.4 Rac1 inhibitor EHT1864 intervention experiment of intracellular H-Ras and Rac1 protein in the logarithmic growth phase of CNE1 and CNE2 cells were inoculated in 6 well plates (1.2 x 105 cells/ml, 2ml/ hole), cell box after 24h incubation were related to drug treatment, cells were observed under inverted microscope at 4h, 8h, 12h, 24h, morphological changes of 48h at different time points. Statistical analysis: data this group of experiments were performed using the IBM SPSS software Statistics 20 Analysis of measurement data, experimental results in mean standard deviation (x + s) said that the two groups if the variance homogeneity of variance with t test, rank sum test for the.P0.05 that the difference was statistically significant. The mapping provided by GraphPadPrism 6 software. Results: 1. the experiment of incense tree fruit order alcohol extract extract rate of main components of extract 4.1%. Platycarya strobilacea sieb.et Zucc alcohol containing flavonoids, and previous experimental results of the same.2. Platycarya strobilacea sieb.et Zucc extracts can induce human nasopharyngeal carcinoma cell line SUNE1 at the concentration of 1.0mg/ml, CNE1 and CNE2 cells with large vacuoles within the cavity continuously mutual integration, increase cell membrane fusion, the final cell rupture, during this period the nucleus had no obvious change. At the same time, 5-8F cells under the same conditions with the concentration of 1.0mg/ml Platycarya strobilacea sieb.et Zucc extracts had significant morphological changes according to the KEGG database for.3. May and methuosis death H-Ras/Arf6/Rac1.4.RT-qPCR pathway results on display, Platycarya strobilacea sieb.et Zucc extracts H-Ras prognosis of CNE1 and CNE2 cells, Rac1 and MAPK1 gene expression increase, Arf6, MAPK3 and FOS gene expression decreased.5.Western Blotting results showed that the extract treated by Platycarya strobilacea sieb.et Zucc alcohol after CNE1, the expression of H-Ras and Rac1 protein in CNE2 cells increased.6.Rac1 protein inhibitor EHT1864 intervention, 1.Omg/ml and Platycarya strobilacea sieb.et Zucc extracts CNE1, CNE2 cells no longer occur methuosis death. Conclusion: 1. ethanol reflux method can get stable component of Platycarya strobilacea sieb.et Zucc extracts. The method of.2. technology as a potential mass industrial production of infructescence of Platycarya strobilacea effective medicines Platycarya strobilacea sieb.et Zucc extracts at the concentration of 1.0mg/ml can induce human nasopharyngeal carcinoma cell line CNE2 SUNE1, CNE1 and met The death of huosis, but not 5-8F cell lines induced by methuosis death.3. Platycarya strobilacea sieb.et Zucc extracts induced human nasopharyngeal carcinoma cell death mechanisms of methuosis may be regulated by RAS/Arf6/Rac1 signaling pathway, thereby affecting the transduction of MAPK signal pathway, eventually induced methuosis death.4.Rac1 may be Platycarya strobilacea sieb.et Zucc extracts induced key methuosis targets the death of human nasopharyngeal carcinoma cell in human nasopharyngeal cancer cells.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.63

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相關(guān)期刊論文 前10條

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本文編號：1556947