化香树果序醇提物诱导人鼻咽癌细胞发生methuosis死亡的机制研究

发布时间：2018-03-02 14:42

本文选题：化香树果序　切入点：鼻咽癌　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:鼻咽癌起源于人鼻咽部粘膜上皮和腺体的恶性肿瘤,发病人群主要分布于我国华南地区,广东省人数尤其较多,因此又称为“广东癌”。目前,鼻咽癌的临床治疗主要以放疗、化疗为主,但这两种方法副作用较大且有约30%的患者即使运用这两种方法进行综合治疗,5年生存率仍较低。近年来研究发现,多种中药具有抗肿瘤作用,且以放化疗为主、中药为辅的综合治疗方案能在一定程度上提高肿瘤患者的5年生存率。因此,中药被认为是一种治疗肿瘤的有效辅助药物。化香树果序是一种主要产于华南和西南各省的中药材。既往研究表明,其水煎物对人鼻咽癌细胞CNE2具有细胞毒性作用,但具体药理机制尚未阐明。Methuosis死亡的发生机制尚未完全阐明,目前部分研究认为methuosis死亡是macropinocytosis过程出现异常时发生的一种细胞死亡方式。在药物和外界刺激下,macropinosomes会在细胞内大量积累,彼此相互融合逐步形成更大的液泡,同时细胞核不会发生明显改变,随后细胞代谢活动会逐渐减少,细胞膜发生破裂,最终细胞死亡。Methuosis细胞死亡方式的发现可为对抗凋亡药物耐药的恶性肿瘤患者提供新的治疗途径。此前,我们课题组通过体内外实验证实,化香树果序醇提物(Alcohol extract of Platycarya strobilacea sieb.et Zucc,PSZ)能抑制CNE1 和CNE2增殖并诱导其发生methuosis死亡,发挥抗肿瘤作用。目前,国内外对methuosis死亡发生的具体机制仍未有明确的阐述。本研究旨在前期研究基础上进一步探究化香树果序醇提物诱导人鼻咽癌细胞发生methuosis死亡的相关信号通路,并初步筛选methuosis死亡发生过程中的关键靶点,为中药治疗恶性肿瘤提供实验室依据。目的:研究化香树果序醇提物诱导人鼻咽癌细胞发生methuosis细胞死亡的范围及methuosis死亡发生的相关信号通路,同时初步筛选导致该死亡方式发生的关键靶点。方法:第1部分化香树果序生药材的提取工艺及其主要成分分析用乙醇回流方法提取化香树果序生药材,随后浓缩、干燥药物,利用高效液相色谱技术(High Performance Liquid Chromatography,HPLC)分析醇提物的主要成分。第2部分检测化香树果序醇提物对不同病理类型人鼻咽癌细胞的作用效果取对数生长期的SUNE1、CNE1、CNE2和5-8F细胞分别接种于6孔培养板(1.2×105cells/ml,2ml/孔),细胞培养箱孵育24h后分别进行相应处理,倒置显微镜下观察细胞在4h、8h、12h、24h、48h这5个时间点的形态学改变。第3部分化香树果序醇提物诱导人鼻咽癌细胞发生methuosis死亡的机制研究3.1生物信息学分析在本课题组前期全基因组测序的基础上,结合相关文献,利用KEGG数据库筛选与methuosis死亡相关的信号通路。3.2荧光实时定量PCR(Real-time qPCR)检测各组相关基因的表达水平化香树果序醇提物(1.0mg/m)干预24h后,分别提取各组RNA,逆转录,扩增,RT-qPCR比较H-Ras、Arf6、Rac1、MAPK1、MAPK3和FOS六个相关基因的表达差异。3.3 Western Blot检测H-Ras和Racl蛋白的表达水平化香树果序醇提物(1.0mg/m)干预24h后,提取各组细胞总蛋白,Bicinchoninic acid(BCA)法蛋白定量检测,Western Blotting(WWB)测定细胞内H-Ras和Rac1蛋白的表达水平。3.4 Rac1抑制剂EHT1864干预实验将处于对数生长期的CNE1和CNE2细胞分别接种于6孔培养板(1.2×105 cells/ml,2ml/孔),细胞箱孵育24h后分别进行相关药物处理,倒置显微镜下观察细胞在4h、8h、12h、24h、48h各时间点的形态学改变。统计学分析:本课题组实验数据均采用IBM SPSS Statistics 20.0软件进行分析,实验结果中的计量资料以均数±标准差(χ±s)表示,两组比较时如果方差齐用t检验,方差不齐用秩和检验。P0.05认为差异有统计学意义。作图由GraphPadPrism 6.0软件提供。结果:1.本次实验化香树果序醇提物出膏率为4.1%。化香树果序醇提物的主要成分含黄酮类物质,与课题组前期实验结果相同。2.化香树果序醇提物在1.0mg/ml浓度下可诱导人鼻咽癌细胞株SUNE1、CNE1和CNE2细胞内出现大量空泡,空泡不断相互融合、增大,细胞膜相互融合,最后细胞破裂,此期间细胞核无明显改变。同时,5-8F细胞相同条件下加入1.0mg/ml浓度的化香树果序醇提物未发生明显的细胞形态学改变。3.根据KEGG数据库找到可能与methuosis死亡方式的发生有关的H-Ras/Arf6/Rac1 信号通路。4.RT-qPCR结果显示,化香树果序醇提物干预后CNE1和CNE2细胞中H-Ras、Rac1和MAPK1基因表达量增加,Arf6、MAPK3和FOS基因表达量降低。5.Western Blotting结果表明经化香树果序醇提物处理后CNE1、CNE2细胞中H-Ras和Rac1蛋白表达量均增加。6.Rac1蛋白抑制剂EHT1864干预下,加了 1.Omg/ml化香树果序醇提物的CNE1、CNE2细胞不再发生methuosis死亡。结论:1.乙醇回流法能够得到成分稳定的化香树果序醇提物,该方法为一种潜在的可批量工业化生产香树果序有效药物成分的技术。2.化香树果序醇提物在1.0mg/ml浓度下时可诱导人鼻咽癌SUNE1、CNE1和CNE2细胞株发生methuosis死亡,但不能诱导5-8F细胞株发生methuosis死亡。3.化香树果序醇提物诱导人鼻咽癌细胞发生methuosis死亡的机制可能是通过调控RAS/Arf6/Rac1信号通路,进而影响MAPK信号通路的转导,最终诱导人鼻咽癌细胞发生methuosis死亡。4.Rac1可能是化香树果序醇提物诱导人鼻咽癌细胞发生methuosis死亡的关键靶点。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.63

【参考文献】