【作者】 郑伟；

【导师】 蔡建春；

【作者基本信息】 厦门大学 ， 外科学， 2020， 硕士

【摘要】 背景:本研究所按照胃肿瘤Ming氏分型的标准,分别成功建立了浸润型(XGC-1)和膨胀型(XGC-2)胃肿瘤细胞株。研究前期对具有不同生长方式来源的细胞系构建小鼠原位移植瘤模型,并剖离胃组织进行脂质组学研究,通过深度挖掘数据信息以及运用多种生物信息学分析方法,我们总结了二者之间在脂代谢方面存在差异性。其中,在肿瘤自噬调节方面我们发现,浸润型相对于膨胀型胃肿瘤具备更大程度的自噬作用,结合实验室已获得的转录组数据,将进一步探究两型胃肿瘤自噬作用的差异及相关分子机制。之前的研究报告表明这两者在临床上具有不同的预后表现,我们推测自噬作用的差异或许可以作为解释这种现象的参考依据。因此,本论文在不同生长方式胃肿瘤组织脂质组学研究基础之上,结合胃肿瘤细胞的分子生物学实验加以验证,以期得到可靠的论证。目的:1.基于不同生长方式胃肿瘤细胞脂质组学差异,研究在药物化疗作用下,XGC-1和XGC-2两者自噬作用发生,并且对比这两种不同生长方式胃肿瘤细胞存在的自噬差异。2.与转录组学数据关联分析,找到自噬差异相关的关键分子Ambral,通过敲低细胞内Ambra1表达,研究自噬作用改变影响胃肿瘤细胞药物作用的效果以及探讨自噬作用是否是造成XGC-1和XGC-2不同耐药表现的原因。方法:1.体外培养XGC-1、XGC-2细胞,分别在1μM的多柔比星(Doxorubicin,DOX)或与100nM巴弗洛霉素(Bafilomycin A1,BafA1)的联合运用下,通过导入mCherry-GFP-LC3荧光质粒,在不同时间点观察自噬的现象。再以Western Blot的实验方法,对自噬相关蛋白Beclin1、LC3、P62以及CathepsinD进行检测,判断药物化疗对自噬的影响,同时对比XGC-1、XGC-2自噬作用的差异性。2.针对自噬调控重要基因Ambral,以RNA干扰的方法分别对XGC-1、XGC-2细胞的Ambra1表达水平进行下调,再以CCK-8细胞增殖-毒性实验,流式细胞凋亡实验以及凋亡相关蛋白PARP和Caspase3的测定对比因自噬改变而产生的细胞药物反应的变化。在此基础上,比较XGC-1、XGC-2胃肿瘤细胞的细胞凋亡实验结果和自噬程度的关系,验证自噬对XGC-1、XGC-2不同肿瘤耐药性的影响。结果:1.多柔比星(Doxorubicin,DOX)对胃肿瘤细胞的化疗作用可以诱导肿瘤细胞产生自噬作用,其自噬程度与药物作用的时间呈现正相关。且XGC-1相对于XGC-2胃肿瘤细胞表现出更大水平的自噬作用。2.针对自噬调控基因Ambra1的干扰敲低,我们发现在XGC-1、XGC-2中,shAmbra1组相较于阴性对照组都可以明显抑制自噬,而且增加了凋亡相关蛋白PARP 和 Caspase3 的表达。3.另外,在对阴性对照组和shAmbra1组的XGC-1、XGC-2胃肿瘤细胞采用的较大剂量10 μM多柔比星(Doxorubicin,DOX)处理作用的CCK-8细胞增殖-毒性实验和流式细胞凋亡实验,结果显示shAmbral组的胃肿瘤细胞表现出更大的细胞凋亡水平。而在阴性对照组的XGC-1、XGC-2胃肿瘤细胞,XGC-1表现出更大的细胞耐药作用。讨论:在运用RNA干扰技术对自噬调节基因Ambra1进行敲低表达可以下调自噬的作用,我们发现抑制自噬可有效增强两种不同胃肿瘤细胞的药物化疗效果。换言之,胃肿瘤细胞在化疗过程中,自噬作用对细胞的耐药起着促进作用。而XGC-1和XGC-2胃肿瘤细胞因其自噬作用的强弱差异,导致了细胞抵抗压力环境的能力不同。这可能也从侧面解释了两型不同生长方式的胃肿瘤临床预后差异的原因,同时也表明自噬作用的差异可能与Ambra1的表达差异密切相关。我们的研究是基于在对两种不同生长方式的胃肿瘤组织关于脂质组学方面的研究基础上进行的,脂质组学在生命科学领域具有宽广的应用价值,无论在临床医学诊断关于生物标记物的发现还是疾病的病理机制以及病因归属的研究应用上都将发挥着独特的作用。更多还原

【Abstract】 Background:According to the criteria of Ming’s classification of stomach neoplasm,we have successfully established invasive(XGC-1)and expansive(XGC-2)stomach neoplasm cell lines.In the early stage of the study,we constructed orthotopic tumor model of mice with different growth patterns of cell lines,and dissected gastric tissue for lipidomics research.Through deep mining of data information and the use of a variety of bioinformatics analysis methods,we summarized the differences in lipid metabolism between the two.In terms of autophagy regulation,we found that invasive stomach neoplasm has a higher degree of autophagy than expansive stomach neoplasm.Combine with the transcriptome data obtained in the laboratory,we will further explore the difference between the two types of autophagy and the related molecular mechanism.Previous studies have shown that the two have different prognosis.We speculate that the difference of autophagy may be a reference for explaining this phenomenon.Therefore,based on the study of lipid metabolism of stomach neoplasm cell lines with different growth patterns,this paper combines with the molecular biological experiments of stomach neoplasm cell lines to verify,in order to get a reliable demonstration.Objective:1.Based on the difference of lipidomics of stomach neoplasm cell lines with different growth modes,the autophagy of XGC-1 and XGC-2 was studied under the effect of drug chemotherapy,and the difference of autophagy between the two growth modes of stomach neoplasm cells was compared.2.By correlation analysis of transcriptome data,we found Ambral which is the key molecule related to difference of autophagy.By knocking down the expression of Ambral in cell lines,we studied the effect of autophagy on drug effect of stomach neoplasm cell lines and whether autophagy is the cause of different drug resistance of XGC-1 and XGC-2.Method:1.XGC-1 and XGC-2 cell lines were cultured in vitro.Under the combination of Doxorubicin(DOX)of 1μM or Baflomycin(Baf Al)of 100nM,the phenomenon of autophagy was observed at different time points by inserting the fluorescence plasmid of mCherry-GFP-LC3.Then western blot was used to detect the autophagy-related proteins such as Beclin1,LC3,P62 and CathepsinD to determine the effect of chemotherapy on autophagy,and to compare the difference of autophagy between XGC-1 and XGC-2.2.In view of the important autophagy-regulated gene Ambral,the expression level of Ambral in XGC-1 and XGC-2 cell lines was down regulated by RNA interference.And then the changes of cell drug response caused by autophagy were compared by the cell proliferation-toxicity experiment of CCK-8,flow cytometry apoptosis experiment and the determination of apoptosis related protein PARP and caspase3.On this basis,the relationship between autophagy and apoptosis of XGC-1 and XGC-2 stomach neoplasm cell lines was compared to verify the effect of autophagy on different tumor drug resistance.Result:1.Doxorubicin(DOX)can induce autophagy in stomach neoplasm cell lines by chemotherapy,and the degree of autophagy is positively correlated with the time of chemotherapy.Compared with XGC-2,XGC-1 show a higher level of autophagy.2.In response to the interference knockdown of the autophagy regulatory gene Ambral,we found that in XGC-1 and XGC-2,the shAmbral group can significantly inhibit autophagy and increase the expression of apoptosis-related proteins PARP and Caspase3 compared to the negative control group.3.In addition,the cell proliferation-toxicity experiment of CCK-8 and flow cytometry apoptosis experiment were treated with high dose of Doxorubicin(DOX)with 10μM in XGC-1 and XGC-2 stomach neoplasm cell lines of negative control group and shAmbral group showed that the stomach neoplasm cell lines of shAmbral group showed higher apoptosis level.In the negative control group,XGC-1 and XGC-2 stomach neoplasm cell lines showed greater cell resistance.Discussion:Using RNA interference technology to knock down the expression of the autophagy regulated gene Ambral and thereby down-regulated the effect of autophagy,we found that inhibit autophagy can effectively enhance the drug chemotherapy effect of two different stomach neoplasm cell lines.In other words,the autophagy effect promotes the drug resistance of stomach neoplasm cell lines during chemotherapy.XGC-1 and XGC-2 stomach neoplasm cell lines differ in their ability to resist stress environments due to the difference in their autophagy.This may also explain the reasons for the difference in clinical prognosis of stomach neoplasm tumors with two different growth modes,and also indicates that the difference in autophagy may be closely related to the difference in the expression of Ambral.Our research is based on the study of two different growth modes of stomach neoplasm tumor tissue on lipidomics.Lipidomics has a broad application value in the field of life sciences,regardless of the clinical diagnosis of the discovery of the biological markers,in the pathological mechanism of the disease and the research and application of the etiology.It will also play a unique role.更多还原