胃癌血清蛋白质组学及免疫逃逸机制的研究
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摘要
背景与目的:
胃癌是临床常见的恶性肿瘤之一,其发病率居全部恶性肿瘤的的第三位,占消化道恶性肿瘤的第一位。2002年的一项调查显示,全球每年胃癌新发病例绝对数目接近100万,每年新发病例约占新发癌症病例总数的9.9%,死亡总数的12.1%。亚太地区是胃癌的高发区,约占50%以上的胃癌患者来自亚洲,中国约占其中的41%。我国是胃癌的高发区,其发病率和死亡率均超过世界平均水平的两倍,每年死于胃癌的人数约为16万人。虽然目前肿瘤的传统治疗模式如手术、化疗及放疗已取得了长足的进展,但由于多数胃癌患者就诊时已到晚期,导致胃癌患者的死亡率仍居高不下。大量的研究证实,早期胃癌患者术后其5年生存率高达90.4%,而晚期的癌症患者治疗后5年存活率均非常低。由此表明,胃癌的早期诊断是提高胃癌临床治疗疗效及5年生存率的最有效的途径。然而科学高效地防癌治癌是世界级的难题,癌症患者临床表现的高度异质性给早期诊断带来了极大的难度。目前临床采用的主要诊断方法包括影像学、病理学、内窥镜、放射免疫学检查等,都必须在肿瘤发展到一定阶段,即形成实体瘤后才能进行,因此通常不能满足早期发现的要求。内窥镜活组织检查对患者而言具有侵入性并造成较大的痛苦,且内镜技术操作较为复杂,所检出的病人多数已到中晚期,而已有的血清胃癌标记物分子如CEA、CA19-9、CA72-4,其诊断敏感性和特异性均不高,如CEA的诊断敏感性为23.8%,CA19-9为45.5%,且CEA和CA19-9并非胃癌的特异性标志物,并不能作为胃癌早期诊断的标志物,到目前为止发展中国家80%的胃癌患者仍然都是在患病晚期才被发现。因此,要达到胃癌的早期发现必须发展新的诊断技术。
近年来随着人类基因组及后基因组研究的飞速发展和生命科学研究的不断进展,目前已经明确,肿瘤是由环境因素与遗传因素相互作用导致的一类疾病,是多种基因突变累积的结果。如何从整体水平认识肿瘤发生的可能病因,寻找肿瘤早期诊断、监测、干预以及治疗等新途径成为当前肿瘤学研究关注的热点,然而如何从复杂的生物样本中高质量地发现差异蛋白并从中提取有用的信息,使其成为敏感性和特异性均高的标志物是研究的重点和难点,蛋白质组学技术的出现解决了这一难题。肿瘤蛋白质组比较研究能动态地反应肿瘤细胞内蛋白表达水平的变化,检测蛋白翻译后的修饰并同时找出多个与肿瘤基因表达和功能相关的蛋白群,从而为寻找肿瘤的特异性标志物及肿瘤的早期诊断提供了极大的可能性,因而成为近年来肿瘤研究的热点领域。近几年来出现的表面增强激光解吸电离飞行时间质谱技术(surface-enhanced laser desorption/ionization time of flightmass spectrometry,SELDI-TOF-MS),结合了生物芯片和质谱的设计理念,把蛋白芯片、层析及质谱等项技术结合应用于蛋白质的检测,达到简便、快速地从各种体液及组织中找出新的蛋白质肿瘤标记物的特定指纹图谱的目的。目前国际上已经采用SELDI-TOF技术初步发现了乳腺癌、肺癌、前列腺癌、卵巢癌、膀胱癌、大肠癌、胰腺癌、鼻咽癌等近十种可用于临床早期检测的肿瘤生物标志物,并显示了良好的应用前景。例如传统的卵巢癌肿瘤标志物CA-125对于卵巢癌的诊断准确率约25%,而SELDI蛋白指纹图谱技术的准确率忆达到99%。该技术比CT、核磁能更早发现肿瘤,从而使得肿瘤的早期诊断、早期治疗成为可能,这标志着一种划时代崭新的诊断模式的开始。本研究拟利用该技术进行胃癌患者血清蛋白质组学研究,并通过建立及分析胃癌患者蛋白质表达指纹谱,筛选有价值的胃癌早期诊断标志物或特征性蛋白质组峰,为胃癌的早期诊断新方法的建立提供有价值的线索。
众所周知,恶性肿瘤的发生不仅有赖于肿瘤本身恶性表型的变化,还与宿主免疫监视机制的变化有关,Burnet于1967年提出了免疫监视学说,认为机体免疫系统通过细胞免疫机制能识别并特异性杀伤突变细胞,使突变细胞在未形成肿瘤之前即被清除。然而许多肿瘤仍能逃避宿主免疫系统的攻击,在机体内大量增殖并可发生浸润与转移。肿瘤逃逸机体免疫系统的监视,发生浸润及转移是导致手术、放化疗治疗失败及患者死亡的主要原因。因此,揭示肿瘤免疫逃逸及浸润转移等机制的研究对肿瘤防治新策略的发现及制订,提高肿瘤的临床治疗疗效具有重要意义。目前认为肿瘤免疫逃逸的机制主要包括:①宿主抗肿瘤效应细胞的变化,如T淋巴细胞数量和功能的异常及活化障碍等;②肿瘤细胞的变化,如协同刺激分子缺失或低表达、凋亡途径异常导致凋亡抵抗等;③细胞因子表达谱变化,如荷瘤机体通常发生由Th1型细胞因子分泌为主,转向Th2型细胞因子分泌为主的漂移,并可导致抗肿瘤效应细胞的迁移障碍致不能有效的歼灭肿瘤细胞。手术疗法是胃癌治疗的主要手段之一,然而手术本身亦可导致肿瘤细胞的入血,因此,血循环中免疫监视功能的主体细胞群T细胞及其分泌的细胞因子在杀伤侵入血流的肿瘤细胞,起着防止其侵袭与转移的关键作用。揭示胃癌患者外周血T细胞表型特征及其细胞因子分泌的变化对揭示胃癌细胞的免疫逃逸机制,发展新的防止术前或术后在血液循环中肿瘤细胞的生存及侵袭与转移提供新的思路。为此,本研究第二部分利用流式细胞术和悬浮芯片技术分析,检测了胃癌患者手术前后外周血T淋巴细胞亚群的表型变化及细胞因子表达类型的变化,初步探讨了胃癌免疫逃逸的机制。
方法:
1.临床资料
共研究35例胃癌标本,26例正常对照。所有标本均取自2006年3月～2007年8月就诊于泰安市中心医院普外科的初治胃癌患者,年龄45～74岁,中位年龄54岁。阴性对照健康人组26例,为2007年4月-2007年10月健康体检者,年龄40～70岁,中位年龄51岁。
2.血样采集
在采取手术、化疗、输血、介入等治疗措施之前以及手术后,采集患者及健康志愿者清晨空腹血。
3.血清样品收集
所有上述样本均为空腹抽血2～5ml。标本采集后30分钟内置入4℃冰箱2h,然后以同样温度3,000rpm离心20分钟,分装血清,-80℃保存。
4.SELDI-TOF-MS检测
所有肿瘤血清标本统一用弱阳离子交换表面(weak cationic exchanger,WCX2)芯片捕获蛋白,便于建立肿瘤统一标准数据库。取出血清样品,冰上融化后,在4℃下以10,000rpm离心5分钟。在标记好的1.5ml离心管中依次加入U9缓冲液20μl和血清样品10μl,混匀,冰浴振荡30分钟,然后向每管加入360μl醋酸钠缓冲液,振荡器上混匀。将WCX2芯片安装于加样器内,向芯片每孔加入100μl上述稀释血清样品,室温振荡孵育1小时后甩去缓冲液。接着每孔加入200μl醋酸钠缓冲液,振荡5分钟,弃缓冲液后,重复加入200μl醋酸钠缓冲液,同法处理再重复1次。最后用200μl去离子水快速冲洗两遍。取出芯片,待芯片表面自然晾干后,每点加0.5μl能量吸附分子SPA 2次。将芯片置入PBSII质谱仪,进行阅读,获得原始数据并绘制蛋白质质谱图。
5.外参照和内参照的设置
以All-in-One标准蛋白质芯片作为外参照。另外,在每一条芯片上都设置1个点内参,即于每条芯片的8点中随机选出1点,上样同一份质控血清(qualitycontrol,QC),并以所有血清图谱中表达较一致的特征性蛋白峰的平均值作为参照,来验证不同批次芯片及同一芯片不同点的重复性。
6.流式细胞仪检测淋巴细胞的比例及CCR7表达检测
在流式细胞仪检测管中加入相应的荧光标记抗体后,分别加入100μl健康人组、胃癌患者术前和术后的血样,室温孵育30分钟后每管加入1ml 1×红细胞裂解液裂解红细胞。PBS缓冲液洗涤样品三次,流式细胞仪检测细胞。Cellquest软件获取分析细胞,每个检测获取10,000个细胞。
7.悬浮芯片系统检测胃癌术前术后血清中IL-8及MCP-1表达的变化
将计算好的微球加入混合管中,混匀后加入待检测的样品以及稀释的标准品,室温下震荡孵育3个小时,每管各加100μl洗涤液进行洗涤1次。然后各加入50μl生物素标记抗体,室温下震荡孵育1个小时后,用100μl洗液进行洗涤,然后每孔加入100μl洗液,震荡重悬微球,悬浮芯片阅读仪进行阅读。
结果:
1.实验重复性分析
利用All-in-One标准蛋白质芯片和QC质控血清,我们对SELDI-TOF-MS产生的蛋白质图谱进行了重复性测试。结果显示,本实验所用WCX2蛋白芯片的intra-assay和inter-assay变异系数(coefficient of variation,CV)分别为10%和12%,提示不同批次芯片、不同芯片点间的蛋白峰值有良好的重复性。
2.筛选胃癌血清特异性标志物
胃癌组与对照组的质谱图比较:应用Biomarker Wizard软件对35例胃癌血清和26例正常血清构建的原始蛋白指纹图谱进行对比分析,在分子量0～50,000Da范围内共检测到75个蛋白峰。结合Biomarker Patterns软件进一步分析,发现胃癌组与对照组间有5个差异蛋白(P＜0.05),其中2个蛋白表达差异显著,其质荷比分别为m/z 4344、m/z4961。m/z 4961在胃癌组明显高于对照组;m/z 4344在胃癌组表达明显降低。
3.筛选胃癌治疗相关生物标志物
手术前后血清蛋白质谱比较:应用Biomarker Wizard软件对20例胃癌术前和术后血清构建的原始蛋白指纹图谱进行对比分析,在分子量0～50,000Da范围内共检测到70个蛋白峰。结合Biomarker Patterns软件进一步分析,发现胃癌术前组与术后组间有7个差异蛋白(P＜0.05),其中2个蛋白表达差异显著,其质荷比分别为m/z 2743、m/z3315。两者在术后表达较术前均明显降低。
4.胃癌患者术前以及术后外周血淋巴细胞亚群检测
胃癌患者与健康人组CD3~+T细胞和CD19~+B细胞所占的比例无显著性差异(CD3~+T:67.62%VS65.69%;CD19~+B:10.8%VS10.44%),胃癌患者术前和术后的外周血中CD3~+T细胞和CD19~+B细胞所占的比例也无明显变化(CD3~+T:67.62%VS67.79%;CD19~+B:10.8%VS10.35%)。
5.胃癌患者术前以及术后外周血淋巴细胞CCR7表达检测
与健康人相比,胃癌患者外周血CD3~+T细胞中CCR7的表达明显降低,术后CCR7的表达有所升高。术后组与健康人组之间CCR7的表达无显著性差异(P＞0.05)。比较CCR7在外周血淋巴细胞的表达与不同病理分期的关系,CCR7在胃癌Ⅲ、Ⅳ期患者外周血淋巴细胞中的表达阳性率明显低于Ⅰ、Ⅱ期胃癌患者。
6.趋化因子IL-8及MCP-1胃癌患者血清中的表达
胃癌患者以及正常对照组血清中IL-8及MCP-1的浓度检测结果显示,与正常对照组相比,胃癌患者术前血清中IL-8以及MCP-1的浓度明显低于正常对照组。
结论:
1.应用SELDI-TOF-MS技术获得胃癌血清蛋白质表达图谱的实验方法稳定可靠,具有良好的重复性。
2.m/z 4344、m/z4961的2个生物标志物可作为筛选胃癌标志物的候选蛋白。
3.胃癌患者外周血淋巴细胞CD3~+T细胞趋化因子受体CCR7表达明显下降,且下降程度与胃癌分期有关。
4.胃癌患者血清中趋化因子MCP-1和IL-8的含量明显降低。
主要创新点:
1.应用WCX2蛋白芯片筛选出2个有别于国际上其他文献报道的血清生物标志物,该标志物可作为筛选胃癌标志物的候选蛋白。
2.首次探讨血清蛋白质图谱在胃癌临床疗效监测方面的应用价值,筛选出2个胃癌治疗相关标志物。
3.检测了胃癌患者外周血淋巴细胞各亚群中CCR7的表达,并且分析了其表达强度与胃癌临床病理分期之间的关系。
4.从趋化因子及其受体方面探讨胃癌免疫逃逸相关机制。
Background and Objectives:
Gastric carcinoma is the most frequent digestive cancer in clinical medicine,the incidence of which is in the third place in all carcinoma and the first place in digestive carcinoma.An investigation in 2002 indicated that the absolute amount of new gastric carcinoma cases was approaching 1,000,000,which takes 9.9%of the total new cancer cases and 12.1%of caner deaths.Asia-Pacific region has a high incidence of gastric carcinoma.Approximately,more than 50%of new cases occur in Asia and about 41%of which come from China.China is an area with high incidence of gastric carcinoma,the morbidity and mortality rates are more than twice of the world average level:there were 160,000 people died from stomach cancer.Although many current traditional treatment approaches for gastric cancer have been developed,the mortality rate is still high since most patients have come to terminal stage when they get diagnosis.A bunch of researches proved that the 5 year survival rate was high to 90.4%in early gastric cancer patients after surgical treatment.While in terminal stage cancer patients,it is rather low.All of these indicated that the early diagnosis is the most effective way for improving the prognosis and 5 year survival rate.However,to scientifically and effectively diagnose gastric carcinoma is still an international difficult problem,and patients' clinical performance heterogeneity brings more difficulties to it.Current clinical diagnosis approaches include Imaging,Pathology, Endoscopy and Radioactive Immunologic Examination,etc.All of these methods can only be put into practice when tumor develops to certain stage which only means when it comes to physical tumor.Thus it is impossible to meet the acquirement of early diagnosis.For patients,the invasion of Endoscopic biopsy brings too much pain and the operation process is complex,at the same time,the patients detected from this way are always in terminal stage.However,the diagnostic sensitivity and specificity of existed biomarker for gastric carcinoma like CEA,CA19-9,CA72-4 are not high (the diagnostic sensitivity of CEA is 23.8%,CA19-9 is 45.5%) and they are not specific,so they can not be treated as early diagnosis biomarkers for gastric carcinoma.Hitherto,about 80%of stomach cancer patients were not found until it came to terminal stage.So it is urgent to explore and set up an early stage diagnosis technology.
With the rapid development of human genome,post-genome research and the progress made in life science research,it is explicited that cancer is a kind of disease induced from the interaction of environmental and genetic factors and cumulative result of various gene mutations.How to recognize the possible reasons from integrity level and find new methods of early tumor diagnosis,monitoring,intervention and treatment becomes a new hot concern point for current oncology research.How to collect high quality proteins with difference from complex samples and get valuable information from them in order to let them become high sensitive and specific biomarkers becomes a key point and difficult problem in research.The appearance of proteomics technology solved this problem.Comparison on tumor proteome can dynamically reflex the changes of proteins in tumor cells;detect the modification of protein after translation and find several proteins which are related to tumor gene expression and function,so that make it possible to find specific biomarkers and make early diagnosis for tumor.The development of surface-enhanced laser desorption / ionization time of flight mass spectrometry technology is to combine the idea of biological chips and mass spectrometry and put these technologies into protein detection,which can detect new tumor biomarkers' fingerprinting from various humors and tissues.Tens of tumor biomarkers for early diagnosis for breast cancer, lung cancer,prostatic cancer,ovarian cancer,bladder cancer,colorectal cancer, pancreatic cancer and nasopharyngeal cancer have been found via SALDI-TOF technology and showed great practical prospects.For example,CA-125 as a traditional biomarker for ovarian cancer can be used to diagnose with accuracy rate of approximately 25%,while the accuracy rate of SELDI protein fingerprinting technology has reached 99%.SELDI-TOF can be used to detect tumor earlier than CT and MIR,which can make it possible to make early diagnosis and treatment for patients.It marks the beginning of a new era of diagnose model.In this project,we suppose to use this technology to research on the proteome of gastric cancer patients' serum.And screen valuable markers or specific protein peeks for gastric carcinoma early diagnosis,in order to shed light on finding new approaches for gastric carcinoma early diagnosis.
It is well known that the development of malignant tumor not only depends on the changes of its malignant phenotype,but also relates to the host's immune surveillance mechanism.The immune surveillance theory that was created by Burnet in 1987 suggested that immune system could recognize and remove the mutant cells through cell immune response before they developed into tumors.However,many tumor cells can escape from the attack of host immune system and grow out of control and even invade or metastasize to other tissues.These are the main reasons for the surgery,radiotherapy and chemotherapy failure.So it is important to investigate the mechanism of immune escape for tumor prevention and treatment.There are three aspects included in the tumor immune escape mechanism:①The changes of host effective cells,such as the abnormality of the T cell amount and function.②The changes of tumor cells,such as lack of co-stimulatory molecules and disorders of apoptosis.③The changes of cytokines,such as the tumor host body always shift from mainly Thl-type cytokine secretion to mainly Th2-type cytokine secretion which can induce that the effective immune cells can not effectively kill tumor cells.Surgery is one of the main treatment models for gastric carcinoma,but the tumor cells can enter blood through the process.So the T cells and cytokines secreted from them are crucial in the process of killing the tumor cells which entered blood.All of these indicated that reveal the T cells phenotypes and the changes of their secretion of cytokines can shed light on reveal immune surveillance mechanism of gastric tumor cells and development of new ways of preventing tumor cells' survival,invasion and metastasis pre- or post-surgery.In the second part of this study,we used flow cytometry and suspension chip technology to detect the changes of peripheral lymphocyte subsets' phenotypes and the variation of the expression of cytokines between pre-and post-surgery in patients in order to further investigate the mechanisms of immune escape of gastric carcinoma.
Methods:
1.Clinical data
35 patients with gastric carcinoma,26 healthy volunteers were included in this study.All samples were collected from the General Surgery Department,TaiAn Central Hospital during March and August in 2006.The middle ages of the two groups are 54 and 51 respectively.There was no statistically significant difference in the ages between the patients and control group.(P＞0.05)
2.The collection of peripheral blood samples
2 ml of peripheral blood was collected from the patients with carcinoma and the control group.For cancer group,the samples were collected before and after treatment.
3.The collection of serum samples
2～5 ml of peripheral blood was collected from the patients with carcinoma and control group.Then the samples were laid-aside at 4℃for 30 minutes and centrifuged at 3,000 rpm for 20 minutes.Serum samples were collected and stored at -80℃.
4.DELDI-TOF-MS analysis
The WCX2 protein chip was used to harvest proteins from serum samples. Serum samples were centrifuged for 5 minutes at 10,000 rpm after melted on ice.20ul of U9 buffer was added into pre-labeled 1.5ml tube and then added 10 ul of serum sample.After vibrate samples at 4℃for 30 minutes,360 ul of 50 mmol/L Na-Acetate wash buffer was added to each tube and then vertex samples.The WCX2 was installed on bioprocessor and 100 ul of diluted serum sample mentioned above was added to each well,then the samples were incubated at room temperature for 60 minutes on shaker,remove the buffer and washed the samples twice with 200 ul Na-Acetate wash buffer for 5 minutes.Two quick rinses with HEPES water followed. Take out the chips and let them air dry.Before SELDI analysis,0.5 ml of saturated EAM solution SPA was applied onto each chip array twice and let the array surface air dry between each EAM application.Place chips on the Protein Biological SystemⅡmass spectrometer reader.
5.Set Quality Controls
All-in-One peptide molecular mass standard was used to calibrate.In addition, the reproducibility of SELDI spectra was determined by using the pooled normal serum quality control samples.We randomly selected two peaks in the range of 5,000～10,000Da to calculate the coefficient of variance.
6.FACS assay
Freshly isolated peripheral lymphocytes were stained immediately with appropriate concentrations of fluorescent-conjugated mAb for cell surface antigen.All Abs,directly conjugated with FITC,Phycoerythrin(PE) or PE-cy5,were obtained from PharMingen(San Diego,CA).The cells were stained with the corresponding FITC-,PE-conjugated or PE-Cy5 mouse anti-human isotype-matched control Abs according to the manufacturer's protocol then incubated for 30 minutes in the dark. Red cell lysis buffer was added to the tube and incubated for 15 minutes at room temperature.Aliquots of 2×to 5×10~6 cells were resuspended in 80ul of PBS containing 0.5%bovine serum albumin.Immunostained cells were then analyzed on flow cytometer using CELLQuest software~(TM)(FACSCalibur,Becton Dickinson,CA). A minimum of 10,000 events per sample were collected for phenotypic analysis as previously described.
7.Suspension Chip Assay
Samples and standards were incubated with pre-mixed beads into the well of a 96-well microplate for 3 hours at room temperature with agitation.The mixture was washed with 100 ul of wash buffer.Then 50 ul of biotin was added to each well and incubated for 1 hour at room temperature with agitation.Wash the plate and add 100 ul of wash buffer to each well and read samples in Bio-Plex Suspension System.
Results:
1.Quality control
We detected the repeatability of protein spectrums got from EELDI-TOF-MS analysis with All-In-One Standard Protein Chip and QC serum.The results indicated that the intra-and inter-assay coefficient of variance of WCX2 Protein Chip were 10% and 12%which suggested that there was a good repeatability in different chips and wells of one chip.
2.Selection of cancer-specific serum protein markers
Comparison of protein spectrum between cancer group and control group:By comparing the original protein spectrum between 35 gastric cancer patients' and 26 healthy volunteers' serum,we found that there were 75 peaks with molecular weigh between 0～50,000 KD.Further analysis with Biomarker Patterns Software,we found that there were 5 proteins with difference in them and two proteins had significant difference.The corresponding m/z rations were m/z 4344 and 4961.The protein with m/z 4961 highly expressed in cancer group,while the protein with m/z 4691 had lower expression in cancer group than in control group.
3.Selection of treatment-related serum biomarkers
Comparison between protein spectrum pre- and after-treatment in cancer group: There were 70 peaks detected in the treated cancer group by Biomarker Wizard software Analysis.Further analysis with Biomarker Pattern revealed that there were 7 proteins which had difference and 3 of them had significant difference.Between untreated cancer sera and treated cancer sera from patients with gastric carcinoma. These two peaks corresponded to m/z ration of 2743 and 3315.Their concentrations were down-regulated after surgery treatment.
4.Peripheral lymphocytes subsets detection
The proportions of CD3~+T and CD19~+B between no-cancer group and gastric carcinoma patients had no significant difference(CD3~+T:67.62%VS 65.69%; CD19~+B:10.8%VS 10.44%).There was no significant difference between untreated cancer group and the treated gastric carcinoma patients either(CD3~+T:67.62%VS 67.79%;CD19~+B:10.8%VS 10.35%).
5.The expression of CCR7 in different groups
Compared with the healthy volunteers group,the expression of CCR7 in gastric carcinoma group significantly decreased.After curative gastrectomy,the expression level of CCR7 increased.The expression level of CCR7 related to TNM stage (P＜0.05).
6.The level of IL-8 and MCP-1 in serum
Compared with the healthy volunteers group,IL-8 and MCP-1 level in gastric carcinoma serum was significantly lower.
Conclusions:
1.Application of SELDI-TOF-MS technology to obtain proteomic profiles in serum for gastric carcinoma is stable and reliable with good reproducibility.
2.Two protein peaks with m/z 4344 and m/z4961 have the potential to act as the candidate biomarker for diagnosis of gastric carcinoma.
3.The expression of chemokine receptor CCR7 decreases significantly in gastric carcinoma.
4.The level of MCP-1 and IL-8 in the serum of gastric carcinoma is significantly lower than normal.
Innovative points:
1.We successfully isolated two biomarkers in gastric carcinoma serum which were different from recently reported.The two protein peaks have the potential to act as the candidate biomarker for diagnosis of gastric carcinoma.
2.It is the first time to investigate the value of proteomic profiles in clinical curative effect for gastric carcinoma,and two treatment-related protein peaks were successfully found.
3.It is the first time to investigate the expression of CCR7 in peripheral lymphocyte subsets of gastric carcinoma and analyze its relationship with the TNM stage.
4.We investigated the mechanism of immune escape in gastric carcinoma from the expression of chemokine and their receptors.
胃癌是临床常见的恶性肿瘤之一,其发病率居全部恶性肿瘤的的第三位,占消化道恶性肿瘤的第一位。2002年的一项调查显示,全球每年胃癌新发病例绝对数目接近100万,每年新发病例约占新发癌症病例总数的9.9%,死亡总数的12.1%。亚太地区是胃癌的高发区,约占50%以上的胃癌患者来自亚洲,中国约占其中的41%。我国是胃癌的高发区,其发病率和死亡率均超过世界平均水平的两倍,每年死于胃癌的人数约为16万人。虽然目前肿瘤的传统治疗模式如手术、化疗及放疗已取得了长足的进展,但由于多数胃癌患者就诊时已到晚期,导致胃癌患者的死亡率仍居高不下。大量的研究证实,早期胃癌患者术后其5年生存率高达90.4%,而晚期的癌症患者治疗后5年存活率均非常低。由此表明,胃癌的早期诊断是提高胃癌临床治疗疗效及5年生存率的最有效的途径。然而科学高效地防癌治癌是世界级的难题,癌症患者临床表现的高度异质性给早期诊断带来了极大的难度。目前临床采用的主要诊断方法包括影像学、病理学、内窥镜、放射免疫学检查等,都必须在肿瘤发展到一定阶段,即形成实体瘤后才能进行,因此通常不能满足早期发现的要求。内窥镜活组织检查对患者而言具有侵入性并造成较大的痛苦,且内镜技术操作较为复杂,所检出的病人多数已到中晚期,而已有的血清胃癌标记物分子如CEA、CA19-9、CA72-4,其诊断敏感性和特异性均不高,如CEA的诊断敏感性为23.8%,CA19-9为45.5%,且CEA和CA19-9并非胃癌的特异性标志物,并不能作为胃癌早期诊断的标志物,到目前为止发展中国家80%的胃癌患者仍然都是在患病晚期才被发现。因此,要达到胃癌的早期发现必须发展新的诊断技术。
近年来随着人类基因组及后基因组研究的飞速发展和生命科学研究的不断进展,目前已经明确,肿瘤是由环境因素与遗传因素相互作用导致的一类疾病,是多种基因突变累积的结果。如何从整体水平认识肿瘤发生的可能病因,寻找肿瘤早期诊断、监测、干预以及治疗等新途径成为当前肿瘤学研究关注的热点,然而如何从复杂的生物样本中高质量地发现差异蛋白并从中提取有用的信息,使其成为敏感性和特异性均高的标志物是研究的重点和难点,蛋白质组学技术的出现解决了这一难题。肿瘤蛋白质组比较研究能动态地反应肿瘤细胞内蛋白表达水平的变化,检测蛋白翻译后的修饰并同时找出多个与肿瘤基因表达和功能相关的蛋白群,从而为寻找肿瘤的特异性标志物及肿瘤的早期诊断提供了极大的可能性,因而成为近年来肿瘤研究的热点领域。近几年来出现的表面增强激光解吸电离飞行时间质谱技术(surface-enhanced laser desorption/ionization time of flightmass spectrometry,SELDI-TOF-MS),结合了生物芯片和质谱的设计理念,把蛋白芯片、层析及质谱等项技术结合应用于蛋白质的检测,达到简便、快速地从各种体液及组织中找出新的蛋白质肿瘤标记物的特定指纹图谱的目的。目前国际上已经采用SELDI-TOF技术初步发现了乳腺癌、肺癌、前列腺癌、卵巢癌、膀胱癌、大肠癌、胰腺癌、鼻咽癌等近十种可用于临床早期检测的肿瘤生物标志物,并显示了良好的应用前景。例如传统的卵巢癌肿瘤标志物CA-125对于卵巢癌的诊断准确率约25%,而SELDI蛋白指纹图谱技术的准确率忆达到99%。该技术比CT、核磁能更早发现肿瘤,从而使得肿瘤的早期诊断、早期治疗成为可能,这标志着一种划时代崭新的诊断模式的开始。本研究拟利用该技术进行胃癌患者血清蛋白质组学研究,并通过建立及分析胃癌患者蛋白质表达指纹谱,筛选有价值的胃癌早期诊断标志物或特征性蛋白质组峰,为胃癌的早期诊断新方法的建立提供有价值的线索。
众所周知,恶性肿瘤的发生不仅有赖于肿瘤本身恶性表型的变化,还与宿主免疫监视机制的变化有关,Burnet于1967年提出了免疫监视学说,认为机体免疫系统通过细胞免疫机制能识别并特异性杀伤突变细胞,使突变细胞在未形成肿瘤之前即被清除。然而许多肿瘤仍能逃避宿主免疫系统的攻击,在机体内大量增殖并可发生浸润与转移。肿瘤逃逸机体免疫系统的监视,发生浸润及转移是导致手术、放化疗治疗失败及患者死亡的主要原因。因此,揭示肿瘤免疫逃逸及浸润转移等机制的研究对肿瘤防治新策略的发现及制订,提高肿瘤的临床治疗疗效具有重要意义。目前认为肿瘤免疫逃逸的机制主要包括:①宿主抗肿瘤效应细胞的变化,如T淋巴细胞数量和功能的异常及活化障碍等;②肿瘤细胞的变化,如协同刺激分子缺失或低表达、凋亡途径异常导致凋亡抵抗等;③细胞因子表达谱变化,如荷瘤机体通常发生由Th1型细胞因子分泌为主,转向Th2型细胞因子分泌为主的漂移,并可导致抗肿瘤效应细胞的迁移障碍致不能有效的歼灭肿瘤细胞。手术疗法是胃癌治疗的主要手段之一,然而手术本身亦可导致肿瘤细胞的入血,因此,血循环中免疫监视功能的主体细胞群T细胞及其分泌的细胞因子在杀伤侵入血流的肿瘤细胞,起着防止其侵袭与转移的关键作用。揭示胃癌患者外周血T细胞表型特征及其细胞因子分泌的变化对揭示胃癌细胞的免疫逃逸机制,发展新的防止术前或术后在血液循环中肿瘤细胞的生存及侵袭与转移提供新的思路。为此,本研究第二部分利用流式细胞术和悬浮芯片技术分析,检测了胃癌患者手术前后外周血T淋巴细胞亚群的表型变化及细胞因子表达类型的变化,初步探讨了胃癌免疫逃逸的机制。
方法:
1.临床资料
共研究35例胃癌标本,26例正常对照。所有标本均取自2006年3月～2007年8月就诊于泰安市中心医院普外科的初治胃癌患者,年龄45～74岁,中位年龄54岁。阴性对照健康人组26例,为2007年4月-2007年10月健康体检者,年龄40～70岁,中位年龄51岁。
2.血样采集
在采取手术、化疗、输血、介入等治疗措施之前以及手术后,采集患者及健康志愿者清晨空腹血。
3.血清样品收集
所有上述样本均为空腹抽血2～5ml。标本采集后30分钟内置入4℃冰箱2h,然后以同样温度3,000rpm离心20分钟,分装血清,-80℃保存。
4.SELDI-TOF-MS检测
所有肿瘤血清标本统一用弱阳离子交换表面(weak cationic exchanger,WCX2)芯片捕获蛋白,便于建立肿瘤统一标准数据库。取出血清样品,冰上融化后,在4℃下以10,000rpm离心5分钟。在标记好的1.5ml离心管中依次加入U9缓冲液20μl和血清样品10μl,混匀,冰浴振荡30分钟,然后向每管加入360μl醋酸钠缓冲液,振荡器上混匀。将WCX2芯片安装于加样器内,向芯片每孔加入100μl上述稀释血清样品,室温振荡孵育1小时后甩去缓冲液。接着每孔加入200μl醋酸钠缓冲液,振荡5分钟,弃缓冲液后,重复加入200μl醋酸钠缓冲液,同法处理再重复1次。最后用200μl去离子水快速冲洗两遍。取出芯片,待芯片表面自然晾干后,每点加0.5μl能量吸附分子SPA 2次。将芯片置入PBSII质谱仪,进行阅读,获得原始数据并绘制蛋白质质谱图。
5.外参照和内参照的设置
以All-in-One标准蛋白质芯片作为外参照。另外,在每一条芯片上都设置1个点内参,即于每条芯片的8点中随机选出1点,上样同一份质控血清(qualitycontrol,QC),并以所有血清图谱中表达较一致的特征性蛋白峰的平均值作为参照,来验证不同批次芯片及同一芯片不同点的重复性。
6.流式细胞仪检测淋巴细胞的比例及CCR7表达检测
在流式细胞仪检测管中加入相应的荧光标记抗体后,分别加入100μl健康人组、胃癌患者术前和术后的血样,室温孵育30分钟后每管加入1ml 1×红细胞裂解液裂解红细胞。PBS缓冲液洗涤样品三次,流式细胞仪检测细胞。Cellquest软件获取分析细胞,每个检测获取10,000个细胞。
7.悬浮芯片系统检测胃癌术前术后血清中IL-8及MCP-1表达的变化
将计算好的微球加入混合管中,混匀后加入待检测的样品以及稀释的标准品,室温下震荡孵育3个小时,每管各加100μl洗涤液进行洗涤1次。然后各加入50μl生物素标记抗体,室温下震荡孵育1个小时后,用100μl洗液进行洗涤,然后每孔加入100μl洗液,震荡重悬微球,悬浮芯片阅读仪进行阅读。
结果:
1.实验重复性分析
利用All-in-One标准蛋白质芯片和QC质控血清,我们对SELDI-TOF-MS产生的蛋白质图谱进行了重复性测试。结果显示,本实验所用WCX2蛋白芯片的intra-assay和inter-assay变异系数(coefficient of variation,CV)分别为10%和12%,提示不同批次芯片、不同芯片点间的蛋白峰值有良好的重复性。
2.筛选胃癌血清特异性标志物
胃癌组与对照组的质谱图比较:应用Biomarker Wizard软件对35例胃癌血清和26例正常血清构建的原始蛋白指纹图谱进行对比分析,在分子量0～50,000Da范围内共检测到75个蛋白峰。结合Biomarker Patterns软件进一步分析,发现胃癌组与对照组间有5个差异蛋白(P＜0.05),其中2个蛋白表达差异显著,其质荷比分别为m/z 4344、m/z4961。m/z 4961在胃癌组明显高于对照组;m/z 4344在胃癌组表达明显降低。
3.筛选胃癌治疗相关生物标志物
手术前后血清蛋白质谱比较:应用Biomarker Wizard软件对20例胃癌术前和术后血清构建的原始蛋白指纹图谱进行对比分析,在分子量0～50,000Da范围内共检测到70个蛋白峰。结合Biomarker Patterns软件进一步分析,发现胃癌术前组与术后组间有7个差异蛋白(P＜0.05),其中2个蛋白表达差异显著,其质荷比分别为m/z 2743、m/z3315。两者在术后表达较术前均明显降低。
4.胃癌患者术前以及术后外周血淋巴细胞亚群检测
胃癌患者与健康人组CD3~+T细胞和CD19~+B细胞所占的比例无显著性差异(CD3~+T:67.62%VS65.69%;CD19~+B:10.8%VS10.44%),胃癌患者术前和术后的外周血中CD3~+T细胞和CD19~+B细胞所占的比例也无明显变化(CD3~+T:67.62%VS67.79%;CD19~+B:10.8%VS10.35%)。
5.胃癌患者术前以及术后外周血淋巴细胞CCR7表达检测
与健康人相比,胃癌患者外周血CD3~+T细胞中CCR7的表达明显降低,术后CCR7的表达有所升高。术后组与健康人组之间CCR7的表达无显著性差异(P＞0.05)。比较CCR7在外周血淋巴细胞的表达与不同病理分期的关系,CCR7在胃癌Ⅲ、Ⅳ期患者外周血淋巴细胞中的表达阳性率明显低于Ⅰ、Ⅱ期胃癌患者。
6.趋化因子IL-8及MCP-1胃癌患者血清中的表达
胃癌患者以及正常对照组血清中IL-8及MCP-1的浓度检测结果显示,与正常对照组相比,胃癌患者术前血清中IL-8以及MCP-1的浓度明显低于正常对照组。
结论:
1.应用SELDI-TOF-MS技术获得胃癌血清蛋白质表达图谱的实验方法稳定可靠,具有良好的重复性。
2.m/z 4344、m/z4961的2个生物标志物可作为筛选胃癌标志物的候选蛋白。
3.胃癌患者外周血淋巴细胞CD3~+T细胞趋化因子受体CCR7表达明显下降,且下降程度与胃癌分期有关。
4.胃癌患者血清中趋化因子MCP-1和IL-8的含量明显降低。
主要创新点:
1.应用WCX2蛋白芯片筛选出2个有别于国际上其他文献报道的血清生物标志物,该标志物可作为筛选胃癌标志物的候选蛋白。
2.首次探讨血清蛋白质图谱在胃癌临床疗效监测方面的应用价值,筛选出2个胃癌治疗相关标志物。
3.检测了胃癌患者外周血淋巴细胞各亚群中CCR7的表达,并且分析了其表达强度与胃癌临床病理分期之间的关系。
4.从趋化因子及其受体方面探讨胃癌免疫逃逸相关机制。
Background and Objectives:
Gastric carcinoma is the most frequent digestive cancer in clinical medicine,the incidence of which is in the third place in all carcinoma and the first place in digestive carcinoma.An investigation in 2002 indicated that the absolute amount of new gastric carcinoma cases was approaching 1,000,000,which takes 9.9%of the total new cancer cases and 12.1%of caner deaths.Asia-Pacific region has a high incidence of gastric carcinoma.Approximately,more than 50%of new cases occur in Asia and about 41%of which come from China.China is an area with high incidence of gastric carcinoma,the morbidity and mortality rates are more than twice of the world average level:there were 160,000 people died from stomach cancer.Although many current traditional treatment approaches for gastric cancer have been developed,the mortality rate is still high since most patients have come to terminal stage when they get diagnosis.A bunch of researches proved that the 5 year survival rate was high to 90.4%in early gastric cancer patients after surgical treatment.While in terminal stage cancer patients,it is rather low.All of these indicated that the early diagnosis is the most effective way for improving the prognosis and 5 year survival rate.However,to scientifically and effectively diagnose gastric carcinoma is still an international difficult problem,and patients' clinical performance heterogeneity brings more difficulties to it.Current clinical diagnosis approaches include Imaging,Pathology, Endoscopy and Radioactive Immunologic Examination,etc.All of these methods can only be put into practice when tumor develops to certain stage which only means when it comes to physical tumor.Thus it is impossible to meet the acquirement of early diagnosis.For patients,the invasion of Endoscopic biopsy brings too much pain and the operation process is complex,at the same time,the patients detected from this way are always in terminal stage.However,the diagnostic sensitivity and specificity of existed biomarker for gastric carcinoma like CEA,CA19-9,CA72-4 are not high (the diagnostic sensitivity of CEA is 23.8%,CA19-9 is 45.5%) and they are not specific,so they can not be treated as early diagnosis biomarkers for gastric carcinoma.Hitherto,about 80%of stomach cancer patients were not found until it came to terminal stage.So it is urgent to explore and set up an early stage diagnosis technology.
With the rapid development of human genome,post-genome research and the progress made in life science research,it is explicited that cancer is a kind of disease induced from the interaction of environmental and genetic factors and cumulative result of various gene mutations.How to recognize the possible reasons from integrity level and find new methods of early tumor diagnosis,monitoring,intervention and treatment becomes a new hot concern point for current oncology research.How to collect high quality proteins with difference from complex samples and get valuable information from them in order to let them become high sensitive and specific biomarkers becomes a key point and difficult problem in research.The appearance of proteomics technology solved this problem.Comparison on tumor proteome can dynamically reflex the changes of proteins in tumor cells;detect the modification of protein after translation and find several proteins which are related to tumor gene expression and function,so that make it possible to find specific biomarkers and make early diagnosis for tumor.The development of surface-enhanced laser desorption / ionization time of flight mass spectrometry technology is to combine the idea of biological chips and mass spectrometry and put these technologies into protein detection,which can detect new tumor biomarkers' fingerprinting from various humors and tissues.Tens of tumor biomarkers for early diagnosis for breast cancer, lung cancer,prostatic cancer,ovarian cancer,bladder cancer,colorectal cancer, pancreatic cancer and nasopharyngeal cancer have been found via SALDI-TOF technology and showed great practical prospects.For example,CA-125 as a traditional biomarker for ovarian cancer can be used to diagnose with accuracy rate of approximately 25%,while the accuracy rate of SELDI protein fingerprinting technology has reached 99%.SELDI-TOF can be used to detect tumor earlier than CT and MIR,which can make it possible to make early diagnosis and treatment for patients.It marks the beginning of a new era of diagnose model.In this project,we suppose to use this technology to research on the proteome of gastric cancer patients' serum.And screen valuable markers or specific protein peeks for gastric carcinoma early diagnosis,in order to shed light on finding new approaches for gastric carcinoma early diagnosis.
It is well known that the development of malignant tumor not only depends on the changes of its malignant phenotype,but also relates to the host's immune surveillance mechanism.The immune surveillance theory that was created by Burnet in 1987 suggested that immune system could recognize and remove the mutant cells through cell immune response before they developed into tumors.However,many tumor cells can escape from the attack of host immune system and grow out of control and even invade or metastasize to other tissues.These are the main reasons for the surgery,radiotherapy and chemotherapy failure.So it is important to investigate the mechanism of immune escape for tumor prevention and treatment.There are three aspects included in the tumor immune escape mechanism:①The changes of host effective cells,such as the abnormality of the T cell amount and function.②The changes of tumor cells,such as lack of co-stimulatory molecules and disorders of apoptosis.③The changes of cytokines,such as the tumor host body always shift from mainly Thl-type cytokine secretion to mainly Th2-type cytokine secretion which can induce that the effective immune cells can not effectively kill tumor cells.Surgery is one of the main treatment models for gastric carcinoma,but the tumor cells can enter blood through the process.So the T cells and cytokines secreted from them are crucial in the process of killing the tumor cells which entered blood.All of these indicated that reveal the T cells phenotypes and the changes of their secretion of cytokines can shed light on reveal immune surveillance mechanism of gastric tumor cells and development of new ways of preventing tumor cells' survival,invasion and metastasis pre- or post-surgery.In the second part of this study,we used flow cytometry and suspension chip technology to detect the changes of peripheral lymphocyte subsets' phenotypes and the variation of the expression of cytokines between pre-and post-surgery in patients in order to further investigate the mechanisms of immune escape of gastric carcinoma.
Methods:
1.Clinical data
35 patients with gastric carcinoma,26 healthy volunteers were included in this study.All samples were collected from the General Surgery Department,TaiAn Central Hospital during March and August in 2006.The middle ages of the two groups are 54 and 51 respectively.There was no statistically significant difference in the ages between the patients and control group.(P＞0.05)
2.The collection of peripheral blood samples
2 ml of peripheral blood was collected from the patients with carcinoma and the control group.For cancer group,the samples were collected before and after treatment.
3.The collection of serum samples
2～5 ml of peripheral blood was collected from the patients with carcinoma and control group.Then the samples were laid-aside at 4℃for 30 minutes and centrifuged at 3,000 rpm for 20 minutes.Serum samples were collected and stored at -80℃.
4.DELDI-TOF-MS analysis
The WCX2 protein chip was used to harvest proteins from serum samples. Serum samples were centrifuged for 5 minutes at 10,000 rpm after melted on ice.20ul of U9 buffer was added into pre-labeled 1.5ml tube and then added 10 ul of serum sample.After vibrate samples at 4℃for 30 minutes,360 ul of 50 mmol/L Na-Acetate wash buffer was added to each tube and then vertex samples.The WCX2 was installed on bioprocessor and 100 ul of diluted serum sample mentioned above was added to each well,then the samples were incubated at room temperature for 60 minutes on shaker,remove the buffer and washed the samples twice with 200 ul Na-Acetate wash buffer for 5 minutes.Two quick rinses with HEPES water followed. Take out the chips and let them air dry.Before SELDI analysis,0.5 ml of saturated EAM solution SPA was applied onto each chip array twice and let the array surface air dry between each EAM application.Place chips on the Protein Biological SystemⅡmass spectrometer reader.
5.Set Quality Controls
All-in-One peptide molecular mass standard was used to calibrate.In addition, the reproducibility of SELDI spectra was determined by using the pooled normal serum quality control samples.We randomly selected two peaks in the range of 5,000～10,000Da to calculate the coefficient of variance.
6.FACS assay
Freshly isolated peripheral lymphocytes were stained immediately with appropriate concentrations of fluorescent-conjugated mAb for cell surface antigen.All Abs,directly conjugated with FITC,Phycoerythrin(PE) or PE-cy5,were obtained from PharMingen(San Diego,CA).The cells were stained with the corresponding FITC-,PE-conjugated or PE-Cy5 mouse anti-human isotype-matched control Abs according to the manufacturer's protocol then incubated for 30 minutes in the dark. Red cell lysis buffer was added to the tube and incubated for 15 minutes at room temperature.Aliquots of 2×to 5×10~6 cells were resuspended in 80ul of PBS containing 0.5%bovine serum albumin.Immunostained cells were then analyzed on flow cytometer using CELLQuest software~(TM)(FACSCalibur,Becton Dickinson,CA). A minimum of 10,000 events per sample were collected for phenotypic analysis as previously described.
7.Suspension Chip Assay
Samples and standards were incubated with pre-mixed beads into the well of a 96-well microplate for 3 hours at room temperature with agitation.The mixture was washed with 100 ul of wash buffer.Then 50 ul of biotin was added to each well and incubated for 1 hour at room temperature with agitation.Wash the plate and add 100 ul of wash buffer to each well and read samples in Bio-Plex Suspension System.
Results:
1.Quality control
We detected the repeatability of protein spectrums got from EELDI-TOF-MS analysis with All-In-One Standard Protein Chip and QC serum.The results indicated that the intra-and inter-assay coefficient of variance of WCX2 Protein Chip were 10% and 12%which suggested that there was a good repeatability in different chips and wells of one chip.
2.Selection of cancer-specific serum protein markers
Comparison of protein spectrum between cancer group and control group:By comparing the original protein spectrum between 35 gastric cancer patients' and 26 healthy volunteers' serum,we found that there were 75 peaks with molecular weigh between 0～50,000 KD.Further analysis with Biomarker Patterns Software,we found that there were 5 proteins with difference in them and two proteins had significant difference.The corresponding m/z rations were m/z 4344 and 4961.The protein with m/z 4961 highly expressed in cancer group,while the protein with m/z 4691 had lower expression in cancer group than in control group.
3.Selection of treatment-related serum biomarkers
Comparison between protein spectrum pre- and after-treatment in cancer group: There were 70 peaks detected in the treated cancer group by Biomarker Wizard software Analysis.Further analysis with Biomarker Pattern revealed that there were 7 proteins which had difference and 3 of them had significant difference.Between untreated cancer sera and treated cancer sera from patients with gastric carcinoma. These two peaks corresponded to m/z ration of 2743 and 3315.Their concentrations were down-regulated after surgery treatment.
4.Peripheral lymphocytes subsets detection
The proportions of CD3~+T and CD19~+B between no-cancer group and gastric carcinoma patients had no significant difference(CD3~+T:67.62%VS 65.69%; CD19~+B:10.8%VS 10.44%).There was no significant difference between untreated cancer group and the treated gastric carcinoma patients either(CD3~+T:67.62%VS 67.79%;CD19~+B:10.8%VS 10.35%).
5.The expression of CCR7 in different groups
Compared with the healthy volunteers group,the expression of CCR7 in gastric carcinoma group significantly decreased.After curative gastrectomy,the expression level of CCR7 increased.The expression level of CCR7 related to TNM stage (P＜0.05).
6.The level of IL-8 and MCP-1 in serum
Compared with the healthy volunteers group,IL-8 and MCP-1 level in gastric carcinoma serum was significantly lower.
Conclusions:
1.Application of SELDI-TOF-MS technology to obtain proteomic profiles in serum for gastric carcinoma is stable and reliable with good reproducibility.
2.Two protein peaks with m/z 4344 and m/z4961 have the potential to act as the candidate biomarker for diagnosis of gastric carcinoma.
3.The expression of chemokine receptor CCR7 decreases significantly in gastric carcinoma.
4.The level of MCP-1 and IL-8 in the serum of gastric carcinoma is significantly lower than normal.
Innovative points:
1.We successfully isolated two biomarkers in gastric carcinoma serum which were different from recently reported.The two protein peaks have the potential to act as the candidate biomarker for diagnosis of gastric carcinoma.
2.It is the first time to investigate the value of proteomic profiles in clinical curative effect for gastric carcinoma,and two treatment-related protein peaks were successfully found.
3.It is the first time to investigate the expression of CCR7 in peripheral lymphocyte subsets of gastric carcinoma and analyze its relationship with the TNM stage.
4.We investigated the mechanism of immune escape in gastric carcinoma from the expression of chemokine and their receptors.
引文
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28. Kasahara K, Strieter RM, Chensue SW, et al. Mononuclear cell adherence induces neutrophil chemotactic factor/interleukin-8 gene expression. J Leukoc Biol. 1991;50(3):287-95.
29.Yoshimura T, Matsushima K, Tanaka S, et al. purification of a human monocyte- derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines. Proc Natl Acad Sci U S A. 1987; 84(24):9233-9237.
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1.Yao X X,Yin L,Sun ZC.The expression of hTERT mRNA and cellular immunity in gastric cancer and precancerosis.World J Gastroenterol.2002;8(4);586-590.
2.Montelli TC,Peracoli MT,Gabarra RC,et al.Familial cancer:depressed NK-cell cytotoxicity in healthy and cancer affected members.Arq Neuropsiquoatr,2001,59(1):6-10.
3.Salvadori S Martinelli G,Zier K.Resection of solid tumors reverses T cell defects and restores protective immunity.J Immunol.2000;164(4);2214-2220.
4.Kim CW,Choi SH,Chung EJ,et al.Alteration of signal-transducing molecules and phenotypical characteristics in peripheral blood lymonocytes from gastric carcinoma patients.Pathobiology.1999;67(3);123-128.
5.Chen XL,Cao XD,Kang AJ,et al.In situ expression and significance of B7costimulatory molecules within tissues of human gastric carcinoma.World J Gastroentero.2003;9(6);1370-1373.
6.Banchereau J,Briere F,Caux C,et al.Immunobiology of dendritic cells.Annu Rev Immunol.2000;18;767-811.
7.Gabrilovich DI,Chen HL,Girgis KR,et al.Production of vascular endothelial growth factor by tumors inhibits the functional maturation of dendritic cells. Nat Med.1996; 2; 1096-1103.
8.Lespagnard L, Gancberg D, Rouas G, et al. Tumor infiltrating dendritic cells in adenocarcinomas of the breast; A study of 143 neoplasms with a correlation to usual prognostic factors and to clinical outcome. Int J Cancer. 1999; 84(3);309-314.
9.Chaux p, Favre N, Martin M, et al. Tumor infiltrating dendritic cells are defective in their antigen presenting function and inducible B7 expression in rats. Int J Cancer.1997;72 (4); 619-624.
10.Rawson P, Hermans IF, Huck SP, et al. Immuno therapy with dendritic cells and tumor major histocompatibility complex class-derived peptid requirs a high density of antigen on tumor cells. Cancer Res. 2000; 60;4493-4498.
11.Cormier JN, Panell MC, Hackett JA, et al. Natural variation of the expression of HLA and endogenous antigen modulates CTL recognition in an in vitro melanoma model. Int J Cancer. 1999; 80;781-790.
12.Allan PS, Colonna M, Lanier LL, et al. Tetrameric complexes of human histocompatibility leukocyte antigen (HLA) G bind to peripheral blood myelomonocytic cells. J Exp Med. 1999; 189(7);1149-1156.
13.Li J, Yang Y, Inoue H, et al. The expression of costimulatory molecules CD80 and CD86 in human carcinoma cell lines; its regulation by interferon gamma and interleukin-10. Cancer Immunol Immunother.1996; 43(4);213-219.
14.Owen SL, Chan H, Cusack JC, et al. Fas and Fas ligand interactions in m alignant disease.Int J Oncol. 2000; 17(1);5-12.
15.Takahama Y, Yamada Y, Emoto K, et al. The prognostic significance of over expression of the decoy receptor for Fas ligand (DcR3) in patients with gastric carcinomas. Gastric Cancer. 2002; 5(2);61-68.
16.Ohno S, Tachibana M, Shibakita M,et al. Prognostics ignificance of Fas and Fas ligand system-associated apoptosis in gastric cancer. Ann Surg Oncol. 2000;7(10);750-757.
17.Zheng HC, SunI M, Wei ZL, et al. Expression of Fas ligand and caspase-3 contributes to formation of immune escape in gastric cancer. World J Gastroenterol. 2003; 9(7);1415-1420.
18.O'Connell J, Bennett MW, O'Sullivan GC, et al. Immunol Today. 1999; 20 (1);46-52.
19.Onishi T, Ohishi Y, Imagawa K, et al. An assessment of the immunological environment based on intratumoral cytokine production in renal cell carcinoma.BJU Int. 1999; 83(4);488-492.
20.Restifo NP. Not so FAS; Re-evaluating the mechanisms of immune privilege and tumor escape. Nat Med; 2000,6(5);493-495
21.Chouaib S, Asselin-Paturel C, Mami-Chouaib F, et al. The host-tumor immune conflict; from immunosuppression to resistance and destruction. Immunology Today. 1997;18;493-497.
22.Ohno S, Tachibana M, Fujii T, et al. Role of stromal collagen in immunomodulation and prognosis of advanced gastric carcinoma. Int J Cancer,2002,97(6):770-774.
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44.Ohno S, Tachibana M, Shibakita M,et al. Prognostics ignificance of Fas and Fas ligand system-associated apoptosis in gastric cancer. Ann Surg Oncol. 2000;7(10):750-757.
45.Zheng HC, SunI M, Wei ZL, et al. Expression of Fas ligand and caspase-3 contributes to formation of immune escape in gastric cancer. World J Gastroenterol. 2003; 9(7):1415-1420.
46.O'Connell J, Bennett MW, O'Sullivan GC, et al. Immunol Today. 1999; 20(1):46-52.
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7.Holcakova J, Hernychova L, Bouchal P, et al. Identification of alphaB-crystallin,a biomarker of renal cell carcinoma by SELDI-TOF MS. Int J Biol Markers.2008; 23(1):48-53.
8.Zhu LR, Zhang WY, Yu L, et al. Proteiomic patterns for endometrial cancer using SELDI-TOF-MS. J Zhejiang Univ Sci B. 2008; 9(4):286-290.
9.Geng X, Wang F, Li YG, et al. SELDI-TOF MS proteinchip technology for screening of serum markers of HBV-induced hepatocellular carcinoma.J Exp Clin Cancer Res. 2007; 26(4):505-508.
10.Cheng L, Zhou L, Tao L, et al. SELDI-TOF MS Profiling of serum for detection of laryngeal squamous cell carcinoma and the progression to lymph node metastasis. J Cancer Res Clin Oncol. 2008; 134(7):769-776.
11.Koopmann J, Zhang Z, White N, et al. Serum diagnosis of pancreatic adenocarcinoma using surface-enhanced laser desorption and ionization mass spectrometry.Clin Cancer Res.2004;10:860-868.
12.Bandera CA,Ye B,Mok SC.New technologies for the identification of markers for early detection of ovarian cancer.Curr Opin Obstet Gynecol.2003;15:51-55.
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17.Li J,Zhang Z,Rosenzweig J,et al.Proteomics and bioinformatics approaches for identification of serum biomarkers to detect breast cancer.Clin Chem.2002;48(8):1296-1304.
18.Paweletz CP,Trock B,Pennanen M,et al.Proteomic patterns of nipple aspirate fluids obtained by SELDI-TOF:Potential for new biomarkers to aid in the diagnosis of breast cancer.Dis Markers.2001;17(4):301-307.
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1.Salvadori S Martinelli G, Zier K. Resection of solid tumors reverses T cell defects and restores Protective immunity. J Immunol. 2000; 164 (4):2214-2220.
2.Kim CW, Choi SH, Chung EJ, et al. Alteration of signal-transducing molecules and phenotypical characteristics in peripheral blood lymonocytes from gastric carcinoma patients. Pathobiology. 1999; 67(3):123-128.
3.Chen XL, Cao XD, Kang AJ, et al. In situ expression and significance of B7 costimulatory molecules within tissues of human gastric carcinoma. World J Gastroentero, 2003; 9(6): 1370-1373.
4.Yao XX, Yin L, Sun ZC. The expression of hTERT mRNA and cellular immunity in gastric cancer and precancerosis. World J Gastroenterol. 2002; 8(4):586-590.
5.Balkwill F. Chemokine biology in cancer. Semin Immuno. 2003; 15(1):49-55.
6.Kodama J, Hasengaowa, Seki N, et al. Expression of the CXCR4 and CCR7 chemokine receptors in human endometrial cancer. Eur J Gynaecol Oncol. 2007;28(5):370-375.
7.Ishigami S, Natsugoe S, Nakajo A, et al. Prognostic value of CCR7 expression in gastric cance. Hepatogastroenterology. 2007; 54(76):1025-1028.
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14.Mashino K, Sadanaga N, Yamaguchi H, et al. Expression of chemokine receptor CCR7 is associated with lymph node metastasis of gastric carcinoma. Cancer Res.2002; 62(10):2937-2941.
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26.Walz A, Baggiolini M. A novel cleavage product of beta-thromboglobulin formed in cultures of stimulated ononuclear cells activates human neutrophils.Biochem Biophys Res Commun. 1989; 159(3):969-975.
27.Baggiolini M, Walz A, Kunkel SL. Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils. J Clin Invest. 1989; 84(4):1045-1049.
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1.Yao X X,Yin L,Sun ZC.The expression of hTERT mRNA and cellular immunity in gastric cancer and precancerosis.World J Gastroenterol.2002;8(4);586-590.
2.Montelli TC,Peracoli MT,Gabarra RC,et al.Familial cancer:depressed NK-cell cytotoxicity in healthy and cancer affected members.Arq Neuropsiquoatr,2001,59(1):6-10.
3.Salvadori S Martinelli G,Zier K.Resection of solid tumors reverses T cell defects and restores protective immunity.J Immunol.2000;164(4);2214-2220.
4.Kim CW,Choi SH,Chung EJ,et al.Alteration of signal-transducing molecules and phenotypical characteristics in peripheral blood lymonocytes from gastric carcinoma patients.Pathobiology.1999;67(3);123-128.
5.Chen XL,Cao XD,Kang AJ,et al.In situ expression and significance of B7costimulatory molecules within tissues of human gastric carcinoma.World J Gastroentero.2003;9(6);1370-1373.
6.Banchereau J,Briere F,Caux C,et al.Immunobiology of dendritic cells.Annu Rev Immunol.2000;18;767-811.
7.Gabrilovich DI,Chen HL,Girgis KR,et al.Production of vascular endothelial growth factor by tumors inhibits the functional maturation of dendritic cells. Nat Med.1996; 2; 1096-1103.
8.Lespagnard L, Gancberg D, Rouas G, et al. Tumor infiltrating dendritic cells in adenocarcinomas of the breast; A study of 143 neoplasms with a correlation to usual prognostic factors and to clinical outcome. Int J Cancer. 1999; 84(3);309-314.
9.Chaux p, Favre N, Martin M, et al. Tumor infiltrating dendritic cells are defective in their antigen presenting function and inducible B7 expression in rats. Int J Cancer.1997;72 (4); 619-624.
10.Rawson P, Hermans IF, Huck SP, et al. Immuno therapy with dendritic cells and tumor major histocompatibility complex class-derived peptid requirs a high density of antigen on tumor cells. Cancer Res. 2000; 60;4493-4498.
11.Cormier JN, Panell MC, Hackett JA, et al. Natural variation of the expression of HLA and endogenous antigen modulates CTL recognition in an in vitro melanoma model. Int J Cancer. 1999; 80;781-790.
12.Allan PS, Colonna M, Lanier LL, et al. Tetrameric complexes of human histocompatibility leukocyte antigen (HLA) G bind to peripheral blood myelomonocytic cells. J Exp Med. 1999; 189(7);1149-1156.
13.Li J, Yang Y, Inoue H, et al. The expression of costimulatory molecules CD80 and CD86 in human carcinoma cell lines; its regulation by interferon gamma and interleukin-10. Cancer Immunol Immunother.1996; 43(4);213-219.
14.Owen SL, Chan H, Cusack JC, et al. Fas and Fas ligand interactions in m alignant disease.Int J Oncol. 2000; 17(1);5-12.
15.Takahama Y, Yamada Y, Emoto K, et al. The prognostic significance of over expression of the decoy receptor for Fas ligand (DcR3) in patients with gastric carcinomas. Gastric Cancer. 2002; 5(2);61-68.
16.Ohno S, Tachibana M, Shibakita M,et al. Prognostics ignificance of Fas and Fas ligand system-associated apoptosis in gastric cancer. Ann Surg Oncol. 2000;7(10);750-757.
17.Zheng HC, SunI M, Wei ZL, et al. Expression of Fas ligand and caspase-3 contributes to formation of immune escape in gastric cancer. World J Gastroenterol. 2003; 9(7);1415-1420.
18.O'Connell J, Bennett MW, O'Sullivan GC, et al. Immunol Today. 1999; 20 (1);46-52.
19.Onishi T, Ohishi Y, Imagawa K, et al. An assessment of the immunological environment based on intratumoral cytokine production in renal cell carcinoma.BJU Int. 1999; 83(4);488-492.
20.Restifo NP. Not so FAS; Re-evaluating the mechanisms of immune privilege and tumor escape. Nat Med; 2000,6(5);493-495
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