| 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 data35 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 samples2 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 samples2～5 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 analysisThe 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 ControlsAll-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 assayFreshly 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 5×106 cells were resuspended in 80ul of PBS containing 0.5%bovine serum albumin.Immunostained cells were then analyzed on flow cytometer using CELLQuest softwareTM(FACSCalibur,Becton Dickinson,CA). A minimum of 10,000 events per sample were collected for phenotypic analysis as previously described.7.Suspension Chip AssaySamples 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 controlWe 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 markersComparison 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 biomarkersComparison 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 detectionThe 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 groupsCompared 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 serumCompared 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. |
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