| BackgroundGastric cancer has always been a serious threat to human health worldwide, and it is our most common gastrointestinal malignancy, its perennial high morbidity and mortality. Gastric cancer prognosis is poor, especially to tumor progression staggering development, and early diagnosis and treatment can significantly improve the prognosis of patients with gastric cancer, reducing the mortality rate. However, most patients with early gastric cancer is no obvious symptoms, or only nausea, vomiting and other non-specific gastrointestinal symptoms, easy and gastritis, gastric ulcer confused, unable to arouse people’s attention, and thus delay the disease, missed best time for treatment. Most often when the patient has been in treatment for locally advanced or distant metastasis has occurred, so early diagnosis and treatment is the key to improve the prognosis of patients with gastric cancer. Currently, the clinical diagnosis of gastric cancer commonly used imaging tests include upper gastrointestinal endoscopy and angiography, but the doctor’s diagnosis is different, patients with early gastric cancer detection rate is not high, but endoscopy is an invasive The inspection will cause patient discomfort, its high cost, and therefore at high risk of gastric cancer is not easy in popularity. Meanwhile CEA, CA19-9, CA72-4, as commonly used in clinical serum tumor markers, due to lack of sensitivity and specificity for early diagnosis of gastric cancer is not big help. In this context, the urgent need to find a new clinical and gastric cancer associated with high sensitivity and high specificity of molecular markers, it has also become a hot spot of gastric cancer-related research. ObjectiveBy proteomics technology to screen and identify possible in the serum of patients with gastric cancer-specific protein markers, to provide more comprehensive serum protein fingerprint model for the early diagnosis of gastric cancer, gastric cancer serum for further study of specific protein markers to lay a solid foundation. Materials and methodsMaterial: First diagnosed 60 cases of gastric cancer patients in this study were collected by the First Affiliated Hospital of Zhengzhou University, General Surgery since May 2013 to January 2014 came to seek treatment, 46 patients were male, 14 cases of female patients, aged 42 to 84 years old The average age of 58 ± 0.5 years. When the 20 cases in which the doctor has confirmed metastatic gastric cancer without surgery, collecting serum samples to metastasis. More than 40 patients diagnosed with primary gastric cancer, and no contraindication for surgery scheduled for surgery, before surgery a day after the first 12 days of each serum samples collected once, were set to preoperative and postoperative group group. All patients before collecting specimens were not receiving chemotherapy, radiation therapy, after surgery and pathological diagnosis of gastric adenocarcinoma, the histopathological classification are in poorly differentiated adenocarcinoma. Pathological diagnosis was confirmed after more than two pathology experts. All 60 patients were made from blood samples drawn under the fasting state, blood drawn after the blood is placed in a dry test tube stand at room temperature for 1 hour natural blood clotting, and then in the centrifuge at 3000 r / min speed centrifugal 15 min, extracting the supernatant after centrifugation moved PE tube placement-80 ℃ refrigerator to save. The experiment by the hospital ethics committee ethics review, subjects have signed informed consent.Methods: Serum thawing ice bath, 4 ℃ 10000 r / min centrifugal 2min. Take 96 placed on ice added to each well U9(9mol / L urea), 1% Bar, 2% CHAPS10μ l, serum total protein and tumor tissue samples 5μ l, 4 ℃ chromatography cabinet 600 r / min oscillations 30 min. 15 min before the end of the shock protein chips do preprocessing chip loaded pipette Bioprocessor, mark the next chip number is added to each well Na AC(100mmol / L, p H4) 200μl, chromatography cabinet 600 r / min oscillation 2min, repeat The above operation once. After 96 treatment after U9 placed on ice, with Volley join Na AC185μ l, chromatography cabinet 4 ℃ 600 r / min shock 2min. 100 μ l samples taken on added processed protein chip, placed chromatography cabinet 4 ℃ 600 r / min combined 60 min, rejection to the residue, fast and pat dry. Then after adding Na AC200μl, 600 r / min oscillations 5min, rejection to the residue, quick pat dry and repeat three times. With 200μl each well rinsed with deionized water twice, drying. After air-dried chips, each well twice with 50% saturation SPA 1μl, after drying machine to be detected. Proteins of known molecular weight chip SELDI-TOF-MS system is corrected, the molecular weight of the protein error is less than 0.1%, then the binding protein WCX2 good protein array was analyzed by mass spectrometry. After the mass spectrometric analysis of the raw data by filtering noise and cluster analysis and processing, the preliminary screening of mass to charge ratio of the peak data do Wilconxon rank sum test, test taking less than 0.01. By P value to determine the extent of the differences of the same m / z peak in expression between groups, P values less prompt the m / z peak between the two groups was statistically significant. Results1. Comparison of the results of preoperative gastric cancer group and the normal groupMass spectrometry data of gastric surgery group and the normal group after subtracting baseline, de-noising and normalization process, cluster analysis to obtain their peak, a peak after two Wilcoxon rank sum test, resulting in 15 specific m / z peak(P <0.01). In which gastric surgery group with low expression of eight, high expression of seven. SVM screened by a combination of the highest index model youden get m / z peak at 6449.1 protein markers. In gastric surgery group showed high expression, the expression intensity of 2299.3 ± 2029.3. Expression was significantly lower in patients with normal group, the expression level of 509.5 ± 168.3. The difference between the two groups was statistically significant(P <0.01).2. The result of the comparison group with preoperative gastric cancer groupMass spectrometry data group and gastric cancer after preoperative group after treatment and statistical analysis, P <0.01 specific m / z peaks 6, wherein after the group of four high expression, low expression of two. Reference youden index highest combined model, obtain m / z peak at 6449.2 protein markers. In gastric surgery group showed high expression, the expression intensity of 1247.9 ± 685.0. Significantly lower expression in normal patients, the expression of the strength of 476.5 ± 157.8. The difference between the two groups was statistically significant(P <0. 01).3. Comparison of the results of the preoperative group and metastasis of gastric cancer groupMass spectrometry data of gastric surgery group and metastasis group after treatment and statistical analysis, P <0.01 specific m / z peak of 12, which is highly expressed metastasis group was a low expression 11. Reference youden index highest combined model, get a protein marker m / z peak at 6448.9. Before expression of metastasis of gastric surgery group was significantly higher than in gastric cancer metastasis expression intensity 1506.9 ± 1036.5. Expression in gastric surgery group strength 649.7 ± 621.0. The difference between the two groups was statistically significant(P <0.01).4. Identification of specific proteinsSerum samples of the target protein be isolated and purified, after the enzymatic hydrolysis, the use of MALDI-TOF / TOF platform peptide mixture was subjected to testing. m / z peak at 6449 after protein identified as Apo CⅢ(apolipoprotein CⅢ). Conclusion1. m / z peak at 6449 after protein identified as Apo CⅢ, consider specific serum protein markers of gastric cancer, to provide more comprehensive serum protein fingerprint model for the early diagnosis of gastric cancer, gastric cancer serum for further study of specific protein markers instructive.2. m / z peak at 6449 protein expression level of tumor progression in gastric cancer and its positive correlation exists for the development of the relationship between post-study-specific protein markers occur with gastric cancer provides a new way of thinking. |
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