| Objective:Colorectal cancer is one of the most common digestive tract tumors. Low rate of early diagnosis and postoperative recurrence or metastasis make it still a highly invasive malignant tumors which have a high mortality rate. Early diagnosis and the monitoring of recurrence or metastasis of colorectal cancer is a current problems to be solved, looking for a convenient and effective tumor markers is particularly important. The purpose of this study is to explore the expression of anti-survivin antibody and survivin mRNA in peripheral blood of patients with CRC and its Clinical Significance, and provide more biological markers for diagnosis and treatment of CRC.Methods:116patients with colorectal cancer were divided into radical surgery group, palliative surgery group and palliative chemotherapy group, and30healthy people were taken as controls.5ml peripheral blood was collected in the morning of1day before surgery and the4th week after surgery form the fasting patients of radical surgery group and palliative surgery group respectively, and the radical surgery group were followed-up every3months, collecting5ml peripheral blood;5ml peripheral blood was collected form palliative chemotherapy group before chemotherapy and after2cycles of chemotherapy respectively, and5ml peripheral blood was collected form fasting healthy people in the morning.2ml of the5ml blood samples was collected with vacuum tube without anticoagulant, and centrifuged at3000r/min for10min, the supernatant was frozen at-20℃for the detection of anti-survivin antibody; Remaining3ml blood samples was collected with vacuum tubes which contain ethylenediaminetetraacetic acid (EDTA) anticoagulant, put it aside for half an hour after collection at room temperature and then extracted total RNA for the detection of survivin mRNA. The level of serum anti-survivin antibody was determined by double antigen sandwich enzyme-linked immunosorbent assay (ELISA). A monoclonal antigen specific for survivin has been pre-coated onto a microplate. Standards, samples and survivin which with horseradish peroxidase (HRP) labeled are pipetted into the wells. After incubation and washing away any unbound substances, followed by adding the substrate A and B, the color turn blue, ultimately transformed into yellow in the action of acid, and there was a positive correlation between the depth of color samples and anti-survivin antibody concentration. According to the standard equation which got by measuring the standards, we can calculate concentration of anti-survivin antibody. The level of survivin mRNA in peripheral blood was determined by semi-quantitative RT-PCR. We can extract the total RNA in peripheral blood firstly, then detect its purity and integrity, then reverse transcription (RT) in progress. The reaction system contains5×Buffer2μl, RT Enzyme Mix I0.5μl, Oligo dT Primer(50M)0.5μl, Random6mers(100M)0.5μl, Total RNA500ng, RNase Free dH2O make up to10μl, then Followed by incubating37℃,15min,85℃,5s to complete the RT. application of software we designed primers with Primer Premier5.0and took (3-actin as control. Forward and reverse primers of survivin mRNA are5’-GTCCCTGGCTCCTCTACTG-3’and5’-GACGCTTCCTATCACTCTATTC-3’respectively, and the size of product is74bp. Forward and reverse primers of β-actin are5’-AGAGCCTCGCCTTTGCCGAT-3’and5’-TGCCAGATTTTCTCCATGTCGT-3’ respectively, and the size of product is313bp. The reaction system contains of PCR contains Premix Ex Taq (2X)12.5μl, forward and reverse primers each0.5μl, template DNA2μl, d2H2O9.5μl. Conditions of PCR were an initial denaturation at95℃for30s, followed by34cycles at94℃for5s,60℃for30s,72℃for50s, and ending with72℃for2min. The cycles and annealing temperature of in survivin group and β-actin group are respectively34and60℃and28and53℃. The products were identified by2.5%agarose gel electrophoresis, and measured the optical density (OD) of β-actin and survivin with Pei-Qing gel imaging analysis system, then worked out the expression coefficient of survivin, the OD ratio of survivin and beta-actin. Analysis of data was performed using the computer software Statistical Package for Social Sciences (SPSS) for Windows (version18.0). Analysis of measurement data using the t test or analysis of variance, and enumeration data using the chi-square test, the difference was significant when P<0.05.Result:1. The positive rates of anti-survivin antibody were respectively40.5%and0, and survivin mRNA were respectively76.7%and0, in peripheral blood of patients with colorectal cancer and healthy people, and differences were significant (P=0.000).2. The levels of anti-survivin antibody and survivin mRNA were closely correlated with the advance of tumor stage and tumor size and lymph node metastasis, but not correlated with the age, gender, tumor location or histological type. The expression levels of anti-survivn antibody and survivin mRNA increased with higher tumor stage; in tumors, diameter>5cm, were significantly higher than those≤5cm (P0.045, P=0.003); in tumors with lymph node metastasis were significantly higher than those without lymph node metastasis (P=0.040, P=0.000).3. After radical surgery, the expression levels of anti-survivin antibody and survivin mRNA in74CRC patients were significantly lower than preoperation (P=0.005, P=0.004).13CRC patients had recurrented or metastasized during follow-up, both of anti-survivin antibody and survivin mRNA significantly increased in the blood of these patients (P=0.024, P=0.048). In addition, the expression levels of anti-survivin antibody and survivin mRNA in recurrent or metastatic tumors were significantly higher than in not recurrent or not metastatic one after radical resection (P=0.001, P=0.004). In14cases with palliative surgery, there was no significant difference between before and after palliative surgery in anti-survivin antibody and survivin mRNA expression levels (P=0.709, P=0.669).4. In palliative chemotherapy cases, the expression levels of anti-survivin antibody and survivin mRNA after chemotherapy were significantly lower than before chemotherapy in the effective group (P=0.043, P=0.032); In the ineffective group, anti-survivin antibody was no significant change (P=0.135), but survivin mRNA expression in cases after chemotherapy was significantly higher than those before chemotherapy (P=0.039). Patients who have low levels of anti-survivin antibody and survivin mRNA are more sensitive to chemotherapy (P=0.001, P=0.006). Conclusion:1. There are high levels of anti-survivin antibodies and survivin mRNA in peripheral blood of CRC patients, survivin in blood maybe one of the incidence indicators of CRC.2. The levels of anti-survivin antibody and survivin mRNA were closely correlated with tumor burden, tumor stage and lymph node metastasis, might serve as one of the indicators of prognosis.3. Survivin in peripheral blood may be one of the indicator to monitor micrometastasis and determine the prognostic. After radical resection, anti-survivin antibodies and survivin mRNA levels can predict the occurrence of metastasis or recurrence; the detection of the anti-survivin antibodies and survivin mRNA levels can monitor metastasis or recurrence during follow-up on a regular basis.4. Anti-survivin antibodies and survivin mRNA expression levels can be used as one of the indicators of sensitivity screening before palliative chemotherapy. Level changes of both them can be used as one of the indicators to evaluate the efficacy of chemotherapy. |
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