Abstract Objective To investigate the colorectal cancer tissues survivin (survivin), vascular endothelial growth factor (VEGF), cell nuclear proliferation-associated antigen Ki67 expression with clinicopathological factors relations, evaluation of survivin, VEGF and Ki67 in colorectal cancer prognosis judgments in value. Methods Immunohistochemistry PowerVision two-step method to detect the relationship between the 40 cases of colorectal cancer tissues of survivin expression of VEGF and Ki67, a retrospective analysis of survivin, VEGF and Ki67 with clinicopathological factors and prognosis. Results survivin, VEGF and Ki67 expression in colorectal cancer, the positive rate was significantly higher than in adjacent normal tissues (P <0.01), and Duke’s stage and lymph node metastasis was significantly related (P <0.05), Duke’s C stage D colorectal cancer tissues was significantly higher than Duke’s A Phase B period, survivin, VEGF and Ki67 positive rate of lymph node metastasis was significantly higher than that without lymph node metastasis. survivin, VEGF and Ki67-positive expression in patients with 3-year and 5-year survival rate was significantly lower than the negative expression (P <0.05). Conclusions in colorectal cancer occurred, accompanied survivin, VEGF and Ki67 expression in the development process, the detection of colorectal cancer tissue of survivin, VEGF and Ki67 might lymph node metastasis of the judgment of colorectal cancer and the progress of the disease have a certain value. survivin, VEGF and Ki67-positive patients with 3-year, 5-year survival rate was significantly lower detection three auxiliary prognostic assessment of patients with colorectal cancer.

Key words survivin VEGF Ki67 colorectal immunohistochemistry

    The formation and development of the malignant tumor apoptosis inhibition, angiogenesis and tumor cell clonal proliferation. survivin is a new member of the inhibitor of apoptosis family of factor IAPs (Inhibitors of apoptosis), is considered in the bcl  2 downstream blocking cysteine ​​protease Caspase  3 and Caspase  7 regulation of apoptosis pathway [1], in the normal not expressed in the tissue, while the selective expression in malignant tissue. In the induction of tumor angiogenesis factor, vascular endothelial growth factor (VEGF) is considered to be the most important angiogenic factor [2]. Tumor cell proliferation activity indicators to reflect a lot to Ki67 most representative [3].

    The experiment is to study the relationship between the expression of the three indicators in colorectal cancer tissues and clinicopathological factors as well as the 3-year and 5-year survival rate, provided the experimental basis for future clinical prognosis.

    1 Data and methods

    1.1 General Information

    Collection Jiangsu Cancer Hospital Surgery from 1999 to 2000, the colorectal surgical resection specimens of 40 cases, including 25 males and 15 females, age 24 to 79 years, with an average age of 56 years, including colon cancer in 20 cases, 20 cases of rectal cancer. All samples were 4% neutral formalin-fixed, paraffin-embedded, HE staining, light microscopy. Pathological types: tubular adenocarcinoma, 23 cases, 12 cases of papillary adenocarcinoma, mucinous adenocarcinoma, 5 cases. Duke’s stage: two cases A, B 17 cases, 12 cases of C, D, 9 cases. Radiotherapy, chemotherapy before surgery had no history. 1 to 60 months of postoperative follow-up, 25 cases of death due to disease progression, mortality was 65.0%.

    1.2 Reagents and Methods

    -survivin mouse anti-human monoclonal antibody (diluted 1:50), VEGF rabbit anti-human polyclonal antibody (working concentration of 1:50), the Ki67 in rabbit anti-human polyclonal antibody (working concentration of 1:50), Universal immunohistochemistry. the PV kit staining kits were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.. Immunohistochemical PowerVision two-step method, the reference to the kit instructions, instead of primary antibody with PBS as a negative control, known positive expression in gastric cancer specimens as a positive control.

    1.3 positive criteria

    Brown granules located in the cytoplasm of the free edge of the tumor cells for the expression of survivin, VEGF positive cells the brown particles located in the nucleus of tumor cells for Ki67-positive cells. Randomly select 10 vision, calculate the percentage of positive cells in the microscope, the cells. Positive staining cells> 10% positive expression.

    1.4 statistical analysis

    The relationship between survivin, VEGF, Ki67 expression status and colorectal cancer clinical and pathological features, univariate survival compared using Pearson chi-square test. All statistical SPSS10.0 statistical package.

    2 Results

    2.1 Immunohistochemical staining results

    survivin, VEGF positive staining was localized in the cytoplasm, including the free edge. 40 cases, 24 cases survivin expression, the positive rate was 60%, and 29 cases of positive expression of VEGF-positive rate of 72.5%, 10 cases of adjacent normal tissues had no survivin, VEGF expression. Ki67 positive staining localized in the nucleus, the 40 cases, 32 cases of Ki67 expression, the positive rate was 80%, two cases Ki67 positive expression in 10 cases of adjacent normal tissue. survivin, VEGF, Ki67 positive expression in the range of 10% to 80%, as shown in Figure 1 to 3.

    2.2survivin the expression of VEGF and Ki67 expression and colorectal cancer clinical pathological features of the relationship between the three and the patient’s gender, age, tumor location, size, histological type, no significant relationship (P> 0.05), and Duke’s staging and lymph node metastasis was significantly related (P <0.05), as shown in Table 1.

    2.3 Analysis of prognostic factors

    Follow-up results of 40 cases of colorectal cancer patients: 3-year survival of 24 cases, 3-year survival rate of 60%, 5-year survival of 14 cases, 5-year survival rate of 35%. survivin, VEGF and Ki67 expression status with the 3-year and 5-year survival rate was significantly related (P <0.05), as shown in Table 2.

    3 Discussion

    Colorectal cancer is one of the common malignant tumors in China, its occurrence and development mechanism has not been fully elucidated. The current study found that tumor apoptosis inhibition, angiogenesis and tumor cell clonal proliferation may have important role in the occurrence and development of colorectal cancer.

    survivin inhibitor of apoptosis protein (Inhibitor of apoptosis protein IAP) family members. survivin may be mainly two ways to inhibit apoptosis: one is blocking the process of apoptosis induced by various stimuli through direct inhibition of apoptosis terminal effector Caspase  3 and Caspase  7 activity; second is survivin cyclin kinase Cdk4 [4], p34cdc2 [5] interaction blocking apoptosis signal transduction pathways. Overseas research shows that, survivin inhibitor of apoptosis, vascular protection, regulation of cell cycle and other biological activity is closely related with the degree of malignancy and prognosis of colorectal [6]. This set of experimental results: 24 cases (60%) survivin positive expression in 40 cases of colorectal cancer tissues, 10 cases of adjacent normal tissues had no expression of survivin Kawasaki et al [6] of 171 cases of colorectal cancer and adjacent normal tissue research results are basically the same. This table in the normal table of survivin, VEGF, Ki67 expression and colorectal cancer clinical pathological features of the relationship between the relationship between the expression of survivin, of VEGF, the expression of Ki67 and 5 year survival rates

    The organization did not express survivin gene expression in tumor tissue characteristics prompted activated stage of colorectal tumorigenesis express survivin protein, can be used as molecular markers of colorectal cancer stage. The research data show survivin expression in colorectal cancer and colorectal cancer, Duke’s stage and lymph node metastasis was positively correlated (P <0.05), and significantly related (P <0.001), especially with Duke’s staging survivin positive expression in colorectal cancer development, and plays an important role in the transfer process. Analysis of survivin and 5 year survival rates of patients: relationship found between survivin positive group and negative group survival rate there are significant differences (P <0.05), survivin positive group, 3-year and 5-year survival rate of the patients was significantly lower with negative group . Prompt positive expression of survivin may be a poor prognosis pointer.

    Vascular endothelial growth factor (VEGF) is the 1989 Ferrara from bovine follicular Star line normal pituitary cells isolated and purified a specific protein. Its main role is to participate in vascular endothelial cell proliferation, angiogenesis and tumor invasion and metastasis [8]. The results of this study show that the expression of VEGF in colorectal Duke’s stage and lymph node metastasis, and no correlation with other clinical indicators, consistent with the Han et al [8] reported, suggesting that VEGF can be used as one of the signs to determine the progress of the disease . VEGF-positive patients with colorectal cancer 3-year and 5-year survival rate was significantly lower than the negative (P <0.05), With the increase in the degree of invasion and metastasis, survival time was significantly shorter.

    Is the most widely used one of the proliferative cell marker Ki67 involved in tumorigenesis and development [9]. This study analyzes the the Ki67 colorectal relationship found in colorectal of Ki67 expression was significantly higher than in adjacent normal tissues (P <0.05), clinical stage and lymph node metastasis of colorectal cancer was positively correlated with The survival time of patients was negatively correlated (P <0.05). From normal mucosa to colorectal cancer and its cell proliferation activity increased colorectal cancer cell proliferation activity of the higher, the greater the likelihood of invasion and metastasis, Ki67 as a prognostic indicator.

    In summary, colorectal upregulation of survivin, VEGF and Ki67 gene expression of colorectal cancer occurrence and development are closely related, to detect the three auxiliary prognostic assessment of patients with colorectal cancer significance.