| Research background and objectiveWorldwide, more than 80% of esophageal cancers happen in developing countries. According to global cancer statistics report for 2008, The incidence of esophageal ranked No.4 in the world. According to the 2012 Annual Report of China Cancer Registry, The incidence of esophageal cancer in China was the fifth in malignant tumor. Esophageal cancer mortality rate ranks No.4 of malignancy. The incidence of malignancy is a variety of factors related to the pathological process of multiple steps. Esophageal cancer is the results of many factors interact to cause malignant transformation of normal cells. The natural environment, social economic status, living conditions, diet, age, immune status, genetic quality, tumorigenic viruses and other factors are closely related with esophageal cancer. In recent years, due to the rapid development of molecular biology techniques, our study of cancer gene is also deepening. At present a more consistent view for tumor occurrence, development is a process of multiple genes, multiple stages, and multiple step incremental evolution. The process involves mutation and inactivation of oncogene and inhibition of activation of many genes. The oncogene and tumor suppressor gene is the study of two kinds of genes most; a variety of key gene change is the basis of human tumor formation and development.PTEN (phosphate and tensin homology deleted on chromose ten) is a novel tumor suppressor gene found in March 1997. The study found that, PTEN gene distribution in many tissues, especially the high expression level of heart, brain, lung, liver and kidney tissues. The PTEN gene plays an important role in maintaining the proliferation, differentiation and apoptosis of cells, and may participate in the regulation of cell growth, invasion, metastasis and angiogenesis of tumor. With further research, there have been a variety of PTEN expressions in tumor tissue, endometrial cancer, glioblastoma, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, thyroid cancer, lung cancer, stomach cancer, colorectal cancer. And found that there is a close relationship between PTEN gene and tumorigenesis, invasion, metastasis and histological grading.The activation of oncogenes and (or) tumor suppressor gene mutation, deletion, inactivation is the basis of all kinds of tumor occurrence, development, mainly for the gene mutation, reduced or no expression of abnormal methylation, mRNA or protein expression. The study found that PTEN gene mutation and inactivation lost to the negative regulation of cell growth, is closely related with a variety of malignant tumors. The biological function of PTEN is mainly in the following points: 1, inhibition of cell proliferation and induce the apoptosis of cells.2, inhibition of cell migration and adhesion of the local.3, participate in the embryo development.4, inhibition of angiogenesis.5, To maintain the stability of the immune system.6, PTEN is a hematopoietic stem cell self replication for gene regulation and gene promoter in leukemia. Now we considered that PTEN gene mechanism mainly completed by the following three ways:1,focal adhesion kinase (FAK) pathway: PTEN has phosphatase activity, can make the dephosphorylation of FAK, inhibiting the activity of FAK, reduce the formation of cell proliferation mediated by integrin and local adhesion, resulting in inhibition of cell invasion and metastasis.2, mitogen activated protein kinase (MAPK) signal pathway:PTEN negatively regulates the MAPK signaling pathway, can inhibit the growth of tumor cells.3, phosphatidylinositol-3-phosphate (PIP3) pathway.PTEN encodes a protein with lipid acid enzyme activity, can decrease the level of PIP3, the cell stops in G1 phase, and induce the apoptosis of tumor cells.Malignant tumor growth and metastasis must rely on the plentiful supply of nutrients. If there is no angiogenesis, tumor growth does not exceed 4mm3 in vitro tissue volume, and does not exceed 1～2mm3 in vivo tumor. Therefore, angiogenesis is essential for tumor growth and metastasis conditions. Angiogenesis is closely related with tumorigenesis, development and metastasis. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) can specifically promote cell division, proliferation and migration plays a critical role in tumor angiogenesis.VEGF is a powerful and can produce a variety of biological effects of cytokines, which are separated in 1989 Ferrarra bovine pituitary follicle stellate cells purified from the culture medium of a class of glycoproteins. VEGF is a member of the platelet-derived growth factor (PDGF) family, is widely distributed in human and animal brains, kidneys, liver, spleen, pancreas, and bone and other tissues. VEGF-related gene family comprises six secreted glycoproteins, namely VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PDGF), and vascular endothelial growth endocrine origin factor (EG-VEGF) and 5 types of vascular endothelial growth factor receptor (VEGFR). VEGF has the following main biological function:1, promote vascular endothelial cell proliferation.2, increased vascular permeability.3, support the generation of content of blood vessels. 4, and inhibit the apoptosis of tumor cells. The related factors of expression and regulation of VEGF mainly include the following:1, hypoxia-inducible factor-1 (HIF-1).2, activator protein-1 (AP-1).3. Signal transducer and activator of transcription3 (STAT3).4, stimulating protein-1 (SP-1).5, stromal cell-derived factor-1 (SDF-1) and CXCR4.6, insulin-like growth factor 1 (IGF-1).7, platelets.8, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). In 1971, Folkman put forward the tumor growth is angiogenesis dependent, increase of the number of tumor cells (more than 106 cells), and the lesions increased, must rely on local vascular tumor of newborn. If there is no angiogenesis, tumor growth will be limited, because there is not enough oxygen and nutrient supply, tumor cells can only obtain nutrients through diffusion, tumor reached 1-2mm diameter or thickness, which is about 107 cells will no longer increase. Once the new blood capillary length human tumor tissues, nutrient supply by diffusion into the perfusion of tumors, tumor growth will accelerate.Tumor growth is divided into two distinct phases:avascular stage and the vascular phase. In stage avascular entity tumor growth can’t more than 2mm in diameter, which is the blood oxygen and nutrients through mass effect can achieve distance. Avascular tumor period no transfer capability. Conversion from vascular phase to the vascular phase is called the "angiogenic switch". Once a tumor through "switch" transformed into vascular phase, capillary formation cause tumors to obtain blood supply and nutrition enough. Tumor vascular structure is lack of integrity, the wall is weak, only a layer of endothelial cells are arranged, the lack of smooth muscle, basement membranes thinner or absent, making them more easily than those of mature vessels by tumor cells to penetrate. In addition, angiogenesis itself has a certain tissue invasion, tumor cell invasion and angiogenesis can along the open collagen fracture, can also enter the blood circulation is formed in the far away from the site of tumor metastasis.Angiogenesis is a complex process, mainly for the normal metabolism of blood vessels to maintain a balance between stimulating factor is broken, and in the pro-angiogenic factor activity increases and (or) angiogenesis inhibitory activity reduction factor. Tumor cells, endothelial cells and macrophages is regulated by hypoxia, system of stimulation that local factors micro environment changes and the synthesis and release of the factor. When the two between the balances is broken, tumor angiogenesis process. Regulations of VEGF factors mainly include angiogenesis factor and formation inhibition factor. Including angiogenesis factor: vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), integrin, angiopoietin (Ang), insulin likes growth factor Ⅱ (IGF-Ⅱ). Angiogenesis inhibiting factor mainly includes:angiostatin, endostatin, platelet-1 reactive protein (TSP-1), tissue inhibitor of metalloproteinase, serine protease inhibitor and tumor suppressor gene product etc.Microvessel density (MVD), or microvessel count (MVC) is a marker of tumor angiogenesis. Weidner reported that has relevance to vascular number at high magnification and tumor growth, and put forward the microvessel density (MVD) of the concept and the method for the determination of the background, namely with distinct endothelial cells or cell clusters, as long as all as one microvessel separate and adjacent vessels, the tumor cells or other connective tissue, lumen area of more than 8 red blood cell diameter and thick muscle layer of the microvessels are not counted in the "hot" area, counting the number of microvessels. At present, MVD count method to Weidner the proposed scheme is the most representative. MVD markers are the following:1, anti-factor Ⅷ antibody.2, platelet endothelial cell adhesion molecule (CD31).3, CD34.4, CD 105. The neovascularization of multi by immunohistochemistry on endothelial cell staining, CD31 marker microvascular sensitive, reliable, and clear background, less cross reaction, are used for markers of vascular endothelial cells, thus the calculation of MVD, and then judge whether the blood supply to tumors, progress may be helpful in determining tumor.At present due to the lack of effective to evaluate the diagnosis and differential diagnosis of esophageal cancer detection means, thus to explore the mechanism of occurrence and metastasis of esophageal and the diagnosis of esophageal cancer, assessment has become one of the hot spot of current research. PTEN, VEGF and MVD are closely associated with the occurrence and development of tumor, but the combined detection of PTEN, VEGF and MVD expression in esophageal squamous cell carcinomas of the research literature at home and abroad is less. To investigate whether PTEN, VEGF, MVD expression and its level of expression with the occurrence, invasion and metastasis in esophageal squamous cell carcinoma in the existence of relationship,SP immunohistochemical method in this paper was used to detect the expression of 50 cases of esophageal carcinoma and tumor adjacent normal tissues of PTEN, VEGF protein and MVD. The relationship between PTEN, VEGF and MVD was also discussed. Through the above experiment, attempting to clarify the expression of PTEN and VEGF protein and MVD occurred in ESCC development, invasion and metastasis of significance for further study of esophageal squamous cell carcinoma, invasion, and metastasis of esophageal squamous cell carcinoma and seek to suppress the occurrence and development of approach provides a theoretical basis.Materials and methodsWe selected 50 cases of Nanfang Hospital and Rizhao People’s Hospital from 2009.1 to 2014.1 complete clinical and pathological data archived tissue specimens of esophageal carcinoma operation.Each specimen in the tumor tissues and the distal normal mucosa (pathology confirmed mucosa) were drawn at. We used immunohistochemical SP method detected 50 cases of squamous cell carcinoma and normal mucosa in the distal PTEN, expression of VEGF and MVD, and the expression and relationship between the three cases were analyzed.Results1. The expression rate of PTEN protein in esophageal squamous cell carcinoma (54%) was significantly lower than that of normal esophageal mucosa PTEN protein expression rate (100%). Through statistical analysis, the difference was statistically significant (P<0.01). The rate of PTEN protein expression in esophageal squamous cell carcinoma is decreasing with decreasing cancer differentiation degree (gradeⅠ 84.2%, gradeⅡ 47.1%, grade Ⅲ 21.4%). Through statistical analysis, the difference was statistically significant (P<0.01). The PTEN protein expression rate (80%) of Shallow layer invasion group was higher than that (40%) of deep layer invasive group. Through statistical analysis, the difference was statistically significant (P <0.01). The PTEN protein expression rates of Lymph node metastasis group (31.6%) were significantly lowers than that (67.7%) of without lymph node metastasis. Through statistical analysis, the difference was statistically significant (P<0.05).2. Adjacent normal mucosa VEGF protein expression rate (100%) was significantly higher than that of squamous cell carcinoma VEGF protein expression rate (64%). Through statistical analysis, the difference was statistically significant (P <0.05). The rate of VEGF protein expression in esophageal squamous cell carcinoma is increasing with decreasing cancer differentiation degree (gradeⅠ 52.6%, gradeⅡ 64.7%, grade Ⅲ 78.6%). After statistical analysis, the difference was not statistically significant (P>0.05). The VEGF protein expression rate of Shallow layer invasion group (73.3%) was higher than that of deep layer invasive group, (60%). After statistical analysis, the difference was not statistically significant (P>0.05). The VEGF protein expression rates of Lymph node metastasis group (63.2%) were lowers than that (64.5%) of without lymph node metastasis. After statistical analysis, the difference was not statistically significant (P>0.05).3. Adjacent normal mucosa MVD value (25.282+3.477) was significantly lower than that of squamous cell carcinoma MVD value (42.224+9.3348). Through statistical analysis, the difference was statistically significant (P<0.01). The MVD value in esophageal squamous cell carcinoma is increasing with decreasing cancer differentiation degree (gradeⅠ 34.8211 ±3.3186, gradeⅡ 40.5647±5.2354, grade Ⅲ 54.2857 ± 6.2518). Through statistical analysis, the difference was statistically significant (P<0.01). The MVD value of deep layer invasion group (43.4943 ± 9.0385) was higher than that of Shallow layer invasive group (39.2600±9.6505). After statistical analysis, the difference was not statistically significant (P>0.05). The MVD values of Lymph node metastasis group (46.0053 ± 8.4854) were higher than that (39.9065 ± 8.4854) of without lymph node metastasis. After statistical analysis, the difference was not statistically significant (P>0.05).4. VEGF protein positive expression rate of PTEN protein expression positive cases (55.6%) is lower than that of the expression of PTEN protein positive expression rate of VEGF protein negative cases (73.9%). After statistical analysis, the difference was not statistically significant (P>0.05). The MVD value among the expression of PTNE protein positive cases (38.76+7.96) is lower than that among the expression of PTEN protein negative cases (46.29+9.34). Through statistical analysis, the difference was statistically significant (P<0.01). The MVD value among the expression of VEGF protein positive cases (46.33 ± 8.99) is lower than that among the expression of VEGF protein negative cases (34.92 ± 3.92). Through statistical analysis, the difference was statistically significant (P<0.01).Conclusion1. The expression of PTEN protein is lower than that of normal esophageal mucosa. The expression of PTEN protein in esophageal squamous cell carcinomas is related with histological grade, invasion depth and lymph node metastasis.2. The expression of VEGF was significantly higher than that of normal esophageal mucosa protein expression of VEGF protein in esophageal squamous cell carcinoma. However, the positive expression of VEGF protein in squamous cell carcinoma of the esophagus is unrelated with the histological grade, invasion depth and lymph node metastasis.3. The MVD in esophageal squamous carcinoma tissues are significantly higher than that of normal esophageal mucosa. MVD expression in esophageal squamous carcinoma tissues related to histological grade and lymph node metastasis, and has nothing to do with the infiltration depth.4. The expression of PTEN protein may be a negative relationship between the expression of VEGF protein, and MVD. The expression of VEGF protein and MVD may be positively correlated relationship.5. The combined detection of PTEN, VEGF and MVD can be used as an objective index for judging the invasion and metastasis of esophageal squamous cell carcinoma. Prognosis of esophageal squamous cell carcinoma has important significance of judgment. |
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