| ObjectivesColorectal carcinoma (CRC) is a common gastrointestinal malignance all over the world. Recently, the incidence of CRC is in increasing in China. There is a close relationship between the immunologic dysfunction and the development of CRC. CRC expresses many tumor associated antigens (TAAs) that can be recognized by human immune system, and host immunity tries to attack CRC cells causing a large number of immune cells to recruit in CRC tissues. Whereas CRC cells will use diverse measures to escape from immune attack, that include the inhibitors of immune cell function via releasing of vary immune suppression factors. When immune cell is inhibited, its function may be changed and promotes tumor progression and metastasis.Macrophage is an important cellular component of the innate immune system, and has a wide range of functions, including phagocytosis, antigen-presenting and secretion of growth factor, cytokines and so on. Macrophage is a frequent observed immune cell in the tumor microenvironment, and had been named as tumor-associated macrophage (TAM). TAM plays a dual function during the development of CRC. On the one hand, it has the ability to directly kill tumor cell and present the tumor antigens to T cells; On the other hand, it can promote tumor growth, invasion and metastasis, advance blood vessel growth and organizational structure reconstruction. At present, there are considerable evidences showing that TAM promotes carcinoma cell growth in a variety of tumors. Some studies show that the development and progression of CRC is closely related to the expression of macrophages. However, its role in CRC is still controversial. Some studies revealed that macrophages possess the function of anti-tumor and are associated with better prognosis in CRC. Other studies showed that the development and growth of the tumor are related with a large number of macrophage infiltration.CD68is I type high-sugar base of the transmembrane protein, belongs to the glycoprotein lysosomal-associated membrane protein family. Generally, CD68is considered to be a selective marker of human monocytes and macrophages. Its expression is mainly adjusted by the macrophage-specific initiation factor, and it is widely used as the specific marker of monocyte-macrophage cell in the diagnosis and research.Traditionally, the expression of macrophages in the tissue is detected by immunohistochemistry staining methods with specific antibodies, but it is limited and impacted by the sensitivity. Real-time PCR is a sensitive technique in quantifying targeted genes and determining the sample DNA or cDNA copy number, and it has been widely used in various fields.In our experiment, real-time PCR associated with CD68was accurately used to quantify the expression of TAMs in the human CRC tissues, and explore the relationships between the expression of macrophages and clinical data of CRC patients. At the same time the expressions of TAMs were confirmed with the CD68monoclonal antibody by immunohistochemistry. And we can establish a new laboratory method that accurately detect the expression of TAMs in human CRC tissues, and can be extended to conduct quantitative detection of macrophages in blood or body fluids, and then provide accurate values for the diagnosis and prognosis of clinical diseases.Materials and methodsThe tissues of CRC were from surgical specimens and the normal colorectal tissues were collected by colonoscopy in the Second Affiliated Hospital of Zhengzhou University. The specimens were immediately preserved in the RNA-later preservation solution after removed, and stored in-80℃ultra-low temperature refrigerator ultimately. Meanwhile, the clinical data of patients were collected.1The mRNA expression of CD68was detected by real-time PCR in colorectal carcinoma and normal colorectal tissues.2The correlation of CD68real-time PCR results were analyzed with the clinical data of patients.3The protein expression of CD68was detected by immunohistochemistry in CRC and normal colorectal tissues.All statistical analysis was performed using SPSS statistical version16.0. The data were expressed as the mean±SEM and the differences between groups were evaluated with the Mann-Whitney U test. P<0.05was considered as statistical significance.Results1The expression of CD68mRNA in CRC was1.78-fold compared with the normal colorectal tissues (P<0.05).2The CD68mRNA expression levels were not significantly associated with the clinicopathological parameters of patients, such as:gender, location, TNM stage, Duke’s staging and involved lymph nodes (.P>0.05).3The CD68protein was observed in the cytoplasm of TAMs. The semi-quantitative results showed that the expression of CD68protein in lamina propria was (2.06±0.19) in CRC, and (1.36±0.13) in the control group. The difference was significantly (P<0.05). The expression of CD68protein in epithelial tissues was (0.83±0.23) in CRC, and (0.09±0.03) in the control group, the difference was significantly (P<0.05).Conclusions1The expression of CD68marked macrophages is significantly increased in CRC tissues than in the nonnal colorectal tissues.2Real-time PCR, combined with CD68, can precisely quantify the expression of tumor-associated macrophage in CRC.3Real-time PCR with CD68is a complementary method in identity TAMs in CRC tissues. |
PDF Full Text Request