Relationship Between Lymph Vessels with Tumor-associated Macrophages in Colorectal Carcinoma Tissues
|School||Guangzhou Medical College|
|Keywords||Colorectal Cancer Lymphangiogenesis Tumor-associated macrophages D2-40 antibody Immunohistochemistry|
Background: colorectal cancer distant metastasis is one of the important factors in patients with treatment failure and death, the blood stream and lymphatic formation of distant metastases. The relationship between the blood vessels and the tumor has been more in-depth study, a long time, however, due to the lack of specific markers of lymphatic, making the study of tumor lymphangiogenesis has lagged far behind in angiogenesis research. D2-40 is a recently discovered marked lymphatic monoclonal antibody its specificity and high sensitivity, does not mark the vascular endothelial cells, it can be used lymphatic (lymph vessel, LV) count. The tumor-associated macrophages (tumor-associated macrophage, TAM) tumor infiltrating macrophages in tumor growth, invasion, metastasis, and play an important role in the recurrence. TAM and the relationship of angiogenesis research more that can promote angiogenesis. TAM and tumor lymphangiogenesis less likely one of the reasons is the lack of marker lymphatic-specific antibodies. The experimental application of new specific the lymphatic antibody (D2-40), using the method of immunohistochemical detection of human colorectal cancer tissues LV TAM expression of colorectal cancer lymphangiogenesis and TAM relationship of tumor The lymphangiogenesis mechanism. MATERIALS AND METHODS: Clinical specimens: colorectal 48 cases of rectal lesions tissue samples taken from December 1998 to December 2000, the Second Affiliated Hospital of Guangzhou Medical College, general surgery or colonoscopy resection and confirmed by pathology archive paraffin . Another 13 cases of normal colorectal tissues from the tumor tissue of colorectal cancer surgery 5cm above normal colorectal tissue pathology HE staining confirmed. Preoperative neither line chemotherapy or radiotherapy. Including 32 cases of colorectal cancer, 16 cases of colorectal benign lesions. 18 cases of colorectal cancer in men and 14 females; aged 38 to 93 years, with an average of 62.3 years old. 17 cases of lymph node metastasis in 15 cases without lymph node metastasis. Nine cases of the 16 cases of benign lesions, polyps, adenomas seven cases; 6 males and 10 females; aged 3.5 to 81 years, with an average of 54.7 years old. 2, antibodies and kits: marked lymphatic mouse anti-human monoclonal antibody D2-40 (type), were purchased from Invitrogen Corporation. D2-40 staining required mouse SP kit, DAB chromogenic kit were purchased from Beijing Zhongshan Biotechnology Company. , DAKO macrophage marker CD68 monoclonal antibody PG-M1 (1:100). CD68 staining process required SP-2001 kit, secondary antibody kit and DAB kit were purchased from Vector. All slices are equipped with positive and negative controls, and routine HE staining. 3, staining results determination and cell counting method: D2-40-positive cells located in the epithelial cell membrane lymphatic brownish yellow. Lymphatic microvessel count standard each positive staining of endothelial cells, endothelial cell clusters or single small vascular considered a micro-lymphatic. The first reference Weidner 100 magnification select the most densely populated areas of the five positive cells, then count under 200 magnification, respectively, calculated the average of each specimen. CD68-positive cells were stained in the cell membrane, was rusty. Reference Leek methods select the five most intensive areas of positive cells under low magnification first, and then counted under 400 magnification, and then calculate the average value of each specimen. 4, the statistical analysis: application SPSS11.5 statistical analysis software package for data analysis of variance, t test and Spearman rank correlation analysis, when the P lt; 0.05 was considered statistically significant. Results: immunohistochemical staining morphologic appearances: D2-40 antibody labeled colorectal lymphatic positive cells were brown, mainly distributed in the interstitium, rounded or oval and cords. Lymphatic vessels of the lymphatic colorectal lesions than normal colorectal tissues. CD68 marker of colorectal tumor-associated macrophages positive cells was rusty, mainly located in between the the mesenchymal cancer and cancer nests. Colorectal cancer lymphatic marker D2-40 is mainly distributed in the tumor the surrounding area, and number of lumen was expansion like in cancer the nest or cancer organization memory is scarce even cancerous tissue within the lymphatic form of bars cable or lockout status. Serial sections stained lymphatic tumor-associated macrophage distribution is almost identical, and are distributed in the tumor stromal and cancer cells has not been positive staining. Immunohistochemical staining cell count analysis: colorectal cancer, colorectal benign lesions LV count was significantly higher than the normal colorectal group LV were statistically significant (P lt; 0.001). Colorectal cancer lymph node metastasis group LV count higher than those without lymph node metastasis, and the difference was statistically significant (P lt; 0.05). Colorectal TAM counts higher than the TAM count colorectal benign lesions, the difference was statistically significant (P lt; 0.05). 3, LV and TAM count of correlation analysis: The Spearman rank correlation analysis, the colorectal cancer group LV count with TAM count was being related (r = 0.58, P lt; 0.01); have lymph node metastasis group LV count with TAM count was being related (r = 0.66, P lt; 0.01); without lymph node metastasis TAM count with LV count showed a positive correlation (r = 0.56, P lt; 0.05). Benign lesions, the LV count and TAM count was close negative correlation (r = -0.66, P lt; 0.01). LV and TAM count and patient survival period: 32 patients with colorectal cancer, 25 cases of complete follow-up results, which have been 23 cases of deaths, the two cases are still alive. The shortest survival time after surgery for five days, the longest survival time of 128.7 months, the average survival time of 72 months. After statistical analysis, the LV counts and survival in patients with colorectal cancer months was a negative correlation (r = -0.53, P lt; 0.01); TAM count and survival in patients with colorectal cancer months was negatively correlated (r = -0.65 , P lt; 0.01). Conclusion: colorectal cancer and benign lesions have the lymphangiogenesis the formation. Patients with colorectal cancer, TAM may promote tumor lymphangiogenesis, which may promote tumor lymph node metastasis; the benign colorectal organization, TAM may play a role in inhibiting lymphangiogenesis.