| All over the world, lung cancer has turned in to the most common malignant tumor since 1980s. Lymphatic metastasis is the most common pathway of cancer dissemination, and the metastatic spread of tumor cells is responsible for the majority of cancer deaths. For most human malignant tumor, tumor metastasis to regional lymph node is a crucial step in the progression of cancer. Detection of tumor cells in lymph nodes is an indication of the clinical stage, and is used clinically as a prognostic tool and a guide to therapy. Angiogenesis and lymphagenesis provide new vessels that malignant cells can use to escape the confines of the primary tumor. Neogenesis of lymphatic vessel and lymphatic invasion is frequently found in the stroma of cancers, but the mechanisms of this phenomenon remain unclear. Lymphangio-genesis has traditionally been overshadowed by the greater emphasis placed on the blood vascular system ( angiogenesis). This is due in part to the lack of identification of lymphangiogenic factors, as well as suitable markers with which to distinguish blood from lymphatic vascular endothelium.In recent years, this status is changing rapidly following the discovery of some lymphangiongenic factors and markers such as VEGF -C and VEGFR - 3. One of the VEGF - C receptor VEGFR - 3 is mainly expressed in lymphatic endothelium of adult human tissues. Inthe present study we detected the VEGF - C and VEGFR - 3 expressions , lymphatic vessel density and microvessel density in NSCLC and explored the correlation between these factors and clinicopathological parameters and prognosis.MATERIALS AND METHODS1. Tissue samples76 NSCLC cases with neighboring noncancerous tissue samples were obtained from patients who had received surgery in the Anshan Tumor Hospital between 1980 and 2001, fixed in 4% paraformalde-hyde, embedded in paraffin. 24 fresh NCSLC tissues with neighboring noncancerous tissue samples were obtained from the First Affiliated Hospital of China Medical University between Sep. 2001 and Mar. 2002.2. ReagentsThe primary antibodies used in this study were an anti - VEGF -C rabbit polyclonal antibody at 1:150 dilution ( Zymed Laboratories Inc. , USA) , an anti - VEGFR - 3 rabbit polyclonal antibody at 1; 1000 dilution (Santa Cruz, USA) , and an anti ?CD34 mouse monoclonal antibody (FuZhou MaiXin company, China). The Ultro ?sensitive S - P kit and DAB agent kit were bought from Fujian Maxin Biological Company. The goat - anti - rabbit second antibody was bought from Huamei Biological Company.3. Methods3. 1 Immunohistochemistry for VEGF - C, VEGFR - 3, and CD34We detected the VEGF - C, VEGFR - 3 expression by immuno-histochemistry S - P method. Microvessel density was determined byimmunohistochemistry for CD34. After the areas of highest vascular-ization had been chosen under low power, vessel count within tumors was carried out in 3 fields at 200 magnification. The average counts of the 3 fields were recorded, and the mean value of the 2 investigators was used for the MVD. The number of VEGFR -3 - positive endothe-lial cells was determined in the same manner. For evaluation of VEGF - C and VEGFR - 3 immunostainings in cancer cells, cases in which at least 10% of cancer cells were immunoreactive were defined as being positive.3. 2 Western blot analysis of VEGF - CRapidly homogenize tissues (1 -2g) in 5 volumes of lysis buffer (50mM Tris - HCL pH 7. 4, 0. 1% SDS, 1% Triton - 100, 1 mM EDTAPH8. 0, Aprotinin 12ul/10ml, PMSF 60ul/10ml) centrifuge the homogenate(10,000rpm, 4℃ ) for 30 minutes to pellet insolublematerial. (1) Electrophoresis (2) transfer proteins from gel to membrane (3) blocking (4) incubation with primary antibody (5) enzyme conjugate incubation (6) substrate incubation.4. Statistical analysisAll statistical calculations were carried out using the Statistical Product and Services Solutions (SPSS) statistical software. The Chi -square test, Linear regression and correlation, and Student t ?test were used to analyse data. Survival curves wer...
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