| IntroductionSimilar to other malignant tumors, the undesirable prognosis in the patients with laryngeal squamous cell carcinomas ( LSCC) has been attributed to cervical lymph nodes metastasis. In LSCC, lymph nodes metastasis was the most important event in the failure of the treatment of LSCC. Therefore, it is a major goal to identify the molecular mechanisms of invasion and metastasis of the LSCC. Metastasis is a complex series of course that involves several gene products. Angio-genesis is essential for tumor growth and metastasis. Increased vascu-larity enhances the growth of primary neoplasms and provides a greater chance for hematogenous metastases. An association between poor prognosis and increases in vascularity has been reported in certain tumors. Angiogenesis is thought to be regulated by a number of growth factors. Vascular endothelial growth factor (VEGF) is an important proangiogenic molecule. VEGF is a disulfide - linked dimeric glyco-protein that increases blood vessel permeability. VEGF and MVD (microvessel Density) have been documented their importance in both the development and progression of tumors in multiple models. App-ling to immunostain analysis might only be a portion of cells or tissues on a slide because of the thickness of the tissue section. RT - PCRtechnique is sensitive and specific in analysis and estimation of tumor micrometastasis by detecting the molecules interrelated with tumors.Experimental MaterialsThe 60 samples of laryngeal carcinoma were acquired from the patients hospitalizing and underwent operation in the ENT department of the first clinical hospital of China Medical University and LiaoNing Tumor Hospital between September, 2001 to February, 2002. Of them , there were 20 supraglotti laryngeal carcinomas patients with cervical lymph node metastasis ( the first group ) , 20 supraglotti laryngeal carcinomas patients with cervical lymph node - negative metastasis ( the second group ) , and 20 glottic carcinoma with cervical lymph node - negative metastasis ( the third group ) . All of the patients had not received either chemotherapy or radiotherapy before surgery. The pathological results were all sqamous cell carcinoma. LSCC were resected and snap - frozen in liquid nitrogen immediately after surgical resection. Specimens were stored at -Experimental methodsWe applied immunohistochemistry and RT - PGR technique to detect the VEGF expression at two level of protein and molecule and applied immunostain method and colorful multiple image analysis system (CMIAS) to measure the MVD. A detection procedure for micro vessels was performed by using anti - CD31 monoclonal antibody. The immunohistochemistry was two steps method. The microvessels were counted by Werner method. VEGF expression was defined through the count of tumor cells being stained. The association of factors was evaluated by using the chi - square test. The significance ofdifferences among means of microvessels density was determined by t test . We used the SPSS 10.0 statistical software system (SPSS Institute, Inc) to do the calculations . P <0. 05 were considered to be statistically significant.ResultThe expression of VEGF mRNA by RT - PCR in the first group, 12 specimens was positive with 60% positive rate ; in the second group 3 specimens was positive, positive rate!5% ; in the third group 4 specimens was positive , positive rate 20% . The statistically significant difference between the first and the second group was observed ( P <0. 01 1,2 ) ,and between the first and the third group was observedImmunostaining for VEGF in the first group 13 specimens was positive with 65% positive rate; in the second group 4 specimens was positive positive rate 20% . The difference were statistically significant (P <0.05 1,2 ) ,and in the third group 5 specimens was positive, positive rate 25% . The difference between the first and the third group were statistically significant (P<0.01113).Immunostaining showed that the microvessl counts was a mean value of 14. 859 microvessels (stan... |
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