| RT-PCR and immunohistochemistry detecting VEGF-C and his receptor Flt-4 in primary non-small lung cancer and the clinical significance Objective Malignant tumor is characterized by the metastasis of tumor cells via vessels and lymphatic ducts. The mechanism of tumor induced formation of lymphatic ducts and its role in the development of metastasis is still unclear. VEGF-C is a member of VEGF family, acts as a highly specific mitogen and serves as the stimulation of vessels and lymphatic ducts formation. Researches have suggested that the combination of VEGF-C and its specific receptor (VEGFR-2/VEGFR-3,Flt-4) can cause signal transfer and play an important regulatory role in tumor genesis, progress and metastasis. This study is to detect the expression of VEGF-C and its receptor Flt-4 in NSCLC, paracancer tissues and metastatic lymphatic nodes by RT-PCR and immunohistochemistry (S-P methods) and to investigate the role of VEGF-C and its receptor in NSCLC genesis, progress and metastasis and the relation between the expression and pathological characters. Materials and methods Detection of VEGF-C and its receptor (Flt-4) by RT-PCR tissue sample: 40 fresh NSCLC specimens confirmed by surgery and pathology and 12 paracancer samples were obtained from Sept. 2003 to Dec 2003. Specimens were put into Eppendorf tube and kept by liquid nitrogen at -70℃ for analyses. Total RNA was extracted by routine method. VEGF-C (152bp) and Flt-4 (259bp) primers were synthesized at Dalian biology Ltd. The internal contrast is β-actin (690bp). This was synthesized by Beijing Auker biology Ltd. Synthesis of cDNA: the toltal volume is 20ul. RT reaction condition is as follows: 65℃ pre-degeneration 1min; 30℃pre-degeneration 5min, 65℃ renaturation 30min, 15'-30'equally heat, 98℃ extension 5min; 5℃ extension 5min. PCR amplification: (1) the total reaction volume of VEGF-C 25ul. PCR reaction: 94℃ pre-degeneration 3min, 94℃ generation 45Second, 51℃renaturation 1min, 72℃ extension 1min, recycle 35; 72℃extension 7min. (2) the total reaction volume of Flt-4 is 25ul. PCR condiction: 94℃ pre-degeneration 3min, 94℃ degeneration 45 Second, 55.5℃renaturation 1min, 72℃ extension 1min, recycle 35; 72℃extension 7min. (3) the total reaction volume ofβ-actin is 25ul. PCR condition: 94℃ pre-degeneration 3min, 94℃ degeneration 1min, 55℃renaturation 1min, 72℃ extension 2min, recycle 35; 72℃ extension 7min. products analysis: after 2% agarose gel electrophoresis (40'-60', 60V) of VEGF-C,Flt-4β-actin PCR% and colouration with ethidium bromid, detect the relative amount. Determine the intensity of ethidium bromid stain. (formula: VEGF-C relative amount=VEGF-C density/β-actin density; F lt-4 relative amount = F lt-4 density/β-actin density). statistical analysis: count variables were expressed by average±standard deviation (±s) and compared by the x2 test and F analysis. Quantitative variables were compared by means of the student t test. All the procedure was performed in SPSS 10.0 software kit. detection of VEGF-C and Flt-4 by immunohistoch- emistry specimen: 60 specimens of NSCLC confirmed by pathology were obtained from Jan 1998 to Oct. 2003. At the same time, 10 cases of normal tissues were obtained from the adjacent tissue. All specimens performed HE and immunohistochemistry. immunohistochemistry: S-P procedure is applied according the instruction. Positive expression of VEGF-C and Flt-4 located in cytoplasm with the formation of brown-yellow granules. We counted 5 fields with a total of 1000 cells. If the positive cells are more than 10 percentages, we defined it as positive. Count Flt-4 positive vessels in 5 fields and take an average. statistical analysis: Data was analyzed by SPSS 10.0 software kit and compared by X2 test,t test and Kendall correlation analysis. Result detection of VEGF-C and Flt-4 by RT-PCR VEGF-CmRNA and Flt-4mRNA detection result: after 2% agarose gel electrophoresis of RT-PCR pr...
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