| Many studies have shown that angiogenesis plays an important role in the growth, progression, and metastasis of solid tumors. Recently , several angiogenic factors have been identified. Vascular endothelial growth factor(VEGF) is thought to be one such angiogenic factor and is also thought to be a selective mitogen for endothelial cells. VEGF acts on endothelial cells to increase microvascular permeability , resulting in the extravasation of plasma proteins, including fibrino-gen,into the extravasation space. Four different isforms of VEGF transcripts encoding peptides of 121,165,189,and 206 amino acids have been reported to be expressed in human cells. VEGFm and VEGF165 are secreted in soluble form, whereas the two large isforms ( VEGF189 and VEGF^) remain associated with cells because of their stronger affinities for cell - surface proteoglycans.VEGF exists in many human tissues, especially in esophagus, lung, grands, kidney and liver etc. It keeps normal vascular density and permeability in these tissues. In recent years,the over ?expression of VEGF was found in a variety of tumor tissues. Because the VEGF expression has been shown to be strictly related to neovascularization and poor prognosis in different types of human cancers, it may be useful to analyze the VEGFmRNA expression with particular regard to its differ-ent isfbrms. The role of VEGF in the behavior of human cancer could be particularly interesting because it might represent an excellent target for the development of new anti - tumor strategies based on inhibition of tumor angiogenesis.Esophageal carcinoma is more and more threatening the health and life of human. In this study, we examined and analyzed the is-forms of VEGFmRNA in primary esophageal carcinoma by reverse transcription polymerase chain reaction (RT -PCR) to define the role of VEGF in this kind of cancer.MATERIALS AND METHODSEsophageal carcinoma tissues and surrounding esophageal tissues from 36 patients who received surgery at the First Affilated Hospital of China Medical University and the Liao Ning Tumor Hospital between July 2000 and Octber 2001 were frozen at - 80℃ The patients ranged in age from 33 to 78 years( average age 59. 3 years) ;24 were men, and 12 were women. They were all squamous cell carcinomas. 14,13 , and 9 of the cases were respectively classified as to TNM stage I ^ II > HI. The patients had no detectable metastases in distant organs at the time of surgery. No paitent had received chemotherapy or radiation therapy before surgery.1. Extraction of total RNA; Total UNA was extracted from tissue samples using a RNA extraction reagent, TRIzol reagent ( GIBCOBRL, Int) . The concentration of total RNA was adjusted to 0. 5ug/ul.2. Reverse Transcription( RT) :2 ul of total RNA were incubated in 12ul of reaction buffer( MgCl22ul;10 RAN buffer 1. 25ul; Rnase Free dH2O 4.5ul; dNTP 1 1; Rnase Inhibitor 0.25 ul; Reverse Tran-scriptase 0. 5 ul; Random 9 mers 0. 5 ul) that was purchased as anUNA PCR kit(AMV)Ver2. 1(TaKaRa biology, China). The RT reaction was carried out in one cycle at 30℃ 10min; 50℃ 30min; 99℃ 5min; 5℃ 5min.3. Polymerase Chain Reaction ( PCR ) : PCR primers ( Sangon, China) for VEGFcDNA can simultaneously detect VEGF,21 (243bp) , VEGF165(375bp), VEGF189(447bp) and VEGF206(498bp) fragments according to the VEGF gene structure. Aliquots of 6pJ first strand cD-NA were mixed with 22ul of PCR mixture. The PCR reaction was carried out in three steps as follows; 95℃ for 2min( 1 cycle) ; 94℃ for 1min; 55℃ for 1min; 72℃ for 1. 5min(28 cycle); and 722 for 7min( 1 cycle)4. PCR fragments were analyzed by electrophoresis on 12% poly-acrylamide gels, and DNA was visualized by ethidium bromide staining.RESULTS1. RT - PCR analysis of VEGFmRNA in esophageal carcinoma and surrounding esophageal tissues; In 36 esophageal carcinoma tissues, there were 28(77. 8%) cases expressed VEGF12,mRNA, 28 (77.8%) cases expressed VEGF165mRNA, and 22(61. 1%) cases expressed VEGF189 mRNA. The there isforms were respectively expressed...
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