| So far, there are few knowledge about etiological and pathophysiological mechanism of astrocytoma. In recent years, reseachers found that tumor angiogenesis is very important to tumor growth, invassiveness, metastasis and prognosis. Clinical studies found that density of microvessels is very related to tumor malignancy and prognosis. Therefore, elucidate pathophysiology of tumor angiogenesis will be likely helpful to the development of therapeutic strategies for astrocytoma.Nitric oxide (NO) is an inorganic free radical gas which is synthesized from L-Arginine by nitric oxide synthase(NOS) and it mainly act as signal and cytotoxic molecule. There are 3 isoform of NOS has been identified: neural NOS(nNOS), inducible NOS(iNOS) and endothelial NOS(eNOS). Among the 3 isoform, eNOS was identified that mainly exist in endothelial cells. NO synthesized by eNOS play a very important role in the regulation of vasodilation and vascular permeability. Excessive express of eNOS was found in several kinds of tumors and it can influence tumor blood supply.Except for NO, VEGF is also very important to tumor angiogenesis. Studies in vitroindicated that VEGF can promote proliferation of endothelial cell and vascular permeability. A series of intracytic signal transduction initiated by NO synthesized by eNOS partially induce the process. And the eNOS is activated by VEGF through PI-3K/Akt kinase. But other studies elucidated that NO can upregulate VEGF expression as well. However, there have been no report about alternation of eNOS in relation to density of microvessels, vascular endothelial growth factor(VEGF), malignancy and prognosis in situ. We immunohistochemically studied the relationship between eNOS, VEGF expression and density of microvessels, malignancy and prognosis in human astrocytoma and the possible affection of NO, VEGF in astrocytoma angiogenesis are discussed.MATERIALS AND METHODS Tissue samplesThirty seven tissue specimens were obtained from patients with astrocytomas in the first affiliated hospital of Medical college, Zhejiang University. Four astrocytic hyperplasia samples were obtained from the other patients as the control group. There were 23 male and 14 female patients for astrocytoma groups with an age ranged from 4 to 71 years(mean 38 years). The tumors were diagnosed as low-grade astrocytoma in 6 cases, anaplastic aslrocytoma in 12 cases, and glioblastoma in 19 cases according to the World Health Organization criteria(1990). Immunohistochemical stainingFormalin-fixed, paraffin-embedded specimens were used for this study. Sections(4-5um) were deparaffinized in xylene and rehydrated through a graded ethanol series to water after a wash in 0.05 mol/L Tris-buffered saline(TBS) (pH7.6), sections were immersed in O.Olmol/L citrate buffer (pH 6.0) and micro waved two times, for 10 minutes each time, for antigen retrieval. Slides were then cooled to room temperature and rinsed in TBS for 5 minutes. Endogenous peroxidase was blocked by a 30-minute incubation in TBS containing 3%hydrogen peroxide at room temperature. After additional washing in TBS, nonspecific binding was blocked with 7.5% normal goat serum at 37 X? temperature for 30 minutes. The samples were incubated overnight at 4'C with rabbit polyclonal antibody against human eNOS (1:50 dilution; Boster Biotechenical Co, Wuhan, China), mouse polyclonal antibody against human VEGF or FVIIIRAg (1:100 dilution; Zhongshan Biotechenical Co, Peking, China). Immunostaining for eNOS and VEGF, FVIIIRAg were performed in serial sections. Sections were washed with TBS three times, for 5 minutes each time, and were then incubated with bioaylated goat anti-rabbit IgG at room temperature for 30 minutes. After two 5 minute rinses with TBS, the sections were treated with peroxidase-conjugated streptavidin (Zhongshan Biotechenical Co, Peking, China) for 30 minutes at 37 "C temperature. Diaminobenzidine/hydrogen peroxide was used as a chromogen, and a hematoxylin counterstain was applied. Sections were dehydrated in alcohol, cleared in xylene, and m...
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