| BackgroundInhibitor of apoptosis is very related to carcinogenesis ,as well as .to shield transformed cells from death induced by chemotherapy or radiotherapy. Survivin is a recently identified member of the inhibitor of apoptosis protein (LAP) famify and is a bifunctional protein that suppresses apoptosis and regulates cell division. The cell-cycle periodicity of survivin expression is transcriptionally controlled, alternative expressed during the G2/M phase. Survivin associates with the microtubules of the mitotic spindle and any disruption results in the loss of apoptosis activity.Cell cycle progression is modulated by a number of cyclin-dependent kinases that consists of catalytic subunits designated Cdks and activating subunits designated cyclins.The p16 gene is multiple tumor suppressor gene determined in recent years, p16 has been found to be a negative regulator of G1-phase of the cell cycle and shown to bind Cdk4 and inhibit the catalytic activity of the Cdk4/cyclin D complex. So far, there are few knowledge about etiological and pathophysiological mechanism of astrocytoma.It was found that survivin is very related to tumor malignancy and prognosis by clinical studies. Atpresent, much attention is paid to the function of survivin and p16 in tumor progression. In this study ,expression of survivin and p16 in human astrocytoma was evaluated by immunohistochemical methods. Whether there is a relationship between tumor phenotype and genotype was demonstrated. Elucidate pathophysiology of survivin and p16 will be likely helpful to the development of therapeutic strategies for astrocytoma.MATERIALS AND METHODS 1.MaterialFifty six tissue specimens were obtained from patients with astrocytomas in the first affiliated hospital of Medical college, Zhejiang University. Seven normal brain tissues were obtained from the other patients as the control group. There were 31 male and 25 female patients for astrocytoma groups with an age ranged from 4 to 84 years (mean 39.7 years). These specimens were classified as Kemohan classification criteria, astrocytoma grade I 9 cases, grade II 20 cases, grade III 17 cases, grade IV 10 cases. There were 4 male and 3 female patients for control groups with an age ranged from 25 to 46 years (mean 36.8 years). 2.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 0.01mol/L citrate buffer (pH 6.0) and microwaved 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 37temperature for 30 minutes. The samples were incubated overnight at 4 with rabbit polyclonal antibody against human survivin (1:150 dilution; Boster Biotechenical Co, Wuhan, China), rabbitpolyclonal antibody against human p16 (1:150 dilution; Boster Biotechenical Co, Wuhan, China). 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 癈 temperature. Diaminobenzidine/hydrogen peroxide was used as a chromogen, and a hematoxylin counterstain was applied. Sections were dehydrated in alcohol, cleared in xylene, and mounted. As additional negative control, primary antibody was omitted. Tissue from a human breast carcinoma known to overexpress survivin and p16 were used as a positive cotrol.In the tumor tissue, positive cell was the cell yellow or brown-yellow. The percentage of positive tumor cell... |
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