| IntroductionWilms' tumor (WT) is the most common primary renal malignancy of childhood. The outcome is generally good, although a poor prognosis is often related to more advanced stages and anaplastic features. Generally, the growth rate of neoplasm depends on the proliferation and death rates of cancer cells. Continuous cell death is an intrinsic feature of malignant tumors in which reduction of cell mass may be achieved through necrosis or apoptosis. Apoptosis is a type of cell death involved in physiological cell deletion during embryonic development and in maintenance of the number of cell during the renewal of mature tissue. Nuclear and cytoplasmatic condensation, formation of membrane - bound apoptotic bodies, and oligonucleosomal DNA degradation without inflammatory infiltrate characterize apoptotic death and differentiate it from necrosis.The inhibition of apoptosis by over expression of the bcl - 2 proto -oncogene causes a decrease in cell death, and the consequent increase in the development of tumors. Over - expression of bcl -2 has been found in epithelial malignancies such as carcinoma of the prostate, breast, and kidney. Recently, an inverse correlation between expression of bcl -2 and the presence of apoptotic cells (AC) , has been observed in neuroblastoma.The present study was designed to analyze the role of apoptosis and bcl - 2 gene expression in WT.Materials and methodsForty patients underwent removal of a WT from January 1995 to February 2001 in the secondary hospital of China medical university. Nine normal tissues near the WT are used for comparison. The mean age at operation was 54 months. After HE staining, two pathologists determined the pathological types of the specimens. AC was detected by the in - situ end - labeling technique ( ISEL). Sections were stained using the in Situ Cell Apoptosis Detection Kit ( Sino - American Biotechnology Co. China). The dewaxed sections were quenched in 3 % hydrogen peroxidase in PBS for 5 min, equilibrated in PBS, incubated with 30(ug/ml proteinase K for 45 min at 37C , washed in distilled water, and then incubated in reaction buffer with TdT enzyme and biotin - 11 - dUTP for 1 h at 37C. Then labeled sections were soaked in 2 x SSC for 15 min, washed by PBS, placed in a stop solution for 30 min and then reacted with Avidin - HRP for 1 h at 37C. After washing, color development was performed using 0. 02% DAB or AEC with 0. 01% hydrogen peroxide in PBS. As a negative control , TdT was omitted. Positive control was generated using the specimens of rat mammary glands taken on the 4th day after weaning.Bcl - 2 expression was detected by ABC immunohistochemistry u-sing Instant SABC Kit ( Wuhan Boster Biological Technology Co. China). The dewaxed sections were quenched in 3% hydrogen peroxidase in PBS for 5 min, equilibrated in PBS, antigens for bcl - 2 were retrieved in the complex digestant liquid for 10 min. After washing by distilled water, sections were blocked by normal goat serum for 20min, and then incubated with anti - human bcl - 2 monoclonal antibody at 4t over night, then washed by PBS, biotin -labeled rabbit anti - mouse antibody was performed at room temperature for 20 min, washed by PBS, soaked in SABC for 20 min; After washing by PBS, color development was performed using 0.02% DAB with 0.01% hydrogen peroxide in PBS. As a negative control, PBS was used to replace 1st antibody. Positive control was generated using the supplied control slides.When DAB was used for action, yellow staining fractions were considered as positive places. When AEC was used, pink staining places were considered as positive cells. ACs were counted as the rati-o of apoptotic to normal cells within a field of view at a magnification of 100 times. A total of ten chosen fields were counted per slide assayed and the mean per field was expressed as the apoptotic index (AI).ResultThe AIs of three kinds of neoplastic WT ( high differentiation, middle differentiation and low differentiation) have no statistic difference. The AIs of blastemal...
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