Eckle Veit-Simon, Buchmann Albrecht, Bursch Wilfried, Schulte-Hermann Rolf, Schwarz Michael
Institute of Pharmacology and Toxicology, Department of Toxicology, University of Tübingen, Wilhelmstr. 56, 72074 Tübingen, Germany.
Toxicol Pathol. 2004 Jan-Feb;32(1):9-15. doi: 10.1080/01926230490260673.
In our study we tested the utility of antibodies that specifically recognize the cleaved large (active) subunits of caspase-3 and caspase-9 for immunohistochemical detection of apoptotic hepatocytes in rat liver sections using archival material from cyproterone acetate (CPA)-treated and control rats. CPA blocks apoptosis of hepatocytes and discontinuation of CPA treatment results in a syncronized wave of hepatocyte apoptosis. By comparing liver sections from CPA-treated and control rats with high and low rates of apoptosis we observed a close correlation between the occurrence of cleaved caspase-positive apoptotic figures and H&E-stained apoptotic bodies when evaluated in parallel sections. Caspase-stained figures were either immuno-positive apoptotic bodies or pre-apoptotic hepatocytes showing cytoplasmic and/or nuclear caspase-staining with otherwise normal cellular appearance. In extension of these observations we developed a double-immunohistochemical staining procedure which enables the detection of caspase-3-positive apoptotic hepatocytes within glutathione-S-transferase-P (GST-P)-positive preneoplastic liver foci. By use of this technique, inhibition of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin as detected by counting of H&E-stained apoptotic bodies was found to correlate with a strong reduction of cleaved caspase-positive hepatocytes in GST-P-positive preneoplastic foci. In summary, this study demonstrates that cleaved caspase-positive apoptotic hepatocytes could be reliably identified and quantified both in normal and neoplastically transformed liver tissue.
在我们的研究中，我们使用醋酸环丙孕酮（CPA）处理的大鼠和对照大鼠的存档材料，测试了特异性识别半胱天冬酶-3和半胱天冬酶-9裂解的大亚基（活性亚基）的抗体在大鼠肝脏切片中免疫组织化学检测凋亡肝细胞的效用。CPA可阻断肝细胞凋亡，停止CPA治疗会导致肝细胞凋亡的同步波。通过比较CPA处理的大鼠和对照大鼠肝脏切片中凋亡率的高低，我们在平行切片评估时观察到，裂解的半胱天冬酶阳性凋亡细胞与苏木精-伊红（H&E）染色的凋亡小体之间存在密切相关性。半胱天冬酶染色的细胞要么是免疫阳性的凋亡小体，要么是凋亡前肝细胞，其细胞质和/或细胞核呈现半胱天冬酶染色，而细胞外观正常。在这些观察结果的基础上，我们开发了一种双重免疫组织化学染色程序，能够在谷胱甘肽-S-转移酶-P（GST-P）阳性的癌前肝灶内检测半胱天冬酶-3阳性的凋亡肝细胞。通过使用该技术，发现通过计数H&E染色的凋亡小体检测到的2,3,7,8-四氯二苯并对二恶英对凋亡的抑制作用与GST-P阳性癌前病灶中裂解的半胱天冬酶阳性肝细胞的显著减少相关。总之，本研究表明，在正常和肿瘤转化的肝组织中，均可可靠地识别和定量裂解的半胱天冬酶阳性凋亡肝细胞。
