Fukui Y, Yamamoto A, Masaki R, Miyauchi K, Tashiro Y
Department of Physiology and Cell Biology of Liver Research Center, Kansai Medical University, Osaka, Japan.
J Histochem Cytochem. 1992 Jan;40(1):73-82. doi: 10.1177/40.1.1729355.
We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A-gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy.
我们研究了是否可以通过免疫金电子显微镜对大鼠肝细胞中细胞色素P450（P450IIB）的苯巴比妥（PB）诱导型进行定量分析。大鼠每24小时腹腔注射PB，在PB诱导的各个阶段，肝脏通过用多聚甲醛（4%）和戊二醛（0.1%）的混合物灌注固定，并包埋在LR White中。切超薄切片，使用亲和纯化的抗P450IIB抗体通过蛋白A-金法进行标记，该抗体先前已用来自对照大鼠（未用PB处理）的肝微粒体进行免疫吸附。我们计算了每微米粗面内质网（ER）膜上的金颗粒数量（颗粒密度）。在PB处理前，大鼠肝细胞中粗面内质网的颗粒密度实际上为零，在PB处理后48小时和72小时显著增加。从这些经PB处理的大鼠肝脏中制备粗面微粒体。通过免疫印迹分析估计P450IIB的量，并通过相同的包埋后免疫金法确定与粗面微粒体膜结合的金颗粒数量。粗面微粒体的颗粒密度与P450IIB量的增加平行增加，表明这两个变量具有良好的相关性。因此，细胞色素P450IIB的诱导可以通过免疫金电子显微镜进行定量且可靠的研究。
