Beer D G, Schwarz M, Sawada N, Pitot H C
Cancer Res. 1986 May;46(5):2435-41.
The appearance of gamma-glutamyl transpeptidase (GGT) in focal areas of hepatocytes is a widely used histochemical marker for the identification of preneoplastic cell populations. The characterization of these GGT-positive preneoplastic cells in relation to possible alterations in protooncogene expression may help define cellular changes occurring during the early stages of hepatocarcinogenesis. Female Sprague-Dawley rats were subjected to a two-thirds partial hepatectomy, followed 18 h later by a single intragastric administration of 30 mg of diethylnitrosamine per kg and subsequent feeding of a diet containing 0.05% phenobarbital for 6 or 11 mo. Primary cell suspensions were obtained after the perfusion of liver with collagenase. Cell debris and nonviable cells were removed with multiple washes and a Percoll gradient step. GGT-positive hepatocytes were enriched from the cell suspension by adherence to an affinity-purified GGT antibody affixed to Petri dishes. These dishes allowed the selective adherence and collection of up to 2.28 X 10(6) GGT-positive cells per liver. The starting cell population and the isolated GGT-positive and -negative cells were then used for subsequent analysis. RNA was prepared from the cell isolates and from hepatocellular carcinomas induced with the same diethylnitrosamine and phenobarbital regimen as that used to induce GGT-positive foci; 10 micrograms of total cellular RNA were used for Northern blot hybridizations. The blots were probed with 32P-labeled c-myc, H-ras, and albumin DNAs. The results indicate that GGT-positive hepatocytes do not differ from the other hepatocyte populations in either the size or amount of mRNA transcripts for the c-myc and H-ras protooncogenes. Increased expression of c-myc and H-ras was observed in some malignant lesions and may represent a secondary alteration occurring during the multistage process of hepatocarcinogenesis.
γ-谷氨酰转肽酶（GGT）在肝细胞局灶区域的出现是用于识别癌前细胞群体的一种广泛使用的组织化学标志物。这些GGT阳性癌前细胞与原癌基因表达可能改变的关系的表征，可能有助于确定肝癌发生早期阶段出现的细胞变化。对雌性Sprague-Dawley大鼠进行三分之二部分肝切除术，18小时后单次胃内给予每千克30毫克二乙基亚硝胺，随后喂食含0.05%苯巴比妥的饲料6或11个月。用胶原酶灌注肝脏后获得原代细胞悬液。通过多次洗涤和Percoll梯度离心去除细胞碎片和非存活细胞。通过粘附到固定在培养皿上的亲和纯化GGT抗体，从细胞悬液中富集GGT阳性肝细胞。这些培养皿允许选择性粘附并收集每只肝脏多达2.28×10⁶个GGT阳性细胞。然后将起始细胞群体以及分离出的GGT阳性和阴性细胞用于后续分析。从细胞分离物以及用与诱导GGT阳性灶相同的二乙基亚硝胺和苯巴比妥方案诱导的肝细胞癌中制备RNA；10微克总细胞RNA用于Northern印迹杂交。印迹用³²P标记的c-myc、H-ras和白蛋白DNA进行探针杂交。结果表明，GGT阳性肝细胞在c-myc和H-ras原癌基因的mRNA转录本大小或数量上与其他肝细胞群体没有差异。在一些恶性病变中观察到c-myc和H-ras表达增加，这可能代表肝癌发生多阶段过程中发生的继发性改变。
