PHENOBARBITAL-DEPENDENT PROLIFERATION OF PUTATIVE INITIATED RAT HEPATOCYTES
PHENOBARBITAL-DEPENDENT PROLIFERATION OF PUTATIVE INITIATED RAT HEPATOCYTES
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DOI：
10.1093/carcin/9.5.779
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发表时间：
1988-05-01
期刊：
影响因子：
4.7
通讯作者：
KAUFMAN, DG
中科院分区：
文献类型：
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作者：
KAUFMANN, WK;RIRIE, DG;KAUFMAN, DG
使用假定的起始肝细胞培养物检查苯巴比妥 (PB) 的促有丝分裂作用，这些肝细胞在正常肝细胞衰老和死亡的条件下增殖并形成集落。在存在PB的情况下，用甲基(乙酰氧基甲基)亚硝胺或苯并[a]芘-7,8-二醇-9,10-环氧化物(抗)启动后2周分离的原代培养物中的集落形成肝细胞的频率在每百万个2-38个范围内。在没有 PB 的情况下，集落形成频率为百万分之 0.1。在含有 PB、表皮生长因子、去甲肾上腺素和胰岛素的无血清培养基中生长的培养物中没有观察到增殖性肝细胞集落。使用启动后 5 周从肝脏中分离出的肝细胞的二次培养物进一步表征了对 PB 的需求。在 2 mM PB 存在的情况下，这些肝细胞的集落形成效率约为 10%，而在不存在 PB 的情况下，这些肝细胞的集落形成效率低于 0.2%。集落形成对 0.5-2 mM 范围内的 PB 浓度呈现线性响应，并在最佳浓度 2 mM 之上下降。放射自显影用于确定在存在或不存在 PB 的情况下合成 DNA 的二次培养物中肝细胞的百分比。到作为单细胞接种后的第三天，肝细胞表现出约 50% 的标记指数，并且这种标记水平在接种后可保持长达 2 周。在没有 PB 的情况下，很少有肝细胞能够合成 DNA，而且大多数肝细胞都已衰老。一小部分集落形成肝细胞在没有 PB 的情况下继续增殖，并形成具有高标记指数的集落。这些结果表明，体内起始肝细胞的增殖可能以肝肿瘤启动子PB的存在为条件。
The mitogenic effects of phenobarbital (PB) were examined using cultures of putative initiated hepatocytes that proliferate and form colonies under conditions in which normal hepatocytes senesce and die. The frequencies of colony-forming hepatocytes in primary cultures isolated 2 weeks after initition with methyl(acetoxymethyl)nitrosamine or benzo[a]pyrene-7,8-diol-9,10-epoxide(anti) were in the range of 2-38 per million in the persence of PB. Colony-formation frequencies were 0.1 per million in the absence of PB. Proliferative hepatocyte colonies were not observed in cultures grown in serum-free medium containing PB, epidermal growth factor, nor-epinephrine and insulin. The requirement for PB was characterized further using secondary cultures of hepatocytes that had been isolated from a liver 5 weeks after initiation. The colony-forming efficiency of these hepatocytes was about 10% in the presence of 2 mM PB and less than 0.2% in its absence. Colony formation displayed a linear response to concentrations of PB in the range of 0.5-2 mM and a decline above the optimal 2 mM concentration. Autoradiography was used to determine the percentages of hepatocytes in secondary cultures that synthesized DNA in the presence or absence of PB. By the third day after seeding as single cells, hepatocytes exhibited a labeling index of about 50% and this level of labeling was preserved for up to 2 weeks after seeding. Very few hepatocytes were found to synthesize DNA in the absence of PB and most senesced. A small fraction of the colony-forming hepatocytes continued to proliferate in the absence of PB and formed colonies with a high labelling index. These results suggest that the proliferation of initiated hepatocytes in vivo may be conditional upon the presence of the hepatic tumor promoter, PB.