Kojima T, Yamamoto M, Mochizuki C, Mitaka T, Sawada N, Mochizuki Y
Department of Pathology, Cancer Research Institute, Sapporo Medical University School of Medicine, Japan.
Hepatology. 1997 Sep;26(3):585-97. doi: 10.1053/jhep.1997.v26.pm0009303487.
In the present study, we determined in detail the changes of liver gap junctions, connexin 26 (Cx26), and connexin 32 (Cx32), during DNA synthesis and redifferentiation of hepatocytes in vitro. We used primary rat hepatocytes that expressed the liver gap junction proteins, which were cultured in the medium containing epidermal growth factor (EGF) with 2% dimethylsulfoxide (DMSO) and 10(-7) mol/L glucagon (a DMSO culture system), as we previously reported. In the present cultures, almost confluent hepatocytes cultured in the medium containing EGF with 2% DMSO and 10(-7) mol/L glucagon, underwent a nearly synchronous wave of DNA synthesis induced by the removal of 2% DMSO and 10(-7) mol/L glucagon, and the addition of 10 mmol/L nicotinamide, after which the DNA synthesis was completely re-inhibited by the re-addition of 2% DMSO and 10(-7) mol/L glucagon. During stimulation of DNA synthesis, both Cx26 and Cx32 messenger RNA (mRNAs) in hepatocytes transiently increased in the G1 phase and then markedly decreased before the onset of the S phase, while only Cx26 messenger RNA (mRNA) increased slightly in the S/M phase. Furthermore, before the onset of the S phase, a disappearance of both Cx26 and Cx32 immunoreactivities and gap junction plaques were observed. Gap junctional intercellular communication (GJIC), as measured by lucifer yellow, which indicated the function of Cx32, decreased markedly from before the onset of the S phase. GJIC measured by propidium iodide, which indicated the function of Cx26, decreased from before the onset of the S phase and then increased slightly in the S/M phase. During the re-inhibition after the stimulation of DNA synthesis, Cx32 mRNA, but not Cx26 mRNA, rapidly returned to the pretreatment control level. Cx32 immunoreactivity and gap junction plaques also recovered. However, the recovery of GJIC measured by lucifer yellow was later than that of Cx32 expression. These results indicated the different changes of expression and function of Cx26 and Cx32 in the hepatocytes during stimulation and re-inhibition of DNA synthesis. This culture system should be useful as a model in which to study liver gap junctions during hepatocyte growth and differentiation in vitro.
在本研究中，我们详细测定了体外培养的肝细胞在DNA合成和再分化过程中肝间隙连接、连接蛋白26（Cx26）和连接蛋白32（Cx32）的变化。我们使用了如我们之前报道的，表达肝间隙连接蛋白的原代大鼠肝细胞，将其培养在含有表皮生长因子（EGF）、2%二甲基亚砜（DMSO）和10⁻⁷ mol/L胰高血糖素的培养基中（一种DMSO培养体系）。在当前培养中，在含有EGF、2% DMSO和10⁻⁷ mol/L胰高血糖素的培养基中培养的几乎汇合的肝细胞，在去除2% DMSO和10⁻⁷ mol/L胰高血糖素并添加10 mmol/L烟酰胺后，经历了由其诱导的近乎同步的DNA合成波，之后DNA合成通过重新添加2% DMSO和10⁻⁷ mol/L胰高血糖素而被完全重新抑制。在DNA合成刺激期间，肝细胞中的Cx26和Cx32信使核糖核酸（mRNA）在G1期短暂增加，然后在S期开始前显著下降，而只有Cx26信使核糖核酸（mRNA）在S/M期略有增加。此外，在S期开始前，观察到Cx26和Cx32免疫反应性以及间隙连接斑块均消失。通过荧光素黄测量的间隙连接细胞间通讯（GJIC）表明Cx32的功能，从S期开始前显著下降。通过碘化丙啶测量的GJIC表明Cx26的功能，从S期开始前下降，然后在S/M期略有增加。在DNA合成刺激后的重新抑制期间，Cx32 mRNA迅速恢复到预处理对照水平，而Cx26 mRNA没有。Cx32免疫反应性和间隙连接斑块也恢复了。然而，通过荧光素黄测量的GJIC的恢复比Cx32表达的恢复要晚。这些结果表明在DNA合成的刺激和重新抑制过程中，肝细胞中Cx26和Cx32的表达和功能发生了不同变化。这种培养体系应作为一种有用的模型，用于研究体外肝细胞生长和分化过程中的肝间隙连接。
