首页> 美国卫生研究院文献>Biochemical Journal >Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations.
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Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations.

机译:Percoll密度梯度离心法分离的大鼠肝细胞亚群显示出与不同腺泡位置一致的特征。

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摘要

Freshly isolated viable rat hepatocytes were separated into five subpopulations on shallow discontinuous Percoll density gradients. The periportal marker enzymes alanine aminotransferase (ALT), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) showed gradients of increasing activity from the subpopulation of least density (band 1, rho = 1.07 g/ml) to the subpopulation of greatest density (band 5, rho = 1.09 g/ml). The perivenous marker enzymes pyruvate kinase (PK) and glutamate dehydrogenase (GDH) showed gradients of decreasing activity from band-1 cells to band-5 cells. Glutamine synthetase (GS), which is confined to the two or three cell layers around the hepatic venule, was almost entirely restricted to band-1 hepatocytes. Band-5: band-1 ratios of enzyme activity were as follows: ALT, 8.0; LDH, 2.1; MDH, 1.6; GDH, 0.7; PK, 0.2; GS, 0.01. Band-5:band-1 ratios for ALT, LDH, PK and GS were maintained after culture of subpopulations in identical conditions for up to 72 h, whereas the ratios for MDH and GDH decreased and increased respectively towards unity. Band-1 hepatocytes exhibited greater cytotoxicity than band-5 cells after incubation with carbon tetrachloride or paracetamol. These perivenous-selective toxins produced greater decreases in cell viability and greater release of ALT and LDH from band-1 hepatocytes than from band-5 hepatocytes. Conversely, band-5 hepatocytes were more susceptible than band-1 hepatocytes to the cytotoxic effects of 1-naphthylisothiocyanate and methotrexate (known periportal-selective toxins). It is concluded that band-5 hepatocytes are enriched in periportal cells, whereas band-1 hepatocytes are enriched in perivenous cells. Isolation of hepatocyte subpopulations by Percoll density-gradient centrifugation has the considerable advantage that periportal and perivenous cells can be obtained from the same liver.
机译:新鲜分离的活大鼠肝细胞在浅的不连续Percoll密度梯度上分为五个亚群。 portportal标记酶丙氨酸转氨酶(ALT),苹果酸脱氢酶(MDH)和乳酸脱氢酶(LDH)表现出从最低密度的亚群(带1,rho = 1.07 g / ml)到最大密度的亚群((带5,rho = 1.09g / ml)。静脉标记酶丙酮酸激酶(PK)和谷氨酸脱氢酶(GDH)表现出从band-1细胞到band-5细胞活性降低的梯度。谷氨酰胺合成酶(GS)局限于肝小静脉周围的两个或三个细胞层,几乎完全局限于带1的肝细胞。酶活性的带5:带1的比例如下:ALT,8.0; Band-1:Band-1。 LDH,2.1; MDH,1.6; GDH,0.7; PK 0.2; GS,0.01。在相同条件下培养多达72小时的亚群后,ALT,LDH,PK和GS的Band-5:band-1比值保持不变,而MDH和GDH的比值则朝着统一方向降低和提高。在与四氯化碳或对乙酰氨基酚孵育后,Band-1肝细胞比Band-5肝细胞表现出更大的细胞毒性。与带5肝细胞相比,这些带静脉选择性毒素从1带肝细胞中产生的细胞活力下降更大,并且ALT和LDH释放更大。相反,带5的肝细胞比带1的肝细胞对1-萘基异硫氰酸盐和氨甲蝶呤(已知的门静脉选择性毒素)的细胞毒性作用更敏感。可以得出结论,带5的肝细胞富含门静脉细胞,而带1的肝细胞富含静脉管细胞。通过Percoll密度梯度离心分离肝细胞亚群具有相当大的优势,即可以从同一肝脏获得门静脉和静脉细胞。

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