Bile acid synthesis in primary cultures of rat and human hepatocytes
Bile acid synthesis in primary cultures of rat and human hepatocytes
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DOI：
10.1002/hep.510270241
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发表时间：
1998-02-01
期刊：
影响因子：
13.5
通讯作者：
Einarsson, C
中科院分区：
文献类型：
--
作者：
Ellis, E;Goodwin, B;Einarsson, C
肝胆汁酸形成的调控尚不完全清楚。哺乳动物肝细胞的原代培养提供了一个相对孤立地检查假定的调节因子的机会。利用原代培养的大鼠和人肝细胞，我们检测了胆汁酸的组成和形成限速酶胆固醇- 7 -羟化酶的表达。对照大鼠肝细胞的胆汁酸产量在4天内下降，从第1天的156 +/- 24 ng/mL（67%胆酸）下降到第4天的55 +/- 11 ng/mL（55%胆酸）。除胆酸外，还形成鹅去氧胆酸、α -胆酸和β -胆酸。单用三碘甲状腺原氨酸（T-3）或地塞米松治疗对胆汁酸产生无显著影响。T-3和地塞米松联合使用显著增加了第4天的总胆汁酸产量（224 +/- 54 ng/mL），并导致其组成显著变化为23%的胆酸和77%的非12 α -羟基化胆汁酸。对照大鼠肝细胞培养4天后胆固醇7 α -羟化酶活性为3.3 +/- 0.6 pmol/mg蛋白/min。地塞米松和T-3联合处理的细胞活性为16.4 +/- 3.6 pmol/mg protein/min。培养4天后，通过溶液杂交测定胆固醇7 α -羟化酶信使RNA （mRNA）水平，结果与活性数据相似；对照细胞的总核酸（tna）为5.3 +/- 0.9 cpm/ μ g。T3或地塞米松处理的细胞与对照细胞没有差异，而T-3和地塞米松联合使用使mRNA水平增加到20.6 +/- 2.8 cpm/mu g tna。从供肝分离的人肝细胞中，胆汁酸的形成从第2天的206 +/- 79 ng/mL增加到第6天的1490 +/- 594 ng/mL，然后略有下降，形成胆酸和鹅去氧胆酸，分别占80%和20%左右。联合添加T-3和地塞米松有减少而不是增加胆汁酸形成的趋势。从第2天到第4天，人肝细胞中胆固醇7 α -羟化酶的mRNA水平增加了数倍，然后下降。添加T-3或地塞米松对mRNA水平没有任何一致的影响。值得注意的是，培养的人肝细胞产生胆汁酸的能力高于培养的大鼠肝细胞，尽管在体内大鼠肝中胆汁酸的产生比人肝高3- 5倍。同样明显的是，虽然激素因素似乎调节了大鼠胆汁酸的合成，但在人类肝细胞中没有发现这方面的证据。由于原代培养的人肝细胞分泌的胆汁酸的组成与体内的胆汁酸的组成非常相似，这为进一步研究胆汁酸的合成和调节提供了一个有用的模型。
The regulation of hepatic bile acid formation is incompletely understood. Primary cultures of mammalian hepatocytes offer an opportunity to examine putative regulatory factors in relative isolation. Using rat and human hepatocytes in primary culture, we examined bile acid composition and the expression of the rate-limiting enzyme of formation, cholesterol 7 alpha-hydroxylase. Control rat hepatocytes showed a declining bile acid production over 4 days, from 156 +/- 24 ng/mL (67% cholic acid) on day 1 to 55 +/- 11 ng/mL (55% cholic acid) on day 4. In addition to cholic acid, chenodeoxycholic acid, alpha-muricholic acid, and beta-muricholic acid were formed. Treatment with triidothyronine (T-3) or dexamethasone alone had no significant effect on bile acid production. A combination of T-3 and dexamethasone significantly increased the total bile acid production on day 4 (224 +/- 54 ng/mL) and resulted in a marked change in composition to 23% cholic acid and 77% non-12 alpha-hydroxylated bile acids. Control rat hepatocytes had a cholesterol 7 alpha-hydroxylase activity of 3.3 +/- 0.6 pmol/mg protein/min after 4 days in culture. Cells treated with the combination of dexamethasone and T-3 had an activity of 16.4 +/- 3.6 pmol/mg protein/min. The cholesterol 7 alpha-hydroxylase messenger RNA (mRNA) levels, determined by solution hybridization after 4 days of culture, showed results similar to those for the activity data; control cells had 5.3 +/- 0.9 cpm/mu g total nucleic acids (tNAs). T3- or dexamethasone-treated cells did not differ from control cells, whereas the combination of T-3 and dexamethasone increased the mRNA levels to 20.6 +/- 2.8 cpm/mu g tNAs. In human hepatocytes, isolated from donor liver, bile acid formation increased from 206 +/- 79 ng/mL on day 2 to 1490 +/- 594 ng/mL on day 6 and then declined slightly, Cholic acid and chenodeoxycholic acid were formed, constituting about 80% and 20%, respectively. The combined addition of T-3 and dexamethasone had a tendency to decrease rather than increase bile acid formation. Also, mRNA levels of the cholesterol 7 alpha-hydroxylase increased severalfold in the human hepatocytes from day 2 to day 4 and then declined. The addition of T-3 or dexamethasone did not effect the mRNA levels in any consistent way. It is noteworthy that the capacity of the cultured human hepatocytes to produce bile acids was higher than that of cultured rat hepatocytes, in spite of the fact that the production of bile acids in rat liver is 3- to 5-fold higher than that in human liver in vivo. It is also evident that while hormonal factors appear to regulate bile acid synthesis in the rat, no evidence for this was found in human hepatocytes. As the composition of bile acids secreted by human hepatocytes in primary culture closely resembles that found in vivo, this represents a useful model for further studies of the synthesis and regulation of bile acids.