Merck
  • Home
  • Search Results
  • An effective protocol for pharmacological defatting of primary human hepatocytes which is non-toxic to cholangiocytes or intrahepatic endothelial cells.

An effective protocol for pharmacological defatting of primary human hepatocytes which is non-toxic to cholangiocytes or intrahepatic endothelial cells.

PloS one (2018-07-26)
Yuri L Boteon, Lorraine Wallace, Amanda P C S Boteon, Darius F Mirza, Hynek Mergental, Ricky H Bhogal, Simon Afford
ABSTRACT

Pharmacological defatting of rat hepatocytes and hepatoma cell lines suggests that the same method could be used to ameliorate macrovesicular steatosis in moderate to severely fatty livers. However there is no data assessing the effects of those drugs on primary human liver cells. We aimed to determine the effectiveness of a pharmacological cocktail in reducing the in vitro lipid content of primary human hepatocytes (PHH). In addition we sought to determine the cytotoxicity of the cocktail towards non-parenchymal liver cells. Steatosis was induced in PHH by supplementation with a combination of saturated and unsaturated free fatty acids. This was followed by addition of a defatting drug cocktail for up to 48 hours. The same experimental method was used with human intra-hepatic endothelial cells (HIEC) and human cholangiocytes. MTT assay was used to assess cell viability, triglyceride quantification and oil red O staining were used to determine intracellular lipids content whilst ketone bodies were measured in the supernatants following experimentation. Incubation of fat loaded PHH with the drugs over 48 hours reduced the intracellular lipid area by 54%, from 12.85% to 5.99% (p = 0.002) (percentage of total oil red O area), and intracellular triglyceride by 35%, from 28.24 to 18.30 nmol/million of cells (p<0.001). Total supernatant ketone bodies increased 1.4-fold over 48 hours in the defatted PHH compared with vehicle controls (p = 0.002). Moreover incubation with the drugs for 48 hours increased the viability of PHH by 11%, cholangiocytes by 25% whilst having no cytotoxic effects on HIEC. These data demonstrate that pharmacological intervention can significantly decrease intracellular lipid content of PHH, increase fatty acids β-oxidation whilst being non-toxic to PHH, HIEC or cholangiocytes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
霍乱毒素 来源于霍乱弧菌, ≥90% (SDS-PAGE), lyophilized powder
Sigma-Aldrich
胰岛素 人, recombinant, expressed in yeast (proprietary host)
Sigma-Aldrich
胶原酶 来源于溶组织梭菌, Type IA, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid, For general use
Sigma-Aldrich
牛血清白蛋白 来源于牛血清, heat shock fraction, lyophilized powder, essentially fatty acid free, ≥98% (agarose gel electrophoresis)
Sigma-Aldrich
油红O, certified by the Biological Stain Commission
Sigma-Aldrich
TERGITOL 溶液, Type NP-40, 70% in H2O
Sigma-Aldrich
油酸-水溶性, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
亚油酸-水溶性, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
酮体检测试剂盒, sufficient for 200 tests (UV)
Sigma-Aldrich
GW501516, ≥98% (HPLC)
Sigma-Aldrich
GW7647, ≥98% (HPLC)
Sigma-Aldrich
抗-EPCAM 兔抗, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Visfatin, recombinant, expressed in E. coli, ≥95% (SDS-PAGE), ≥95% (HPLC)