RESUMO
BACKGROUND/AIMS: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep) cells directly reprogrammed from mice fibroblast cells were fully characterized. METHODS: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α) and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed. RESULTS: The expression and function of p-glycoprotein (P-gp), bile salt efflux pump (Bsep), multidrug resistance-associated protein 2 (Mrp2), Na+-dependent taurocholate cotransporting polypeptide (Ntcp), and organic anion transporter polypedtides (Oatps) in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α) and treatment with selected inducers. In vitro intrinsic biliary clearances (CLb,int) of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid) and DPDPE ([D-Pen2,5] enkephalin hydrate) correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs) (r2 = 0.984). Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes. CONCLUSION: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening.
Assuntos
Anticolesterolemiantes/metabolismo , Reprogramação Celular , Fator 4 Nuclear de Hepatócito/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Anticolesterolemiantes/análise , Anticolesterolemiantes/toxicidade , Ácidos e Sais Biliares/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , D-Penicilina (2,5)-Encefalina/análise , D-Penicilina (2,5)-Encefalina/metabolismo , D-Penicilina (2,5)-Encefalina/toxicidade , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Proteínas de Membrana Transportadoras/genética , Metotrexato/análise , Metotrexato/metabolismo , Metotrexato/toxicidade , Camundongos , Camundongos Endogâmicos ICR , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Rosuvastatina Cálcica/análise , Rosuvastatina Cálcica/metabolismo , Rosuvastatina Cálcica/toxicidade , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Simportadores/genética , Simportadores/metabolismoRESUMO
Based on the structural characteristics of Cycas trunk, including vegetative leaf base scars, sporophyll concave rings, and average occurrence probabilities of vegetative leaf and sporophyll, a method for the age estimation of Cycas fairylakea population was developed, and the age of each individual was calculated. Three approaches, i.e., age structure diagram, age distribution curve and curve estimation were used to study the age structure of C. fairylakea population at genet and clone population levels. The age structure diagram showed that the clone population of C. fairylakea was stable, but the genet population was in declining. However, both of the clone and genet populations were in declining when using the other two approaches. It was considered that the C. fairylakea population was in declining, and needed an urgent protection.