| Human embryonic stem cells(h ESCs)have different metabolic phenotypes from other somatic cells.The role of glucose and amino acid metabolism in h ESCs pluripotency has been well studied,but little is known about the fatty acids(FAs)metabolism in h ESCs.FAs are directly or indirectly involved in the regulation of energy and metabolism.In this study,several metabolism-related exogenous chemicals were selected to interfere h ESCs culture,and the growth of cells,the expression of plutipotency genes,the alteration of FA combination,and the change of expression of related gene were investageted to figure out the effects of FAs on h ESCs.Sodium chloride,L-ascorbic acid,L-glutamine,calcium chloride,D-glucose,ethanol and vitamin B6 were selected as exogenous stimulants,and a concentrated storage solution of 1 mg/m L were prepared,and then diluted to 0.1%,0.5%,1%,5%,10% and30% in culture medium for h ESCs culture.Then,5% sodium chloride,5% L-ascorbic acid,5% L-glutamine,10% calcium chloride,10% D-glucose,0.5% ethanol,1% vitamin B6 and 5% vitamin B6 were screened out as the maximum concentration of cell culture,which did not affect the proliferation of h ESCs.The cells were collected and cultured after15 generations for pluripotency gene detection.The results showed that the pluripotency gene OCT4 was upregulated in all the treated groups,NANOG was upregulated in ethanol,sodium chloride and 1% vitamin B6,but there was no significant change under other conditions,REX1 was down-regulated,KLF4 was down-regulated in all conditions except sodium chloride,and SOX2 had no significant change.DNMT3 A,DNMT3B,and DNMT3 L also changed.In addition,37 kinds of fatty acids were detected by gas chromatograph mass spectrometry and the expression of related genes were detected by RT-PCR.The results showed that the total fatty acids,saturated fatty acids and long-chain fatty acids in all treatment groups were basically unchanged except the sodium chloride treated group,but the polyunsaturated fatty acids in all treatment groups were generally lower than that in the control group.At the same time,the saturated fatty acids were increased and other fatty acids were decreased after ethanol treatment.The total fatty acid content in the sodium chloride treatment group was significantly increased,while that in the glucose treatment group was dramatically decreased.Glutamine had no significant effect on the total fatty acid content,but the monounsaturated fatty acid content in the glutamine treatment group was significantly increased,which also confirmed that glucose may inhibit the synthesis of free fatty acids in h ESCs.The expression of genes related to FA metabolism also changed at the same time.In the ethanol and sodium chloride treatment group,the FA synthase gene such as ACLY,ACC1 and FASN were up-regulated,but significantly down-regulated in the glucose treatment group,while no significant changes were found in the glutamine treatment group.In addition,the triglyceride synthase and degrading genes DGAT1 and HSL were upregulated only by ethanol.The expression of FA oxidation related enzymes HADH and ACSS1 were up-regulated.FA synthesis transcription factor genes C/EBP?,ADD1 and PPAR? were upregulated only in ethanol treatment group.These results were corresponded to the FA content alteration,which indicate that glucose inhibited FA synthesis,sodium chloride promoted total fatty acid synthesis,and glutamine had little effects on FA content alteration.In conclusion,the pluripotency of h ESCs under the stimulation of exogenous chemicals is closely related to FA content and the expression of related genes,while the undrelized mechanism need further investigation. |
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