The Main Proteins And Amino Acids Of Egg Regulate Caco-2 Cellular Cholesterol Absorption And Mechanism Research

Whether the concentration of human serum cholesterol and egg cholesterol intake was a positive correlation has been controversial. In order to solve this problem this study chooses the main protein of eggs for the research object, through the cell experiment to study the effect of Caco-2 cellular cholesterol absorption. This study used the technology of molecular biology to explore the regulation of egg protein on cholesterol metabolism pathways. The contents and results are as follows:(1)This study cultured Caco-2 cells according to an optimized protocol. In the optimized protocol, Caco-2 cells were cultured with the classical Caco-2 medium supplemented with L-ascorbic acid. The accelerated Caco-2 model has been compared to the traditional model in terms of tightness of the junctions, ability to rank chemical compounds for apparent permeability, and to discriminate P-gp substrates.Caco-2 cell monolayer established with the L-ascorbic acid culture system showed good integrality of cellular morphology and cell monolayer reflected by TEER > 220 Ω?cm2; Papp value of compound fluorescein was( 0.39 ± 0.15)×10-6 cm/s; and obvious polar differentiation of cell monolayer reflected by enhanced AKP activity on the apical side.Short-term culture had the similar RNA level of P-gp and MRP2 to 21-day culture. It had been possible to accelerate the cell culture process in only 9 days.(2)We Explored the regulation of egg protein on cholesterol absorption and transport, and applyed the method of CCK-8 to determine the effect of OVA, OTf, OV and casein on the activity of Caco-2 cell. Caco-2 cells incubated with OVA, OTf, OV and casein respectively, then the cellular uptake experiments. The results showed that after incubation with egg protein significant inhibit absorption of cholesterol in Caco-2 cell. Caco-2 cells were cultured on Transwell 12 well plates to simulate intestinal epithelial cells, which studyed the effect of egg protein on cholesterol transport.The results showedthat add OVA, OTf can inhibit cholesterol transport and OV had no effect on cholesterol transport.(3)We studyed the micellar solubility of cholesterol after adding OVA、OTf、OV and Casein. Cholesterol micellar solutios with OVA、OTf and OV in cultural medium which were add in Caco-2 cell, expression of transcriptional and protein levees of key proteins in the pathway of cholesterol in Caco-2 cell by RT-q PCR and Western-bolt. The results showed that OVA and OTf could reduce the solubility of cholesterol, but OV had no significant effect. Egg proteins could significantily inhibit NPC1L1 levels, which suggest that the cholesterol-inhibitory effect be mediated in part by their action at the NPC1L1 gene and protein level. OVA、OTf、OV increased ABCG5/8 and ABCA1 m RNA transcription level, decresed transcription of MTP.(4)We Explored amino acids affect cholesterol solubility in the micellar, the regulation of amino acids on cholesterol absorption and transport and changes in key gene m RNA expression level of cholesterol absorption pathway. When serine at 50 μg/m L and cysteine at 100 μg/m L, the concentration of cholesterol in the mixed micelle was significant decreased. Cys and Ser increased ABCG8 and MTP m RNA transcription level, decresed transcription of ABCA1 and had no significant effect on ABCG-5.