| Lipid raft is located on the cell membrane and is rich in cholesterol,phospholipids and target proteins.It is a microdomain structure that is insoluble in nonionic detergents at low temperatures.The components of lipid rafts are closely related to human diseases and can be involved in signal transduction and protein transport,which are associated with diseases such as cancer,diabetes and Alzheimer’s disease.Lipid raft chromatography is based on the principle of affinity chromatography and cell membrane chromatography.Through the specific affinity adsorption of biomolecules,the active components can be screened,separated and enriched,and the structure is identified.Currently,there are single-target and multiple-target lipid raft chromatograms,which have proved that the active ingredients can be screened from complex traditional Chinese medicines with high efficiency and high selectivity.The process is quick and simple,highly specific,it improves the time-consuming process of traditional Chinese medicine extraction,and also extends the use time of partial affinity chromatography and cell membrane chromatography.However,there are still no systematic preparation and evaluation methods,and there is still a problem of poor repeatability.P-glycoprotein(P-gp)has the function of pumping drugs out of the cell and is one of the causes of tumor drug resistance and low bioavailability of drugs.More and more researchers are investing in vitro screening studies of P-gp substrates and inhibitors.At present,there are many reports on lipid raft function and affinity chromatography,but there are few studies on the optimization of lipid raft extraction,characterization methods and the determination of lipid raft components.There are few studies on the construction and adsorption properties of lipid raft chromatography.In this paper,the commonly used detergent method and non-detergent method were used to extract lipid rafts from cells,and the effects of different extraction methods on lipid rafts in Caco-2 cells and U251 cells were compared.The cholesterol and total protein in lipid rafts were analyzed.Quantitative methods were studied to quantify and control the lipid rafts.The lipid raft chromatogram rich in P-gp protein was constructed,and the lipid raft stationary phase was characterized.The relationship between the content of lipid raft and the adsorption performance of lipid raft was studied.The adsorption performance of lipid raft chromatography and its influencing factors were investigated.It provides reference and thinking for solving the problem of poor reproducibility.It also provides a new means for screening P-gp substrates and inhibitors in vitro.It provides new ideas for the efficient screening of active ingredients and the discovery of new targets for various diseases.Chapter Ⅰ ReviewThis chapter mainly introduces the lipid raft and lipid raft chromatogram of the research subjects.Firstly,the definition,structure,function and methods of extraction and characterization of lipid rafts were summarized,which provided reference for the optimization of lipid raft extraction and characterization.At the same time,the research on the composition of lipid rafts was introduced.The functions of cholesterol,total protein and characteristic proteins of lipid rafts were introduced,as well as their methods of quantification.Next,the physiological function of P-gp and its in vitro screening method for substrates and inhibitors are introduced.;finally introduced the principles and applications of affinity chromatography and lipid raft chromatography,as well as the affinity chromatography mechanism and the influencing factors of adsorption performance,in order to provide reference and consideration for the wide application of lipid raft chromatography.Chapter Ⅱ Lipid extraction and characterizationIn this chapter,Caco-2 cells and U251 cells were cultured,and lipid rafts were extracted by different methods and compared.Caco-2 cells and U251 cells were lysed by detergent method 1,detergent method 2 and non-detergent method,and then lipid rafts were extracted and collected by sucrose density gradient centrifugation.Lipid rafts were characterized by Western Blot and immunofluorescence with β-cyclodextrin as the negative control group.The characteristics of lipid rafts were determined by Western Blot method for the characteristic proteins of caveolin-1 and Flotillin-1,P-gp and Trka.The effects of different methods on lipid raft extraction were analyzed.The results showed that the lipid raft layering and protein expression in the lipid rafts extracted by different methods were different: the detergent method was more abundant than the non-detergent method and the extraction was more adequate,while the detergent method 2 has better extraction effect than the detergent method 1,and the lipid raft layer is more,and the target protein P-gp expression is higher.Therefore,the appropriate extraction method should be selected according to the experimental needs and cell characteristics.This laid the foundation for the subsequent study of lipid raft components and the construction of lipid raft chromatography.Chapter Ⅲ Lipid component quantificationIn this chapter,we quantitatively studied the total protein in the lipid rafts of different Caco-2 cells and the cholesterol in the two cell lipid rafts.Total protein was determined by BCA(detergent compatible)kit method;cholesterol was quantified by HPLC and kit methods.It can be seen from the protein content that the total amount of lipid raft protein extracted by the detergent method is more than that of the nondetergent method,and the content of the β-cyclodextrin control protein which is destroyed by the lipid raft structure is the lowest;The total protein content of lipid rafts was consistent with the expression of Flotillin-1 and P-gp in this layer..This not only provides a quantitative supplement for the optimization of lipid raft extraction conditions,but also provides new data for the analysis of protein content in lipid rafts.The results of cholesterol content indicate that the sample preparation process of the high-performance liquid phase method easily causes sample loss,the impurity peak is large,and the sample peak is small,which is difficult to distinguish.The kit method can quickly and accurately quantify cholesterol,and found that the cholesterol content of the lipid raft extracted by U251 cells is higher than that of the lipid raft extracted by Caco-2.The quantification of lipid raft components is expected to quantify and control lipid rafts,providing experience and reference for the quantitative study of lipid raft components.Chapter Ⅳ Preparation and Characterization of Lipid-Phase Stationary PhaseThe method and characterization method of the combination of lipid raft and silica gel were studied.We used the method that the research group was used,and the method of modifying the silica gel using carbon nanotubes was explored.The stationary phase was characterized by immunofluorescence,scanning electron microscopy and energy spectrum analysis.The methods of sample preparation in scanning electron microscopy were compared in detail.At the same time,the type of stationary phase silica gel was optimized.This chapter aims to prepare and optimize lipid raft chromatographic stationary phases,providing prerequisites for better application of lipid raft chromatography.Chapter Ⅴ Lipid chromatographic construction and adsorption performance evaluationIn this chapter,a chromatographic model of Caco-2 cells rich in P-glycoprotein was constructed,and the adsorption performance of lipid raft chromatography was evaluated with verapamil as a positive drug.The relationship between the content of lipid rafts and the adsorption properties of lipid raft chromatography was studied.The specificity,stability and time limit of lipid raft chromatography were also investigated.The factors affecting the adsorption performance of lipid rafts were investigated.The composition of the mobile phase,flow rate,temperature and the ratio of lipid rafts to silica gel were further studied.It was determined that the lipid raft extracted by detergent method 2 was combined with the particle size of 5 μm and-NHS modified silica gel to prepare a lipid raft chromatographic model rich in P-gp protein,the ratio of lipid rafts to silica gel was 1:1 and the lipid raft column prepared by this method has good specificity.The adsorption performance is stable within 3 months,and the adsorption performance is still maintained after 200 hours of continuous use.The chromatographic conditions were as follows: the mobile phase was 10 mmol/L ammonium acetate(pH 7.4),the flow rate was 0.3 mL/min,the temperature was 37 ℃.. |
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