Development And Applications Of New Cell Membrane Chromatography Covalently Decorated By APTES

Cell membrane chromatography,CMC for short,is a kind of biological active chromatography to immobilize cell membranes on solid phase.It has combined the advantages of traditional chromatography method in high throughout-put and online analysis and the advantages of biological materials in biological activities,making it possible,simple and convenient to screen potential biological active components from a complex system,such as TCM.The characterization of integrality and complexity of traditional Chinese medicine,known as TMC,has endowed it with the advantages of multiple targets and multiple regulation pathways.But it is with these advantages that lead to the ambiguity of its drug effect,modern scientific theory and active components.Up to day,the practice of using TMC still mainly relies on the experience and the dialectical TCM theory we obtained from our ancestors in the past thousands of years.Thus,as a new-born biological chromatography technology,CMC has been widely applied in the screening of potential active components in TCM.This research mainly focuses on the investigation and improvements of CMC methodology.A new CMC model after decoration with APTES has been established.And after method validation,a series of applications in the screening of potential biological active components from TCM have been developed.These applications have provided scientific,normative and logical evidence to use CMC and have also further extended the range of use of CMC.1.Establishment and method validation of APTES decorated CMCAs a new affinity biological chromatography,CMC is prepared by immobilizing cell membranes on solid phase.But its short life span and biological activity loss have prevented it from being more widely used.Besides,low reproducibility between different labs resulting from different preparation procedures has also been a main obstacle for CMC.Thus,in this study,to solve the problem of short life span,a kind of new silica,which is decorated with(3-aminopropyl)triethoxysilane(APTES),is used in the preparation of CMC.The theory of this procedure is that with the help of APTES,the surface of silica gel can be covered with abundant aldehyde groups,which can be bonded to the massive amino groups on cell membranes covalently.By this way,the bond strength between silica gel and cell membranes has been greatly improved and the phenomenon of cell membrane falling off or activity loss has been relived to some extent.Thus the CMC columns prepared under this method have shown higher column efficiency and longer column life span.To solve the problem of low reproducibility of CMC,a method validation of the newly established APTES decorated CMC has been carried out.Key procedures in the preparation of CMC have been investigated,such as cell quant and cell disruption.And a series of defined usage quant and standard protocols have also been established.2.Establishment of 2D MCF7/CMC and its application in the screening of potential anti-breast cancer components from Corydalis yanhusuoTo further broaden the range of application of the newly established model in the first chapter and to investigate the main anti-breast cancer components in Corydalis yanhusuo,a 2D MCF7 cell membrane chromatography model is established to screen potential active components in Corydalis yanhusuo online.Having fully investigated the selectivity and stability of the 2D MCF7/CMC system,three potential active components are screened out,namely oxoglaucine,protopine and berberine.These three components can effectively kill MCF7 cells in a dose dependent way,with the IC50 of 36.16 ?M?29.30 ?M and 45.52 ?M respectively.Besides,by carrying out cell apoptosis and death assay,we find that these three potential active components generate medical effects mainly through inducing the apoptosis of MCF7 cells.In all,the study in this chapter shows that the 2D MCF7/CMC model we established is fast,sensitive and of high recognition ability,which is very suitable for screening potential anti-breast cancer components from TCM.3.Establishment of 2D DU145/CMC and its application in the screening of potential anti-prostate cancer components from Rheum palmatum L and Scutellaria baicalensis GeorgiTo further confirm the validation of the newly established APTES decorated CMC model in the first and second chapter and to broaden the range of its application,a 2D-DU145/CMC system is developed and used to screen potential anti-prostate cancer components from Rheum palmatum L and Scutellaria baicalensis Georgi.Having thoroughly investigated the stability and selectivity of the 2D-DU145/CMC model,three potential active components are screened out,namely oroxylin A,rhapontigenin and emodin.These three components can effectively kill DU145 cells in a dose dependent way,with the IC50 of 151.1 ?M?55.66 ?M and 85.81 ?M respectively.Besides,having carried out cell apoptosis and death assay,we find that these three potential active components generate medical effects mainly through inducing the apoptosis of DU145 cells.In all,the study in this chapter shows that the APTES decorated 2D-DU145/CMC model we established is fast,sensitive and of high recognition ability,which is very suitable for screening potential active components from TCM.4.Establishment of 2D HepG2 cancer stem cell membrane chromatotraphy and its application in the screening of potential anti-liver cancer components from Salvia miltiorrhizaDuring recent years,cancer stem cells,CSCs for short,have aroused wide attention in the field of anti-cancer research.CSC is a kind of special cancer cell with the ability of self-refresh,which plays vital role in the recurrence,transfer and migration of cancer progress.Thus killing CSC will not only improve the cure rate of cancer,but also reduce the rate of recurrence.However,it is difficult and time-consuming to separate and culture CSCs,resulting the CSC-related researches hard to be carried out.While the APTES decorated CMC model we established in this research takes advantages in longer life span and higher efficiency,which is very suitable to be applied in the research of CSC.Thus in this study,based on the APTES decorated CMC model we established in the first chapter,a new HepG2 cancer stem cell membrane chromatography(HepG2-CSC/CMC)is established.And a famous TCM,Salvia miltiorrhiza,is selected as an ideal target to screen potential active components.There has been report that 70% newly approved drugs in clinical has been discovered from natural products.Therefore,it will be a promising field to screen potential anti-cancer-stem-cell drugs from TCM.Additionally,a comprehensive tow dimensional chromatography method is also applied to improve the separation ability of CMC.Under these conditions,an APTES decorated 2D HepG2-CSC/CMC model is established and used to screen potential anti-cancer-stem-cell components from Salvia miltiorrhiza.Finally,three active components are targeted,namely tanshinone IIA,cryptotanshinone,dihydrotanshinone I.By carrying out cell inhibition assay,these three components can inhibit the growth of HepG2-CSCs in a dose dependent manner,with the IC50 of 10.300 ?M,17.850 ?M,2.53 ?M respectively.Their drug effects are further confirmed by carrying out cell apoptosis and death assay.Besides,a virtual molecular docking method is applied and VEGFR2 is targeted as a potential target.It will be an effective method to use APTES decorated CMC to screen potential active components from TCM,especially when the target we loaded on the CMC is of high biological value or hard to obtain.In all,this research focuses on the problems of short life span and low reproducibility of CMC and a covalent decoration method is innovatively and firstly applied.By decorating the silica gel with APTES,the surface of gels can be covered with abundant aldehyde groups,which can be able to from covalent bond with the massive amino groups on cell membranes.In this way,the binding force between cell membranes and solid phase is further strengthened,resulting in improved column life span and higher column efficiency.This is the first time that CMC is strengthened by convalent bonding.Besides,a series of protocol and standards are investigated and established to guarantee the reproducibility of the new APTES decorated CMC model.Based on all this,a 2D MCF7/CMC model,a 2D DU145/CMC model and a 2D HepG2-CSC/CMC model are established and used to screen potential active components from TCM.Multiple components targeting at breat cancer,prostate cancer and liver cancer have been identified and confirmed.To sum up,this research has improved the methodology of CMC by developing a brand new APTES-decorated CMC model and broadened the range of CMC applications,which may provide TCM screening with new ideas.