CN110201419B - Cell membrane chromatographic column with polyvinyl alcohol microspheres as carrier and preparation method thereof - Google Patents
Cell membrane chromatographic column with polyvinyl alcohol microspheres as carrier and preparation method thereof Download PDFInfo
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- G01N2030/889—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 monitoring the quality of the stationary phase; column performance
Abstract
The invention discloses a cell membrane chromatographic column taking polyvinyl alcohol microspheres as a carrier and a preparation method thereof, belonging to the technical field of cell membrane chromatographic columns. The cell membrane chromatographic column is prepared by wrapping cell membranes with polyvinyl alcohol microspheres to prepare a cell membrane stationary phase and then filling the cell membrane stationary phase into the column by adopting a wet method. The cell membrane stationary phase is prepared by wrapping the cell membrane with the polyvinyl alcohol microspheres, and the polyvinyl alcohol microspheres have abundant hydroxyl and aldehyde groups on the surfaces, so that the polyvinyl alcohol microspheres can be combined with the cell membrane through hydrophobic action, and the aldehyde groups can also react with abundant phospholipid amino groups on the membrane, so that the aim of covalently connecting the polyvinyl alcohol microspheres and the cell membrane is fulfilled, the fixation of the cell membrane on the stationary phase is more stable, and the service life of a cell membrane chromatographic column is prolonged. Meanwhile, the problems that the cell membrane is easy to fall off and the pH application range is narrow when silica gel is used as a carrier are improved to a certain extent.
Description
Technical Field
The invention belongs to the technical field of cell membrane chromatographic columns, and particularly relates to a cell membrane chromatographic column taking polyvinyl alcohol microspheres as a carrier and a preparation method thereof.
Background
The cell membrane chromatography is an effective new technology for directly screening and identifying target components from a complex system. The cell membrane chromatography is to fix the cell membrane of active tissue or cell on the surface of silica gel to prepare a cell membrane stationary phase, and then to prepare a cell membrane chromatographic column by wet column packing. The interaction between the medicine and cell membrane and receptor on the stationary phase is studied by liquid chromatography under dynamic condition by using buffer solution as mobile phase and medicine as solute or added in the mobile phase.
The chromatographic packing is a core material for separating components by liquid chromatography, and directly influences the separation efficiency of the chromatography and the accuracy of detection data. Therefore, research and development of novel chromatographic packing materials are always the focus of attention of chromatographs. Chromatography packing can now be divided into three major classes depending on its matrix material: inorganic matrix fillers, organic matrix fillers and composite materials. The silica gel matrix chromatographic packing has the advantages of high mechanical strength, high column efficiency, controllable packing grain size, controllable pore size and other advantages, and is one kind of inorganic packing widely used at present. However, the chromatographic packing has the following two obvious defects: firstly, the pH application range is narrow, and silica gel can be dissolved in strong acid or strong alkali solution, so that the application of the silica gel is limited; and secondly, residual silicon hydroxyl on the surface of the silica gel has adsorption activity, particularly shows a bee-shaped tailing effect on the separation of an alkaline compound, influences the separation effect and limits the further application of the silica gel.
The high molecular polymer matrix has the advantages of good biocompatibility, chemical stability, easy derivatization and the like. Polyvinyl alcohol (PVA) is a water-soluble high polymer material with wide application, is generated by performing base-catalyzed alcoholysis on Polyvinyl acetate, and has a relative molecular mass of 20000-200000. The physical properties of polyvinyl alcohol are mainly determined by molecular weight and alcoholysis degree, and different polymerization degrees result in different molecular weights. The polyvinyl alcohol has good water solubility because the molecules contain more hydroxyl groups; the molecular polymerization has good film forming property, adhesion force, emulsibility, oil resistance, solvent resistance and the like. Therefore, it is widely used as biomedical materials, polymer materials, etc. However, there are few reports on cell membrane chromatographic columns using polyvinyl alcohol microspheres as carriers and methods for preparing the same.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a cell membrane chromatographic column using polyvinyl alcohol microspheres as a carrier and a preparation method thereof, and the cell membrane chromatographic column can effectively solve the problems that cell membranes are easy to fall off when silica gel is used as the carrier and the service life of the cell membrane chromatographic column is short.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a cell membrane chromatographic column taking polyvinyl alcohol microspheres as a carrier, which is prepared by wrapping the polyvinyl alcohol microspheres with cell membranes to prepare a cell membrane stationary phase and then filling the cell membrane stationary phase into a column by a wet method.
Preferably, the polyvinyl alcohol microspheres are prepared by taking polyvinyl alcohol as a raw material and glutaraldehyde as a cross-linking agent and adopting an inverse suspension-chemical cross-linking method under the catalysis of acidic conditions.
Further preferably, the rigidity, particle size and dispersibility of the polyvinyl alcohol microspheres can be adjusted by controlling the concentration of polyvinyl alcohol, the amount of glutaraldehyde and the acid catalysis conditions.
Preferably, the polyvinyl alcohol microspheres have an average particle size of 4 μm.
Preferably, the cell membrane is an EGFR cell membrane.
The invention discloses a preparation method of a cell membrane chromatographic column with polyvinyl alcohol microspheres as carriers, which comprises the following steps:
1) polyvinyl alcohol is used as a raw material, glutaraldehyde is used as a cross-linking agent, and a reversed phase suspension-chemical cross-linking method is adopted under the catalysis of acid conditions to prepare the polyvinyl alcohol microspheres with regular spherical shapes and uniform sizes;
2) culturing the cells until the cell count is not less than 107In one occasion, the culture medium is removed to obtain cells, and cell membranes are separated;
3) preparing cell membranes into cell membrane suspension, adding the cell membrane suspension into the polyvinyl alcohol microspheres prepared in the step 1) under a vacuum condition, uniformly stirring, and standing overnight to obtain a cell membrane stationary phase taking the polyvinyl alcohol microspheres as a carrier;
4) and (3) filling the cell membrane stationary phase taking the polyvinyl alcohol microspheres as the carrier into a column by adopting a wet method to obtain the cell membrane chromatographic column taking the polyvinyl alcohol microspheres as the carrier.
Preferably, in step 1), the average particle size of the prepared polyvinyl alcohol microspheres is 4 μm.
Preferably, in step 2), the obtained cells are suspended with Tris-HCl and then disrupted in a cell disruptor, followed by differential centrifugation to separate the cell membranes.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a cell membrane chromatographic column taking polyvinyl alcohol microspheres as a carrier, wherein a cell membrane wraps the polyvinyl alcohol microspheres to prepare a cell membrane stationary phase, and the surface of the polyvinyl alcohol microspheres is provided with abundant hydroxyl and aldehyde groups, so that the polyvinyl alcohol microspheres can be combined with cell membranes through hydrophobic action, and the aldehyde groups can also react with abundant phospholipid amino groups on the membranes, thereby achieving the purpose of covalently connecting the polyvinyl alcohol microspheres with the cell membranes, ensuring that the cell membranes are more stably fixed on the stationary phase, and prolonging the service life of the cell membrane chromatographic column. Meanwhile, the problems that the cell membrane is easy to fall off and the pH application range is narrow when silica gel is used as a carrier are improved to a certain extent.
Furthermore, the polyvinyl alcohol microspheres are prepared by taking polyvinyl alcohol as a raw material and glutaraldehyde as a cross-linking agent and adopting an inverse suspension-chemical cross-linking method under the catalysis of acidic conditions. Therefore, the rigidity of the prepared polyvinyl alcohol microspheres can be regulated and controlled by controlling the concentration of the polyvinyl alcohol, the dosage of glutaraldehyde and the acid catalysis conditions, so that the prepared polyvinyl alcohol microspheres meet the requirements of chromatographic packing.
Drawings
FIG. 1 is a schematic representation of the cross-linking reaction of polyvinyl alcohol and glutaraldehyde.
FIG. 2 is an infrared spectrum of a polyvinyl alcohol microsphere (PVA) stationary phase and an EGFR cell membrane (EGFR/PVA) stationary phase using the polyvinyl alcohol microsphere as a carrier.
FIG. 3 is a graph showing the relationship between the flow rate and the back pressure of a polyvinyl alcohol microsphere (PVA) column and an EGFR cell membrane (EGFR/PVA) column using the polyvinyl alcohol microsphere as a carrier.
FIG. 4 is a chromatogram of gefitinib on an EGFR cell membrane (EGFR/PVA) chromatographic column using polyvinyl alcohol microspheres as carriers at different flow rates.
FIG. 5 shows the selectivity of an EGFR cell membrane (EGFR/PVA) chromatographic column using polyvinyl alcohol microspheres as carriers (AI: captopril, II: diazepam, III: tamsulosin hydrochloride, IV: gefitinib).
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
Referring to fig. 1, the cell membrane chromatographic column using polyvinyl alcohol microspheres as a carrier provided by the invention uses polyvinyl alcohol as a raw material and adopts an inverse suspension-chemical crosslinking method to prepare spherical polyvinyl alcohol microspheres. The cell membrane is prepared by digesting and centrifuging cells growing adherent to the walls, then the cell membrane is fully mixed with the polyvinyl alcohol microspheres, the cell membrane is coated on the surfaces of the polyvinyl alcohol microspheres by utilizing the adsorption effect of the polyvinyl alcohol microspheres and the fluidity of the cell membrane, and the aldehyde group can also react with abundant phospholipid amino groups on the membrane, so that the aim of covalently connecting the polyvinyl alcohol microspheres and the cell membrane is fulfilled. Then the fixed phase of the cell membrane chromatogram is filled into a chromatographic column through a wet column filling method to form the cell membrane chromatographic column taking the polyvinyl alcohol microspheres as a carrier. The cell membrane chromatographic column solves the problems that the cell membrane is easy to fall off when silica gel is used as a carrier, the cell membrane chromatographic column has short service life, the pH application range is narrow and the like, and provides a novel cell membrane loading material.
Specifically, the preparation method of the cell membrane chromatographic column with the polyvinyl alcohol microspheres as the carrier comprises the following steps:
1) preparation of polyvinyl alcohol microspheres
Under the condition of stirring, adding 1g of surfactant Span-80 into 20mL of liquid paraffin to form a continuous phase oil phase; after stirring for 10min, adding 5mL of 5% polyvinyl alcohol aqueous solution into the oil phase, setting the rotating speed at 400rpm/min, stirring for 3h, adding 1mL of glutaraldehyde as a cross-linking agent, and immediately adding 500 μ L of concentrated HCl as a catalyst. The rotation speed is set to be 400rpm/min, and stirring is carried out for 2 hours. After the crosslinking reaction was completed, the mixture was allowed to stand for 30 min. Adding a small amount of absolute ethyl alcohol, placing the mixture into a centrifuge for centrifugation (12000rpm, 20min), taking out supernatant, repeatedly washing precipitates with the absolute ethyl alcohol, isopropanol and pure water, and filtering the microspheres by using a G5 sintered funnel to obtain polyvinyl alcohol microspheres with uniform size (4 mu m). And finally, drying the mixture in a blast drying oven for 24 hours to obtain white powder, namely the polyvinyl alcohol microspheres.
2) Preparing cell membrane chromatographic stationary phase using polyvinyl alcohol microsphere as carrier
The cultured cells were counted at not less than (10)7And then digesting with 0.25% trypsin, centrifuging for 10min at 1000g and 4 ℃, removing the culture medium to obtain precipitates, adding 10mmol/L PBS buffer solution for resuspension, centrifuging for 10min at 1000g and 4 ℃, sucking the culture medium remained on the cell surface, repeating for 3 times, and separating the cultured cells. Then, 5mL of 50mmol/L Tris-HCl suspension cells are placed in a cell ultrasonic crusher to crush the cells, the cells are centrifuged for 10min at the temperature of 1000g and 4 ℃, the supernatant is taken and placed in a centrifuge tube to be centrifuged for 10min at the temperature of 12000g and 4 ℃, the precipitate is the cell membrane, and the cell membrane is washed for 1 time by using 10mmol/L PBS. Then the cells are culturedAdding the membrane suspension into 0.05g of polyvinyl alcohol microspheres under the condition of vacuum oscillation, placing the mixture on a magnetic stirrer, stirring the mixture for 30min at the temperature of 4 ℃, standing the mixture overnight after ensuring that cell membranes and the polyvinyl alcohol microspheres are fully mixed, and fully wrapping the cell membranes and the polyvinyl alcohol microspheres by utilizing the fluidity of the cell membranes and the adsorption effect of the polyvinyl alcohol microspheres to obtain the cell membrane chromatographic stationary phase taking the polyvinyl alcohol microspheres as a carrier.
3) Establishment of cell membrane chromatographic column
And (3) filling the obtained cell membrane chromatographic stationary phase taking the polyvinyl alcohol microspheres as the carrier into a 10mm (L) multiplied by 2.0mm (I.D.) column core by using an RPL-ZD10 column filling machine in a wet method to obtain the cell membrane chromatographic column taking the polyvinyl alcohol microspheres as the carrier.
The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.
In the case of Epidermal Growth Factor Receptor (EGFR), EGFR is an N-linked glycoprotein having tyrosine kinase activity widely distributed on the surface of mammalian epithelial cells, glial cells, fibroblasts, and keratinocytes. The expression of EGFR is closely related to the generation, proliferation, metastasis and angiogenesis of tumors, so that the EGFR-mediated signal transduction process becomes a new target for the research of antitumor drugs. The cell membrane chromatography can screen out target components from a complex system, has double functions of chromatographic separation and receptor affinity, adopts HEK293 cells with high expression of EGFR receptors and takes polyvinyl alcohol microspheres as carriers to prepare a cell membrane chromatography stationary phase, and can screen out specific components acting on the EGFR receptors.
1. The cell membrane chromatographic column with polyvinyl alcohol microsphere as carrier is prepared through the following steps:
1) preparation of polyvinyl alcohol microspheres
Under the condition of stirring, adding 1g of surfactant Span-80 into 20mL of liquid paraffin to form a continuous phase oil phase; after stirring for 10min, 5mL of 5% PVA aqueous solution is added into the oil phase, the rotation speed is set to be 400rpm/min, 1mL of glutaraldehyde is added as a cross-linking agent after stirring for 3h, and 500 μ L of concentrated HCl is immediately added as a catalyst. The rotation speed is set to be 400rpm/min, and stirring is carried out for 2 hours. After the crosslinking reaction was completed, the mixture was allowed to stand for 30 min. Adding a small amount of absolute ethyl alcohol, placing the mixture into a centrifuge for centrifugation (12000rpm, 20min), taking out supernatant, repeatedly washing precipitates with the absolute ethyl alcohol, isopropanol and pure water, and filtering microspheres by using a G5 sintering funnel to obtain polyvinyl alcohol microspheres with uniform sizes. And finally, drying the mixture in a blast drying oven for 24 hours to obtain white powder, namely the polyvinyl alcohol microspheres.
2) Preparation of EGFR cell membrane stationary phase
Selecting EGFR cell adhered to the bottle wall, counting the cells, and counting the number of the cells not less than 107In this case, the cell suspension was obtained by digesting with 0.25% trypsin digestion solution. And centrifuging the cell suspension at 4 ℃ and 1000rpm for 10min, removing the culture solution of the supernatant, and obtaining the precipitate, namely the EGFR cell. Adding physiological saline to resuspend, centrifuging again under the above conditions, washing off residual culture medium on cell surface, discarding supernatant, and repeating the cleaning process with physiological saline for 2 times. And adding 5mL of precooled Tris-HCl hypotonic buffer solution into the obtained cell sediment, placing the cell sediment in an ultrasonic instrument for 30min of ice bath ultrasonic disruption, and ensuring the cell sediment to be below 4 ℃ during ultrasonic disruption so as to ensure the activity of cell membranes. After the cells are broken by ultrasonic, the cell breaker is used for breaking membranes, the operation is carried out for 3s, the interval is 1s, the operation is repeated for 6 times, and the program is reset. Centrifuging 1000g at 4 ℃ for 10min, suspending the cell membrane in supernatant, sucking the supernatant into another centrifuge tube, centrifuging 12000g at 4 ℃ for 20min to obtain a precipitate, namely the cell membrane, resuspending the precipitate with normal saline, centrifuging 12000g at 4 ℃ for 20min again, discarding the supernatant, suspending the obtained cell membrane precipitate with 5mL of normal saline, sucking into a 5mL syringe, uniformly mixing with polyvinyl alcohol microspheres under the condition of vacuum vortex oscillation, placing on a magnetic stirrer, stirring at 4 ℃ for 30min, and standing overnight to obtain the EGFR cell membrane stationary phase taking the polyvinyl alcohol microspheres as a carrier.
3) Establishment of EGFR cell membrane chromatographic column using polyvinyl alcohol microspheres as carrier
And (2) uniformly mixing the cell membrane stationary phase suspension in a vortex manner, transferring the cell membrane stationary phase suspension to a 10mL centrifuge tube, centrifuging for 5min at 1000g under the condition of 4 ℃, discarding the supernatant, adding 5mL of physiological saline into the precipitate, uniformly mixing in a vortex manner, repeating the operation for 2 times, removing redundant cell membranes which are not wrapped on the polyvinyl alcohol microspheres, adding about 5mL of physiological saline into the precipitate, uniformly mixing in a vortex manner, pouring the precipitate into a column filling machine which is washed in advance, filling the cell membrane stationary phase into a column core, filling the filled cell membrane chromatographic column core into a stainless steel column sleeve, filling the column into a liquid chromatograph for use, or placing the column filling machine into triple-distilled water, and storing in a refrigerator at 4 ℃ for later use.
2. Characterization of the EGFR cell Membrane stationary phase prepared above
Taking a proper amount of dried polyvinyl alcohol microspheres and cell membrane-wrapped polyvinyl alcohol microspheres, respectively grinding and uniformly mixing with KBr, drying, and testing by adopting a Fourier infrared spectrometer (FT-IR) at the wavelength range of 4000-500 cm < -1 >. The samples were thoroughly dried and kept clean before testing. Measuring the infrared spectrum, and the detection result is shown in FIG. 2, wherein 2955.67cm is shown-1In the terminal CH corresponding to lipids in the cell membrane3And CH in proteins3Thus, the successful coating of the cell membrane and the surface of the polyvinyl alcohol microsphere are demonstrated.
3. Investigating the permeability of an EGFR cell membrane chromatographic column using polyvinyl alcohol microspheres as carriers
The permeability of the column is an important parameter in its practical application, and the mechanical properties of the microspheres and the permeability of the packed column can be characterized by the flow rate-back pressure relationship of the packed column. Polyvinyl alcohol microspheres and cell membrane-coated polyvinyl alcohol microspheres were packed in a stainless steel column of 10mm (l) × 2.0mm (i.d.) by wet column packing, and column pressures at different flow rates were examined with water as a mobile phase, and the results are shown in fig. 3. The flow rate backpressure of the polyvinyl alcohol microsphere chromatographic column and the EGFR cell membrane chromatographic column using the polyvinyl alcohol microspheres as the carrier keep a good linear relation in the flow rate range of 0.05-0.30 mL/min, and the phenomenon of column bed collapse does not occur, so that the polyvinyl alcohol microsphere chromatographic column and the EGFR cell membrane chromatographic column using the polyvinyl alcohol microspheres as the carrier have good permeability and connectivity, have high mechanical strength, have the capability of separation under high pressure and high flow rate, and are beneficial to screening active ingredients from a traditional Chinese medicine complex system.
4. Investigating the flow rate of an EGFR cell membrane chromatographic column using polyvinyl alcohol microspheres as carriers
An EGFR cell membrane chromatographic column using polyvinyl alcohol microspheres as a carrier maintains a good linear relation of flow rate and back pressure within a flow rate range of 0.05-0.30 mL/min, and retention graphs of the positive drug gefitinib are respectively considered when the flow rates are respectively 0.05, 0.10, 0.15, 0.20, 0.25 and 0.30mL/min, as shown in FIG. 4. As the flow rate is increased, the retention time of gefitinib is shortened, the peak width is narrowed, but the pressure of the chromatographic column is increased, so that 0.2mL/min is selected as the optimal flow rate of the EGFR cell membrane chromatographic column using the polyvinyl alcohol microspheres as the carrier.
5. Verifying the effect of the EGFR cell membrane chromatographic column using the polyvinyl alcohol microspheres as the carrier
Selective investigation of an EGFR cell membrane chromatographic column, 5 muL of 0.1mg/mL gefitinib, 5 muL of 0.1mg/mL captopril, 5 muL of 0.1mg/mL diazepam and 5 muL of 0.1mg/mL tamsulosin hydrochloride are injected in sequence, the result shows that the gefitinib positive drug is reserved on the EGFR cell membrane, and the captopril, the diazepam and the tamsulosin hydrochloride as negative control drugs are not reserved on the cell membrane, as shown in figure 5.
The results show that the selectivity of the EGFR cell membrane chromatographic column is good. The intra-column difference of the EGFR cell membrane chromatographic column mainly takes the retention time of gefitinib on the EGFR cell membrane chromatographic column as an index, the intra-column difference is that an EGFR/CMC chromatographic column is prepared, placed in a liquid chromatogram, and continuously fed with 5 mu L0.1mg/mL gefitinib samples for 5 times after full balance, and the retention time of the gefitinib of each sample injection is respectively recorded. The difference among the columns is that 3 EGFR/CMC chromatographic columns are prepared simultaneously according to the same method, and after being placed in a liquid chromatograph and fully balanced by applying the same chromatographic conditions, 5 mu L of 0.1mg/mL gefitinib samples are respectively put in, and the retention time of gefitinib on each EGFR/CMC chromatographic column is respectively recorded, and the results are shown in Table 1: the intra-column and inter-column variability was good. The cell membrane chromatographic column not only has the function of chromatographic separation, but also has biological activity, so the activity time of the cell membrane chromatographic column is one of the important indexes of the cell membrane chromatographic column. According to the research, 3 EGFR2 cell membrane chromatographic columns are prepared simultaneously by utilizing the preparation method of the EGFR cell membrane chromatographic column, then the EGFR cell membrane chromatographic column is placed in a liquid chromatogram, after full balance, 5 mu L of 0.1mg/mL gefitinib sample is continuously injected, after 72 hours, gefitinib still obviously remains on the EGFR cell membrane chromatographic column, and the results are shown in Table 1, which indicates that the EGFR cell membrane chromatographic column still has good activity after 72 hours of application.
TABLE 1 reproducibility and Activity examination of EGFR cell membrane chromatographic column Using polyvinyl alcohol microspheres as Carrier
In summary, the EGFR cell membrane chromatographic column using the polyvinyl alcohol microspheres as the carrier can specifically identify the target component gefitinib acting on the EGFR cell membrane receptor, the repeatability in and among columns of the EGFR cell membrane chromatographic column is good, and the activity time of the EGFR cell membrane chromatographic column can meet the requirements of qualitative identification tests, so the EGFR cell membrane chromatographic column using the polyvinyl alcohol microspheres as the carrier can be applied to screening and retaining components from a traditional Chinese medicine complex system.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (5)
1. A cell membrane chromatographic column taking polyvinyl alcohol microspheres as a carrier is characterized in that the cell membrane chromatographic column is prepared by wrapping cell membranes on the surfaces of the polyvinyl alcohol microspheres by utilizing the fluidity of the cell membranes and the adsorption effect of the polyvinyl alcohol microspheres, reacting aldehyde groups on the surfaces of the polyvinyl alcohol microspheres with phospholipid amino groups on the cell membranes to covalently connect the polyvinyl alcohol microspheres with the cell membranes to prepare a cell membrane stationary phase taking the polyvinyl alcohol microspheres as the carrier, and then filling the cell membrane stationary phase into the column by adopting a wet method;
the average grain diameter of the polyvinyl alcohol microspheres is 4 mu m, and EGFR cell membranes are adopted as cell membranes.
2. The cell membrane chromatographic column taking the polyvinyl alcohol microspheres as the carrier according to claim 1, wherein the polyvinyl alcohol microspheres are prepared by taking polyvinyl alcohol as a raw material and glutaraldehyde as a cross-linking agent and adopting an inverse suspension-chemical cross-linking method under the catalysis of acidic conditions.
3. The cell membrane chromatographic column using the polyvinyl alcohol microspheres as the carrier according to claim 2, wherein the rigidity, the particle size and the dispersibility of the polyvinyl alcohol microspheres can be adjusted by controlling the concentration of polyvinyl alcohol, the dosage of glutaraldehyde and the acid catalysis conditions.
4. A method for preparing a cell membrane chromatographic column by using polyvinyl alcohol microspheres as a carrier is characterized by comprising the following steps:
1) polyvinyl alcohol is used as a raw material, glutaraldehyde is used as a cross-linking agent, and a reverse phase suspension-chemical cross-linking method is adopted under the catalysis of acid conditions to prepare polyvinyl alcohol microspheres with regular spherical shapes and average particle sizes of 4 microns;
2) culturing EGFR cell until cell count is not less than 107In each case, removing the culture medium to obtain cells, and separating the EGFR cell membrane;
3) preparing an EGFR cell membrane into an EGFR cell membrane suspension, adding the EGFR cell membrane suspension into the polyvinyl alcohol microspheres prepared in the step 1) under a vacuum condition, uniformly stirring, and standing overnight to obtain a cell membrane stationary phase taking the polyvinyl alcohol microspheres as a carrier;
4) and (3) filling the cell membrane stationary phase taking the polyvinyl alcohol microspheres as the carrier into a column by adopting a wet method to obtain the cell membrane chromatographic column taking the polyvinyl alcohol microspheres as the carrier.
5. The method for preparing a cell membrane chromatographic column with polyvinyl alcohol microspheres as a carrier according to claim 4, wherein in the step 2), the obtained cells are suspended with Tris-HCl and then placed in a cell ultrasonication instrument for disruption, and then cell membranes are separated by differential centrifugation.
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| WO2015035125A1 (en) * | 2013-09-06 | 2015-03-12 | Arizona Board Of Regents For The University Of Arizona | Cholesteryl succinyl silane bound proteins and methods for producing and using the same |
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| WO2015035125A1 (en) * | 2013-09-06 | 2015-03-12 | Arizona Board Of Regents For The University Of Arizona | Cholesteryl succinyl silane bound proteins and methods for producing and using the same |
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