CN107349636B

CN107349636B - Capillary tube with biological material as interaction phase and preparation and application thereof

Info

Publication number: CN107349636B
Application number: CN201710564781.0A
Authority: CN
Inventors: 凌笑梅; 武瑞君; 李晨; 朱凯; 张晓丹; 张素芳; 任进宇; 刘燕萌; 杨东辉; 叶敏
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2017-07-12
Filing date: 2017-07-12
Publication date: 2020-01-10
Anticipated expiration: 2037-07-12
Also published as: CN107349636A

Abstract

The invention provides a capillary with a column screen and using biological materials as an interaction phase, a preparation method thereof and application in researching interaction between biomacromolecules and active ligands or compounds and drug screening by a capillary electrophoresis method. Aiming at the problems that the separation and purification of certain bioactive macromolecules from cells or isolated biological tissues are difficult and can cause the change of natural conformation, the invention develops a Capillary Electrophoresis method which is used for injecting biological materials (such as cells, mitochondria, isolated tissues, tumor tissues, biological target-carrying solid substances and the like with the overexpression of the biological macromolecules) into capillaries with column screens, intercepting the biological materials at the rear ends of the column screens to serve as an interaction phase, establishing Non-immobilized biological Capillary Electrophoresis (NIBCE) of the biological materials, and researching the interaction of the biological macromolecules and active ligands or compounds, drug screening and the like under the condition close to physiological environment.

Description

Capillary tube with biological material as interaction phase and preparation and application thereof

Technical Field

The invention relates to a Capillary tube, a preparation method and application thereof, in particular to a Capillary chromatographic column with a column screen for intercepting biological materials serving as an interaction phase, a preparation method thereof and application of the Capillary chromatographic column in the aspects of researching interaction between biomacromolecules and active ligands or compounds, screening drugs and the like in the field of biomedicine through High Performance Capillary Electrophoresis (HPCE).

Background

Most biomolecules exert their physiological functions by specifically interacting with their target molecules, such as enzymes and substrates, antigens and antibodies, receptors and hormones, lectins and polysaccharide proteins, and the like. These specific interactions are a widespread phenomenon in chemical and biochemical systems and are the basis for the occurrence of life phenomena. Characterization of interaction between biological molecules and their functional ligands has become one of the key points of biochemical research, which helps to deeply understand the structural basis and action mechanism of various functions between biological macromolecules and helps to discover new drugs. Therefore, it becomes important to develop a method for studying the interaction between biomolecules or between a biomacromolecule and other ligands.

Methods for studying the interaction between biomolecules or between biomacromolecules and other ligands can be mainly divided into two main categories: one is a direct assay, including UV absorption, raman spectroscopy, fluorescence intensity, potentiometry, thermal analysis, Nuclear Magnetic Resonance (NMR), fourier transform infrared spectroscopy (FTIR), Mass Spectrometry (MS), Surface Plasmon Resonance (SPR), etc.; another class is separation assays, including membrane filtration assays, ultrafiltration, ultracentrifugation, chromatography (thin layer and liquid), electrophoresis (plate and HPCE). The latter has the advantages that: can provide information of two molecules and compounds interacting with each other at the same time, and avoids the interference of coexisting substances. The membrane analysis, ultrafiltration, ultracentrifugation, chromatography and flat electrophoresis, which are included in the separation analysis, are often used to study the ligand-receptor interaction, but they have the disadvantages of leakage of binding molecules from the membrane, volume change, Donnan effect, nonspecific adsorption, long analysis time, large sample consumption, etc.; in recent years, HPCE-based techniques have become the primary method of studying interactions between biomolecules or biomacromolecules and other ligands.

HPCE is used for researching the interaction between biomolecules or between biomacromolecules and other ligands, and has the following advantages: high separation efficiency up to 10⁵-10⁶Plate/meter; the analysis time is short, and the analysis can be completed within dozens of seconds to dozens of minutes; the sample consumption is less, the volume required by sample injection can be as small as 1 mu L, and the consumption volume is between 1 and 50 nL; the method can be carried out in a buffer solution under physiological conditions or close to physiological conditions, and can obtain relatively real information of interaction between biomolecules; the binding behavior of a plurality of molecules to one molecule can be studied simultaneously; the application is flexible, and different modes can be selected to be suitable for different separation objects; is economical; cleaning; the automation degree is higher. Among the forms of research of interaction, HPCE is used for pre-equilibrium zone electrophoresis, kinetic equilibrium zone electrophoresis and immobilized affinity electrophoresis. Immobilized Cell Capillary Electrophoresis (ICCE) and Cell Membrane Chromatography (CMC) have been developed in recent years, allowing ligand screening of receptors that cannot be separated directly by HPCE or CMC. The research on the interaction of receptor ligands by fixing cells in a chromatographic column and applying the research to drug screening is a new technology developed in recent years, and the technology has the outstanding advantage of immobilizing inseparable cell membrane receptors, thereby overcoming the problems of low accuracy, high toxicity, high cost and the like of the traditional biological method. The cell immobilization technology utilizes a physical or chemical method to fix cells on a proper carrier, so that the cells exert biological activity, and the method is simpler, more convenient, quicker and more economical than an enzyme immobilization method. However, the method still has the defects of complex operation, difficult preparation, long period, short service life, capability of screening high-expression receptor cells and the like.

Therefore, on the basis of ICCE, the method is innovated, and the establishment of the method which can directly screen various biomaterial surface receptors such as cells, mitochondria, isolated tissues, tumor tissues, carrying biological target solid substances and the like with biological macromolecule overexpression (such as GLUT1) becomes important.

Disclosure of Invention

The invention provides a capillary tube and a capillary electrophoresis system (namely non-immobilized biological material capillary electrophoresis, NIBCE) with biological materials as interaction phases, and preparation and application thereof.

The invention provides a novel capillary for the first time, prepares a column screen with controllable pore diameter, injects a fixed biological material as an interaction phase, and ensures that a receptor on the surface of the interaction phase keeps natural conformation and biological activity, and researches the application of the novel capillary in the fields of biotechnology such as the research of the interaction of biological macromolecules and active ligands or compounds, the medicine screening and the like through HPCE.

The invention provides a capillary tube with a column screen and an interaction phase, wherein the pore diameter of the column screen is controllable and can be used for intercepting biological materials serving as the interaction phase.

The column screen may be disposed at the middle or end of the capillary tube, or the column screen may be attached to the end of the capillary tube.

The biological material comprises cells, mitochondria, isolated biological tissues, tumor tissues, solid matter carrying biological targets and the like. Wherein the cell is a biological macromolecule over-expression cell or a tumor cell, and the macromolecule receptor on the cell surface is highly expressed and keeps the natural conformation and the biological activity.

A column sieve with controllable pore diameter is arranged in the capillary or among the capillaries, and biological materials are fixed and then injected into the capillaries as an interaction phase, so that receptors on the surface of the interaction phase keep natural conformation and biological activity. Wherein the material of the column screen is inorganic material, organic material, composite material, biological material or any other suitable material. In another aspect of the present invention, there is provided a method of preparing the capillary, which comprises:

pretreating the capillary tube;

pretreating the column screen material and forming a column screen, and enabling the column screen to be arranged in the middle or at the end part of the capillary tube, or enabling the column screen to be connected to the end part of the capillary tube, so that the capillary tube with the column screen is formed; if the column screen is arranged at the end part of the capillary or connected to the end part of the capillary, the end part of the capillary and the end part of the capillary with the detection window need to be connected through a connector;

culturing or treating and immobilizing the biological material as an interaction phase; and injecting the immobilized biological material into the capillary tube with the column screen, so that the biological material is intercepted at the rear end of the column screen as an interaction phase.

In addition, the present invention also relates to an HPCE system, comprising: the invention relates to a capillary tube with a pore diameter controllable column screen; means for introducing biological material and a sample into the capillary; means for performing an electrophoresis operation on said sample introduced into said capillary; and a device for detecting the sample.

Meanwhile, the invention also provides an HPCE method, which comprises the steps of providing a capillary with a pore-size-controllable column screen, fixing biological materials such as cells, mitochondria, isolated tissues, tumor tissues and the like, and injecting the fixed biological materials into the capillary to serve as an interaction phase; providing an electrophoretic device; introducing a biological material or sample into the capillary; performing HPCE analysis on the sample; and carrying out detection analysis on the sample.

The HPCE method is suitable, for example, for capillary electrophoresis for studying intermolecular interactions, including array capillary electrophoresis, chip capillary electrophoresis, array chip capillary electrophoresis, capillary liquid chromatography, and the like.

Furthermore, the invention also provides the application of the capillary or the capillary chromatographic column in the field of biotechnology. Such fields of biotechnology include, for example, drug screening, the interaction of biological macromolecules with active ligands or with compounds, the analysis of pharmaceutical compositions, the purification and identification of pharmaceutical active ingredients, etc.

The column screen established by the invention intercepts and fixes biological materials (cells, mitochondria, isolated tissues, tumor tissues and the like) as an interaction phase, is used for researching intermolecular interaction and a method for screening drugs, and can be used for qualitatively and quantitatively researching the interaction of a compound and a receptor. In phase with ICCE and other methods for immobilizing cells in capillariesCompared with the prior art, the method provided by the invention has the advantages that: (1) when cells and the like are taken as an interaction phase, the cells injected into the capillary have two states of dynamic state and steady state, wherein the dynamic state can be used for simulating the interaction between the drugs and the tumor cells in the physiological environment; (2) the interaction phase can be updated at any time by reversely beating the capillary, so that a false negative result caused by saturation of an active site is avoided, the capillary column can be repeatedly used, and the cyclic utilization rate is high; (3) the biological material is intercepted and can not reach the detector, thereby eliminating the interference of the biological material on the detector, being particularly beneficial to quickly finding the bound substance from the mixture by adopting HPCE-MS and selecting the substance with the strongest binding activity through the calculation of the binding constant; (4) the method has the advantages of less sample consumption, high sensitivity, good reproducibility and ideal chromatographic peak shape, and can be used for quantitatively analyzing the interaction of the receptor and the ligand by using NLC technology to give kinetic parameters K, K', K_a，k_d(ii) a (5) The preparation method is very simple and rapid, the cost is low, and the experimental period is short; (6) the method widens the application range, introduces different biological materials into the capillary as an interaction phase, is used for researching the interaction between molecules, and has practical application value.

The invention realizes the immobilization of macromolecular receptor protein which is difficult to separate and purify by intercepting biological materials (cells, mitochondria, isolated tissues, tumor tissues and the like) injected into capillaries through a column screen, and further realizes the affinity chromatography ACE research of the macromolecular receptor protein. Secondly, the method has the advantages of rapidness, high efficiency, economy, sensitivity, small sample consumption and the like of HPCE, and can be used for researching the interaction between a mixture or a single compound and a biological material and is different from other drug screening methods. Some currently available drug screening technologies can only provide IC₅₀The method of the invention can provide kinetic parameters such as binding constants.

The capillary and related methods of the present invention are also applicable to other fields and have significant technical advantages, such as: the high-flux medicine screening method of array capillary electrophoresis is used in screening combined chemical library or precursor medicine in traditional Chinese medicine.

Drawings

Fig. 1 shows a correlation diagram established for the preparation of a capillary column screen (taking silica gel as the column screen material as an example) and the screening method according to one embodiment of the present invention. Wherein: a is electron microscopy scan of capillary column screen (SEM, JSM-5600LV, JEOL, Japan); b is an image of HEK293 cells transfected with GLUT1-eGFP for 36h collected by confocal microscopy (GLUT1-eGFP is localized on the cell membrane and shows green fluorescence, and the nuclei of HEK293 cells of GLUT1-eGFP stain blue fluorescence); c is an image of the immobilized cell chromatographic column collected by a confocal microscope; d images of the cell status within the capillary tube collected by the optical microscope (left "dynamic" cells, right "steady" cells); e is the HPCE plot of EGCG at different GLUT1-HEK293 cell densities; f is HPCE diagram of DMSO and EGCG under the state of dynamic and static GLUT1-HEK293 cells; g is an electrophoretogram of Non-immobilized biological material capillary electrophoresis (NIBCE) established according to the invention, and DMSO and EGCG are on different capillary chromatographic columns (column screen capillaries, common HEK293 cells as an interaction phase, HEK293 cells overexpressing GLUT1 as an interaction phase).

Fig. 2 is a table comparing kinetic parameters of EGCG measured by both NICCE and ICCE methods according to an embodiment of the present invention.

FIG. 3 is a HPCE diagram of the total flavonoids of scutellaria (A) and part of the compounds (B) screened and a glucose competition experiment electrophoresis diagram of the part of the active compounds screened according to the embodiment of the present invention (C).

FIG. 4 illustrates the structures of 22 compounds screened according to embodiments of the present invention.

FIG. 5 is a table showing the screening results of 22 compounds shown in FIG. 4 and the comparison of NICCE and ICCE results.

FIG. 6 is a graph of HPCE of a portion of the compounds when A549 tumor cells are in the interaction phase, according to an embodiment of the invention.

FIG. 7 is a graph of HPCE of a portion of the compounds when ex vivo lung tissue and tumor tissue are in interacting phase, according to an embodiment of the invention.

FIG. 8 shows the results of experiments on MTT cell level antitumor activity of EGCG, FSB-1, FSB-2, FSB-3, SL-3, and SL-4.

FIG. 9 shows the results of the anti-tumor activity test at animal level. Wherein: A. b is the tumor growth of the animals in the normal saline group (NS group), EGCG group (80mg/kg), FSB-1 group (80mg/kg) and FSB-2 group (80mg/kg) at the time of sacrifice on day 27 after administration; c is the change of body weight of each group of animals after administration; d is the change of the tumor volume of each group of animals after administration; e is the weight of the tumors in each group at the time of sacrifice of the animals on day 27 after administration. Represents significant differences from NS group (. P <0.05,. P <0.01,. P < 0.001).

Detailed Description

The invention is further described, and illustrated, in connection with certain embodiments, with the preparation of a column screen in or between capillaries, the culture and immobilization of cells, the extraction of mitochondria, the immobilization of ex vivo tissue and tumor tissue, the injection of biological material such as cells, mitochondria, ex vivo tissue, tumor tissue, etc. into capillaries as an interactive phase, and the use of such novel capillaries by HPCE in biotechnology areas such as studying the interaction of biological macromolecules with active ligands or compounds and drug screening.

In one embodiment of the present invention, a material powder, such as silica gel powder with a certain particle size, is fully treated with a solution, and then filled into the middle or end of the capillary tube, and after a certain time of heat treatment, the column-sieve capillary tube with a wide pH tolerance range, good reproducibility and controllable pore size is prepared.

In one embodiment, biological material, e.g. cells, mitochondria, ex vivo tissue, tumor tissue, etc., is injected into a capillary chromatography column with a column screen, thereby obtaining a capillary chromatography column with biological material as an interaction phase.

In one embodiment, a cell, e.g., a normal cell, a cell over-expressing a biological macromolecule, a tumor cell, is fixed and injected into a capillary tube, thereby obtaining a capillary tube having the cell as an interaction phase.

In one embodiment, the cells are cultured appropriately (e.g., RPMI 1640 medium containing 10-20% FBS at 37 ℃ in 5% CO)₂Culturing in an incubator for a period of, e.g., 10-48 h), suspending the cells in PBS to a density of, e.g., 1.0X 10⁴-1.0×10⁷one/mL, followed by treatment of the layer of living cells with, for example, 4% paraformaldehyde, which may be for, for example, 5-25 min.

In one embodiment, the body tissue and tumor tissue are treated with 4% paraformaldehyde for 12-48h, and then stored in 75% ethanol for further use.

While providing the novel capillary preparation methods described above, embodiments of the present invention also include the use of the capillary chromatography columns in biotechnology, such as procedures involving drug screening. In one embodiment of the present invention, the method is validated with EGCG as a positive control and Dimethylsulfoxide (DMSO) as a negative control, depending on the specific operating conditions. The results show that the chromatographic behaviors of EGCG and DMSO in a capillary chromatographic column which takes HEK293 cells over-expressing GLUT1 as an interaction phase are completely different, so that whether the interaction exists between the compound and the receptor can be qualitatively judged.

An embodiment provided by the present invention further includes the use of the capillary in an HPCE system and method thereof. The HPCE method comprises the following steps: providing a capillary tube, wherein a column sieve is arranged at the middle part or the end part of the capillary tube, and biological materials serving as interaction phases are intercepted; providing an electrophoretic device; introducing a biological material or sample into the capillary; performing electrophoretic analysis on the sample; and carrying out detection analysis on the sample.

In other embodiments of the invention, in order to truly, reliably and rapidly reflect the interaction between receptor and ligand, the invention establishes a method for studying the intermolecular interaction by immobilizing HEK293 cells overexpressing GLUT1 receptor and intercepting the cells at the back end of a column sieve as an interaction phase. And performing binding activity screening on the total flavone extract of the scutellaria baicalensis, the flavone monomeric compound in the scutellaria baicalensis, the metabolite of streptomyces and the metabolite of bacillus. On the basis, in order to further verify the interaction between the screened active substances and the actual tumor cells and tumor tissues, the invention establishes a method for fixing the A549 tumor cells, the isolated lung tissues and the A549 cell transplantation tumor tissues as interaction phases for the first time, and is used for researching the interaction between the binding active compounds and the binding active compounds.

In other embodiments of the present invention, in order to determine whether there is competitive binding between the positive compound and glucose, the present invention targets GLUT1 to perform competitive binding experiments between the positive compound and glucose, for example, glucose buffers with different masses are added to PB buffer to prepare glucose buffers with 0-10mM, and the behavior of HPCE of the positive compound in these buffers is observed to determine whether they occupy the glucose binding site when they bind to GLUT 1.

In addition, the Non-linear Chromatography (NLC) theory is adopted to perform quantitative calculation of binding constant, binding rate constant, dissociation rate constant and capacity factor on the screened active compounds, and simultaneously perform MTT experiment on the compounds to perform cell level anti-tumor activity research, and further perform animal level anti-tumor activity research on the compounds with strong activity (including compounds without competitive binding and with competitive binding with glucose). The results show that the active compounds screened by the method can inhibit the growth of tumor cells to different degrees, lay a foundation for screening antitumor drugs and treating tumors, provide scientific basis and new thought, and also show that the newly established method is used for screening the biological activity of the compounds at the molecular level, and the results of the method are basically consistent and effective with the results of the ICCE method established at the earlier stage for screening the compounds at the molecular level.

Examples

The invention will be further elucidated with reference to the following specific examples. It should be understood that these descriptions are not intended to limit the scope of the claims herein.

Example 1 creation of a New method for HPCE with biomaterials as interaction phase, i.e. non-immobilized biomaterial capillary electrophoresis (NIBCE) (taking GLUT1 overexpression HEK293 cells as interaction phase as example)

Preparing a capillary column screen and verifying the performance: washing a fused quartz capillary tube with the length of 30.2cm and the I.D. of 200 mu M with methanol, ultrapure water, 0.1M HCl, ultrapure water, 0.1M NaOH and ultrapure water in sequence, drying, heating a certain amount of silica gel, SE-30 or ODS powder in the capillary tube to prepare a bonded column sieve, obtaining the capillary tube with the column sieve, and storing at room temperature for later use. An electron microscope scan of a capillary column screen prepared with silica gel as the starting material is shown in FIG. 1 (A).

Performance verification of the capillary column screen: in this example, the HPCE used was a Beckman P/ACETM MDQ system (Beckman-Coulter, Fullerton, Calif., USA) equipped with a secondary array detector and 32KaratSoftware workstation (version 5.0, Beckman), and an outer polyimide coated fused silica capillary chromatography column with a screen. The experiment verifies the pH tolerance range and reproducibility of the column screen. NaOH or HCl solutions with different pH values are prepared in the experiment, a negative compound DMSO is injected under the same condition, alkali or acid is respectively used for backflushing for 5-10min between two times of injection, and the change of DMSO peak appearance conditions is observed to judge the pH tolerance range of the column screen. Each group was repeated twice, and the reproducibility of the results was good. The results demonstrate that the capillary column sieves prepared in the experiments can tolerate a pH range of 1-14. In addition, three capillary column sieves were prepared under the same conditions, DMSO was injected over five consecutive days, and the reproducibility of each was investigated. Wherein the RSD of the precision within the retention time day, the precision within the day and the precision between batches is respectively 4.90 percent, 6.56 percent and 9.04 percent, and the RSD within the retention time day, the precision within the day and the precision between batches is respectively 2.50 percent, 4.13 percent and 6.77 percent, which indicates that the prepared column screen capillary tube has good reproducibility and stability.

Culture of GLUT1 overexpressing HEK293 cells: HEK293 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS, Canada GIBCO) containing penicillin (100U/ml) and streptomycin (100. mu.g/ml). 400 μ l HEK293 cells (4X 10)⁶) GLUT1-eGFP/pcDNA-DEST47 plasmid 10-20. mu.g was transiently transferred by an electric pulse generator (Electro Square PoratorECM830, BTX, San Diego, Calif.) at a voltage of 123V for a pulse time of 20 ms. After 36-48hThe distribution of green fluorescence on HEK293 cell membrane was observed by fluorescence microscopy, as shown in FIG. 1(B), demonstrating that GLUT1 is expressed on most cell surfaces.

Preparation of capillary chromatography column as interaction phase after GLUT1-HE293K cell immobilization: after (t-36-48h) the GLUT1-HEK293 cells were digested and centrifuged from the culture dish, the cells were counted, fixed with 4% paraformaldehyde for 15-25min in the dark, and the cell density was adjusted to 1.0X 10 with PBS based on the counting results⁴-1.0×10⁷one/mL. Storing in a refrigerator at 4 ℃ for later use. In the experiment, the fixed cells are injected into the capillary, and due to the existence of the column screen, the GLUT1 overexpression cells are injected into the capillary in two existing modes: "dynamic" and "steady state", i.e., a state of movement in which the cells have not reached the posterior end of the lamina cribosa in a period of time just after the cells were injected into the capillary, and a state of steady state in which all the cells have moved to the posterior end of the lamina cribosa, are shown in FIG. 1 (D). After the cells in the method are fixed by 4% paraformaldehyde, the cells are stored in PBS for 30 days at 4 ℃ and still maintain the drug screening activity, the cells can be injected into a capillary tube when being used, the capillary tube is reversely punched after the cells are used, the operation is simple and convenient, the cells are updated at any time, the false negative result caused by saturation of an active site is avoided, and the recycling utilization rate is high.

The effect of different numbers, different states of cells and living cells on binding: the present invention investigates the influence of different numbers of cells, different states of cells and living cells on the binding. As can be seen in fig. 1(E), the peak shape of EGCG varied in capillary chromatography columns of different density cells. When the cell density is 10⁴And 10⁵At the time of one/mL, the peak height of the EGCG only shows a certain reduction, and the broadening is not obvious; and as the cell density increased to 10⁶Per mL, the peak shape of EGCG showed significant tail broadening; when increasing to 10⁷One per mL, the peak shape is represented by a broad flat peak attached to the base line. The result shows that for the compound with the binding effect, when the cell number reaches a certain density, the result is more true and credible. And (3) comparing the electrophoresis behaviors of DMSO and EGCG in different cell states by taking GLUT1 as a target point to judge whether the cell state has influence on a binding constant. As seen in FIG. 1(F), NICThe CE method is applicable to both cell dynamics and homeostasis, and strong positive compounds are more suitable for experiments in cell dynamics. It should be noted that the dynamic condition of the cells can better simulate the environment of the drug in blood, and the obtained binding constant is closer to the physiological condition. In the subsequent experiments, no special description is given, and the experiments are carried out under the dynamic condition of cells.

In addition, the invention also considers whether the living cells can not be fixed under the method and the research of the intermolecular interaction is directly carried out. The results show that the baseline is unstable and the peak shape is irregular when living cells are in the interaction phase. Therefore, the method is not suitable for studying the interaction between molecules in living cells (i.e., cells that are not fixed with 4% paraformaldehyde).

The HEK293 cells overexpressing GLUT1 were used as capillary chromatography columns for the interaction phase for validation of compound screening methods: the transport substrates of GLUT1 include glucose, galactose and mannose, but the sensitivities of the three are low, so that EGCG is used as a positive compound and DMSO is used as a negative compound in the experiment, the chromatographic behaviors of the GLUT1 in a column screen capillary, a normal HEK293 cell as a capillary of an interaction phase and a GLUT1 overexpression HEK293 cell as a capillary of an interaction phase are respectively researched, and the experimental results are shown in fig. 1 (G). Comparing the electrophorograms of DMSO on different chromatographic columns, the peak heights of the DMSO do not have significant difference. The response value of 100 mu M EGCG on the column screen capillary almost reaches 25mAU, but when the EGCG is injected into the capillary injected with common cells at the same concentration, the response value of the peak shows obvious reduction of tailing broadening, which indicates that the EGCG and the common cells have nonspecific interaction; in capillaries overexpressing GLUT1 cells as the interacting phase, 100 μ MEGCG exhibited a broad equatorial peak attached to the baseline, with strong specific interactions. Similarly, we conducted comparative studies using the mature ICCE method that had been established earlier, and the capillary chromatography column used in the ICCE method is shown in FIG. 1(C), and the electrophoresis behaviors of DMSO and EGCG on different capillary columns in the two methods are substantially the same, further demonstrating the feasibility of the method of the present invention.

Colour in affinity chromatographyThe change in spectral peak shape is the result of specific and non-specific interactions between the compound and the stationary phase. In general, the chromatographic peak appears as a broadened, trailing non-gaussian peak shape due to the higher dissociation rate than association rate of the compound with the stationary phase. The degree to which the peak shape of a compound deviates from a gaussian peak at a given chromatographic capacity correlates with the concentration of the compound. The NLC theory explains the relation between concentration and peak shape change and the calculation method of various kinetic parameters. The invention uses GLUT1 super-expressed HEK293 cells as a capillary chromatographic column of an interaction phase to carry out electrophoretic analysis on EGCG. The resulting asymmetric peak profile can be interpreted using a non-linear chromatographic model and software fitted to the peak profile to calculate the binding constant (K), binding rate constant (K)_a) Dissociation rate constant (k)_d) And a capacity factor (k'). The results obtained by the method and the results obtained by the ICCE method are respectively calculated and compared, and particularly shown in FIG. 2, so that the results of the kinetic parameters of the EGCG and GLUT1 determined by the method are basically consistent with the results obtained by the ICCE method.

Example 2 application of GLUT 1-overexpressed HEK293 cells as capillary chromatography column of interaction phase in Compound screening

The new method for screening drugs established by the invention is firstly used for screening the scutellaria total flavone extract, and the result is shown in figure 3 (A). The chromatographic behavior results of the total flavone extract show that when HEK293 cells over-expressed by GLUT1 are used as interaction phases, the retention time of main components in the total flavone extract is delayed, the peak height is reduced, and the peak shapes of certain components are seriously trailing, thereby providing scientific basis for further discovering which substances and GLUT1 have specific interaction.

On the basis, the new method is used for screening 22 compounds shown in fig. 4, mainly comprising flavone extract, streptomyces metabolite and bacillus metabolite in scutellaria baicalensis, and the chromatographic behaviors of the compounds in a column screen capillary chromatographic column, a capillary chromatographic column with normal HEK293 cells as an interaction phase and a capillary chromatographic column with GLUT1 overexpression HEK293 cells as an interaction phase are respectively researched. The chromatographic peak shapes of FSB-4, FSB-6, FSB-7, FSB-8, SL-1, SL-2, SL-5, SL-6, SL-7 and STR-1-7 in three capillary chromatographic columns are basically kept unchanged, and are consistent with the peak shape change of DMSO, and the chromatographic peak shapes belong to negative compounds, and the comparative peak shapes of representative FSB-4 in three capillaries are shown in FIG. 3 (B); in contrast, the peak shape changes of FSB-1, FSB-2, FSB-3, FSB-5, SL-3 and SL-4 in three capillary chromatographic columns are similar to EGCG, and as shown in FIG. 3(B) showing comparative peak shapes of representative FSB-1 in three capillaries, the compounds are seen to have delayed retention time and reduced peak height and severe tailing in capillaries over-expressing GLUT1 cells as an interaction phase, and are judged to have specific binding with GLUT1 and belong to positive compounds. We screened 22 compounds and calculated kinetic parameters of the screened active compounds by NLC theory. In this regard, we also conducted comparative studies by the ICCE method, and the results showed (fig. 5) that the capillary electrophoresis behavior of the 22 compounds obtained by the two methods is consistent, i.e. consistent with the screening result of whether there is an interaction between GLUT1, and the kinetic parameters of the obtained positive compounds are consistent.

Glucose competitive binding assay: to verify whether the active site of the glucose molecule bound to GLUT1 was occupied by the binding activity of the compound to GLUT 1. The experiment designs a glucose competitive combination experiment of EGCG and active compound FSB-1, FSB-2, FSB-3, FSB-5, SL-3 and SL-4 targeting GLUT1, and uses HEK293 cells over expressing GLUT1 as capillary tubes of an interaction phase to perform the glucose competitive HPCE experiment. GLUT1 is used as a target point, glucose with different masses is added into buffer solution respectively to prepare 40mM PB buffer solution containing 0mM, 1mM, 5mM and 10mM glucose, and positive compounds with the same concentration are injected into the buffer solution respectively. Comparing the electrophoresis behavior of the positive compounds in glucose buffers with different concentrations to judge whether competitive binding exists between the positive compounds and glucose, and thus judging whether the positive compounds occupy the binding site of the glucose when being bound with the GLUT 1. The results of the competition experiment of EGCG and glucose are shown in FIG. 3(C), which shows that as the concentration of glucose added into the buffer increases, EGCG is gradually released, the peak height increases, and the peak shape changes from a tail-broadening peak shape with binding characteristics to a narrow and sharp peak shape without binding characteristics. This is a good indication that EGCG competes with glucose for binding to the active site on GLUT1, i.e. EGCG occupies the glucose binding site when bound to GLUT 1. Similarly, FSB-1 and FSB-3 also exhibited a phenomenon in which the peak height increased and the compound was released as the concentration of glucose added to the buffer increased. In contrast, the peak heights and peak shapes of FSB-2 and FSB-5, SL-3 and SL-4 did not change significantly as the concentration of added glucose in the buffer increased, indicating that the sites of GLUT1 binding differ from glucose, with the results for the representative compound FSB-5 shown in FIG. 3 (C). In this case, we also conducted comparative studies of competitive binding experiments by the ICCE method, showing whether the active compounds obtained by both methods occupy the same site of glucose when binding to GLUT 1.

Example 3 screening of capillary chromatography columns of tumor cells as interaction phase for partially bound active Compounds

The preparation method of the capillary chromatographic column taking the A549 tumor cells as the interaction phase is the same as the preparation method of the capillary chromatographic column taking HEK293 cells over-expressed by GLUT1 as the interaction phase. The patent verifies whether DMSO, EGCG, FSB-1 and FSB-2 have interaction with A549 tumor cells or not by using the A549 tumor cells as a capillary chromatographic column of an interaction phase. As can be seen from fig. 6, when a549 tumor cells were used as the interaction phase, the DMSO peak heights were not significantly different, and the electrophoresis showed the typical negative compound characteristics. The EGCG, FSB-1 and FSB-2 are characterized by positive compounds, the retention time is delayed, the peak height is reduced, the peak shape is obviously reduced and trailed, the result is consistent with the result that the HEK293 cell over-expressed by GLUT1 is used as an interaction, and meanwhile, the GLUT1 receptor on the surface of the A549 cell is highly expressed.

Example 4 preparation of capillary chromatography columns with other biomaterials such as isolated lung tissue or tumor tissue as interaction phase and screening of partially active Compounds

According to the novel drug screening method established by the invention, the interaction phase can be not only cells (including cells overexpressing GLUT1, tumor cells and the like), but also other biological materials such as isolated tissues or tumor tissues and the like. Stripping the tumor tissue of the mouse normal lung tissue and the tumor tissue of the xenograft tumor established by the human A549 lung cancer cell, fixing the stripped tumor tissue for 12 to 48 hours in a dark place by using 4 percent paraformaldehyde, placing the stripped tumor tissue in 75 percent ethanol, and storing the stripped tumor tissue in a refrigerator at 4 ℃ for later use. In the experiment, the fixed in vitro tissue or the fixed tumor tissue is injected into a capillary tube of a column screen, the length of the capillary tube is 0.5-5mm, and the capillary tube is washed and balanced by a phosphate buffer solution with the pH value of 40 mM7.4 for later use.

Fig. 7 is a graph of HPCE when ex vivo lung tissue (b) and tumor tissue (c) are in interaction phase. The result shows that DMSO is on a capillary chromatographic column with a column screen capillary, an isolated lung tissue as an interaction phase and a tumor tissue as an interaction phase, and the isolated lung tissue and the tumor tissue adopt a steady-state form, so that the pressure of the system is increased to a certain extent, and the peak-out time is delayed. But other electrophoretic behaviors are basically consistent, the peak heights and peak shapes are not obviously different, and the characteristics of typical negative compounds are shown. By comparing the peak shapes of EGCG, FSB-1 and FSB-2 in three capillary chromatographic columns, the peak shapes of the compounds on the capillary chromatographic columns with isolated lung tissues as interaction phases show certain broadening, and certain nonspecific interaction exists; compared with the isolated lung tissue, the capillary tube with the tumor tissue as the interaction phase has the advantages of delayed retention time, more obvious reduction of peak height and serious tailing, judges that the capillary tube and the tumor tissue have specific interaction, and further explains the high expression of GLUT1 in the tumor tissue. Because the ICCE method can not determine the combination effect of the compound and the isolated tissue, and the part is not verified by the ICCE method, the method widens the application range, introduces different biological materials into the capillary column as interaction phase for researching the interaction between molecules, has more practical application value, can provide a treatment target medicament with optimal combination kinetic parameters by researching the interaction between the human tumor tissue and the target medicament, lays a foundation for optimizing the treatment target medicament for clinically treating tumors, and provides a brand new method and a scientific basis.

Example 5 cell level and animal level antitumor Activity experiments

The experiment was performed on a strong positive compound obtained by primary screening of HPCE6 concentration points of 0 mu M, 20 mu M, 40 mu M, 60 mu M, 80 mu M and 100 mu M are respectively selected for EGCG, FSB-1, FSB-2, FSB-3, SL-3 and SL-4, and the effect of the compound on the growth of A549 tumor cells is researched by adopting an MTT method. The absorbance of each well was measured at the position of enzyme linked immunosorbent assay OD490nm, and t-test was performed on drug groups of different concentrations and drug-free groups, and the difference indicates that the drug had inhibitory effect on A549 cells at the corresponding concentration. Meanwhile, the average inhibition rate at each concentration was calculated, and an inhibition rate-drug concentration curve was plotted, as shown in fig. 8. The experimental result is obtained by statistics according to the experimental conditions of parallel operation for more than three times. The result shows that 100 mu M EGCG has obvious difference with the drug-free group, and other concentration groups have no obvious difference; 20 mu M, 40 mu M, 60 mu M, 80 mu M, 100 mu M FSB-1 and SL-4 have significant differences with the non-drug group; 80 mu M and 100 mu M FSB-2 and FSB-3 have obvious difference with the medicine-free group, and other concentrations have no obvious difference; SL-3 at each concentration did not differ significantly from the drug-free group. Calculating IC of compound by using Excel to fit linear equation₅₀See figure 5 for values. IC of the Compound₅₀The smaller the value, the stronger the inhibitory effect of the drug. Therefore, the molecular level screening result of the compound is basically consistent with the cell level anti-tumor activity experiment result.

Aiming at a strong positive compound obtained by HPCE primary screening and the experimental result of in vitro anti-tumor activity, the effect of the compounds EGCG, FSB-1 and FSB-2 on the tumor growth of human non-small cell lung cancer cells (A549) in mice is researched. A549 and 6-8 week-old Balb/nu-nu mice in logarithmic growth phase are selected to construct a nude mouse tumor-bearing model, a normal saline group is used as a blank control group, EGCG (80mg/kg) is taken as a positive control group, and the pharmacodynamic evaluation of an FSB-1 group (80mg/kg) and an FSB-2 group (80mg/kg) on a tumor model is researched. Nude mice were weighed on day 1 of the experiment and injected with A549 cells (5X 10) in the right underarm of nude mice⁶One/one), 10d after injection of cells (tumors reached about 35 mm)³) The administration was started by randomly dividing the nude mice into 4 groups of 6 mice per group. The administration was performed once every three days, and the weight of the nude mice and the tumor volume were measured before each administration. A total of 9 doses were administered, mice were sacrificed on day 27 post dose,the tumor was removed and weighed. The average tumor weight and tumor inhibition rate of each group were calculated, and tumor inhibition rate (average tumor weight of blank group-average tumor weight of experimental group)/average tumor weight of blank group × 100%. FIG. 9 (A-E) shows that EGCG, FSB-1 and FSB-2 all had significant differences in tumor suppression (. about.P) compared to saline group<0.001,**P<0.01,*P<0.05), the tumor inhibition rates are respectively as follows: 71.51%, 64.69% and 61.73%. FSB-1 and FSB-2 have no significant difference on the weight influence of mice, which indicates that FSB-1 and FSB-2 have less toxicity; however, the weight influence of the EGCG group on mice is significantly different from that of the physiological saline group, which indicates that the EGCG may have certain side effects.

Specific embodiments are described in detail herein, however, this is by way of example for purposes of illustration only and is not intended to limit the scope of the claims which follow. It should be understood that various substitutions, alterations and modifications to the embodiments described herein may be made without departing from the spirit and scope of the invention as defined by the appended claims and shall therefore fall within the scope of the invention as hereinafter claimed.

Claims

1. A capillary tube having a column screen and an interactive phase, wherein the pore size of the column screen is controllable and is useful for intercepting biological material as the interactive phase; wherein the biological material comprises cells, ex vivo biological tissue, tumor tissue, and the biological material is fixed with 4% paraformaldehyde.

2. The capillary tube of claim 1, wherein the pillar screen is disposed at a middle portion or an end portion of the capillary tube, or the pillar screen is connected to an end portion of the capillary tube.

3. The capillary of claim 1, wherein said cell is a biological macromolecule overexpressed cell or tumor cell, with high expression of a macromolecular receptor on the cell surface and retention of native conformation and biological activity; wherein the biological macromolecule is overexpressed GLUT 1.

4. The capillary of claim 1, wherein the capillary is a fused silica capillary.

5. A method of making the capillary tube of any one of claims 1-4, comprising the steps of:

pretreating the capillary tube;

pretreating the column screen material and forming a column screen, and enabling the column screen to be arranged in the middle or at the end part of the capillary tube, or enabling the column screen to be connected to the end part of the capillary tube, so that the capillary tube with the column screen is formed; wherein, if the pillar screen is arranged at the end of the capillary or connected to the end of the capillary, the end of the capillary and the end of the capillary with the detection window need to be connected through a connector;

6. The method of claim 5, wherein the pre-treating step comprises rinsing the capillary tube with methanol, ultrapure water, 0.1M HCl, ultrapure water, 0.1M NaOH, ultrapure water in that order.

7. The method of claim 5, wherein an inner diameter of the connector matches an outer diameter of the capillary.

8. The method of claim 5, wherein the step of immobilizing the biological material comprises treating the biological material with 4% paraformaldehyde.

9. A capillary electrophoresis system comprising:

the capillary tube of any one of claims 1-4;

means for introducing a biological material or sample into the capillary;

means for performing an electrophoresis operation on said sample introduced into said capillary; and

and (c) equipment for detecting the sample.

10. A method of capillary electrophoresis comprising:

providing a capillary tube according to any one of claims 1 to 4;

providing an electrophoretic device;

introducing a biological material or sample into the capillary;

performing electrophoretic analysis on the sample; and

and carrying out detection analysis on the sample.

11. Use of the capillary tube according to any one of claims 1 to 4 in the field of biotechnology, comprising the use of said capillary tube for electrophoretic analysis.

12. Use according to claim 11, wherein the biotechnology includes drug screening, interaction of biological macromolecules with active ligands or with compounds, analysis of drug components, purification and identification of drug active components.

13. Use according to claim 11, wherein the electrophoretic analysis is capillary electrophoresis.

14. Use according to claim 13, wherein the capillary electrophoresis is array capillary electrophoresis or chip capillary electrophoresis.