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Capillary tube with fixed phase formed by culturing cell layer through adhering cell layer on inner wall of capillary tube and manufacturing method and application thereof

A capillary, cell layer technology, applied in capillary and its preparation, capillary chromatographic column through capillary electrophoresis in the field of biomedicine, can solve the problem of difficult separation and purification and maintain molecular conformation, achieve ideal chromatographic peak shape, less sample consumption , the effect of stationary phase stability

Inactive Publication Date: 2012-03-21
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] It has high selectivity to CCR4, high chemotaxis inhibition and strong binding ability, but due to a series of related problems in its synthesis process and in vivo toxicity, it is necessary to seek compounds with better effects and simpler synthesis. Appropriate drug screening methods need to be found
[0009] In addition, it is worth noting that CCR4, like many biological macromolecules, is difficult to isolate and purify from cells and maintain molecular conformation

Method used

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  • Capillary tube with fixed phase formed by culturing cell layer through adhering cell layer on inner wall of capillary tube and manufacturing method and application thereof
  • Capillary tube with fixed phase formed by culturing cell layer through adhering cell layer on inner wall of capillary tube and manufacturing method and application thereof
  • Capillary tube with fixed phase formed by culturing cell layer through adhering cell layer on inner wall of capillary tube and manufacturing method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the culture of CCR4 overexpression cell

[0036] HEK293 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS, Canada GIBCO), which contained penicillin (100 U / mL) and streptomycin (100 μg / mL). 400 μL HEK293 cells (4 × 10 6 ) transiently transfected 30 μg of CCR4-EGFP plasmid; this process was carried out by an electric pulse generator (Electro Square Porator ECM830, BTX, San Diego, CA), and its operating conditions included a voltage of 123V and a pulse time of 20sm. After electrotransfection of CCR4-EGFP in HEK293 cells, the expression of CCR4-EGFP protein on the cell surface can be seen figure 2 (A, B, C). It was proved that the receptor CCR4 was expressed on the surface of most cells.

Embodiment 2

[0037] Example 2, Preparation of Quartz Capillary Chromatography Column Attached to CCR4 Overexpression Living Cells

[0038] Quartz capillary columns were rinsed and sanitized sequentially with methanol, 0.1M NaOH, and deionized water. After the capillary was rinsed dry under sterile conditions, 0.2 mg / mL polylysine (PLL) was injected and incubated for 1.5-4.0 hours before use. After electroporation, the cells were returned to the medium to suspend the cells to a density of 1.5×10 7 cells / mL, and injected into the pretreated capillary chromatography column, the capillary chromatography column was immersed in RPMI 1640 medium containing 10% FBS, at 37°C, 5% CO 2 Incubate for 12 hours in the incubator. In order to maintain the structure of the cells, the capillary chromatographic column attached to the cells needs to be fixed with 4% paraformaldehyde for 10-40min, and then rinsed with PBS before use. These CCR4 overexpression cells grow adherently on the inner wall of the ca...

Embodiment 3

[0039] Embodiment 3, verification of new screening method

[0040] Lactam compound A, as a novel CCR4 antagonist, has strong affinity for CCR4 and good chemotaxis inhibitory activity. Therefore, in this experiment, compound A was used as a positive compound, and DMSO was used as a negative compound. The chromatographic behaviors of the two in uncoated capillary chromatographic columns and CCR4 expressing cell-coated capillary chromatographic columns were studied respectively. The experimental results are as follows: image 3 As shown in A. Comparing the electropherograms of DMSO on the two chromatographic columns, it was found that the peak height remained unchanged and the peak shape was slightly broadened. The peak height of the electrophoresis peak of compound A in the cell-coated capillary column was significantly reduced, and the peak was broadened and tailed, indicating that the positive compound A had a strong affinity with the stationary phase. There is no significan...

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Abstract

The invention provides a capillary tube with a fixed phase formed by culturing cells through adhering the cells on the inner wall of the capillary tube, a manufacturing method thereof, and application thereof to medicament screening with a capillary tube electrophoresis method. Specific to the change of native conformation caused by separation and purification of certain biologically active macromolecules from cells, the invention develops the capillary tube electrophoresis method for researching the coaction between biologically active macromolecules and an active ligand or a compound and for screening medicaments by using a fixed phase prepared by culturing cells overexpressed by the biologically active macromolecules in a capillary tube chromatographic column and by fixing after adherent growth of the cells.

Description

technical field [0001] The present invention relates to a capillary and its preparation method and application, in particular to a capillary chromatographic column whose inner wall is attached with living cells as a stationary phase, its preparation method and the use of the capillary chromatographic column in the field of biomedicine such as drug screening through capillary electrophoresis . Background technique [0002] Most biomolecules interact specifically with their target molecules when performing their physiological functions, such as enzymes and substrates, lectins and polysaccharide proteins, receptors and hormones, antigens and antibodies, regulatory proteins and DNA, etc. These specific interactions are the basis for the generation of life phenomena, and are widespread phenomena in chemical and biochemical systems. Biomolecules are inseparable from biochemical cyclic cascade reactions and highly selective catalytic properties of enzymes, the transmission, expres...

Claims

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Application Information

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IPC IPC(8): B01J20/281B01J20/30B01D15/22G01N27/453
Inventor 凌笑梅帕孜来提·亚库甫王应綦辉张颖妹
Owner PEKING UNIV
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