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Method for in vitro collecting Midkine proteins and treating cells with collected Midkine proteins

A protein and cell technology, applied in the field of cell biology and translational medicine, to achieve the effect of reducing total concentration, facilitating ultrafiltration operation, and enriching flexibility

Active Publication Date: 2020-06-09
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on the function of Midkine in cells, which is a major deficiency in the study of the mechanism of Midkine's function and nuclear action

Method used

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  • Method for in vitro collecting Midkine proteins and treating cells with collected Midkine proteins

Examples

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Effect test

example 1

[0029] The Midkine protein secreted by LM3 cells was collected in vitro and used to treat MHCC97H cells

[0030] 1) Using Western blot method to detect the expression level of Midkine in different cells, it was found that the expression level of Midkine protein in LM3 and HepG2 cells was much higher than that of normal cell THLE-2 ( figure 1 A), meanwhile, a large amount of secreted Midkine protein can also be detected in the culture medium of LM3 and HepG2 cells ( figure 1 B), indicating that these two cells can be used as donors of Midkine protein, and in 7402 and MHCC97H cells, the expression level of Midkine is much lower than that of normal cell THLE-2, which can be used as recipient cells of Midkine protein;

[0031] (2) Select Midkine high-expressing cell LM3 for culture, according to 2X10 6 The number of cells / dish LM3 cells were inoculated into a 10 cm culture dish and cultured with DMEM medium (gibco, C11995500BT) containing 10% fetal bovine serum (SERANA, S-FBS-SA-...

example 2

[0043] Midkine protein secreted by LM3 cells was collected in vitro and combined with heparin to treat MHCC97H cells

[0044] (1) Western blot method was used to detect the expression level of Midkine in different cells, and it was found that the expression level of Midkine protein in LM3 and HepG2 cells was much higher than that in normal cells THLE-2 ( figure 1 A), meanwhile, a large amount of secreted Midkine protein can also be detected in the culture medium of LM3 and HepG2 cells ( figure 1 B), indicating that these two cells can be used as donors of Midkine protein, and in 7402 and MHCC97H cells, the expression level of Midkine is much lower than that of normal cell THLE-2, which can be used as recipient cells of Midkine protein;

[0045] (2) Select Midkine high-expressing cell LM3 for culture, according to 2X10 6 The number of cells / dish LM3 cells were inoculated into a 10 cm culture dish and cultured with DMEM medium (gibco, C11995500BT) containing 10% fetal bovine se...

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Abstract

The invention discloses a method for in vitro collecting Midkine proteins and treating cells with the collected Midkine proteins. Cells for highly expressing Midkine proteins are cultured, through thesecretion characteristics of the Midkine proteins, the Midkine proteins are collected in culture mediums, and the secretion quantity of the Midkine proteins is detected through Western blot experiment; through detection with the Western blot experiment, the inventor finds that MK proteins can be transferred into the cells, and the phosphorylation level of AMPK is restrained; when the Midkine proteins are applied outside the cells, heparin (heparin) is added, through detection with the Western blot experiment, the inventor finds that Heparin restrains the Midkine proteins from entering the cells, and the activity of the AMPK proteins is improved. The method provides a novel method for researching the action mechanism of the Midkine.

Description

technical field [0001] The invention belongs to the fields of cell biology and translational medicine, and in particular relates to a method for constructing and identifying a cell line with stable and high expression of midkine protein. Background technique [0002] Midkine protein is a secretable protein. After the translation is completed, the molecular weight of Midkine without the signal peptide is about 13kD. Studies have shown that Midkine is widely expressed in human tissues and can be transported in vivo through secretion into the circulatory system. Midkine is widely involved in the regulation of various physiological processes such as immune inflammatory response, neurodevelopment, and blood pressure regulation. The latest clinical sample data show that in breast cancer, prostate cancer, liver cancer, pancreatic cancer and other malignant tumors, the expression level of Midkine presents an up-regulation trend, and the survival rate of the patient group with high ...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K14/475C12N5/10
CPCC12P21/02C07K14/475C12N5/0693C12N2501/10
Inventor 朴海龙夏天
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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