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Application of interleukin-22 in preparing medicine for treating hepatopathy and preparation method thereof

A technology for interleukin and human interleukin, which is applied in the field of preparation of recombinant human interleukin-22, can solve the problems of high cost, small expression amount, low cost and the like, and achieves the effects of low production cost, high expression amount and expanding application range

Inactive Publication Date: 2008-04-30
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there are two main methods for preparing IL22: (1) prokaryotic expression system (Ronaldo Alves PintoNagem et al., Crystal Structure of Recombinant Human Interleukin-22 Structure, Vol.10, 1051-1062, August, 2002), the prokaryotic expression system is the use of pET A series of vectors, whose induction method is chemical induction (IPTG induced expression), has the disadvantage of high cost; (2) eukaryotic expression system (Jing Li et al., International Immunopharmacology 4(2004) 693-708), using eukaryotic expression system to express hIL22 is soluble, and its disadvantage is that the expression level is small, which cannot meet the needs of animal experiments; therefore, it is necessary to find a preparation method with high expression level and low cost

Method used

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  • Application of interleukin-22 in preparing medicine for treating hepatopathy and preparation method thereof
  • Application of interleukin-22 in preparing medicine for treating hepatopathy and preparation method thereof
  • Application of interleukin-22 in preparing medicine for treating hepatopathy and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Preparation of hIL22

[0073] (1) Capture of hIL-22 gene and construction of recombinant expression vector

[0074] a. Extraction of total RNA Dilute 1ml of human fresh peripheral blood by 2 times with PBS containing 2mmol / L EDTA, spread it on an equal volume of lymphocyte separation medium (purchased from Shanghai Huajing Biological Co., Ltd.), and then run it at 800r / min Centrifuge to make layers, and then use capillaries to suck out the cells of the gray and white coat; wash the gray and white coat cells with Hanks solution (purchased from Tianrun Shanda Co., Ltd.) cells, and at 37°C, 5% CO 2 Cultivate in the incubator for 24 hours, and then add ConA (2 mg / ml, purchased from Xiasi Biological Company) and anti-CD respectively 3 (4mg / ml purchased from Shanghai Wowu Biotechnology Co., Ltd.) to co-stimulate the cells, and then at 37 ° C, 5% CO 2 Continue culturing in the incubator for 24 hours, and then extract the total RNA with a total RNA extraction kit (Promega Co...

Embodiment 2

[0086] Proliferation-promoting activity of hIL22 on LO2 cells

[0087] LO2 cells were cultured with RPMI1640 culture medium containing 10% fetal bovine serum (purchased from Gibco BRL Company) until the cells grew well, and the cells were collected and divided into 1 × 10 5 cells / mL were inoculated in 96-well cell culture plates, 50 μl / well; then 50 μl of the hIL-22 protein obtained in Example 1 were added to each well, and the final concentrations were 0.1, 1, 2.5, 10, 50, 100, 1000, 2000, 4000 and 8000ng / ml, set up a blank control without cells and a negative control with cells but no factors, and do three repetitions for each concentration; at 37°C, 5% CO 2 After culturing in the incubator for 72 hours, add 20 μl of 5 mg / ml MTT to each well; continue to cultivate for 5 hours, then add 100 μl of 10% SDS (dissolved in 0.01N HCl) to each well, and measure the absorbance at 570 nm after the purple formazan crystals are dissolved value.

[0088] The concentration of hIL-22 aft...

Embodiment 3

[0090] Effect of hIL22 on the expression level of c-myc in LO2 cells

[0091] LO2 cells were cultured in a 60 mm culture dish for 2 h, and then grouped into groups and sequentially added the recombinant IL-22 protein solution prepared in Example 1 so that the final concentrations of various proteins were 20, 200, 400, and 800 ng / mL, respectively. 37°C, 5% CO 2 After culturing for 2 hours, RNA was extracted and quantitatively performed by RT-PCR. The results (see Figure 8) showed that after being stimulated by exogenous hIL-22, the expression level of c-myc gene gradually increased from 20 to 800 ng / mL, which was dependent on the concentration of hIL-22, indicating that hIL22 can increase the c - expression level of myc.

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Abstract

The invention discloses the usage of interleukin-22 during the preparative medicine for treating hepatic diseases, in particular to the application of medicine in preparing to treat alcoholic liver diseases and nonalcoholic fatty liver diseases. The range of application of the interleukin-22 is enlarged, bringing comfort to hepatopath. The invention also discloses a process for preparing the recombinant human interleukin-22, and the recombinant human interleukin-22 is produced by employing the method with high expression level and low costs, providing the foundation of mass production and application of the interleukin-22.

Description

technical field [0001] The present invention relates to the medical use and preparation method of interleukin-22, in particular to the use of interleukin-22 in the preparation of medicines for treating liver diseases, and also to the preparation method of recombinant human interleukin-22. Background technique [0002] Interleukin-22 (Interleukin 22 in English, abbreviated as: IL22) is a cytokine discovered in 2000, which is secreted by activated T cells and NK cells (Wolk K et al., J Immunol 2002; 168: 5397~402) , exert biological effects through heterologous receptor complexes (IL22R1, IL10R2) (XieMH et al. J Biol Chem 2000; 275; 31335~9), IL10R2 is widely distributed, and the expression of IL22R1 is highly restricted, mainly expressed in the pancreas, skin , liver, lungs and kidneys. Neither resting nor activated immune cells expressed IL-22 R1 (Wolk K et al., Immunity 2004; 21:241-54). The medical uses of interleukin-22 mainly include: (1) in the treatment of hyperlipid...

Claims

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Application Information

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IPC IPC(8): A61K38/20A61P1/16C12N15/24
Inventor 徐东刚邢微微蔡欣邹民吉徐涛刘深王园园吴伯灵杨旗
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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