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Monoclonal cell strain and application of monoclonal cell strain for measuring relative biological activity of IL-6R inhibitors

A technology of biological activity and cell line, applied in the field of measuring the relative biological activity of IL-6R inhibitors, monoclonal cell line and its construction, can solve the problems of unstable measurement results, high culture requirements, poor repeatability, etc. The effect of short assay cycle, simple cell culture, and low reagent requirements

Inactive Publication Date: 2017-11-24
SHANGHAI INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, KT-3 cells are suspension cells, and their growth depends on specific cytokines, which requires high culture requirements. Moreover, this method takes a long time and has poor repeatability, resulting in unstable measurement results.
However, there is no report on the application of this principle to drug screening of IL-6R inhibitors and the determination of the relative biological activity of IL-6R inhibitors.

Method used

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  • Monoclonal cell strain and application of monoclonal cell strain for measuring relative biological activity of IL-6R inhibitors
  • Monoclonal cell strain and application of monoclonal cell strain for measuring relative biological activity of IL-6R inhibitors
  • Monoclonal cell strain and application of monoclonal cell strain for measuring relative biological activity of IL-6R inhibitors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] This embodiment is the construction of monoclonal cell line 293T-SIE, which includes the following steps:

[0074] (1) Determination of the optimal screening concentration of hygromycin b;

[0075] 293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). Cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 1×10 6 / ml, 2ml per well was inoculated in a 6-well plate, and placed in a cell culture incubator for routine culture. After 24 hours of cell inoculation, dilute hygromycin b, establish a total of 10 concentration gradients of 0-1 mg / ml, add it to a 6-well plate, observe the growth of cells every day, and the lowest hygromycin that causes all cells to die within 10-14 days The b concentration is the working concentration for screening and transfecting 293T cells. In the human kidney epithelial cell line 293T-SIE stably expressing luciferase regulated by SIE, the optimal working concentration of hygromy...

Embodiment 2

[0087] This embodiment is the condition optimization of the luciferase reporter gene method for determining the relative biological activity of IL-6R inhibitors, which includes the following steps:

[0088] (1) Recovery and subculture of cells: Take out the frozen 293T-SIE cells from the liquid nitrogen tank, thaw the cells quickly, transfer them to 5ml DMEM medium containing 10% FBS, and store them at 37°C and 5% CO 2 cultured in an incubator.

[0089] (2) Cell inoculation: the cells in the logarithmic growth phase were digested with 0.25% trypsin, centrifuged at 1000rpm for 2min, the supernatant was discarded, and the cells were resuspended in the cell culture medium (containing 10% fetal bovine serum in phenol red-free DMEM) base), count. Dilute the cell suspension to 0.8×10 6 cells / ml, 0.4×10 6 cells / ml, 0.2×10 6 cells / ml, inoculated in 96-well white plate at 50 μl / well.

[0090](3) Add IL-6 gradient solution: take 100 μg of human recombinant IL-6 powder, add 1 ml of ...

Embodiment 3

[0096] This example is a luciferase reporter gene method to determine the relative biological activity of tocilizumab.

[0097] The method for measuring the relative biological activity of the IL-6R inhibitor tocilizumab using 293T-SIE cell lines comprises the following steps:

[0098] (1) Recovery and subculture of cells: Take out the frozen 293T-SIE cells from the liquid nitrogen tank, thaw the cells quickly, transfer them to 5ml DMEM medium containing 10% FBS, and store them at 37°C and 5% CO 2 cultured in an incubator.

[0099] (2) Cell inoculation: the cells in the logarithmic growth phase were digested with 0.25% trypsin, centrifuged at 1000rpm for 2min, the supernatant was discarded, and the cells were resuspended in the cell culture medium (containing 10% fetal bovine serum in phenol red-free DMEM) base), count. Dilute the cell suspension to 0.8 x 10 6 After cells / ml, inoculate 96-well white plate with 50 μl / well.

[0100] (3) Preparation of IL-6 working solution: ...

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Abstract

The invention discloses a monoclonal cell strain. The monoclonal cell strain is a human renal epithelial 293T cell carrying an SIE-regulated luciferase gene and capable of expressing luciferase stably and is named as 293T-SIE (preservation No. CGMCC No. 13816, and the depositary unit is the General Microbiology Center of the China General Microbiological Culture Collection Center); the invention further relates to a construction method and application for the monoclonal cell strain and a method for measuring relative biological activity of IL-6R inhibitors by adopting the monoclonal cell strain. According to the Monoclonal cell strain and application for measuring relative biological activity of the IL-6R inhibitors, the activation situation of IL-6 related signal pathway is taken as a research object, a human renal epithelial cell strain capable of expressing SIE-luciferase reporter gene stably is designed, culturing is simple, repeatability is good, actual action situation of sample in a body is closer, and stability and accuracy of the measuring result are guaranteed; meanwhile, the established method for measuring the activity of the IL-6R inhibitor established is short in measuring period, researching and developing, quality controlling and clinical application of IL-6R inhibitor medicine are facilitated, and high application value is obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a monoclonal cell line and a construction method thereof, and a method for measuring the relative biological activity of an IL-6R inhibitor using the monoclonal cell line. Background technique [0002] Interleukin-6 (IL-6), a pleiotropic cytokine that regulates immune and inflammatory responses, binds to the IL-6 receptor (IL-6R) and induces chondrocytes and synoviocytes to produce matrix Metalloproteinase (MMP)-1, MMP-3 and MMP-13 (Hashizume M, Mihara M.Osteoarthritis Cartilage, 2009, 17(11): 1513-1518) can also induce the expression of RANKL, thereby promoting osteoclast Osteoclasticity, causing damage to cartilage (Hashizume M, Hayakawa N, Mihara M. Rheumatology, 2008, 47(11): 1635-1640), has been shown to be powerful in the destruction of articular bone and cartilage tissue in patients with rheumatoid arthritis role. IL-6R inhibitors inhibit IL-6 from transducing signals into c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85G01N33/68
CPCC12N5/0686C12N15/85C12N2510/00G01N33/6869G01N2333/5412
Inventor 王自强陈钢王灿董闪闪邵泓吴利红
Owner SHANGHAI INST FOR FOOD & DRUG CONTROL
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