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Application of Raw264.7 cell line capable of stably expressing different human ApoE genotypes and preparation method of Raw264.7 cell line

A raw264.7, stable expression technology, applied in the application and preparation of Raw264.7 cell line, can solve the problem of no in vitro immune cell model and the like

Pending Publication Date: 2022-04-15
THE FIRST AFFILIATED HOSPITAL OF ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, so far there is no good in vitro immune cell model, which can provide a good cytological research platform for studying the impact of different ApoE gene subtypes on inflammatory immunity

Method used

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  • Application of Raw264.7 cell line capable of stably expressing different human ApoE genotypes and preparation method of Raw264.7 cell line
  • Application of Raw264.7 cell line capable of stably expressing different human ApoE genotypes and preparation method of Raw264.7 cell line
  • Application of Raw264.7 cell line capable of stably expressing different human ApoE genotypes and preparation method of Raw264.7 cell line

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Establishment of Raw264.7 cell lines stably expressing different human ApoE genotypes

[0034] 1. Materials and methods

[0035] 1.1 Cell culture

[0036] The Raw264.7 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai). at 5% CO 2 , Raw264.7 cells were cultured in DMEM medium containing 10% FBS and 1% double antibody in a 37°C incubator.

[0037] 1.2 Reagents

[0038] Lipofectamine 3000 transfection kit was purchased from Invitrogen, DMEM, G418 and mammalian DNA kits were purchased from Sigma, opti-MEM and FBS were purchased from Gibico, and Elisa kit was purchased from Wuhan Cloud Clone Company , the fluorescent secondary antibody was purchased from Jackson ImmunoResearch Laboratories, USA, and the primary ApoE antibody was purchased from Calbiochem, USA.

[0039] 1.3 Establishment of Raw264.7 cell lines stably transfected with different human ApoE genotypes

[0040] The Raw264.7 cell line was planted in a 60mm cultu...

Embodiment 2

[0047] Example 2: Application of Raw264.7 cell lines stably expressing different human ApoE genotypes

[0048] 2.1 Effects of different human ApoE genotypes on the activation of NLRP3 inflammasome in Raw264.7 cell line

[0049] Raw264.7 cells stably expressing different human ApoE genotypes were stimulated with or without LPS (1 μg / ml) or Staphylococcus aureus (infection ratio of 1:10) for 8 hours, and the ELISA method was used to detect the stable transfected cell culture The levels of NLRP3-related cytokines IL-1β and IL-18 in the supernatant. IL-1β, IL-18 monoclonal antibodies were pre-coated onto microwell plates, 50ul samples were added to each well, the incubation was stopped by washing three times to remove unbound conjugates, and horseradish peroxidase-conjugated di Antibody incubation for 30 minutes, the substrate solution was added, and the color development was performed after the reaction was terminated; Western Blotting was used to detect the changes in the prote...

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Abstract

The invention discloses an application of a Raw264.7 cell line capable of stably expressing different human ApoE genotypes in researching the influence of the ApoE genotypes on inflammation immunity, and a preparation method of the stably transfected cell line. The preparation method comprises the following steps: transfecting different human ApoE genotype plasmids ApoE2, ApoE3 and ApoE4 with EGFP labels and a control plasmid N1 into a Raw264.7 cell by using a transfection reagent; after a positive screening agent G418 is added for selective culture, a stably transfected cell strain is obtained through multiple times of sorting in a flow cytometry single cell sorting mode, and ApoE genotype and expression identification is carried out; the stably transfected cell is used for researching the influence of the ApoE genotype on NLRP3 inflammasome activation, endogenous melatonin synthesis, mitochondrial energy metabolism, pathogen removal and the like. The cell strain provides a good tool for further research on biological characteristics of different ApoE genotypes in the field of inflammation immunity and research and development of drugs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application and preparation method of a Raw264.7 cell line stably expressing different human ApoE genotypes. Background technique [0002] Human ApoE, also known as apolipoprotein E, is an arginine-rich polymorphic basic protein composed of 299 amino acids with a molecular weight of 34KDa. It is mainly synthesized by the liver. In recent years, it has been found that the brain, kidney, bone, Adrenal glands and macrophages can also synthesize. The ApoE gene is polymorphic and contains three common alleles: ε2, ε3 and ε4, resulting in six phenotypes E2 / 2, E3 / 3, E4 / 4, E2 / 3, E2 / 4, E3 / 4 . There are three common isoforms of ApoE protein in humans, namely ApoE2, ApoE3 and ApoE4. They differ only at positions 112 / 158. ApoE3 has cysteine ​​at position 112 and arginine at position 158, while ApoE2 has cysteine ​​at both positions, and ApoE4 has cysteine ​​at both positions. Each of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/12C12N15/85C12N15/65C12Q1/02
Inventor 吴惠梅谢邱梦章海云沙家凤阮雅
Owner THE FIRST AFFILIATED HOSPITAL OF ANHUI MEDICAL UNIV
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