阅读说明：本技术 检测巨噬细胞中rfx1表达水平的试剂在制备巨噬细胞分型检测制剂中的应用 (Application of reagent for detecting RFX1 expression level in macrophage in preparing macrophage typing detection preparation ) 是由 贾素洁 赵明 杨爽 堵培 于 2020-07-02 设计创作，主要内容包括：本发明公开了一种检测巨噬细胞中RFX1表达水平的试剂在制备巨噬细胞分型检测制剂中的应用。通过巨噬细胞中RFX1的表达水平判断巨噬细胞分化类型,在M1型巨噬细胞中RFX1的表达水平高于M0和M2型巨噬细胞。提示该基因可作为M1型巨噬细胞的生物标志物,在巨噬细胞的分型鉴定中具有较好的应用价值。(The invention discloses an application of a reagent for detecting the expression level of RFX1 in macrophages in preparing a macrophage typing detection preparation. Macrophage differentiation type was judged by the expression level of RFX1 in macrophages, with RFX1 expression levels in M1-type macrophages higher than M0 and M2-type macrophages. The gene can be used as a biomarker of M1 type macrophages and has good application value in the typing identification of the macrophages.)

1. Use of a reagent for detecting the expression level of RFX1 in macrophages in the preparation of a macrophage typing detection formulation.

2. The use of claim 1, wherein detecting the expression level of RFX1 in the macrophage cell comprises detecting the expression level of RFX1mRNA and/or protein in the macrophage cell.

3. The use according to claim 2, wherein the PCR amplification primer pair sequences in the reagent for detecting the expression level of macrophage RFX1mRNA are as follows:

RFX1-F：5'-GATCCAAGGCGGCTACAT-3'；

RFX1-R：5'-CAGCCGTCTCATAGTTGTCC-3'。

4. the use of claim 2, wherein the agent for detecting the expression level of RFX1mRNA in macrophages further comprises: RNA extraction reagent, reverse transcription reagent and PCR amplification reagent.

5. The use of claim 2, wherein the agent for detecting the expression level of RFX1 protein in macrophages comprises a protein extraction reagent, an RFX1 specific antibody and a luminescent detection reagent.

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to an application of a reagent for detecting RFX1 expression level in macrophages in preparation of a macrophage typing detection preparation.

Background

The macrophage is an inherent immune cell of a mononuclear phagocyte system and plays an important role in multiple aspects of stress defense, tissue growth and development, homeostasis balance and the like of a body, researches show that the macrophage has obvious plasticity which can be polarized into a phenotype promoting inflammation or a phenotype inhibiting inflammation, and the plasticity enables the body to effectively resist infection and perform self-repair after infection, the macrophage promoting inflammation is a classical inflammatory macrophage induced by Interferon gamma (IFN-gamma), lipopolysaccharide (L ipopyysacriline, L PS), namely an M1 type macrophage, the M1 type cell releases proinflammatory cytokines such as Interleukin (Interleukin, I L0) -1L, I L-6, I L-12, tumor necrosis factor (tumor necrosis factor) α - α), Inducible nitric oxide synthase (Inducible nitric oxide synthase) and the like to activate inflammatory response, and the phenotype inhibiting inflammation is a low-level of a substitute for the proinflammatory cytokine expressed by MRM 12-11-7-11-6, I-6, I-11-12, I-III-.

Abnormal macrophage polarization is involved in the development of various diseases. Research finds that M1 macrophage can secrete proinflammatory cytokine to promote the development process of inflammatory bowel disease, and M2 macrophage promotes tissue repair and inflammation regression to reduce the development degree of inflammatory bowel disease. There are studies showing increased macrophage infiltration in epicardial adipose tissue of patients with coronary heart disease and polarization towards a pro-inflammatory state of the M1 type. The polarization direction of macrophages influences the development and prognosis of chronic inflammatory diseases such as inflammatory bowel diseases, atherosclerosis and the like. Therefore, accurate identification of macrophage subtypes is important in the study of chronic inflammatory diseases.

The morphologies of the macrophage subtypes are not obviously different, the subtypes of the macrophages are mainly identified by biomarkers at present, common molecules mainly comprise CD68, iNOS, CD206 and the like, detection and identification are usually carried out by flow cytometry, certain requirements are required on instruments of an experimental platform, and the identification of the macrophage subtypes by the molecules has certain limitation. Therefore, the discovery of novel markers and detection reagents for identifying macrophage subtypes, which are convenient to detect and high in applicability, has great significance for identifying macrophage subtypes.

The Regulatory Factor X (RFX) family is an important transcription factor, and RFX1 is a member of the family and has the dual capacity of inhibiting and activating the transcription of target genes. The expression of RFX1 was found to be inversely related to the proliferation, survival and invasive capacity of glioblastoma cells. Currently, there is no study report that the expression of RFX1 is related to macrophage differentiation.

Disclosure of Invention

The invention aims to provide a macrophage typing detection method. The method is simple to operate and accurate in result, and provides a new way for identifying macrophage differentiation types.

A macrophage typing detection method detects the expression level of macrophage RFX 1.

The detection method judges the differentiation type of the macrophages according to the expression level of RFX1 in the macrophages, and the expression level of RFX1 in M1 type macrophages is higher than that of M0 and M2 type macrophages.

In the above detection method, the detecting the expression level of RFX1 in the macrophage comprises detecting the expression level of RFX1mRNA and/or protein in the macrophage.

The result detected by the macrophage typing detection method is the expression level of RFX1 in the macrophage, and the macrophage differentiation type is judged or researched only by the intermediate result.

The second purpose of the invention is to provide a macrophage typing detection reagent, namely, a reagent which is matched with the detection method and is used for detecting the expression level of RFX1 in macrophages.

The detection reagent for detecting the expression level of RFX1 in the macrophage comprises the step of detecting the expression level of RFX1mRNA and/or protein in the macrophage.

The detection reagent comprises the following PCR amplification primer pair sequences in the reagent for detecting the expression level of macrophage RFX1 mRNA:

RFX1-F：5'-GATCCAAGGCGGCTACAT-3'；

RFX1-R：5'-CAGCCGTCTCATAGTTGTCC-3'。

the detection reagent, the reagent for detecting the expression level of RFX1mRNA in macrophage further comprises: RNA extraction reagent, reverse transcription reagent and PCR amplification reagent.

The detection reagent for detecting the expression level of the RFX1 protein in the macrophage comprises a protein extraction reagent, an RFX1 specific antibody and a luminescence detection reagent.

The third purpose of the invention is to provide the application of the reagent for detecting the expression level of RFX1 in the macrophage in preparing a macrophage typing detection preparation.

In the invention, the expression of RFX1 in M0, M1 and M2 macrophages induced by human monocytes in vitro is detected by fluorescence real-time quantitative PCR and Western blotting, the expression level of mRNA and protein of the gene in M1 macrophages is obviously higher than that of M0 and M2 macrophages, the expression level of RFX1 in M0, M1 and M2 macrophages induced by C57B L/6 mouse marrow-derived cells in vitro is detected by Western blotting, and the expression level of protein of the gene in M1 macrophages is obviously higher than that of M0 and M2 macrophages, so that the result indicates that RFX1 can be used as a biomarker of M1 macrophages, and the detection method has good application value in macrophage typing identification, is simple and convenient and has universal applicability.

Drawings

FIG. 1 shows the verification results of the RFX1 amplification primer specificity and the amplification product fragment size designed by the present invention;

a is the lysis profile of human monocyte-induced macrophages amplified with RFX1 primer;

b agarose gel electrophoresis result of the product of QPCR amplification of macrophage induced by human monocyte by RFX1 primer.

FIG. 2 is a graph showing the results of measurement of RFX1mRNA and protein expression levels in M0, M1 and M2-type macrophages induced by human monocytes in vitro;

a is a statistical result chart of the mRNA relative expression detection of RFX1 in M0, M1 and M2 macrophages induced by human monocytes in vitro;

b is SDS-PAGE gel electrophoresis picture of RFX1 protein in M0, M1 and M2 type macrophages induced by human monocytes in vitro;

c is a statistical result chart of the detection of the equivalent expression level of RFX1 protein in M0, M1 and M2 type macrophages induced by human monocytes in vitro.

FIG. 3 is a graph showing the results of measuring the expression level of RFX1 protein in M0, M1 and M2-type macrophages induced in vitro by C57B L/6 mouse bone marrow-derived cells;

a is an SDS-PAGE gel electrophoresis picture of RFX1 protein in M0, M1 and M2 type macrophages induced in vitro by C57B L/6 mouse bone marrow-derived cells;

b is a statistical result chart of relative expression detection of RFX1 protein in M0, M1 and M2 type macrophages induced in vitro by C57B L/6 mouse bone marrow-derived cells.

FIG. 4 is a graph showing the results of calculating the area under the curve (AUC) when comparing the relative expression amounts of RFX1mRNA and protein of M1 type macrophages with M0 and M2 type macrophages by a Receiver Operating Characteristic (ROC) curve analysis;

a is a graph of AUC results obtained when relative expression of M1 type macrophages and M0 and M2 type macrophages RFX1mRNA is calculated by analysis of a Receiver Operating Characteristic (ROC) curve;

b is a graph of AUC results obtained when relative expression of M1 type macrophages and M0 and M2 type macrophages RFX1 protein is calculated by analysis of a Receiver Operating Characteristic (ROC) curve.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as described in the claims.

The following examples relate to DMEM, Gibco, FBS fetal bovine serum, Gibco, rabbit derived RFX1 antibody, GeneTex, centrifuge manufacturer, Freeze Rapid centrifuge model FRESC017, Applied Biosystem, T7900HT Fast Real-Time PCR machine, BIO-RAD, PowerPac and Mini-Sub cell GT, Membrane Transducer manufacturer, Bio-Rad, MiniTrans-Blot cell, chemiluminescence gel imaging System manufacturer, ImageQuant L AS4000 Mini.