CN111500693B

CN111500693B - Application of reagent for detecting RFX1 expression level in macrophage in preparing macrophage typing detection preparation

Info

Publication number: CN111500693B
Application number: CN202010625128.2A
Authority: CN
Inventors: 贾素洁; 赵明; 杨爽; 堵培
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2020-07-02
Filing date: 2020-07-02
Publication date: 2020-09-22
Anticipated expiration: 2040-07-02
Also published as: CN111500693A

Abstract

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.

Description

Application of reagent for detecting RFX1 expression level in macrophage in preparing macrophage typing detection preparation

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

Macrophages are innate immune cells of the mononuclear phagocyte system and play an important role in various aspects such as stress defense, tissue growth and development, homeostasis and the like. Studies have shown that macrophages have significant plasticity-polarizable into either an inflammation-promoting phenotype or an inflammation-inhibiting phenotype. This plasticity allows the body to both effectively fight infection and to undergo self-repair after infection. Macrophages that promote inflammation are the classical inflammatory macrophages induced by Interferon gamma (Interferon gamma, IFN-gamma), Lipopolysaccharide (LPS), i.e. M1 type macrophages. The M1-type cell releases proinflammatory cytokines such as Interleukin (IL) -1 β, IL-6, IL-12, tumor necrosis factor (TNF- α), Inducible nitric oxide synthase (iNOS), and the like to activate the inflammatory response. The phenotype of inhibiting inflammation is the replacement of activated macrophages, also called M2 type macrophages, induced by IL-4 and IL-13. M2 macrophages express low levels of proinflammatory cytokines, high levels of Arginase-1 (ARginase-1, ARG-1), Chitinase-like protein 3 (Chitinase-like protein 3, Chil3 or YM 1), IL-10, TGF-beta, Mannose receptor-1 (Mannose receptor-1, MRC-1), and the like, and participate in tissue repair and remodeling.

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 C57BL/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 C57BL/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 C57BL/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 C57BL/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 refer to: growth medium was DMEM purchased from Gibco; FBS fetal bovine serum, purchased from Gibco; rabbit-derived RFX1 antibody, purchased from GeneTex, usa; the centrifuge manufacturer is a Thermo company in America, and the model is an FRESC017 high-speed freezing centrifuge; the fluorescent quantitative PCR instrument manufacturer is American Applied biosystems company, and the model is T7900HT Fast Real-Time PCR instrument; the electrophoresis apparatus manufacturer is BIO-RAD company in America, and the model is PowerPacTMand Mini-Sub cell GT; the film transfer instrument manufacturer is American Bio-Rad company, and the model is MiniTrans-Blot cell; the chemiluminescent gel imaging system manufacturer is GE corporation, USA, model ImageQuantLAS4000 mini.

Example 1: isolation of human peripheral blood mononuclear cells

Obtaining 4 healthy volunteers (the inclusion standard of the healthy volunteers is that no serious infectious diseases exist within 1.3 months, no large-scale surgery is performed, 2. the healthy volunteers have healthy life style and good eating habits, 3. the BMI is in the normal range (18.5-23.9), 4. the previous patients have no chronic diseases, no autoimmune diseases or immunosuppression states and malignant disease history), extracting about 200 mL of peripheral venous blood after informed consent, and separating the peripheral blood CD14 by adopting a density gradient centrifugation method and a magnetic bead sorting method⁺A monocyte. The method comprises the following specific steps:

a. adding 5mL of heparin solution into each 50 mL of centrifuge tube, adding 20 mL of peripheral venous blood into the centrifuge tube, supplementing the volume to 35 mL by recovered sterile PBS, and gently mixing by using a pipette with the measuring range of 10 mL;

b. taking 15mL of rewarming human lymphocyte separation solution into a 50 mL centrifuge tube;

c. b, slowly adding the diluted peripheral venous blood obtained in the step a onto the liquid level of the lymphocytes along the tube wall by using a pipette, keeping the layered interface of the lymphocytes and the blood clear, and preventing the interface from being damaged (paying attention to the gentle action of the whole process);

d. in order to ensure that the slow deceleration does not damage the layered liquid level and hemolysis, the centrifugation condition adopts 18 ℃, the rotating speed is 2000rpm, the acceleration is 8, the deceleration is 0, and the horizontal centrifugation is carried out for 30 min;

e. the liquid level in the tube after centrifugation is divided into three layers: the uppermost layer is mixed liquid of blood plasma and PBS, the bottommost layer is red blood cells and blood platelets, and the middle layer is white nebulous peripheral blood mononuclear cells. The middle cloud layer is sucked by a capillary suction pipe and transferred to another sterile 50 mL centrifuge tube;

f. adding sterile PBS (phosphate buffer solution) with the volume more than 2 times of that of the peripheral blood mononuclear cell solution transferred into a new centrifuge tube, washing and centrifuging, wherein the centrifugation condition is 4 ℃, 2000rpm, and horizontally centrifuging for 10 min;

g. repeating step f (washing again), centrifuging and discarding the supernatant;

h. taking 480. mu.L of buffer (4 ℃) to resuspend the cell sediment, adding 120. mu.L of CD14 magnetic beads (4 ℃, keeping out of the light), mixing evenly, and incubating for 15 min at 4 ℃ in the absence of the light;

i. after the incubation is finished, adding buffer into the centrifuge tube to 20 mL, horizontally centrifuging for 10 min under the conditions of 4 ℃, 1200rpm, 8 acceleration and 9 deceleration, and discarding the supernatant after centrifugation;

j. taking 1000. mu.L of buffer (4 ℃) to resuspend the cells, fixing the LS separation column on a Midi MACS sorter, and rinsing the LS column with 3 mL of precooled sterile buffer;

k. adding 1 mL of cell suspension into a separation column, adding 3 mLbuffer to wash the column when no liquid flows out of the bottom of the LS separation column, and repeating for 3 times;

taking down the LS separation column, adding 5mL buffer into the column, and rapidly pushing the cells into a 15mL centrifuge tube by adopting a matched push handle;

plating after cell counting.

Example 2: in vitro induction of M0, M1 and M2 type macrophages by human monocytes

a. With macrophages at a final concentration of 50 ng/mLColony stimulating factor (M-CSF) induces CD14⁺Differentiating the monocyte to macrophage, changing the liquid every two days, and culturing for 6 days;

b. the cells were induced to differentiate into M1 type by adding LPS at a final concentration of 100 ng/mL to the human macrophage supernatant (see: Mediators Inflamm. 2016;2016: 6986175.), induced to differentiate into M2 type by adding IL-4 at a final concentration of 20 ng/mL (see: Exp Cell Res. 2017 Aug 15; 357(2): 155-162.), and the unstimulated control group was M0 type macrophages, which were harvested 24 h for subsequent experiments.

Example 3: RNA extraction and reverse transcription

(1) Extraction of sample RNA

a. Spraying 95% alcohol on the table, taking out the sample dissolved in Trizol and frozen in-80 deg.C refrigerator, and dissolving on ice;

b. adding 200 μ L chloroform into 1000 mL Trizol, turning upside down, mixing for 15 s, and standing on ice for 10 min;

c. rotating at 12000 rpm at 4 deg.C, centrifuging for 30 min;

d. approximately 400. mu.L of the supernatant was removed in another new EP tube, and 400. mu.L of isopropanol was added to each tube, mixed by inverting it upside down, and incubated at room temperature for 15 min, taking care not to insert the pipette tip into the white surface and into the following portions:

e. centrifuging at 12000 rpm and 4 deg.C for 20 min, and gently discarding supernatant;

f. adding 500 μ L of precooled enzyme-free 75% ethanol into each tube, turning upside down, mixing gently, centrifuging at 7500 rpm and 4 deg.C for 5 min;

g. gently removing the supernatant, and airing on a floating plate for 5-10 min;

h. adding 20 mu L of non-enzyme water for dissolving, dissolving for 3-4 h at 4 ℃, and measuring the concentration;

(2) reverse transcription

The specific steps of the method are as follows:

a. genomic DNA was removed and the reaction system is shown in Table 1 below:

TABLE 1 DNA removal reaction System

b. The reaction mixture was prepared as shown in table 2 below and dispensed into enzyme-free PCR tubes:

TABLE 2 reverse transcription Mix formulation system

c. The synthesized cDNA was reverse-transcribed, diluted 5-fold with enzyme-free water, and stored at-20 ℃.

Example 4: real-time fluorescent quantitative PCR detection of RFX1mRNA expression level

Taking the sequence of the human RFX1 gene as a template, designing the following primers, wherein the sequences of the primers are as follows: 1, SEQ ID No:

RFX1-f：5'-GATCCAAGGCGGCTACAT-3'

SEQ ID No:2

RFX1-R：5'-CAGCCGTCTCATAGTTGTCC-3'

the results of the primer specificity and the size verification of the amplified product fragment are shown in FIG. 1,

from the results, fig. 1A shows the lysis curve of human monocyte-induced macrophages amplified by RFX1 primer, which shows that the lysis curve is a single peak, the amplified product is single, and the primer specificity is good; and the size of the amplified product fragment is 107bp through a result graph of primer amplified product fragment size compared by the BLAST function of the NCBI website; FIG. 1B is the agarose gel electrophoresis result of the qPCR amplification product of human monocyte-induced macrophage with RFX1 primer, the band is single, and the size of the product fragment is 107bp, which indicates that the qPCR amplification product is the target fragment;

a. operating according to the specification of SYBR PrimeScriptTM RT-PCR Kit of Takara, taking beta-actin as an internal reference, wherein the reaction system is shown in Table 3;

TABLE 3 Real time-PCR amplification System

b. The reaction conditions are shown in Table 4 below:

TABLE 4 Real time-PCR reaction conditions

c. Carrying out real-time fluorescent quantitative PCR by using a LightCyclery 7900 PCR instrument of Roche, and repeating the experiment for more than three times;

the results of the experiment are shown in figure 2,

the results show that the relative expression quantity of RFX1mRNA in M0, M1 and M2 type macrophages induced by human monocytes in vitro is obviously higher for M1 than for M0 and M2 type macrophages,P=0.0035, the results were statistically significant.

Example 4: total protein extraction

a. Freezing at-80 deg.C 2 × 10⁶Thawing the cell samples (4 each of M0, M1 and M2) on ice, and preparing protein lysate by using strong protein lysate (RIPA) and protease inhibitor (PMSF) according to a ratio of 100: 1;

b. adding 50 μ L of prepared lysis solution into each tube of cells, blowing, mixing, and placing on ice for lysis for 30 min;

c. performing ice ultrasound on cells in the EP tube by using an ultrasonic probe for 5 min/time, and repeating the steps for three times;

d. centrifuging the ultrasonic cells at 4 ℃, 10000-12000 rpm for 10 min, and transferring the supernatant into another new EP tube;

e. detecting protein concentration by BCA method, subpackaging according to 30 μ g protein amount per tube, and freezing at-80 deg.C.

Example 5: western blotting method for detecting RFX1 protein expression level

(1) Preparation of SDS-PAGE gels

a. Preparing 8% separating glue solution, mixing completely, injecting into the assembled glass plywood with a suction pipe, and injecting 3/4 with final volume of separating glue;

b. sucking deionized water and slowly injecting into the splint to avoid large fluctuation of the liquid level until the separation gel is completely isolated from the air, and standing for 30 min;

c. pouring out deionized water on the upper layer of the separation gel, slightly sucking residual water by using filter paper, preparing a 5% concentrated gel solution, completely mixing uniformly, transferring the mixture to the upper layer of the separation gel by using a suction pipe, and slightly inserting an 8-hole comb (slightly shaking the comb before completely inserting the mixture to ensure that no air bubbles exist around the comb);

d. standing for 1 h to ensure that the concentrated gel is completely solidified, placing the gel plate in an electrophoresis solution, and storing at 4 ℃ for later use;

(2) SDS-PAGE gel electrophoresis

a. Taking out the subpackaged protein samples, dissolving on ice, measuring 20 mu g of protein solution, mixing with 2 xSDS-loading buffer according to the volume ratio of 1:1, boiling for 7 min (denatured protein) at 100 ℃ in a metal bath, and centrifuging at 12000 rpm for 5 min;

b. and (5) installing an electrophoresis frame, and adding electrophoresis liquid to check whether leakage exists. After no leakage is confirmed, filling the electrophoresis frame and pulling out the comb of the gel plate to wash the gel hole (remove impurities in the gel hole);

c. sequentially adding the Marker and the denatured protein sample into the sample adding hole, adding electrophoresis liquid into an electrophoresis box, adjusting the voltage to 80V (constant voltage electrophoresis) for 40-60 min, and when the protein is moved from the concentrated gel to the separation gel and is electrophoresed into a line, adjusting the voltage to 100V and continuing electrophoresis until the electrophoresis is finished;

(3) rotary film

a. Preparing a membrane transferring solution, taking out the electrophoresis rack, and recovering the electrophoresis solution for next use;

b. taking out the gel plate, carefully prying the glass plate, taking out SDS-PAGE gel, cutting off redundant gel according to the position of a marker strip (ensuring that RFX1 and beta-actin protein at the positions of 130 kd and 42 kd are not cut off), and placing the gel plate in a membrane transferring solution for later use;

c. cutting the PDVF membrane and the filter paper with the same size according to the size of the glue, putting the PDVF membrane into methanol for soaking for 5 min, taking out, and putting the sponge mat, the filter paper and the PDVF membrane into a membrane transferring liquid for soaking;

d. clamping a sponge pad, three layers of filter paper, SDS-PAGE gel, a PDVF membrane, three layers of filter paper and a sponge pad (ensuring that each layer has no bubbles) in sequence from bottom to top, plugging the clamped sponge pad into a transfer printing clamp, and adopting a membrane transfer instrument to perform constant current 300mA in a chromatography cabinet at 4 ℃ and transfer the membrane for 90 min;

(4) sealing of

a. Weighing about 2.5 g of skimmed milk powder, placing into a 50 mL centrifuge tube, adding PBST solution, turning upside down, mixing uniformly until the mixture is completely dissolved, and metering to 50 mL;

b. taking out the blotting membrane from the transfer printing clamp, enabling the front side of the blotting membrane to face upwards, cutting off small corner marks at the upper left corner, enabling the blotting membrane to face backwards, then placing the blotting membrane in a washing box, adding enough 5% of skimmed milk powder sealing liquid to cover the blotting membrane, and sealing the blotting membrane on a vertical shaking table at room temperature for 1 h;

(5) hatching-resisting

a. Cutting the blotting membranes according to the marker position to enable RFX1 and beta-actin to be positioned on the two blotting membranes, respectively diluting an RFX1 antibody, a beta-actin antibody and a confining liquid according to a ratio of 1:500 and a ratio of 1:2000, uniformly mixing, adding the mixture into an antibody incubation box in which the two blotting membranes are positioned, placing the incubation box in a shaking table of a chromatography cabinet at 4 ℃, and incubating overnight;

b. washing the membrane with PBST solution three times after overnight, 10 min each time;

(6) hatching secondary antibody

a. Placing the two washed blotting membranes in a secondary antibody diluent (the dilution ratio is 1: 2000) for resisting a mouse and a rabbit respectively, and incubating for 1 h on a vertical shaking table at room temperature;

b. transferring the membrane into sufficient PBST, washing for 10 min/time on a horizontal shaking table, and washing for three times;

(7) and (3) developing: opening the developing instrument in advance, preparing the developing solution according to the specification of the developing solution, uniformly wetting the blotting membrane by using a liquid transfer gun, and then placing the blotting membrane in a dark room of the developing instrument for developing;

the results are shown in figure 2 of the drawings,

the results show that the relative expression quantity of RFX1 protein in M0, M1 and M2 type macrophages induced by human monocytes in vitro is obviously higher for M1 than for M0 and M2 type macrophages,P=0.0062, the results were statistically significant.

Example 6: isolation of bone marrow-derived cells from C57BL/6 mice

a. Preparing a 1% sodium pentobarbital solution, carrying out abdominal anesthesia on the mouse according to 50mg/kg, waiting for the mouse to enter a deep anesthesia state, and killing the mouse after no response is caused to nociceptive clamping;

b. cutting the skin of a mouse, dissociating a tibia and a fibula, removing muscles and tissues outside the bones with absorbent paper sprayed with 75% alcohol, and putting the bones into a clean 15mL centrifuge tube;

c. washing with 75% alcohol, washing off residual alcohol with pure DMEM medium, shearing off two ends of bone with sterilized scissors, washing out mouse bone marrow with pure DMEM medium into a 10 cm culture dish, mixing, centrifuging at 1500 rpm for 5 min;

d. preparing a mouse bone marrow macrophage induction culture medium by using a 50% DMEM culture medium, a 30% sterile L929 cell culture supernatant and 1% double antibody, and 20% FBS (20% FBS), re-suspending bone marrow cells by using the mouse bone marrow macrophage induction culture medium, paving two 10 cm culture dishes for each mouse bone marrow cell, wherein each culture dish contains 6 mL of induction culture medium, and replacing the culture medium once every two days;

e. EDTA macrophage digest was prepared using 20 mL of 0.5M EDTA solution, 960 mL of PBS solution, and 20 mL of fetal bovine serum. Cells were cultured until day six (supernatant was directly decanted), cells were digested with formulated EDTA macrophage digest, and plated by counting.

Example 7: m0, M1 and M2 type macrophages induced in vitro by C57BL/6 mouse bone marrow-derived cells

After macrophage is cultured overnight, LPS with the final concentration of 100 ng/mL is added into cell culture supernatant to induce 24 hours, and then the macrophage is differentiated to M1 type; after 24 h of induction by the addition of IL-4 to the cell culture supernatant at a final concentration of 20 ng/mL, macrophages differentiated towards M2 type.

Example 8: total protein extraction

Example 9: western blotting method for detecting RFX1 protein expression level

(1) Preparation of SDS-PAGE gels

(2) SDS-PAGE gel electrophoresis

(3) rotary film

(4) sealing of

(5) hatching-resisting

(6) hatching secondary antibody

the results are shown in figure 3 of the drawings,

the results show that the relative protein expression of RFX1 in M0, M1 and M2 type macrophages induced by C57BL/6 mouse bone marrow-derived cells in vitro is obviously higher for M1 than for M0 and M2 type macrophages,P=0.0165, the results were statistically significant.

Example 10: specific analysis of relative expression of mRNA and protein of RFX1 as biomarker of M1 type macrophage

Calculating the area under the curve (AUC) when the relative expression quantity of M1 type macrophages is compared with M0 and M2 type macrophages RFX1mRNA and protein by adopting the Receiver Operating Characteristic (ROC) curve analysis;

the results are shown in figure 4 of the drawings,

as can be seen from the results, the AUC value of the relative expression amount of M1, M0 and M2 type macrophage RFX1mRNA induced in vitro by human monocytes in 11 healthy volunteers was 0.839 (95% CI: 0.658-1.000) (n = 33); the relative expression of RFX1mRNA indicated that: according to the 'you' n index ', namely sensitivity- (1-specificity), the index value is the optimal threshold value at the maximum value, the maximum value of the' you 'n index' is R0.773, the optimal threshold value of the relative expression quantity of M1 and M0 and M2 type macrophages RFX1mRNA is a =1.2491, namely the sample a value is compared with the threshold value (a = 1.2491), a is greater than 1.2491 and judged as M1 type macrophages, a is less than or equal to 1.2491 and judged as non-M1 type macrophages, and particularly judged as M2/M0 type macrophages according to IL-4 induction/non-induction. The sensitivity and specificity of M1-type macrophages were identified as 81.8% and 95.5% using this optimal cut-off value, respectively;

AUC values for relative expression of M1 and M0, M2-type macrophage RFX1 protein in human monocytes induced in vitro in 4 healthy volunteers and in 4C 57BL/6 mouse bone marrow-derived cells were 0.938 (95% CI: 0.842-1.000) (n = 24); the relative expression level of RFX1 protein shows that: the maximum value of the ' you ' n index ' is R0.75, the optimal threshold value of relative expression of M1 and M0 and M2 type macrophage RFX1 protein is b =30.55, namely the b value of the sample is compared with the threshold value (b = 30.55), the macrophage with b >30.55 is judged as M1 type macrophage, the macrophage with a less than or equal to 30.55 is judged as non-M1 type macrophage, and the macrophage with IL-4 induction/non-induction is judged as M2/M0 type macrophage. The sensitivity and specificity of M1-type macrophages were identified using this optimal cut-off value as 87.5% and 87.5%, respectively.

The results indicate that the expression of RFX1 can be used as a biomarker of M1 type macrophages, has higher accuracy and specificity, and has better application value for identifying M1 type macrophages.

Sequence listing

<110> university of south-middle school

<120> application of reagent for detecting RFX1 expression level in macrophage in preparing macrophage typing detection preparation

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Claims

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 the agent that detects the expression level of RFX1 in macrophages comprises an agent that detects the expression level of RFX1mRNA and/or protein in macrophages.

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.