阅读说明：本技术 多肽在调控巨噬细胞体外转型中的应用 (Application of polypeptide in regulating and controlling macrophage in vitro transformation ) 是由 宋淑亮 陈锚 潘炳旗 王珂 马士玉 关浩 陈晓东 尹宪利 于 2021-11-09 设计创作，主要内容包括：本发明涉及生物医药技术领域,尤其是涉及多肽在调控巨噬细胞体外转型中的应用。本发明提供的由序列SEQ ID No.1所示氨基酸序列组成的多肽来源于水蛭,发明人经研究发现,该多肽能够在体外高效地促进M1型巨噬细胞向M2型巨噬细胞转化,转化效果显著且稳定,适于体外批量转化,同时由序列SEQ ID No.1所示氨基酸序列组成的多肽在体外制剂中能够稳定存在,适于制备成相关调控产品,进行长期运输、保存和大批量使用。将由序列SEQ ID No.1所示氨基酸序列组成的多肽用于以M1型巨噬细胞转型为M2型巨噬细胞为检测指标的药效评价或科学研究中。(The invention relates to the technical field of biological medicines, in particular to application of polypeptide in regulating and controlling macrophage in-vitro transformation. The polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 is derived from leeches, and the inventor finds that the polypeptide can efficiently promote M1-type macrophages to be converted into M2-type macrophages in vitro, has obvious and stable conversion effect and is suitable for in vitro batch conversion, and meanwhile, the polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 can stably exist in an in vitro preparation and is suitable for being prepared into related regulation and control products for long-term transportation, storage and mass use. The polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 is used for the drug effect evaluation or scientific research by taking the M1 type macrophage transformed into the M2 type macrophage as a detection index.)

1. The application of the polypeptide in regulating and controlling macrophage in vitro transformation or preparing a product for regulating and controlling macrophage in vitro transformation is disclosed, wherein the polypeptide is a polypeptide consisting of an amino acid sequence shown in a sequence SEQ ID No. 1;

the macrophage in vitro transformation includes a transformation between M1-type macrophages and M2-type macrophages.

2. A method for regulating and controlling macrophage in vitro transformation is characterized by comprising the steps of adding polypeptide with the final concentration of 200-800 mu g/mL into cell suspension of M1 type macrophages, and carrying out cell culture to obtain the cell suspension of M2 type macrophages;

the polypeptide is composed of an amino acid sequence shown in a sequence SEQ ID No. 1.

3. The method for regulating and controlling M1-type macrophages, wherein the suspension of M1-type macrophages is cultured to obtain a suspension of M1-type macrophages after adding inflammation inducing factors to the suspension of macrophages.

4. The method of claim 3, wherein the macrophage suspension comprises a logarithmic growth phase macrophage suspension obtained by culturing macrophages in a DMEM high-glucose medium.

5. The method according to claim 3, wherein the inflammatory inducing factor comprises LPS, and the final concentration of LPS added to the macrophage suspension is 0.5-1.5 μ g/mL.

6. A preparation for regulating and controlling macrophage in vitro transformation is characterized by comprising a polypeptide for regulating and controlling macrophage in vitro transformation, a M1 type macrophage molecular marker detection reagent and a M2 type macrophage molecular marker detection reagent;

the polypeptide for regulating and controlling the in vitro transformation of the macrophage is a polypeptide consisting of an amino acid sequence shown in a sequence SEQ ID No. 1.

7. The formulation of claim 6, wherein the M1-type macrophage molecular marker detection reagent comprises a forward amplification primer ACTCAGCCAAGCCCTCACCTAC that amplifies a gene encoding iNOS and a reverse amplification primer TCCAATCTCTGCCTATCCGTCTCG that amplifies a gene encoding iNOS; and forward amplification primer ATGTCTCAGCCTCTTCTCATTC that amplifies the gene encoding TNF- α, and reverse amplification primer GCTTGTCACTCGAATTTTGAGA that amplifies the gene encoding TNF- α.

8. The preparation of claim 6, wherein the M2-type macrophage molecular marker detection reagent comprises a forward amplification primer CATATCTGCCAAAGACATCGTG that amplifies the Arg-1-encoding gene and a reverse amplification primer GACATCAAAGCTCAGGTGAATC that amplifies the Arg-1-encoding gene; and a forward amplification primer TTCTTTCAAACAAAGGACCAGC that amplifies the gene encoding IL-10 and a reverse amplification primer GCAACCCAAGTAACCCTTAAAG that amplifies the gene encoding IL-10.

9. An in vitro macrophage transformation regulating kit, which comprises the preparation and consumable of any one of claims 6-8.

10. Use of the kit according to claim 9 for evaluation of drug efficacy or scientific research using the conversion of macrophage M1 into macrophage M2 as a detection marker.

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of polypeptide in regulating and controlling macrophage in-vitro transformation.

Background

Macrophages are widely distributed in tissues and organs of the body and play an important role in pathogen defense, inflammatory response, homeostasis maintenance, and injury repair. Macrophages are a population of immune cells that are highly heterogeneous in phenotype and function, and can alter their own polarization state to adapt to complex external conditions. Macrophages usually comprise two polarized types of action functions of M1 and M2, wherein the M1 type can promote inflammatory reaction, secrete ROS, TNF-alpha, IL-6 and MCP-1 after stimulation by LPS, IFN-gamma and TNF-alpha, and improve the body defense capacity. The M2 type can reduce inflammatory reaction, and secrete IL-10, TGF-beta and IL-1Ra after being stimulated by IL-4 and IL-13, and is beneficial to tissue repair. Therefore, the relative amount of M1 and M2 macrophages in the body plays an important role in the regulation of inflammatory diseases such as atherosclerosis, tumors and obesity. In the process of developing and producing drugs depending on macrophage transformation mechanism, synchronous induction transformation is required to be carried out by using a comparative induction transformation product in the evaluation process of drug effect to serve as the evaluation standard of drug effect, but no such comparative method or product is reported at present.

Macrophages of the M1 type, which are macrophages that produce proinflammatory cytokines, are known as classical macrophages and have strong microbial killing properties, but these specificities are also likely to cause tissue destruction. For example, Inflammatory Bowel Disease (IBD), Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), and Rheumatoid Arthritis (RA) are associated with macrophages of type M1.

Macrophages of the M2 type, also known as surrogate activated macrophages, play a central role in response to parasites, tissue remodeling, angiogenesis and allergic diseases. Research on macrophages of the M2 type is becoming increasingly intensive, and diseases that have been identified as being associated therewith include allergies, asthma, and helminth infections.

Although the macrophages are the same, the mechanism of action of the M1 type macrophages and the M2 type macrophages is different, the related indications are obviously different, and the M1 type macrophages and the M2 type macrophages are transformed mutually, so that the damage of the macrophages to normal cells or tissues of the organism under the condition that the indications are weakened or disappeared can be relieved, and the method has extremely important research value.

It has been found that a plurality of signal pathways are related to M1 and M2 transformation of macrophages, and a plurality of regulatory molecules exist for different signal pathways, so that the corresponding signal pathways are closed or activated to realize the transformation of the macrophages, and the regulatory molecules comprise various substances such as miRNA and small molecular proteins, but the regulatory molecules are only reported in scientific research documents, and whether the effectiveness of the induction actually applied in vitro can be used for evaluating the corresponding drug effect is unknown.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide application of polypeptide in regulation and control of macrophage in vitro transformation or preparation of a product for regulation and control of macrophage in vitro transformation, and provides a method for effectively inducing M1 type macrophages to transform to M2 type macrophages in vitro based on the polypeptide, so that evaluation on prevention and/or treatment effects of a medicament depending on a macrophage transformation mechanism or development of related scientific research can be smoothly carried out.

The invention also aims to provide a product for regulating and controlling the in vitro transformation of the macrophage, which comprises a preparation or a kit, and provides an in vitro regulating and controlling product which is simple and convenient to operate and visual in effect for realizing the in vitro transformation of the macrophage so as to meet the requirement of evaluating the effect of medicaments on a large scale.

In order to solve the technical problems and achieve the purpose, the invention provides the following technical scheme:

in a first aspect, the invention provides the use of a polypeptide in regulating macrophage in vitro transformation or in preparing a product for regulating macrophage in vitro transformation, wherein the polypeptide is a polypeptide consisting of an amino acid sequence shown in a sequence SEQ ID No. 1;

the macrophage in vitro transformation includes a transformation between M1-type macrophages and M2-type macrophages.

In a second aspect, the invention provides a method for regulating macrophage in vitro transformation, which comprises the steps of adding polypeptide with the final concentration of 200-800 mug/mL into cell suspension of M1 type macrophages, and then carrying out cell culture to obtain the cell suspension of M2 type macrophages;

the polypeptide is composed of an amino acid sequence shown in a sequence SEQ ID No. 1.

In an alternative embodiment, the cell suspension of the M1 type macrophage cell comprises a cell suspension of the M1 type macrophage cell obtained by cell culture after adding the inflammation inducing factor to the macrophage cell suspension.

In alternative embodiments, the macrophage suspension comprises a log phase macrophage suspension obtained by culturing macrophages in a DMEM high glucose medium.

In an alternative embodiment, the inflammation inducing factor comprises LPS, and the final concentration of LPS added to the macrophage suspension is 0.5-1.5 μ g/mL.

In a third aspect, the invention provides a preparation for regulating macrophage in vitro transformation, which comprises a polypeptide for regulating macrophage in vitro transformation, a M1 type macrophage molecular marker detection reagent and a M2 type macrophage molecular marker detection reagent;

the polypeptide for regulating and controlling the in vitro transformation of the macrophage is a polypeptide consisting of an amino acid sequence shown in a sequence SEQ ID No. 1.

In alternative embodiments, the M1-type macrophage molecular marker detection reagent comprises a forward amplification primer ACTCAGCCAAGCCCTCACCTAC that amplifies a gene encoding iNOS and a reverse amplification primer TCCAATCTCTGCCTATCCGTCTCG that amplifies a gene encoding iNOS; and/or the presence of a gas in the gas,

a forward amplification primer ATGTCTCAGCCTCTTCTCATTC that amplifies the gene encoding TNF- α, and a reverse amplification primer GCTTGTCACTCGAATTTTGAGA that amplifies the gene encoding TNF- α.

In alternative embodiments, the M2-type macrophage molecular marker detection reagent comprises a forward amplification primer CATATCTGCCAAAGACATCGTG that amplifies the Arg-1-encoding gene and a reverse amplification primer GACATCAAAGCTCAGGTGAATC that amplifies the Arg-1-encoding gene; and/or the presence of a gas in the gas,

a forward amplification primer TTCTTTCAAACAAAGGACCAGC that amplifies the gene encoding IL-10 and a reverse amplification primer GCAACCCAAGTAACCCTTAAAG that amplifies the gene encoding IL-10.

In a fourth aspect, the present invention provides a kit for modulating macrophage in vitro transformation, the kit comprising a formulation according to any one of the preceding embodiments and a consumable.

In a fifth aspect, the present invention provides a use of the preparation according to any one of the preceding embodiments or the kit according to any one of the preceding embodiments for evaluating a drug effect or scientific research using conversion of macrophage M1 into macrophage M2 as a detection index.

The polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 is derived from leeches, and the inventor finds that the polypeptide can efficiently promote M1-type macrophages to be converted into M2-type macrophages in vitro, has obvious and stable conversion effect and is suitable for in vitro batch conversion, and meanwhile, the polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 can stably exist in an in vitro preparation and is suitable for being prepared into related regulation and control products for long-term transportation, storage and mass use. The polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 is used for macrophage in vitro induction transformation, and provides a powerful and feasible evaluation standard for the evaluation of the prevention and/or treatment effect of related medicaments.

The invention provides a method for regulating and controlling macrophage in vitro transformation, wherein the final concentration of a polypeptide consisting of an amino acid sequence shown in a sequence SEQ ID No.1 is gradually increased within the range of 200-800 mu g/mL, and the transformation degree of M1 type macrophages to M2 type macrophages is also gradually increased, so that the final concentration equivalent of the polypeptide can be used for evaluating the prevention and/or treatment effect of related medicines, namely the prevention and/or treatment effect of a certain medicine is equivalent to the regulation and control effect of the polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 at a certain final concentration, and the medicine effects of different medicines are compared and evaluated, and the method can also be used in scientific research for quantitatively evaluating the influence of a certain compound on the transformation of M1 type macrophages to M2 type macrophages.

The invention also provides a preparation and a kit for regulating and controlling macrophage in vitro transformation, which combines the polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 with a M1 type macrophage molecular marker detection reagent and a M2 type macrophage molecular marker detection reagent for use, and realizes the in vitro stable and accurate detection of the macrophage transformation degree.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the result of expression of Arg-1 gene mRNA in effect example 1 of the present invention;

FIG. 2 shows the results of expression of mRNA of iNOS gene in example 1 in which the present invention is effective;

FIG. 3 shows the results of expression of mRNA of IL-10 gene in example 1 of the effects of the present invention;

FIG. 4 shows the result of expression of mRNA of TNF-. alpha.gene in example 1 in accordance with the present invention;

FIG. 5 shows the results of electrophoretic bands of the respective sets of embodiments in Effect example 2;

FIG. 6 shows the result of expression of Arg-1 gene mRNA in effect example 2 of the present invention;

FIG. 7 shows the results of expression of iNOS gene mRNA in example 2 in which the present invention is effective;

FIG. 8 shows the results of expression of mRNA of IL-10 gene in example 2 of the effects of the present invention;

FIG. 9 shows the results of expression of mRNA of TNF-. alpha.gene in example 2 showing the effects of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

In a first aspect, the present invention provides the use of a polypeptide synthesized by a solid phase total synthesis technique, having an amino acid sequence of EAGSAKELEGDPVAG (shown as SEQ ID No. 1) and a molecular weight of 1428.6731Da for regulating macrophage in vitro transformation and for preparing a product for regulating macrophage in vitro transformation in a specific embodiment. The addition of the polypeptide is proved to be capable of obviously increasing the expression of the M2 type macrophage marker and simultaneously inhibiting the expression of the M1 type macrophage marker.

Based on the first aspect, the invention provides a method for regulating macrophage in vitro transformation, which comprises the steps of adding 200-800 mu g/mL polypeptide into a cell suspension of M1 type macrophages, and carrying out cell culture to obtain a cell suspension of M2 type macrophages;

the polypeptide is composed of an amino acid sequence shown in a sequence SEQ ID No. 1.

In an alternative embodiment, the cell suspension of the M1 type macrophage cell comprises a cell suspension of the M1 type macrophage cell obtained by cell culture after adding the inflammation inducing factor to the macrophage cell suspension.

In alternative embodiments, the macrophage suspension comprises a log phase macrophage suspension obtained by culturing macrophages in a DMEM high glucose medium.

In an alternative embodiment, the inflammation inducing factor comprises LPS, and the final concentration of the LPS added to the macrophage suspension is 0.5-1.5 mu g/mL, and the preferred final concentration is 1 mu g/mL.

Based on the second aspect, the invention provides a preparation for regulating macrophage in vitro transformation, which comprises a polypeptide for regulating macrophage in vitro transformation, a M1 type macrophage molecular marker detection reagent and a M2 type macrophage molecular marker detection reagent.

For ease of standardisation, the formulation may also include DMEM high glucose medium and LPS as described in the second aspect.

In a fourth aspect, the invention provides a kit for modulating macrophage in vitro transformation, which comprises a polypeptide for modulating macrophage in vitro transformation, a M1 type macrophage molecular marker detection reagent, a M2 type macrophage molecular marker detection reagent and a consumable.

To facilitate efficient operation, the consumables include well plates, pipetting devices, reagent containers, and the like.

The M1-type macrophage molecular marker detection reagent in the above three aspects and the fourth aspect includes a forward amplification primer ACTCAGCCAAGCCCTCACCTAC (shown in SEQ ID No. 2) for amplifying a gene encoding iNOS and a reverse amplification primer TCCAATCTCTGCCTATCCGTCTCG (shown in SEQ ID No. 3) for amplifying a gene encoding iNOS; and/or, a forward amplification primer ATGTCTCAGCCTCTTCTCATTC (shown as SEQ ID No. 4) that amplifies a gene encoding TNF- α, and a reverse amplification primer GCTTGTCACTCGAATTTTGAGA (shown as SEQ ID No. 5) that amplifies a gene encoding TNF- α;

the M2 type macrophage molecular marker detection reagent comprises a forward amplification primer CATATCTGCCAAAGACATCGTG (shown as SEQ ID No. 6) for amplifying an Arg-1 encoding gene and a reverse amplification primer GACATCAAAGCTCAGGTGAATC (shown as SEQ ID No. 7) for amplifying an Arg-1 encoding gene; and/or the presence of a gas in the gas,

a forward amplification primer TTCTTTCAAACAAAGGACCAGC (shown as SEQ ID No. 8) for amplifying the gene encoding IL-10 and a reverse amplification primer GCAACCCAAGTAACCCTTAAAG (shown as SEQ ID No. 9) for amplifying the gene encoding IL-10.

Through research, the inventor can confirm that M1 type macrophages formed by DMEM high-glucose medium culture and LPS induction can be induced to be converted into M2 type macrophages by adding the polypeptide provided by the invention, and the conversion process can be accurately detected by adopting the combination of primers with nucleotide sequences shown in SEQ ID No. 2-SEQ ID No. 9.

In combination with the above aspects, the present invention provides the use of the preparation according to the foregoing embodiment or the kit according to the foregoing embodiment for evaluating the prophylactic and/or therapeutic effect of a drug accompanied by the process of transforming M1-type macrophages into M2-type macrophages.

Some embodiments of the invention are described in detail below with reference to the accompanying drawings. The embodiments described below and the features of the embodiments can be combined with each other without conflict.

Example 1

The embodiment provides an in vitro macrophage transformation regulating preparation, which comprises polypeptide (HE-D) consisting of an amino acid sequence shown in a sequence SEQ ID No.1, a primer for amplifying a gene coding for iNOS, a primer for amplifying a gene coding for TNF-alpha, a primer for amplifying a gene coding for Arg-1, a primer for amplifying a gene coding for IL-10 and an amplification primer for a control gene beta-actin, wherein the specific primer components are shown in Table 1.

TABLE 1 primer composition in the preparation for modulation of macrophage in vitro transformation as described in example 1

Example 2

This example provides an in vitro macrophage-modulating transforming preparation, which differs from example 1 in that the primer composition is shown in Table 2.

TABLE 2 primer composition in the preparation for modulation of macrophage in vitro transformation as described in example 2

Examples 3 and 4

The group of embodiments provide a kit for regulating macrophage in vitro transformation on the basis of the preparations for regulating macrophage in vitro transformation provided by the embodiments 1 and 2, wherein the kits provided by the embodiments 3 and 4 respectively comprise a pore plate, a pipette gun and a reagent solvent, and further comprise the preparations for regulating macrophage in vitro transformation provided by the embodiments 1 and 2.

Example 5

This example provides a method of modulating macrophage in vitro transformation using the modulation formulation provided in example 1 or using the modulation kit provided in example 3, comprising the steps of:

(1) taking cells in logarithmic growth phase, and preparing Raw264.7 cells in logarithmic growth phase into 5 x 10 cells by using DMEM high-glucose medium with 10% FBS by volume⁵Cell suspension with density of one/mL is inoculated in a six-well plateInternal, at 5% CO₂And culturing at 37 ℃.

(2) The experiment is divided into five groups including a blank group, an LPS model group, a low dose group (LHED) LPS +200 mu g/mL HE-D group, a medium dose group (MHED) LPS +400 mu g/mL HE-D group, and a high dose group (HHED) LPS +800 mu g/mL HE-D group, wherein 3 biological replicates are taken in each group. After the cells of Raw264.7 in the step (1) are cultured for 24 hours, LPS is respectively added into the LPS model group, the low dose group, the medium dose group and the high dose group to enable the final concentration to be 1.0 mu g/mL, HE-D is added into each dose group to enable the final concentration to be 200 mu g/mL, 400 mu g/mL and 800 mu g/mL after 1 hour of treatment, and the cells are continuously cultured for 24 hours.

(3) And (3) absorbing the culture solution in each hole, slowly washing the culture solution for three times by using PBS (phosphate buffer solution), adding 1mL of TRIZOL into each hole to blow and beat the cells uniformly so as to fully crack the cells, immediately putting the cells into liquid nitrogen to quickly freeze for 20min, and then transferring the cells to a low temperature of-80 ℃ for storage to be delivered.

Effect example 1

mRNA level of macrophage transformation related gene is detected by qRT-PCR, and mRNA level of related gene of five groups of cell culture solution in the embodiment 5 is detected by the following method:

(1) preparation of RNA samples

1. The cell culture medium of each group was discarded, washed with PBS and TRIZOL was added to lyse the cells, transferred to RNase-free EP tubes, and left to stand for 10 min.

2. The nucleic acid was extracted by adding chloroform to an EP tube, 0.2mL of chloroform was added to 1mL of TRIZOL lysate, and the mixture was thoroughly mixed, left to stand for 10min, and centrifuged at 12000rpm at 4 ℃ for 15 min.

3. Sucking supernatant of colorless supernatant into RNase-free EP tube, adding 500 μ L isopropanol, mixing, standing for 10min, and centrifuging at 4 deg.C at 12000rpm for 10 min.

4. The supernatant was discarded, 1mL of 75% ethanol in DEPC water was added, the mixture was gently blown up, the mixture was inverted by gentle shaking for 15 seconds, and the mixture was centrifuged at 7000 rpm at 4 ℃ for 5 min.

6. Carefully aspirate the supernatant, open the tube cap, dry at room temperature for 10min to volatilize the residual ethanol, then add 50 μ L of DEPC water, dissolve well and store at-80 ℃.

(2) Determination of RNA concentration

Zeroing the spectrophotometer, mixing 1 μ L sample solution with 99 μ L EPC water, and measuring A₂₆₀/A₂₈₀This value, which is in the range of 1.8-2.1, can be used in subsequent experiments.

(3) Sample cDNA Synthesis

1. The following components were added to the RNase Free tube, and the reaction for removing genomic DNA was first carried out:

mixing, water bathing at 42 deg.C for 5min, and placing on ice.

2. The same tube was continued for cDNA synthesis with the following components:

centrifuging instantly, mixing, heating in 50 deg.C water bath for 15min, heating in 85 deg.C water bath for 5min to inactivate SPAPKscript HRTase and gDNA Eraser, cooling, and storing at-80 deg.C.

（4）qRT-PCR

1. The following reaction was carried out in a PCR tube (25. mu.L reaction):

and (4) performing instantaneous centrifugation, uniformly mixing, and performing operation on ice in a dark place.

2. The following program was run using a qRT-PCR instrument:

denaturation at 1.95 deg.C for 2 min;

denaturation at 2.95 deg.C for 15s, annealing at Tm of-5 deg.C for 15s, and extension at 72 deg.C for 30s, and circulating for 40 times;

3. melting section, 95 ℃ 1min → 65 ℃ 1min → 95 ℃ 20s → 40 ℃ 1 min.

The detection results of the Arg-1 gene, the iNOS gene, the IL-10 gene and the TNF-alpha gene are respectively shown in figures 1-4, and as shown in the figure, the qRT-PCR result shows that after the HE-D intervention, the iNOS and TNF-alpha genes which are typical markers of M1 type macrophages are down-regulated, and the Arg-1 and IL-10 genes which are typical markers of M2 type macrophages are up-regulated. 1 ug/mL LPS significantly promoted the mRNA levels of iNOS and TNF- α, and 200-800 ug/mL HE-D significantly reduced the mRNA levels of iNOS and TNF- α with a concentration dependence (maximum inhibition rates at 800 ug/mL HE-D concentrations of 40.3% and 53.5%, respectively). Meanwhile, 200-800. mu.g/mL HE-D significantly increased the mRNA level of Arg-1 (the maximum promotion rate at the concentration of 800. mu.g/mL HE-D was 94.5%), and 400-800. mu.g/mL HE-D significantly increased the mRNA level of IL-10 (the maximum promotion rate at the concentration of 800. mu.g/mL HE-D was 44.5%) as compared with the LPS group. The above results indicate that HE-D can promote LPS-induced conversion of M1-type macrophages to M2-type macrophages.

Effect example 2

Western blot is adopted to detect the mRNA level of the macrophage transformation related gene, and the mRNA level of the related gene of the five groups of cell culture solution in the embodiment 5 is detected by the following method:

(1) preparation of protein samples

1. Each set of cell culture media was discarded and washed three times slowly with PBS.

2. Adding a proper amount of cell lysate into cells to be detected, lysing the cells at 4 ℃ for 30 min, then centrifuging the cells at 4 ℃ and 12000rpm for 5min, and taking supernatant for later use.

And 3, measuring and leveling the protein concentration of the sample by using a BCA method, adding a loading buffer to 1 x, carrying out water bath at 95 ℃ for 5min, cooling, and placing at-20 ℃ for later use.

(2) SDS-PAGE electrophoresis

The glue-making glass plate was washed and fixed in a jig, and a separation glue was prepared according to the following criteria. When the separation glue is prepared, each component needs to be fully and uniformly mixed, glue pouring needs to be carried out immediately after TEMED is added, the separation glue is subjected to liquid sealing by using ultrapure water, and after an obvious two-phase boundary appears, the water phase is absorbed.

10% separation gel (two pieces of gel, 10 mL) added sequentially:

5% concentrated gum (two pieces of gum, 4 mL), added sequentially:

adding concentrated gel, inserting into electrophoresis comb, adding electrophoresis buffer after gel is solidified, and pulling out electrophoresis comb. Adding a proper amount of Marker and a sample to be detected, adjusting the voltage to 80V, carrying out electrophoresis for 15-20 min, then changing the voltage to 120V, and carrying out electrophoresis for 70-80 min.

(3) Rotary film

The film transfer device and the PVDF film are prepared in advance, the target band is cut out, the target band is placed in the film transfer device, and the PVDF film is placed on the adhesive tape. After the electrophoresis is finished, cutting the target protein from the separation gel according to the size of the protein of the Marker, and soaking in 1 x membrane transferring liquid for 5 min. And then transferring the adhesive tape with the target protein to the position above the filter paper, placing the adhesive tape and the filter paper in the sequence from bottom to top, slightly extruding bubbles in the 1 multiplied membrane transferring solution, turning on the membrane transferring clamp, transferring the membrane transferring clamp into a membrane transferring groove, adding sufficient 1 multiplied membrane transferring solution, and transferring the membrane for 110min at 100V.

(4) Immune response

After the membrane transfer was completed, the PVDF membrane was blocked overnight with the side in contact with the protein strip facing up, and the PVDF membrane was washed with 1 XTBST, followed by incubation of the primary antibody for 2 h and washing with 1 XTBST. The PVDF membrane was then placed in the secondary antibody, incubated for 2 h at room temperature on a shaker, and finally washed five times for 5min each at room temperature on a shaker using 1 XTSST.

(5) Development

And (3) opening the chemiluminescence imager in advance, dripping ECL luminescent reagent, and then covering the imager cover for exposure, wherein the exposure time is set to be 1-2 min. After exposure, the strip picture is saved, and then the strip is analyzed using Image J.

The banding result is shown in figure 5, the protein expression amounts of the Arg-1 gene, the iNOS gene, the IL-10 gene and the TNF-alpha gene are respectively shown in figures 6-9, and the Western blot result shows that HE-D can inhibit the protein expression of iNOS and TNF-alpha induced by LPS, and increase the protein expression of Arg-1 and IL-10, which indicates that HE-D can promote the macrophage to be converted from M1 type to M2 type.

400-800. mu.g/mL HE-D significantly increased the Arg-1 protein level (maximum promotion rate of 52.8% at 800. mu.g/mL HE-D concentration), and 800. mu.g/mL HE-D significantly increased the IL-10 protein level (promotion rate of 77.4%). Meanwhile, 400-800 μ g/mLHE-D can significantly reduce iNOS (the maximum inhibition rate is 72.7% under the concentration of 800 μ g/mL HE-D), and 800 μ g/mLHE-D can significantly reduce the protein level of TNF-alpha (the inhibition rate is 16.3%). The above results indicate that HE-D can promote LPS-induced conversion of M1-type macrophages to M2-type macrophages.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Hui Tai Bohai sea aquatic product Limited liability company Shandong university Weihai Industrial and technical research institute

Application of <120> polypeptide in regulation and control of macrophage in-vitro transformation

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<170> PatentIn version 3.5

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<213> Artificial sequence

<400> 8

ttctttcaaa caaaggacca gc 22

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence

<400> 9

gcaacccaag taacccttaa ag 22

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<400> 10

ctacctcatg aagatcctga cc 22

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence

<400> 11

cacagcttct ctttgatgtc ac 22