CN112028967B

CN112028967B - Sea cucumber polypeptide with macrophage proliferation inhibition activity and application thereof

Info

Publication number: CN112028967B
Application number: CN201910412343.1A
Authority: CN
Inventors: 宋淑亮; 吉爱国
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2019-05-17
Filing date: 2019-05-17
Publication date: 2022-07-15
Anticipated expiration: 2039-05-17
Also published as: CN112028967A

Abstract

The invention provides a sea cucumber polypeptide with macrophage proliferation inhibition activity and application thereof, belonging to the technical field of biological medicines. The experiment shows that the sea cucumber polypeptide artificially synthesized by the invention has obvious inhibition effect on macrophage proliferation and presents obvious dose effect relationship, and the sea cucumber polypeptide can be used for preventing or treating diseases related to macrophage proliferation by inhibiting macrophage proliferation and can also be used as a macrophage proliferation inhibitor to inhibit the in vitro culture of the macrophage, thereby providing materials for the macrophage and related scientific research thereof. Therefore, it has good prospect of practical application.

Description

Sea cucumber polypeptide with macrophage proliferation inhibition activity and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a sea cucumber polypeptide with macrophage proliferation inhibition activity and application thereof.

Background

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Sea cucumber belongs to the class holothuria (Holothuroidea), is a marine echinoderm living in seaside to 8000 m, has been six hundred million years old today, grows full of meat stings all over the body, and is widely distributed in various oceans in the world. Sea cucumber is named together with ginseng, bird's nest and shark's fin, and is one of eight major treasures in the world. Sea cucumber is not only a precious food, but also a rare medicinal material. According to records in Ben Cao gang mu Shi Yi: the sea cucumber is sweet and salty in taste, can tonify the kidney, replenish essence and marrow, control urine, strengthen yang and treat flaccidity, is warm in nature and tonify, is full of enemy ginseng, and is named as the sea cucumber. It has been reported that sea cucumber has effects of improving memory, delaying gonadal aging, preventing arteriosclerosis, and resisting tumor. Sea cucumber is a typical high protein, low fat, low cholesterol food, with proteins as high as 55%. Modern research shows that: the sea cucumber polypeptide has the activity effects of resisting oxidation, reducing blood pressure, resisting tumors, regulating blood sugar, regulating immunity and the like.

Macrophages are widely distributed in tissues and organs of the body, play an important role in pathogen defense, inflammatory response, homeostatic maintenance and injury repair, and can also be used as professional antigen presenting cells to process and present antigens to activate adaptive immune response. It is usually a terminally differentiated cell that cannot undergo further cell division. However, modern biomedical research has shown that macrophage proliferation is involved in certain serious proliferative diseases, such as lymphoma, cardiovascular disease, and nephrosclerosis, among others. Gabrielian et al also reported a role for macrophage penetration in traumatic proliferative vitreoretinopathy. However, the inventors found that, to date, there have been few reports on the effect of sea cucumber polypeptides on macrophages.

Disclosure of Invention

Based on the above-mentioned deficiencies of the prior art, the present invention provides a sea cucumber polypeptide having macrophage proliferation inhibiting activity, which can be used for preventing or treating diseases related to macrophage proliferation by inhibiting macrophage proliferation, including but not limited to: such as lymphoma, cardiovascular diseases, nephrosclerosis, and traumatic proliferative vitreoretinopathy; meanwhile, the sea cucumber polypeptide can also be used as a macrophage proliferation inhibitor to inhibit the in vitro culture of macrophages, thereby providing materials for the macrophages and related scientific researches. The invention has good practical application value.

In one aspect of the invention, the sea cucumber polypeptide with macrophage proliferation inhibiting activity is provided, and the amino acid sequence of the sea cucumber polypeptide is as follows:

WDGVGVSGR(SEQ ID NO.1)。

the Stichopus japonicus polypeptide has effect in inhibiting macrophage proliferation activity in vitro, and can be synthesized by solid phase polypeptide synthesis method.

In a second aspect of the present invention, there is provided a nucleotide encoding the sea cucumber polypeptide, comprising any one of the following groups:

(a) a nucleotide encoding a polypeptide having the amino acid sequence;

(b) a nucleotide complementary to the nucleotide of (a).

In a third aspect, the invention provides an application of the sea cucumber polypeptide in preparing a medicament for inhibiting macrophage proliferation.

Further, the medicament can prevent or treat diseases related to macrophage proliferation by inhibiting macrophage proliferation, preferably, the diseases include but are not limited to: such as lymphoma, cardiovascular disease, nephrosclerosis and traumatic proliferative vitreoretinopathy.

In a fourth aspect of the present invention, there is provided the use of the sea cucumber polypeptide as a non-therapeutic macrophage proliferation inhibitor. According to the invention, the non-therapeutic purpose such as inhibiting macrophage proliferation in vitro is beneficial to researching macrophage proliferation signal path and gene expression interaction by applying the sea cucumber polypeptide to macrophages, thereby providing raw materials for further researching macrophage proliferation related diseases and laying a foundation.

In a fifth aspect of the present invention, there is provided a method for inhibiting macrophage proliferation in vitro, the method comprising administering the sea cucumber polypeptide as described above to macrophages cultured in vitro.

Further, the macrophage is RAW 264.7;

furthermore, the amino acid sequence of the sea cucumber polypeptide is WDGVGVSGR (SEQ ID NO. 1).

Further, the concentration of the sea cucumber polypeptide is 10-1000 mug/ml, and further is 25-400 mug/ml; the concentration of the sea cucumber polypeptide includes but is not limited to 25 mug/ml, 100 mug/ml and 400 mug/ml.

The invention has the beneficial technical effects that:

the invention obtains the sea cucumber polypeptide with the activity of inhibiting the proliferation of macrophages from sea cucumbers, and the artificial synthesis is carried out, and then the MTT method is adopted to detect the effect of the artificial synthesis sea cucumber polypeptide on the macrophages. Therefore, it has good prospect of practical application.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing the optimum concentration of cells for inoculation as determined in the preliminary MTT assay of the present invention.

FIG. 2 is a graph showing the effect of different concentrations of the sea cucumber polypeptide P1 on the macrophage proliferation rate in example 1 of the present invention.

FIG. 3 is a graph showing the effect of different concentrations of Stichopus japonicus polypeptide P2 on macrophage proliferation rate in Experimental example 1 of the present invention.

FIG. 4 is a graph showing the effect of different concentrations of Stichopus japonicus polypeptide P3 on macrophage proliferation rate in Experimental example 2 of the present invention.

FIG. 5 is a graph showing the effect of different concentrations of Stichopus japonicus polypeptide P4 on macrophage proliferation rate in Experimental example 3 of the present invention.

FIG. 6 is a graph showing the effect of different concentrations of Stichopus japonicus polypeptide P5 on macrophage proliferation rate in Experimental example 4 of the present invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In one exemplary embodiment of the present invention, a holothurian polypeptide having macrophage proliferation inhibiting activity is provided, wherein the holothurian polypeptide comprises the amino acid sequence:

WDGVGVSGR(SEQ ID NO.1)。

In another embodiment of the present invention, there is provided a nucleotide encoding the sea cucumber polypeptide, comprising any one of the following groups:

(a) a nucleotide encoding a polypeptide having the amino acid sequence;

(b) a nucleotide complementary to the nucleotide of (a).

In another embodiment of the present invention, the application of the sea cucumber polypeptide in preparing a medicament for inhibiting macrophage proliferation is provided.

In yet another embodiment of the present invention, the medicament is capable of preventing or treating a disease associated with macrophage proliferation by inhibiting macrophage proliferation, preferably, the disease includes but is not limited to: such as lymphoma, cardiovascular disease, nephrosclerosis and traumatic proliferative vitreoretinopathy.

In another embodiment of the present invention, there is provided a use of the sea cucumber polypeptide as a macrophage proliferation inhibitor for non-therapeutic purposes. According to the invention, the non-therapeutic purpose is to inhibit macrophage proliferation in vitro, for example, by applying the sea cucumber polypeptide to macrophages, the research on macrophage proliferation signal pathways and gene expression interaction is facilitated, so that an original material is provided for further research on diseases related to macrophage proliferation, and a foundation is laid.

In another embodiment of the present invention, there is provided a method for inhibiting macrophage proliferation in vitro, the method comprising administering the sea cucumber polypeptide described above to macrophages cultured in vitro.

In yet another embodiment of the present invention, the macrophage is RAW 264.7;

in yet another embodiment of the present invention, the amino acid sequence of the Stichopus japonicus polypeptide is WDGVGVSGR (SEQ ID NO. 1).

In another embodiment of the present invention, the concentration of the sea cucumber polypeptide is 10 to 1000. mu.g/ml, and further 25 to 400. mu.g/ml; the sea cucumber polypeptide concentration includes but is not limited to 25 mug/ml, 100 mug/ml and 400 mug/ml.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

1. Experimental materials and apparatus

1.1 Experimental materials

(1) Cell culture and sample processing-related materials

Mouse mononuclear macrophage RAW 264.7: shanghai cell bank of Chinese academy of sciences

Artificially synthesizing sea cucumber polypeptide: biotechnology engineering (Shanghai) Ltd

Cell culture plates (96 wells, 24 wells, 6 wells): nest Ltd

Disposable filter (0.22 μm): german Minisart

Filter paper: western Minghuang filter paper manufacturer, domestic product

DMEM (Dulbecco's Modified Eagle Medium) Medium: gibco, USA

Penicillin and streptomycin: invitrogen, USA

Trypsin (Solarbio corporation): solarbio, Beijing

Fetal Bovine Serum (Fetal Bovine Serum, FBS): gibco Corp, USA

(2) MTT experiment related material

Dmso (dimethyl sulphoxide): sigma, USA

MTT: sigma, USA

1.2 Experimental instruments

Air-blast drying cabinet

Ultrasonic cleaning only (KQ skilful 00 type): kunshan ultrasonic Instrument Co Ltd

Superclean work laboratory bench: hardong Union Inc

Pipette (2.5 μ L, 10 μ L, 100 μ L, 200 μ L, 1000 μ L): german eppendorf

Blood cell count plate: shanghai refining Biochemical reagent Instrument Co., Ltd

An enzyme-labeling instrument: sunrise, Tecan, Austria

Electric heating constant temperature water bath: german IKA

High-pressure steam sterilization pot: english Astell

Vacuum pump filtration equipment: vzcubrand, Germany

Inverting the biological microscope: leica DMIRB in Germany

Electronic analytical balance (AB 204-S, PB403-S, PB 3002-S): mettler OLEDO of Switzerland

An ultrapure water system: Millipore-Q, Millipore, USA

Carbon dioxide constant temperature cell incubator: MCO-15AC, SANYO, Japan

Desk-top high-speed refrigerated centrifuge: 5810R, eppendorf, Germany

High speed tissue homogenizer (T18 basic): IKA, Germany

Ultraviolet spectrophotometer (8453E): agilent Inc., USA

Vortex appearance: german IKA

A water bath kettle: heidolphin, Germany

1.3 Main reagents and preparation method

(1) 10% DMEM:

taking 100mL of basic culture medium and 10mL of fetal calf serum, mixing the two under aseptic condition, adding 5mL of double antibody and 6mL of HEPES, subpackaging, and storing at 4 ℃ for later use.

(2) Phosphate buffered saline (PBS, pH 7.2):

weighing 18.00g NaCl, 0.20g KCl and Na₂HPO₄.12H₂O 3.47g，KH₂PO₄0.20g, dissolved in an appropriate amount of ultrapure water, and chargedDissolving, diluting to 1000mL, filtering with 0.22 μm disposable filter membrane in ultra-clean bench for sterilization, packaging, and storing at 4 deg.C.

(3) Preparation of MTT:

weighing 100mg MTT powder, dissolving in 20mL PBS (pH7.2), ultrasonic vibrating for dissolving, filtering with 0.22 μm disposable filter membrane for sterilization after complete dissolution to obtain 5mg/mL MTT dye solution, subpackaging with 4mL sterile EP tube, and storing in dark at-20 deg.C for use.

2. Test method

2.1 cell Resuscitation

(1) The cryovial was taken out of the liquid nitrogen and quickly placed into a 37 ℃ water bath to melt, and after the cells had melted (about 1min) they were removed from the 37 ℃ water bath as soon as possible. The prolonged water bath time at 37 ℃ can improve the cell death rate, and the cell death rate is generally between 20 and 25 percent in the recovery process.

(2) The cell freezing tube was opened aseptically, and the frozen cell suspension was transferred to a 15ml sterile centrifuge tube, and 5ml DMEM medium containing 10% FBS was added.

(3) Centrifuging at 1000rpm/min for 5min, and discarding the supernatant.

(4) Adding 5-6ml complete culture medium into centrifuge tube, blowing and beating uniformly to obtain cell suspension, transferring into T75 culture flask, placing at 37 deg.C and 5% CO₂Culturing in an incubator.

(5) And after 24h, replacing the culture solution and then continuing to culture, and when the adherent cells cover 80% of the bottom area of the culture bottle, carrying out passage.

2.2 passage of cells

(1) Cell density was observed under a microscope and passaging was performed when the cells covered about 80% of the flask bottom area.

(2) Under sterile conditions, the culture medium in the cell culture flask was decanted. Add 2ml PBS, wash three times, and discard PBS by aspiration.

(3) Adding 2-3ml of 10% DMEM, removing the walls of the cells at the bottom of the culture flask by using a scraper, and uniformly mixing by blowing a pipette for multiple times to disperse the cells.

(4) The cells are seeded at a suitable cell density (typically 1:4 split for cell subculture) in another flask.

(5) Recording cell name, passage number, operator name and passage date, placing at 37 deg.C and 5% CO₂And continuing culturing in the incubator.

2.3 cell cryopreservation

(1) Preparation of cell cryopreservation solution (50% DMEM, 40% serum, 10% DMSO)

(2) Selecting cells which are in a logarithmic growth phase after passage for 3 times, washing with PBS, adding 2ml of pancreatin digestive juice for digestion for 3min, adding an equal volume of culture medium to stop digestion, lightly blowing and beating by using a pipettor to ensure that the cells are detached and dispersed into single cells, and transferring the cells into a 15ml sterile centrifuge tube.

(3) Centrifuge at 1000rpm for 5 min.

(4) Discarding supernatant, adding cell freezing solution, blowing and beating uniformly, adjusting cell density in the freezing solution to (5-10) × 10⁶/ml。

(5) The cell suspension was dispensed into a cryopreservation tube (1 ml/tube).

(6) The tube cap is screwed down, the sealing film is sealed, and RAW264.7 macrophage, operator and freezing date are marked.

(7) Placing the freezing tube at 4 deg.C for 30min, and at-40 deg.C for 48h, and finally placing into liquid nitrogen tank for long-term freezing.

2.4 selection of optimal concentration for cell inoculation in preliminary experiments with MTT method

(1) Log phase cells were collected and cell suspension concentrations were adjusted to 5000, 6000, 7000, 8000, 9000/ml, 100ul per well, with marginal wells filled with sterile PBS.

(2) Culturing the cells: 5% CO2, incubation in 37 ℃ incubator.

(3) Color generation: after 48h, 10ul of MTT solution was added to each well and incubated in an incubator for 4-6 h. The incubation was terminated and the supernatant from the wells was aspirated off, 100ul DMSO per well. Shaking for 10min to dissolve the crystals.

(5) Color comparison: OD value was measured at a wavelength of 570nm by a microplate reader.

(6) And (6) data processing and analysis.

The optimum cell inoculation concentration is determined according to the MTT method preliminary experiment, and the experimental result is shown in figure 1: when the cell concentration was 5000 cells/ml, the absorbance value was 0.2512; when the cell concentration was 6000 cells/ml, the absorbance value was 0.3473; when the cell concentration was 7000 cells/ml, the absorbance value was 0.3903; when the cell concentration was 8000 cells/ml, the absorbance value was 0.4468; when the cell concentration was 9000 cells/ml, the absorbance value was 0.6632. As can be seen from the data, the absorbance is between 0.2 and 0.8 to ensure that the relative error of the measurement is small. However, since the state and amount of macrophages are different in each experiment, the absorbance should be kept between 0.4 and 0.5 to avoid too small (<0.2) or too large (>0.8), and the optimal concentration of cell suspension should be in the range of 7000 plus 8000 cells/ml, and 7500 cells/ml was selected for the experiment.

2.5 research of detecting immunomodulatory effect of artificially synthesized sea cucumber polypeptide on RAW264.7 macrophage by MTT method

(1) Inoculating cells: log phase cells were collected, cell suspension concentrations were adjusted to 7500/ml, 100ul per well, and marginal wells filled with sterile PBS.

(2) Culturing the cells: 5% CO₂Incubate at 37 ℃.

(3) Adding medicine: after 24h, the 8 synthetic polypeptides were replaced at drug concentrations of 25, 100, 400ug/ml, 100ul per well.

(4) Color generation: after 48h, 10ul of MTT solution was added to each well and incubated in an incubator for 4-6 h. The incubation was stopped and the supernatant from the wells was aspirated off, 100ul DMSO per well. Shaking for 10min to dissolve the crystals.

(6) And (6) data processing and analysis.

The experimental results of artificially synthesized sea cucumber polypeptides are shown in various embodiments and experimental examples.

Example 1P 1 (peptide sequence 5 '-3' WDGVGVSGR, SEQ ID No.1) Stichopus japonicus synthetic peptide on macrophage proliferation rate

The MTT method is adopted to detect the influence of P1 on the macrophage proliferation activity, five groups of parallel experiment results show that P1 has obvious biological activity, and compared with a blank control group, the increment rates of P1 at 25 mug/ml, 100 mug/ml and 400 mug/ml are 23.13%, 39.52% and 57.39% respectively. When t-test is carried out, P is less than 0.001, and the difference is very significant. This indicates that P1 has obvious inhibiting effect on RAW264.7 macrophage.

Experimental example 1 research results of P2 (peptide sequence 5 '-3' WVDGVGSGR, SEQ ID NO.2) on macrophage proliferation rate by reference to synthetic peptide

The MTT method was used to examine the effect of P2 on macrophage proliferation activity. The results are shown in FIG. 3, and no significant difference is observed by t-test, which indicates that P2 has no biological activity on macrophage.

Experimental example 2P 3 (peptide sequence 5 '-3' WDVGVGSGR, SEQ ID No.3) Stichopus japonicus synthetic peptide research results on cell proliferation rate

The MTT method is adopted to detect the influence of P3 on the proliferation activity of macrophages. The results are shown in FIG. 4, and no significant difference was observed by t-test, and P3 has no biological activity on macrophages.

Experimental example 3P 4 (peptide sequence 5 '-3' WDGVGSGVR, SEQ ID No.4) Stichopus japonicus synthetic peptide on macrophage proliferation rate

The MTT method is adopted to detect the influence of P4 on the proliferation activity of macrophages, and five groups of parallel experiment results show that the proliferation rates of P4 at 25 mug/ml, 100 mug/ml and 400 mug/ml are respectively 4.13%, 16.32% and 7.60%. And when the concentration of P4 is 100 mug/ml, P is less than 0.05, and the difference is significant. It is therefore believed that the proliferative effects of P4 on macrophages are not significant.

Experimental example 4P 5 (peptide sequence 5 '-3' WDGVGSGRV, SEQ ID No.5) Stichopus japonicus selenka synthesis method for investigating macrophage proliferation rate

The MTT method is adopted to detect the influence of P5 on the proliferation activity of macrophages, five groups of parallel experiment results show that the proliferation rates of P5 at 25 mug/ml, 100 mug/ml and 400 mug/ml are respectively 11.00%, 17.16% and 11.06%, and P5 has significant difference at 25 mug/ml, but the proliferation effect is not obvious on the whole. The overall variance is too small to fit the MTT experimental results.

Finally, it should be noted that, although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

SEQUENCE LISTING

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Claims

1. A sea cucumber polypeptide with activity of inhibiting macrophage proliferation is characterized in that the amino acid sequence of the sea cucumber polypeptide is SEQ ID NO. 1.

2. A polynucleotide encoding the sea cucumber polypeptide of claim 1, consisting of any one of the following groups:

(a) a polynucleotide encoding a polypeptide having the amino acid sequence;

(b) a polynucleotide complementary to the polynucleotide of (a).

3. Use of a sea cucumber polypeptide according to claim 1 for the preparation of a non-therapeutic macrophage proliferation inhibitor.

4. A method for inhibiting macrophage proliferation in vitro, comprising administering a sea cucumber polypeptide according to claim 1 to macrophages cultured in vitro.

5. The method of claim 4, wherein the macrophage cell is RAW 264.7.

6. The method of claim 4, wherein the concentration of the sea cucumber polypeptide is 10-1000 μ g/mL.

7. The method of claim 4, wherein the concentration of the sea cucumber polypeptide is 25-400 μ g/mL.

8. The method of claim 4, wherein the concentration of the sea cucumber polypeptide is 25 μ g/mL.

9. The method of claim 4, wherein the concentration of the sea cucumber polypeptide is 100 μ g/mL.

10. The method of claim 4, wherein the concentration of the sea cucumber polypeptide is 400 μ g/mL.