阅读说明：本技术 一种海洋生物多肽在制备治疗骨质疏松症药物中的应用 (Application of marine biological polypeptide in preparation of medicine for treating osteoporosis ) 是由 吴双俊 周鹏 孙晓敏 罗昕迪 霍雪琴 胡晓雪 于 2021-11-25 设计创作，主要内容包括：本发明提供了一种海洋生物多肽在制备治疗骨质疏松症药物中的应用,属于海洋生物学技术领域。本发明所要解决的技术问题是提供一种治疗骨质疏松的新药物。为解决该技术问题,本发明提供了一种可以用于制备骨质疏松药物的海洋多肽,本发明所提供的海洋生物多肽为皱纹蛤多肽,本发明通过实验发现,该多肽能够有效的促进骨髓间充质干细胞的成骨分化,并且可以促进骨髓间充质干细胞的增殖,因此后续可以将该多肽用于制备治疗骨质疏松的药物。(The invention provides application of marine biological polypeptide in preparing a medicine for treating osteoporosis, and belongs to the technical field of marine biology. The invention aims to solve the technical problem of providing a new medicament for treating osteoporosis. In order to solve the technical problem, the invention provides a marine polypeptide for preparing an osteoporosis medicine, the marine polypeptide provided by the invention is a wrinkle clam polypeptide, and experiments show that the polypeptide can effectively promote osteogenic differentiation of bone marrow mesenchymal stem cells and can promote proliferation of the bone marrow mesenchymal stem cells, so that the polypeptide can be subsequently used for preparing the medicine for treating osteoporosis.)

1. An application of wrinkle clam polypeptide in preparing osteoporosis medicine is provided.

2. The use according to claim 1, wherein the preparation method of the wrinkle clam polypeptide comprises the following steps:

(1) cleaning the clam, drying the clam meat, and putting the dried clam meat into a grinder for grinding to obtain clam meat powder;

(2) placing the wrinkled clam powder into a container, adding distilled water of which the mass is 5 times that of the powder, uniformly mixing, adjusting the pH to 8, adding trypsin of which the mass is 0.5% that of the solution, and carrying out water bath enzymolysis for 6 hours at 37 ℃;

(3) after enzymolysis, heating to 100 ℃, and heating for 20min to inactivate enzyme;

(4) centrifuging to collect supernatant, filtering with 3000Da ultrafiltration membrane, collecting permeate, and freeze drying to obtain Concha Meretricis Seu Cyclinae polypeptide.

3. An application of wrinkle clam polypeptide in preparing the medicines for increasing the osteogenic differentiation of bone marrow mesenchymal stem cells is disclosed.

4. The use according to claim 3, wherein the preparation method of the wrinkle clam polypeptide comprises the following steps:

(1) cleaning the clam, drying the clam meat, and putting the dried clam meat into a grinder for grinding to obtain clam meat powder;

(2) placing the wrinkled clam powder into a container, adding distilled water of which the mass is 5 times that of the powder, uniformly mixing, adjusting the pH to 8, adding trypsin of which the mass is 0.5% that of the solution, and carrying out water bath enzymolysis for 6 hours at 37 ℃;

(3) after enzymolysis, heating to 100 ℃, and heating for 20min to inactivate enzyme;

(4) centrifuging to collect supernatant, filtering with 3000Da ultrafiltration membrane, collecting permeate, and freeze drying to obtain Concha Meretricis Seu Cyclinae polypeptide.

5. An application of a wrinkle clam polypeptide in preparing a medicament for enhancing expression of a bone marrow mesenchymal stem cell osteogenic differentiation promoting factor RUNX2 protein.

6. The use according to claim 5, wherein the preparation method of the wrinkle clam polypeptide comprises the following steps:

(1) cleaning the clam, drying the clam meat, and putting the dried clam meat into a grinder for grinding to obtain clam meat powder;

(2) placing the wrinkled clam powder into a container, adding distilled water of which the mass is 5 times that of the powder, uniformly mixing, adjusting the pH to 8, adding trypsin of which the mass is 0.5% that of the solution, and carrying out water bath enzymolysis for 6 hours at 37 ℃;

(3) after enzymolysis, heating to 100 ℃, and heating for 20min to inactivate enzyme;

(4) centrifuging to collect supernatant, filtering with 3000Da ultrafiltration membrane, collecting permeate, and freeze drying to obtain Concha Meretricis Seu Cyclinae polypeptide.

7. An application of short necked clam polypeptide in preparing medicine for enhancing ALP enzyme activity of mesenchymal stem cells of bone marrow.

8. An application of the polypeptide of cockle in preparing the medicine for increasing the calcification of bone marrow mesenchymal stem cells is disclosed.

9. An application of wrinkle clam polypeptide in preparing the medicine for increasing the proliferation of bone marrow mesenchymal stem cells is disclosed.

10. The use according to any one of claims 7 to 9, wherein the method for preparing the wrinkle clam polypeptide comprises the following steps:

(1) cleaning the clam, drying the clam meat, and putting the dried clam meat into a grinder for grinding to obtain clam meat powder;

(2) placing the wrinkled clam powder into a container, adding distilled water of which the mass is 5 times that of the powder, uniformly mixing, adjusting the pH to 8, adding trypsin of which the mass is 0.5% that of the solution, and carrying out water bath enzymolysis for 6 hours at 37 ℃;

(3) after enzymolysis, heating to 100 ℃, and heating for 20min to inactivate enzyme;

(4) centrifuging to collect supernatant, filtering with 3000Da ultrafiltration membrane, collecting permeate, and freeze drying to obtain Concha Meretricis Seu Cyclinae polypeptide.

Technical Field

The invention belongs to the technical field of marine biology, and particularly relates to application of marine biological polypeptide in preparation of a medicine for treating osteoporosis.

Background

Osteoporosis is a disease characterized by a sustained loss of bone mass, accelerated bone metabolism and susceptibility to fracture. Currently, the main treatment mode of osteoporosis still is drug therapy, which is not only expensive, but also requires long-term treatment for patients. Meanwhile, in the treatment process, the medicine generates toxic and side effects, so that the treatment effect is not ideal, and therefore, a new treatment mode needs to be found at present.

Dysfunction of mesenchymal stem cells is one of the pathogenesis of osteoporosis. The mesenchymal stem cell is a cell having a multipotentiality, which can differentiate toward bone, cartilage or fat, and thus, it is a seed cell commonly used in bone tissue engineering. When the osteogenic differentiation and the adipogenic differentiation of the bone marrow mesenchymal stem cells are unbalanced and the osteogenic differentiation of the bone marrow mesenchymal stem cells is less, the fat content in the bone marrow is increased, the bone absorption is increased, and the osteoporosis is caused. Therefore, the effective promotion of the osteogenic transformation of the mesenchymal stem cells is helpful for realizing the treatment of osteoporosis.

The Venerupis philippinarum is an animal of Venerupis philippinarum in Venerupis of Venerupis, and at present, the function of the polypeptide of Venerupis philippinarum in osteogenic differentiation of mesenchymal stem cells of bone marrow is not reported.

Disclosure of Invention

The invention aims to provide a marine biological polypeptide capable of effectively promoting osteogenic differentiation of bone marrow mesenchymal stem cells, so that the marine biological polypeptide can be further used for preparing a medicine for treating osteoporosis.

In order to achieve the above purpose, the present invention provides the following technical solutions:

the invention provides an application of a wrinkle clam polypeptide in preparation of an osteoporosis medicine.

Preferably, the preparation method of the wrinkle clam polypeptide comprises the following steps:

(1) cleaning the clam, drying the clam meat, and putting the dried clam meat into a grinder for grinding to obtain clam meat powder;

(2) placing the wrinkled clam powder into a container, adding distilled water of which the mass is 5 times that of the powder, uniformly mixing, adjusting the pH to 8, adding trypsin of which the mass is 0.5% that of the solution, and carrying out water bath enzymolysis for 6 hours at 37 ℃;

(3) after enzymolysis, heating to 100 ℃, and heating for 20min to inactivate enzyme;

(4) centrifuging to collect supernatant, filtering with 3000Da ultrafiltration membrane, collecting permeate, and freeze drying to obtain Concha Meretricis Seu Cyclinae polypeptide.

Secondly, the invention provides an application of the wrinkle clam polypeptide in preparing a bone marrow mesenchymal stem cell osteogenic differentiation enhancing medicine.

Preferably, the preparation method of the wrinkle clam polypeptide comprises the following steps:

(1) cleaning the clam, drying the clam meat, and putting the dried clam meat into a grinder for grinding to obtain clam meat powder;

(2) placing the wrinkled clam powder into a container, adding distilled water of which the mass is 5 times that of the powder, uniformly mixing, adjusting the pH to 8, adding trypsin of which the mass is 0.5% that of the solution, and carrying out water bath enzymolysis for 6 hours at 37 ℃;

(3) after enzymolysis, heating to 100 ℃, and heating for 20min to inactivate enzyme;

(4) centrifuging to collect supernatant, filtering with 3000Da ultrafiltration membrane, collecting permeate, and freeze drying to obtain Concha Meretricis Seu Cyclinae polypeptide.

Thirdly, the invention provides an application of the wrinkle clam polypeptide in preparing a medicament for enhancing the expression of a bone marrow mesenchymal stem cell osteogenic differentiation promoting factor RUNX2 protein.

Preferably, the preparation method of the wrinkle clam polypeptide comprises the following steps:

(1) cleaning the clam, drying the clam meat, and putting the dried clam meat into a grinder for grinding to obtain clam meat powder;

(2) placing the wrinkled clam powder into a container, adding distilled water of which the mass is 5 times that of the powder, uniformly mixing, adjusting the pH to 8, adding trypsin of which the mass is 0.5% that of the solution, and carrying out water bath enzymolysis for 6 hours at 37 ℃;

(3) after enzymolysis, heating to 100 ℃, and heating for 20min to inactivate enzyme;

(4) centrifuging to collect supernatant, filtering with 3000Da ultrafiltration membrane, collecting permeate, and freeze drying to obtain Concha Meretricis Seu Cyclinae polypeptide.

The invention also provides application of the clam polypeptide in preparing a medicament for enhancing the ALP enzyme activity of the mesenchymal stem cells.

The invention also provides application of the clam polypeptide in preparation of a medicine for enhancing cell calcification of mesenchymal stem cells.

The invention further provides an application of the wrinkle clam polypeptide in preparation of a proliferation enhancing medicine of the bone marrow mesenchymal stem cells.

Preferably, the preparation method of the wrinkle clam polypeptide comprises the following steps:

(1) cleaning the clam, drying the clam meat, and putting the dried clam meat into a grinder for grinding to obtain clam meat powder;

(2) placing the wrinkled clam powder into a container, adding distilled water of which the mass is 5 times that of the powder, uniformly mixing, adjusting the pH to 8, adding trypsin of which the mass is 0.5% that of the solution, and carrying out water bath enzymolysis for 6 hours at 37 ℃;

(3) after enzymolysis, heating to 100 ℃, and heating for 20min to inactivate enzyme;

(4) centrifuging to collect supernatant, filtering with 3000Da ultrafiltration membrane, collecting permeate, and freeze drying to obtain Concha Meretricis Seu Cyclinae polypeptide.

The invention has the beneficial effects that:

the wrinkle clam polypeptide is prepared by the invention, and experiments show that the wrinkle clam polypeptide prepared by the invention can effectively promote the protein expression of osteogenic differentiation promoting factor RUNX2 of the bone marrow mesenchymal stem cells, can effectively promote the ALP enzyme activity and the cell calcification level of the bone marrow mesenchymal stem cells, and can effectively promote the proliferation of the bone marrow mesenchymal stem cells. Therefore, the wrinkle clam polypeptide prepared by the invention can be used for preparing medicines for promoting osteogenic differentiation of bone marrow mesenchymal stem cells and further treating osteoporosis.

Drawings

FIG. 1 shows the effect of cockle polypeptides on the expression of protein of bone marrow mesenchymal stem cell osteogenic differentiation promoting factor RUNX 2;

FIG. 2 shows the results of an alkaline phosphatase staining experiment;

FIG. 3 shows the results of alizarin red staining experiments;

FIG. 4 shows the effect of cockle polypeptides on the proliferation of mesenchymal stem cells.

Detailed Description

In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.

Example 1

(1) Cleaning the clam, drying the clam meat, and putting the dried clam meat into a grinder for grinding to obtain clam meat powder;

(2) placing 100g of the wrinkled clam powder into a container, adding 500g of distilled water, uniformly mixing, adjusting the pH to 8, adding 3g of trypsin, and carrying out water bath enzymolysis at 37 ℃ for 6 hours;

(3) after enzymolysis, heating to 100 ℃, and heating for 20min to inactivate enzyme;

(4) centrifuging to collect supernatant, filtering with 3000Da ultrafiltration membrane, collecting permeate, and freeze drying to obtain Concha Meretricis Seu Cyclinae polypeptide.

Example 2

Regulation effect of ruga clam polypeptide on RUNX2 protein in osteogenic differentiation process

(1) 1.5X 10 of good growth state⁴The P3 generation human bone marrow mesenchymal stem cells are inoculated in a 6-well plate;

(2) when the cell density reached 80%, the control group: complete medium culture was used, experimental group 1: culturing in osteogenic differentiation medium, wherein in the experimental group 2, the osteogenic differentiation medium added with 250ug/ml wrinkle clam polypeptide is cultured; experimental group 3: culturing in osteogenic differentiation medium supplemented with 500ug/ml wrinkle clam polypeptide;

(3) after culturing for 7 days, removing the culture medium, adding cell lysate, scraping the cells by using a cell scraper, and then collecting the lysate into an EP tube;

(4) after cracking for 30min, placing the mixture in a centrifuge for centrifugation, collecting supernatant, and preparing a protein sample after measuring the protein concentration;

(5) preparing concentrated gel and split gel, loading a protein sample, performing constant pressure of 80V until bromophenol blue reaches the split gel, and adjusting to 120V until electrophoresis is finished;

(5) soaking the PVDF membrane in methanol for 15s, activating, then carrying out electric rotating clamp installation, and adjusting a power supply to be a constant current of 250mA for 1.5 h;

(6) after the electrotransformation is finished, taking out the PVDF membrane, putting the PVDF membrane in 5% skimmed milk powder, and sealing for 1 h;

(7) incubating RUNX2 and GAPDH primary antibody after closing, incubating overnight at 4 ℃, and washing the PVDF membrane for 3 times (10 min each time) by TBST after the incubation is finished;

(8) then, incubating corresponding secondary antibodies, incubating at room temperature for 1h, and washing the PVDF membrane for 3 times by using TBST, wherein each time is 10 min;

(9) the PVDF membrane is developed under the condition of keeping out of the light.

The results obtained by the experiment are shown in fig. 1, and it can be seen that after the wrinkle clam polypeptide is added, the protein expression level of an osteogenic differentiation promoting factor RUNX2 is obviously improved in the osteogenic differentiation process of human bone marrow mesenchymal stem cells, which indicates that the wrinkle clam polypeptide provided by the invention can effectively promote the protein expression of RUNX 2.

Example 2

Regulation of ALP enzyme activity in osteogenic differentiation process by cripple clam polypeptide

(1) 1.5X 10 of good growth state⁴The P3 generation mesenchymal stem cells are inoculated in a 6-well plate;

(2) when the cell density reaches 80%, adding osteogenic differentiation culture medium into the control group for culture, adding osteogenic differentiation culture medium into the experimental group for culture, adding 500ug/ml wrinkle clam polypeptide into the experimental group for culture, changing the culture solution once every 2-3 days, and setting 3 repeats in each group;

(3) after culturing for 7 days, removing the culture medium, gently cleaning cells by using PBS, and adding 4% paraformaldehyde to fix the cells for 30 min;

(4) removing paraformaldehyde, washing cells with PBS, and treating cells according to the alkaline phosphatase kit instructions;

(5) after the processing is finished, photographing is carried out.

The results obtained by the experiment are shown in fig. 2, and it can be seen that the ALP staining degree in the experimental group is significantly higher than that in the control group, and the results show that the wrinkle clam polypeptide prepared by the invention can effectively promote the activity of ALP enzyme in mesenchymal stem cells.

Example 3

Regulation of cell calcification in osteogenic differentiation process by wrinkle clam polypeptide

(1) 1.5X 10 of good growth state⁴The P3 generation mesenchymal stem cells are inoculated in a 6-well plate;

(2) when the cell density reaches 80%, adding osteogenic differentiation culture medium into the control group for culture, adding osteogenic differentiation culture medium into the experimental group for culture, adding 500ug/ml wrinkle clam polypeptide into the experimental group for culture, changing the culture solution once every 2-3 days, and setting 3 repeats in each group;

(3) after culturing for 7 days, removing the culture medium, gently cleaning cells by using PBS, and adding 4% paraformaldehyde to fix the cells for 30 min;

(4) removing paraformaldehyde, washing cells with PBS, and incubating for 30min at room temperature in a dark place with alizarin red staining solution;

(6) alizarin red staining solution was removed, and cells were washed 2 times with PBS and photographed.

The results obtained by the experiment are shown in fig. 3, and it can be seen that the description of the cell mineralized nodules in the experimental group is obviously more than that in the control group, and the results show that the wrinkle clam polypeptide prepared by the invention can effectively promote the calcification of the mesenchymal stem cells in the bone marrow.

Example 4

(1) 1X 10 prepared from P3 generation mesenchymal stem cells with good growth state⁴Each hole is connected with 100ul of single cell suspension of ml, and 3 multiple holes are arranged;

(2) after inoculation, putting the culture medium into a cell culture box, changing the culture medium after 24h, adding a complete culture medium into a control group for culture, and adding a complete culture medium of 500ug/ml wrinkle clam polypeptide into an experimental group;

(3) taking out after culturing for 1,3 and 5 days respectively, adding 10ul of CCK-8 into each hole, and placing in a cell culture box for further incubation for 4 hours;

(4) after the incubation is finished, the OD average value of each group is measured by using an enzyme-linked immunosorbent assay instrument, and the detection wavelength is 450 nm.

The results obtained by the experiment are shown in fig. 4, and it can be seen from the figure that the OD values of the experimental group are significantly higher than those of the control group when the cells are cultured for 3d and 5d, and the difference has statistical significance, and the results show that the proliferation capacity of the mesenchymal stem cells can be effectively promoted by adding the wrinkle clam polypeptide.