Umbilical cord mesenchymal stem cell preparation and application thereof in treating ovarian diseases

Abstract

The invention relates to the technical field of medicines, in particular to an oligopeptide capable of being specifically combined with integrin alpha v beta 5 obtained by screening through a phage display technology, the oligopeptide serves as a ligand of the integrin alpha v beta 5, the oligopeptide is further anchored on the surface of human umbilical cord mesenchymal stem cells through a click chemistry method, and then an umbilical cord mesenchymal stem cell preparation is prepared. The method is applied to precise targeted therapy of ovarian diseases.

Description

Technical Field

[0001] The present invention relates to a preparation containing umbilical cord mesenchymal stem cells, and specifically relates to a preparation of umbilical cord mesenchymal stem cells for treating ovarian diseases, a preparation method thereof, and an application thereof. Background Art

[0002] Ovarian cancer (OC) is the most common gynecological tumor. Due to the insidious onset, poor prognosis and high mortality rate of OC, early diagnosis and treatment are of great significance for OC. Mesenchymal stem cells can secrete a large number of cytokines and exosomes, etc. Among them, exosomes are extracellular vesicles with a diameter between 30 - 100 nm. They are spherical particles enclosed by a phospholipid bilayer released by different types of cells, and contain various proteins, lipids, genetic materials, etc. These inclusions can be transferred from one cell to another cell and play a role in target cells. In recent years, studies have found that exosomes can be used as biomarkers of OC and drug carriers for immunotherapy of OC. Therefore, exosomes are inhibitors of OC progression, and they can play an anti-OC role as ideal drug carriers and therapeutic targets.

[0003] However, the targeting specificity of exosomes secreted by umbilical cord mesenchymal stem cells is poor, and its application is severely restricted. Therefore, the targeting of umbilical cord mesenchymal stem cells is an urgent problem to be solved.

[0004] Integrin is an adhesion factor on the cell surface. It participates in various physiological activities of cells by mediating the interaction between cells and between cells and the extracellular matrix. Integrin is closely related to the occurrence, development, invasion and metastasis of tumors. Existing studies have shown that integrin αvβ5 is highly expressed in ovarian cancer and can be used as a biomarker for ovarian cancer, providing a new idea for the treatment of ovarian cancer. Summary of the Invention

[0005] In order to solve the problem of the targeting of umbilical cord mesenchymal stem cells, the present invention uses integrin αvβ5 highly expressed in ovarian cancer as a target to guide the binding of umbilical cord mesenchymal stem cells to ovarian cancer cells, and finally treats ovarian cancer through umbilical cord mesenchymal stem cells.

[0006] One aspect of the present invention is to provide a short peptide that specifically binds to integrin αvβ5. Preferably, the short peptide of the present invention is obtained by screening through phage display technology. Specifically, the present invention uses phage display technology to screen a peptide segment with high binding specificity to integrin αvβ5 from a polypeptide library. Specifically, the amino acid sequence of the short peptide of the present invention is shown as SEQ ID No.1: RGDLGRPA.

[0007] This short peptide can act as an integrin ligand, bind to the corresponding integrin receptor on the ovarian cancer cell membrane, and anchor umbilical cord mesenchymal stem cells to ovarian cancer cells.

[0008] Another aspect of the present invention is to provide a kind of mesenchymal stem cells derived from human umbilical cord blood, and its preparation method is: (1) Select human umbilical cord blood stem cells and carry out routine culture in a serum-free manner; (2) Carry out culture by passage, and passage to the 5th generation to amplify a large number of human umbilical cord mesenchymal stem cells.

[0009] Another aspect of the present invention is to provide a preparation of mesenchymal stem cells derived from human umbilical cord blood, and the preparation contains umbilical cord mesenchymal stem cells.

[0010] Furthermore, the short peptide with high binding specificity is anchored to the surface of umbilical cord mesenchymal stem cells by click chemistry.

[0011] Furthermore, the click chemistry method is to introduce complementary click chemistry functional groups on the surface of the short peptide and umbilical cord mesenchymal stem cells respectively. The click chemistry functional groups are azide compounds and alkynes, and the short peptide is anchored to the surface of umbilical cord mesenchymal stem cells through click chemical reaction.

[0012] Furthermore, the short peptide with high binding specificity of the present invention specifically binds to integrin αvβ5 highly expressed on the surface of ovarian cancer cells through ligand binding, and guides umbilical cord mesenchymal stem cells to the surface of ovarian cancer cells to achieve targeted therapy.

[0013] Furthermore, the preparation method of the preparation includes the following steps: Step (1): Screen and obtain a short peptide that specifically binds to integrin αvβ5 through phage display technology; Step (2): Isolate mesenchymal stem cells from human umbilical cord blood for routine culture and passage. After passage to the 5th generation, amplify and culture to harvest a large number of umbilical cord mesenchymal stem cells; Step (3): Anchor the short peptide in step (1) to the surface of the umbilical cord mesenchymal stem cells in step (2) by click chemistry to make a preparation of umbilical cord mesenchymal stem cells.

[0014] Furthermore, the click chemistry method is to introduce complementary click chemistry functional groups on the surface of the short peptide and umbilical cord mesenchymal stem cells respectively. The click chemistry functional groups are azide compounds and alkynes, and the short peptide is anchored to the surface of umbilical cord mesenchymal stem cells through click chemical reaction.

[0015] Another aspect of the present invention is to provide a drug for treating ovarian cancer, the drug comprising human umbilical cord mesenchymal stem cells, wherein a short peptide with high specific binding to integrin αvβ5 is anchored on the surface of the umbilical cord mesenchymal stem cells, and the amino acid sequence of the short peptide is as shown in SEQ ID No.1.

[0016] Another aspect of the present invention is to provide an application of a preparation in the treatment of ovarian cancer, the preparation comprising human umbilical cord mesenchymal stem cells, wherein a short peptide with high specific binding to integrin αvβ5 is anchored on the surface of the umbilical cord mesenchymal stem cells, and the amino acid sequence of the short peptide is as shown in SEQ ID No.1.

[0017] Another aspect of the present invention is to provide an application of a drug in the treatment of ovarian cancer, the drug comprising human umbilical cord mesenchymal stem cells, wherein a short peptide with high specific binding to integrin αvβ5 is anchored on the surface of the umbilical cord mesenchymal stem cells, and the amino acid sequence of the short peptide is as shown in SEQ ID No.1.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) A short peptide that only binds to integrin αvβ5 is screened and obtained by phage display technology in the present invention, showing the characteristics of high binding specificity, providing a basis for the precise targeted therapy of subsequent umbilical cord mesenchymal stem cells.

[0019] (2) The short peptide of the present invention is anchored on the surface of umbilical cord mesenchymal stem cells by click chemistry. The principle is to utilize the high specific binding of the short peptide to αvβ5 highly expressed on the surface of ovarian cancer cells, realizing the guidance of umbilical cord mesenchymal stem cells to the surface of ovarian cancer cells and achieving precise targeted therapy.

[0020] (3) Compared with the prior art, the preparation of umbilical cord mesenchymal stem cells containing the short peptide of the present invention can achieve precise targeted therapy for ovarian cancer cells, effectively kill ovarian cancer cells in in vitro cell experiments, and effectively reduce the lesion area and inhibit the growth of ovarian tumors in in vivo animal experiments. Description of the Drawings

[0021] Figure 1 In vivo animal experiment - tumor volume measurement results; Figure 2 In vivo animal experiment - WB map of CA125 protein. Detailed Embodiments

[0022] The present invention will be further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0023] Example 1 Preparation of a Short Peptide with High Specific Binding to Integrin αvβ5 (1) Library construction and amplification A series of random DNA sequences were designed and cloned into the coding region of the coat protein of bacteriophage M13 to construct an 8-peptide library; (2) Target capture The integrin αvβ5 molecule highly expressed on the surface of ovarian cancer cells is fixed on the surface of a solid phase carrier, and the library in step (1) is incubated with the solid phase carrier for 4 hours to allow the two to bind; (3) Washing Repeated washing to remove unbound, non-specific phages to remove unbound and non-target specific phages; (4) Elution After a brief incubation with a low-pH buffer or by competitive elution, the target-bound phages are separated; (5) Infection stage The eluted phages were used to infect bacteria to amplify the selected phages, thus forming a new phage library for the next round of biopanning.

[0024] After 5 rounds of bio-panning, a short peptide with high binding specificity was screened out. After sequencing, the amino acid sequence of the short peptide was shown as SEQ ID No.1.

[0025] Example 2 Preparation of human umbilical cord mesenchymal stem cell preparation Human umbilical cord tissue was obtained from a healthy full-term fetus and stored in a sterile PBS solution containing 1% double antibody. Before use, the umbilical cord was cleaned with saline and moved into a new culture medium, and 75% medical alcohol was added to soak for 2 minutes. After disinfection, it was added to a culture dish containing saline, and the washing was repeated 2-3 times to remove the serum. First, the umbilical cord was unfolded with two tissue forceps, two veins and one artery were removed, and the umbilical cord was cut into pieces with tissue forceps and cultured in a T75 culture bottle. 4 ml of complete culture medium was added to each bottle, and the cell bottle was placed flat to make the tissue as evenly distributed as possible on the entire bottom surface, and placed in an incubator at 37 ° C and 5% CO2 for culture. On the 5th day after the primary preparation, the full amount of medium was replaced every 3 days. When the cell fusion reached 85%-90%, it was passaged until the 5th generation. The 5th generation was expanded and a large number of umbilical cord mesenchymal stem cells were harvested.

[0026] The short peptide of Example 1 and the above-mentioned umbilical cord mesenchymal stem cells are anchored to the surface of the umbilical cord mesenchymal stem cells by a click chemistry method. The specific steps are to introduce an azide functional group on the surface of the short peptide to prepare a compound, introduce an alkynyl group on the surface of the umbilical cord mesenchymal stem cells, complete the reaction under copper catalysis to anchor the short peptide to the surface of the umbilical cord mesenchymal stem cells, and prepare an umbilical cord mesenchymal stem cell preparation containing the short peptide.

[0027] Example 3 Application of the preparation of Example 2 in treating ovarian cancer 1. In vitro experiment Ovarian cancer cells SK-OV-3 were taken for routine culture, passaged to a 96-well plate and continued to be cultured. They were divided into 4 groups, namely the normal culture group, the PBS treatment group, the conventional umbilical cord mesenchymal stem cell treatment group, and the umbilical cord mesenchymal stem cell treatment group containing short peptides. After culturing for 3 days until the cell density reached 80%, the culture medium was changed. The normal culture group was not treated. The PBS treatment group was added with 100 μl of PBS. The conventional umbilical cord mesenchymal stem cell treatment group was added with 100 μl of umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cell treatment group containing short peptides was added with 100 μl of the umbilical cord mesenchymal stem cells containing short peptides of Example 2. After incubating for 24 h in each group, they were washed with PBS, and the cell viability was detected by the MTT method.

[0028] The test results showed that both the umbilical cord mesenchymal stem cell treatment group containing short peptides and the conventional umbilical cord mesenchymal stem cell treatment group had a significant effect on inhibiting the proliferation of tumor cells, and the umbilical cord mesenchymal stem cell treatment group containing short peptides had a better inhibitory effect than the conventional umbilical cord mesenchymal stem cell treatment group. There was no difference between the normal culture group and the PBS treatment group, and they had no inhibitory effect on tumors (Table 1).

[0029] Table 1

[0030] 2. In vivo experiment Ovarian cancer cells SK-OV-3 were injected into the lower axilla of the hind limb of rats to construct an animal model. Subsequently, rats with similar tumor volumes were randomly divided into four groups, namely the non-treatment group, the PBS treatment group, the conventional umbilical cord mesenchymal stem cell treatment group, and the umbilical cord mesenchymal stem cell treatment group containing short peptides. The non-treatment group was not treated. The PBS treatment group was injected with PBS via the tail vein. The conventional umbilical cord mesenchymal stem cell treatment group was injected with conventionally extracted umbilical cord mesenchymal stem cells via the tail vein. The umbilical cord mesenchymal stem cell treatment group containing short peptides was injected with the umbilical cord mesenchymal stem cells containing short peptides of Example 2 via the tail vein. Each group was given drug intervention 14 days after constructing the animal model. On the 21st day after drug intervention, the tumors of each group of rats were taken out for volume detection, and proteins were extracted for WB quantitative analysis of ovarian cancer marker proteins.

[0031] The results showed that the umbilical cord mesenchymal stem cell treatment group containing short peptides had an obvious inhibitory effect on ovarian tumors in rats. Secondly, it was the conventionally extracted umbilical cord mesenchymal stem cell treatment group. There was almost no difference in tumor volume between the PBS treatment group and the non-treatment group ( Figure 1). WB analysis was performed on the ovarian cancer marker protein CA125, and the results were consistent with the tumor suppression effect observed with the naked eye. The experimental results showed that by anchoring umbilical cord mesenchymal stem cells with short peptides, the umbilical cord mesenchymal stem cells were precisely targeted to ovarian cancer tissues, achieving precise targeted therapy and effectively improving the therapeutic effect of umbilical cord mesenchymal stem cells ( Figure 2 ).

[0032] The basic principles, main features, and advantages of the present invention have been described above. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection required by the present invention is defined by the appended claims and their equivalents.

Claims

1. A short peptide that specifically binds to integrin αvβ5, characterized in that: The short peptide comprises the amino acid sequence shown in SEQ ID No.

1.

2. An umbilical cord mesenchymal stem cell preparation, characterized in that: The preparation comprises umbilical cord mesenchymal stem cells, wherein the short peptide specifically binding to integrin αvβ5 according to claim 1 is anchored on the surface of the umbilical cord mesenchymal stem cells.

3. The preparation according to claim 2, characterized in that The short peptide is anchored on the surface of umbilical cord mesenchymal stem cells by click chemistry.

4. The preparation according to claim 3, characterized in that The click chemistry method is to introduce complementary click chemistry functional groups on the surface of short peptides and umbilical cord mesenchymal stem cells respectively, wherein the click chemistry functional groups are azide compounds and alkyne groups, and the short peptides are anchored to the surface of umbilical cord mesenchymal stem cells through click chemistry reactions.

5. The method for preparing the preparation according to claim 2, characterized in that: The method comprises the following steps: Step (1): Screening and obtaining a short peptide that specifically binds to integrin αvβ5 through phage display technology; Step (2): culturing and expanding umbilical cord mesenchymal stem cells; Step (3): Anchoring the short peptide of step (1) on the surface of the umbilical cord mesenchymal stem cells of step (2) by click chemistry to prepare an umbilical cord mesenchymal stem cell preparation.

6. The method according to claim 5, characterized in that The click chemistry method is to introduce complementary click chemistry functional groups on the surface of short peptides and umbilical cord mesenchymal stem cells respectively, wherein the click chemistry functional groups are azide compounds and alkyne groups, and the short peptides are anchored to the surface of umbilical cord mesenchymal stem cells through click chemistry reactions.

7. Use of the preparation according to claim 2, characterized in that: The application is for preparing medicine for treating ovarian diseases, and specifically, the ovarian disease is ovarian cancer.

8. The use according to claim 7, characterized in that: The treatment is achieved by specifically binding a short peptide to the integrin αvβ5 expressed on the surface of ovarian cancer cells, and then targeting the umbilical cord mesenchymal stem cells to the ovarian cancer cells through the short peptide, thereby achieving the effect of targeted treatment of ovarian cancer.

9. A drug for treating ovarian diseases, characterized in that: The medicine comprises the preparation according to claim 2.

10. The medicament according to claim 9, characterized in that The medicine further comprises a pharmaceutically acceptable carrier.

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