An umbilical cord mesenchymal stem cell preparation and its use in ovarian targeting

Abstract

The present application relates to the technical field of medicine, and specifically, a peptide capable of specifically binding to ovary-specific protein-inhibin alpha protein (INHA, alpha-inhibin) is obtained through phage display technology, the peptide is anchored on the surface of human umbilical cord mesenchymal stem cells through the method of click chemistry, and then a preparation of umbilical cord mesenchymal stem cells is prepared. The peptide in the preparation of umbilical cord mesenchymal stem cells of the present application serves as a bridge and is applied to the targeted treatment of ovary-related diseases by anchoring the ovary organ.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation containing umbilical cord mesenchymal stem cells, in particular to an umbilical cord mesenchymal stem cell preparation and its preparation method and application in ovarian targeting. BACKGROUND

[0002] Umbilical cord mesenchymal stem cells can be isolated from the whole umbilical cord or umbilical cord blood, which has the advantages of rich source, non-invasive collection, small ethical controversy, low immunogenicity, strong in vitro expansion ability, etc. Compared with MSCs from bone marrow, fat and other sources, umbilical cord mesenchymal stem cells are more likely to obtain high-purity, high-proliferation activity and biologically stable cell populations, laying a foundation for large-scale clinical application.

[0003] Recent studies have shown that umbilical cord mesenchymal stem cells have therapeutic effects on early ovarian dysfunction, chemotherapy or radiotherapy-induced ovarian damage and physiological ovarian aging. Generally through the following ways: (1) homing-paracrine: after intravenous or local transplantation, hUC-MSCs migrate to the ovarian stroma, highly express VEGF, HGF, IGF-1, EGF and other cytokines, promote angiogenesis, inhibit granulosa cell apoptosis, and maintain the follicle pool; (2) exosome-miRNA pathway: hUC-MSC exosomes are rich in miR-21, miR-126, miR-10a, etc., which can down-regulate PTEN / Bax signals, reduce cyclophosphamide-induced DNA damage and ovarian fibrosis; (3) immune-inflammation regulation: reduce the infiltration of myeloid-derived suppressor cells (MDSCs) in ovarian tissue, reduce inflammatory factors IL-6, TNF-α, and block the fibrosis process, thereby delaying ovarian aging; (4) restore the overall hormonal environment: animal models show that after hUC-MSC treatment, serum AMH and estradiol are significantly increased, FSH is decreased, the estrous cycle is restored to normal, and the number of oocytes and blastocyst formation rate are increased.

[0004] Although umbilical cord mesenchymal stem cells have certain therapeutic effects on ovarian-related diseases. However, the targeting of umbilical cord mesenchymal stem cells is poor, which makes it difficult to effectively exert its function, limiting the depth of clinical application. Therefore, there is an urgent need for umbilical cord mesenchymal stem cells with ovarian targeting function to treat ovarian-related diseases. SUMMARY

[0005] In order to improve the targeting problem of umbilical cord mesenchymal stem cells, the present application uses the ovarian-specific protein INHA as a target to guide the combination of umbilical cord mesenchymal stem cells and ovaries, and ultimately treats ovarian-related diseases through umbilical cord mesenchymal stem cells.

[0006] One aspect of the present application is to provide a peptide specifically binding to INHA, preferably, the peptide of the present application is obtained by screening a polypeptide library using phage display technology, specifically, the peptide with high binding specificity to INHA is obtained by screening a polypeptide library using phage display technology, and specifically, the amino acid sequence of the peptide of the present application is shown in SEQ ID No. 1: RSSGPVPLPTQDHGQ. The peptide of the present application binds to the ovary-specific protein INHA, and anchors the umbilical cord mesenchymal stem cells to the ovary.

[0007] Another aspect of the present application is to provide a mesenchymal stem cell derived from human umbilical cord blood, and the preparation method comprises the following steps: (1) selecting human umbilical cord blood stem cells for conventional culture in a serum-free manner; and (2) culturing by subculturing to the 5th generation to expand the human umbilical cord mesenchymal stem cells.

[0008] Another aspect of the present application is to provide a mesenchymal stem cell preparation derived from human umbilical cord blood, wherein the preparation comprises umbilical cord mesenchymal stem cells.

[0009] Further, the peptide with high specificity to INHA is anchored to the surface of the umbilical cord mesenchymal stem cells by a click chemistry method.

[0010] Further, the click chemistry method is to introduce complementary click chemistry functional groups, namely azide and alkyne, to the peptide and the surface of the umbilical cord mesenchymal stem cells, respectively, and to anchor the peptide to the surface of the umbilical cord mesenchymal stem cells by a click chemistry reaction.

[0011] Further, the peptide with high specificity to INHA of the present application specifically binds to the ovary-specific protein INHA, and guides the umbilical cord mesenchymal stem cells to the ovary to achieve targeted therapy.

[0012] Further, the preparation method comprises the following steps:

[0013] Step (1): screening a peptide specifically binding to INHA by phage display technology;

[0014] Step (2): isolating mesenchymal stem cells from human umbilical cord blood, performing conventional culture and subculturing, expanding and culturing after subculturing to the 5th generation to obtain a large amount of umbilical cord mesenchymal stem cells;

[0015] Step (3): anchoring the peptide of step (1) to the surface of the umbilical cord mesenchymal stem cells of step (2) by a click chemistry method to prepare a umbilical cord mesenchymal stem cell preparation.

[0016] Further, the method of click chemistry is to introduce complementary click chemistry functional groups, namely azide and alkyne, on the surface of the peptide and the umbilical cord mesenchymal stem cells respectively, and to anchor the peptide to the surface of the umbilical cord mesenchymal stem cells through click chemistry reaction.

[0017] Another aspect of the present application is to provide a drug for treating ovarian-related diseases, which comprises human umbilical cord mesenchymal stem cells, wherein the surface of the umbilical cord mesenchymal stem cells is anchored with a peptide with high specificity to INHA, and the amino acid sequence of the peptide is shown as SEQ ID No. 1.

[0018] Another aspect of the present application is to provide a preparation for treating ovarian-related diseases, which comprises human umbilical cord mesenchymal stem cells, wherein the surface of the umbilical cord mesenchymal stem cells is anchored with a peptide with high specificity to INHA, and the amino acid sequence of the peptide is shown as SEQ ID No. 1.

[0019] Another aspect of the present application is to provide a drug for treating ovarian-related diseases, which comprises human umbilical cord mesenchymal stem cells, wherein the surface of the umbilical cord mesenchymal stem cells is anchored with a peptide with high specificity to INHA, and the amino acid sequence of the peptide is shown as SEQ ID No. 1.

[0020] The ovarian-related diseases of the present application include, but are not limited to, acute / chronic oophoritis, ovarian cancer, polycystic ovary syndrome (PCOS), ovarian abscess, premature ovarian failure, premature ovarian aging, physiological ovarian aging and follicular cyst, corpus luteum cyst, and luteinized cyst.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] (1) The present application obtains a peptide that only binds to INHA through phage display technology, which shows high binding specificity, and provides a basis for subsequent precise targeted treatment of umbilical cord mesenchymal stem cells.

[0023] (2) The peptide of the present application is anchored on the surface of umbilical cord mesenchymal stem cells through click chemistry method, which realizes the guidance of umbilical cord mesenchymal stem cells to the ovary by using the combination of peptide and ovarian specific protein INHA, and realizes precise targeted treatment.

[0024] (3) Compared with the prior art, the preparation of umbilical cord mesenchymal stem cells containing the peptide of the present application can realize precise positioning of the ovary, thereby realizing precise targeted treatment, and can effectively treat ovarian-related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 In vitro experiment - WB diagram of inflammatory factor expression;

[0026] Figure 2 In vivo experiment - WB figure of inflammatory factor expression. DETAILED DESCRIPTION

[0027] The application will be further described in conjunction with specific implementation cases, but the application is not limited to these examples.

[0028] Example 1 Preparation of a peptide with high specificity to bind INHA

[0029] (1) Library construction and amplification

[0030] A series of random DNA sequences are designed and cloned into the coding region of the coat protein of bacteriophage M13 to construct a 15-peptide library.

[0031] (2) Target capture

[0032] The ovary-specific protein INHA is immobilized on the surface of a solid carrier, and the library of step (1) is incubated with the solid carrier for 4 h to allow binding;

[0033] (3) Washing

[0034] Repeated washing removes unbound non-specific phages to remove unbound and non-target specific phages.

[0035] (4) Elution

[0036] After brief incubation with low-ph buffer or by competitive elution, the target-bound phages are separated out.

[0037] (5) Infection stage

[0038] The eluted phages are used to infect bacteria to amplify the selected phages, thereby forming a new phage library for the next round of biopanning.

[0039] After 5 rounds of biopanning, a peptide with high binding specificity is screened, and its amino acid sequence is shown in SEQ ID No. 1.

[0040] Example 2 Preparation of human umbilical cord mesenchymal stem cell preparation

[0041] The human umbilical cord tissue is taken from a healthy full-term vaginal delivery fetus, is placed in a sterile PBS solution containing 1% of a double antibody for storage, is washed with normal saline before use to remove the surface and is moved into a new culture medium, and is soaked with 75% medical alcohol for 2 minutes. After sterilization, the umbilical cord is added to a culture dish containing normal saline, is washed for 2-3 times, and serum is removed. First, the umbilical cord is unfolded by using two tissue forceps, two veins and an artery are removed, and the tissue is cut into a T75 culture bottle by using a tissue forceps, 4 ml of complete culture medium is added to each bottle, the cell bottle is placed flat to make the tissue evenly distributed on the entire bottom surface, and the cell bottle is placed in a culture box for culture at 37°C and 5% CO2. After primary preparation, full-volume liquid replacement is performed every 3 days, and when the cell fusion degree reaches 85%-90%, subculture is performed until the 5th generation. The 5th generation is expanded for culture, and a large number of umbilical cord mesenchymal stem cells are harvested.

[0042] The peptide of example 1 and the 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 introducing an azide functional group on the surface of the peptide to prepare a compound, introducing an alkyne group on the surface of the umbilical cord mesenchymal stem cells, and completing the reaction under copper catalysis to anchor the peptide to the surface of the umbilical cord mesenchymal stem cells, thereby preparing a preparation of umbilical cord mesenchymal stem cells containing the peptide.

[0043] Example 3 Application of umbilical cord mesenchymal stem cell preparation in treatment of oophoritis

[0044] 1. In vitro experiment

[0045] Human ovarian granulosa cells COV434 are taken for routine culture, are subcultured into a 6-hole plate for continuous culture, are divided into 4 groups, and are respectively a normal culture group, a PBS treatment group, a conventional umbilical cord mesenchymal stem cell treatment group and an umbilical cord mesenchymal stem cell preparation treatment group of example 2. After 3 days of culture, when the cell density is 80%, liquid replacement is performed, LPS 1 μg / mL is added for induction of inflammation, 4 hours after induction, the normal culture group is not treated, the PBS treatment group is added with 100 μl of PBS, the conventional umbilical cord mesenchymal stem cell treatment group is added with 100 μl of umbilical cord mesenchymal stem cells, and the umbilical cord mesenchymal stem cell preparation treatment group of example 2 is added with 100 μl of the umbilical cord mesenchymal stem cell preparation of example 2. After 24 hours of incubation of each group, the cells are washed with PBS, are lysed, total cell protein is collected, and expression of inflammatory factors is analyzed by WB.

[0046] The WB results show that the expression of inflammatory factors in the normal culture group and the PBS treatment group is significantly higher than that in the conventional umbilical cord mesenchymal stem cell treatment group and the umbilical cord mesenchymal stem cell preparation treatment group of example 2. Since the umbilical cord mesenchymal stem cell preparation of example 2 has the function of targeting the ovary, it can accurately target the ovary, and the expression of inflammatory factors is the lowest, proving that the umbilical cord mesenchymal stem cell preparation has a good effect of inhibiting inflammation of the ovary.

[0047] 2. In vivo experiment

[0048] Intraperitoneal injection of 20 mg / kg LPS can cause acute oophoritis within 48 hours to construct an oophoritis animal model. Then, the animals are randomly divided into four groups, which are untreated group, PBS treatment group, conventional umbilical cord mesenchymal stem cell treatment group and the umbilical cord mesenchymal stem cell preparation of Example 2 treatment group. The untreated group is not treated, the PBS treatment group is injected with PBS through the tail vein, the conventional umbilical cord mesenchymal stem cell treatment group is injected with conventional extracted umbilical cord mesenchymal stem cells through the tail vein, and the umbilical cord mesenchymal stem cell preparation of Example 2 treatment group is injected with the umbilical cord mesenchymal stem cell preparation of Example 2 through the tail vein. Each group is intervened with drug administration once a day 1 day after the animal model is constructed, and the volume of each injection is 100 μl. The ovarian tissues of rats in each group are taken out and proteins are extracted 28 days after the drug administration intervention, and the expression of inflammatory factors is analyzed by WB.

[0049] The results show that the WB results show that the expression of inflammatory factors in the untreated group and the PBS treatment group is significantly higher than that in the conventional umbilical cord mesenchymal stem cell treatment group and the umbilical cord mesenchymal stem cell preparation of Example 2 treatment group. Since the umbilical cord mesenchymal stem cell preparation of Example 2 has the function of targeting the ovary, it can accurately target the ovary, and the expression of inflammatory factors is the lowest, which proves that the umbilical cord mesenchymal stem cell preparation of the application has a good effect of inhibiting oophoritis.

[0050] The above describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above examples, and the above examples and descriptions in the specification are only the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection required by the present application is defined by the appended claims and their equivalents.

Claims

1. A peptide that specifically binds to INHA, characterized in that, The amino acid sequence of the peptide is shown as SEQ ID No.

1.

2. A preparation of umbilical cord mesenchymal stem cells, characterized in that, The preparation comprises umbilical cord mesenchymal stem cells, wherein the umbilical cord mesenchymal stem cells are anchored on the surface with the peptide specific to INHA according to claim 1.

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

4. The preparation according to claim 3, characterized in that, The click chemistry method is to introduce complementary click chemistry functional groups, namely azide and alkyne, to the peptide and the surface of the umbilical cord mesenchymal stem cells respectively, and to anchor the peptide to the surface of the umbilical cord mesenchymal stem cells by a click chemistry reaction.

5. Use of a preparation according to claim 2, characterized in that, The application is used for preparing a drug for treating oophoritis by targeting the ovary.

6. Use according to claim 5, characterized in that, The treatment is to target the umbilical cord mesenchymal stem cells to the ovary by using the specific binding property of the peptide according to claim 1 to the ovary specific protein INHA, so as to achieve the effect of targeted treatment of oophoritis.

7. A medicament for treating oophoritis, characterized by, The drug comprises the preparation according to claim 2.

8. The medicament according to claim 7, characterized in that, The drug further comprises a pharmaceutically acceptable carrier.

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