WO2020228743A1 - Combination of stem cells and cytokines and use thereof in improving sperm motility - Google Patents

Abstract

The present invention relates to the field of biopharmaceuticals, and provided thereby is a pharmaceutical composition or kit used for improving low sperm motility in a subject, and a use of adult stem cells and cytokines in the preparation of a pharmaceutical composition or kit used for improving low sperm motility in a subject, respectively. The pharmaceutical composition or kit used for improving low sperm motility in a subject comprises: adult stem cells; one, two or three kinds of cytokines selected from among monocyte chemoattractant protein-1, glial cell line-derived neurotrophic factor and osteoprotegerin; and an optional pharmaceutically acceptable carrier.

Description

Combination of stem cells and cytokines and their use in improving sperm motility Technical field

The invention belongs to the field of biopharmaceuticals, and specifically relates to the use of a combination of stem cells and cytokines in improving sperm motility.

Background technique

The research on bone marrow transplantation started after the Second World War, which is related to the urgent need for treatment of massive radiation damage caused by the atomic bomb explosion. During the 1960s, bone marrow transplantation was used to treat patients with leukemia and Hodgkin's disease. However, practice shows that most transplants fail due to rejection. During the 1970s, the progress of HLA research brought about a turning point in the pessimistic results of bone marrow transplantation. HLA-matched relatives bone marrow transplantation was used to treat aplastic anemia and leukemia. At the same time, platelet transfusion technology and new antibacterial were used. Various cooperative measures such as drugs have ensured the increase in the success rate of bone marrow transplantation. In the 1980s, the area where bone marrow transplantation was carried out was expanded to the whole world. Several countries established a marrow donor registry and began worldwide cooperation. Due to the improvement of chemotherapy regimens and the improvement of freezing technology, the number of autologous bone marrow transplantation has increased exponentially.

In recent years, hematopoietic stem cells from the collected peripheral blood have been used for intravenous infusion. This convenient and safe transplantation method can be used as a substitute for bone marrow transplantation.

Stem cell transplantation technology has been applied to some indications alone or in combination with other treatment methods (such as chemotherapy). Stem cells are widely used because when they are administered to a subject, they can regenerate one or more tissue-specific cells in the subject.

Many stem cell transplants use stem cells provided by matched donors (allogeneic). However, allogeneic transplantation has many disadvantages, such as immune rejection and graft-versus-host disease. Moreover, the economic cost of allotransplantation is also high.

The advantage of autologous transplantation is that autologous cells (including peripheral blood stem cells) are reintroduced into the subject, thereby reducing the risk of rejection by the immune system. Stem cells from a foreign body may cause transplant rejection and have the risk of so-called graft-versus-host disease, that is, the transplanted cells attack the cells in the recipient's body. This risk can usually only be suppressed by immunosuppressive drugs.

Sources of stem cells usually include embryonic stem cells, stem cells collected from cord blood, and adult stem cells. For embryonic stem cells, although they are relatively easy to grow in culture, these cells are rarely found in adult tissues and the method to expand their numbers in cell culture is not mature, and their cell sources cannot match the needs of stem cell transplantation therapy. The actual demand for a large number of cells. In addition, if allogeneic embryonic stem cells are transplanted into the subject, it will increase the risk of transplant rejection. On the other hand, stem cells collected from cord blood can be used for some treatments, but their cells are immature and the number of cells is small. The current clinical application requires HLA matching and culture amplification technology.

Adult stem cells or somatic stem cells are undifferentiated cells found among differentiated cells in tissues or organs. Adult stem cells can self-renew and can differentiate to produce the main specific cell types of the tissue or organ. In organisms, the main function of adult stem cells is to maintain and repair the tissues they are in. Adult stem cells are a source of stem cell autologous transplantation, which can minimize the risk of transplant rejection. Using the subject's own adult stem cells will mean that the cells will not be rejected by the immune system. This advantage is unmatched by allograft. Therefore, the use of autologous transfer of adult stem cells has important practical significance.

There are also many stem cell components in peripheral blood after mobilization injection, such as hematopoietic stem cells, circulating fibroblasts, vascular endothelial progenitor cells, and mesenchymal stem cells. Compared with adult stem cells from other parts of the body, their biggest advantages are that they are convenient to obtain materials, the large number of cells causes less pain to patients, and there are no viral infections and immune rejections. They are ideal sources of stem cells.

If couples of normal childbearing age do not take any contraceptive measures, the woman has the normal ability to conceive. However, in recent years, there has been a high incidence of diseases in which the woman cannot get pregnant due to the man’s reasons. The reason is that asthenospermia or idiopathic asthenospermia accounts for a large proportion. In addition, according to data, infertility caused by low sperm motility accounts for 60%-80% of all male infertility, and one third of them are unknown.

CN107693624A discloses a Chinese medicinal composition for clinical treatment of idiopathic asthenospermia and male infertility, which comprises rehmannia glutinosa, dodder seed, astragalus, epimedium, salvia, achyranthes bidentata, leeches, spatholobi and Passepartout.

CN105395841A discloses a medicinal combination for treating oligoasthenospermia and its preparation method; its raw materials include 20-50 parts by weight, 15-30 parts of curculio, 15-30 parts of myrtle, and 10 parts by weight. ～30 parts, 10～30 parts of Cistanche, 15～50 parts of Chinese Yam, 15～40 parts of Rehmannia vulgaris, 10～30 parts of Ligustrum lucidum, 10-30 parts of Leek Seed, 10-30 parts of Xianling Spleen, 15-30 parts of Licorice . Its dosage form is decoction. The preparation process includes: weighing the above-mentioned raw medicinal materials in parts by weight, and soaking in water; first frying: decocting on a fire until boiling, then decocting on a slow fire for 20-25 minutes, second frying: after the first frying, filtering the concoction Add water again, decocting until boiling, change to slow decocting for 15-20 minutes. Combine the first decoction and the second decoction to obtain the mixture. The mixture is preserved as a decoction.

However, due to the different growth environment of its raw materials, Chinese herbal medicines have great differences in their efficacy, which in turn leads to greatly reduced therapeutic effects of Chinese herbal medicines.

WO2011048806A1 discloses a medicament for improving sperm motility and fertility, which contains gamma-linolenic acid. When this agent is administered to a male and the female is inseminated with semen from the male, the female's fertility can be improved. The agent can improve the sperm motility of males, and can improve the fertility of females inseminated by male semen. However, this program is mainly used to improve the fertility status of domestic animals. Whether it is applicable to humans is still unknown.

Therefore, researching a drug that can effectively improve human sperm motility and has a stable curative effect is of great significance for solving the current infertility problem.

Summary of the invention

In order to better improve sperm motility and provide another feasible option, the inventors of the present invention have conducted a large number of persistent screenings and found that the combination of adult stem cells (preferably autologous peripheral blood stem cells) and specific cytokines has significant effects on improving sperm motility. The effect, and then provides a new therapy or means for the treatment of low sperm motility. To this end, the inventors of the present invention provide a pharmaceutical composition or kit for improving hypospermia in a subject, and adult stem cells and cytokines are prepared for improving hypospermia in a subject. Use in pharmaceutical composition or kit. The pharmaceutical composition or kit of the present invention has a significant therapeutic or improvement effect on asthenospermia or low sperm motility, and has small side effects and no immune rejection reaction.

Specifically, the first aspect of the present invention provides a pharmaceutical composition or kit for improving hypospermia in a subject, which comprises adult stem cells and selected from monocyte chemotactic protein-1 (MCP-1) , Glial cell-derived neurotrophic factor (GDNF) and osteoprotegerin (OPG) one, two or three cytokines, and optionally a pharmaceutically acceptable carrier.

In the pharmaceutical composition or kit, the cytokine can be selected from monocyte chemotactic protein-1 (MCP-1), glial cell-derived neurotrophic factor (GDNF) and osteoprotegerin (OPG) For example, the cytokine can be selected from any one of the following substances or combinations: MCP-1, a combination of GDNF and OPG, a combination of MCP-1 and GDNF, a combination of GDNF and OPG, The combination of MCP-1 and OPG, MCP-1, GDNF, OPG.

According to a preferred embodiment of the present invention, the pharmaceutical composition or kit contains adult stem cells, MCP-1 and GDNF.

Preferably, the dosage of the pharmaceutical composition or kit of this specific embodiment includes: relative to 1 kg of body weight, the content of the adult stem cells is 10 ⁶ -10 ⁸ and the content of the MCP-1 is 0.01-10 mg. The content of GDNF is 0.01-10 mg.

More preferably, the dosage of the pharmaceutical composition or kit of this specific embodiment includes: relative to 1 kg of body weight, the content of the adult stem cells is 10 ⁶ -10 ⁸ and the content of the MCP-1 is 0.02-5 mg, The content of the GDNF is 0.02-5 mg.

Further preferably, the dosage of the pharmaceutical composition or the kit of this specific embodiment includes: relative to 1 kg of body weight, the content of the adult stem cells is 10 ⁶ -10 ⁸ and the content of the MCP-1 is 0.1-0.5 mg The content of the GDNF is 0.1-0.5 mg.

Most preferably, the dosage of the pharmaceutical composition or kit of this specific embodiment includes: relative to 1 kg of body weight, the adult stem cells are peripheral blood stem cells with a content of (1-5)×10 ⁶ cells, and the MCP-1 The content of GDNF is 0.2-0.4mg, and the content of GDNF is 0.2-0.4mg.

According to another preferred embodiment of the present invention, the pharmaceutical composition or kit comprises adult stem cells, MCP-1, GDNF and OPG.

Preferably, the dosage of the pharmaceutical composition or kit of this specific embodiment includes: relative to 1 kg of body weight, the content of the adult stem cells is 10 ⁶ -10 ⁸ and the content of the MCP-1 is 0.01-10 mg. The content of GDNF is 0.01-10 mg, and the content of OPG is 0.01-10 mg.

More preferably, the dosage of the pharmaceutical composition or kit of this specific embodiment includes: relative to 1 kg of body weight, the content of the adult stem cells is 10 ⁶ -10 ⁸ and the content of the MCP-1 is 0.02-5 mg, The content of the GDNF is 0.02-5 mg, and the content of the OPG is 0.02-5 mg.

Further preferably, the dosage of the pharmaceutical composition or the kit of this specific embodiment includes: relative to 1 kg of body weight, the content of the adult stem cells is 10 ⁶ -10 ⁸ and the content of the MCP-1 is 0.1-0.5 mg The content of the GDNF is 0.1-0.5 mg, and the content of the OPG is 0.1-0.5 mg.

Most preferably, the dosage of the pharmaceutical composition or kit of this specific embodiment includes: relative to 1 kg of body weight, the adult stem cells are peripheral blood stem cells with a content of (1-5)×10 ⁶ cells, and the MCP-1 The content of GDNF is 0.2-0.4mg, the content of GDNF is 0.2-0.4mg, and the content of OPG is 0.2-0.4mg.

In the present invention, preferably, the monocyte chemoattractant protein-1, glial cell-derived neurotrophic factor and osteoprotegerin are recombinant proteins.

In the present invention, the storage method of adult stem cells and cytokines in the pharmaceutical composition or kit is not particularly limited, and they can be stored separately or mixed.

In the present invention, preferably, the adult stem cell is an autologous stem cell, more preferably a subject's autologous peripheral blood stem cell.

In the present invention, the pharmaceutical composition or kit may also contain other active pharmaceutical ingredients for treating or improving male sperm motility, as long as the other ingredients do not counteract the activity of the aforementioned cytokines and adult stem cells.

The cytokines of MCP-1, GDNF and OPG of the present invention can be obtained according to conventional methods in the art, or commercially available. The cDNA sequence information of MCP-1, GDNF and OPG can be obtained by logging into the NCBI database.

For the adult stem cells and cytokines of the present invention, those skilled in the art can make any modifications to them, provided that the modifications do not negatively affect their activities. For example, the protein can be modified or loaded on other carriers to increase its half-life in the body; or can be linked to a known penetrating peptide to promote transdermal absorption of the compound of the present invention or cross the blood-brain barrier. In short, those skilled in the art can make various modifications to the compounds of the present invention to improve delivery efficiency or for other purposes while maintaining their activity.

Adult stem cells and cytokines as active ingredients in the present invention can be used together with a pharmaceutically acceptable carrier. In addition to the active ingredients, the methods, uses and products of the present invention may also contain suitable pharmaceutically acceptable carriers, including excipients and auxiliaries that facilitate the processing of the active ingredients into preparations (for example, preparations suitable for injection or infusion) .

Formulations suitable for injection or infusion may include aqueous and non-aqueous sterile injections and aqueous and non-aqueous sterile suspensions. The sterile injections may optionally contain antioxidants, buffers, bacteriostatic agents and A solute capable of making the preparation equal to the blood of the intended recipient. The sterile suspension may include a suspending agent and a thickening agent. The preparations can be presented in unit-dose or multi-dose containers, such as sealed ampoules, and can be stored in freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use.

The active ingredient of the present invention may optionally be combined with solid excipients, and the resulting mixture may optionally be ground, and if necessary, after adding suitable auxiliaries, the mixture of granules is processed to obtain the desired dosage form . Suitable excipients are especially fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose or starch preparations, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, carboxylate Sodium methylcellulose and/or polyvinylpyrrolidone (PVP). If necessary, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

The second aspect of the present invention provides the use of adult stem cells (preferably peripheral blood stem cells) in preparing the pharmaceutical composition or kit described in the first aspect of the present invention for treating or improving male sperm motility in a subject.

The third aspect of the present invention provides that one, two or three of MCP-1, GDNF and OPG are used for the preparation of any one of claims 1-8 for improving the patient’s health. The use in a pharmaceutical composition or a kit for the subject's sperm motility is low.

With regard to the use of the second and third aspects of the present invention, the following description will be made together.

The present invention provides a method for treating or improving male sperm motility in a subject, which comprises simultaneously or sequentially administering adult stem cells and cytokines to the subject. Optionally, in the method of the present invention, other active pharmaceutical ingredients for treating or improving male sperm motility can also be administered simultaneously or sequentially.

For the subject of the present invention, it is preferably a mammal, more preferably a human.

The amount of adult stem cells and cytokines administered in the present invention can be any amount that can synergistically treat the subject or improve male sperm motility. For example, the dosage unit includes: relative to 1 kg body weight, 0.01-10 mg of each cytokine and 10 ⁶ -10 ⁸ adult stem cells; preferably 0.02-5 mg of each cytokine and 10 ⁶ -10 ⁸ adult stem cells; more preferably 0.1-0.5 mg of each cytokine and 10 ⁶ -10 ⁸ adult stem cells; most preferably 0.2- 0.4 mg of each cytokine and (1-5)×10 ⁶ peripheral blood stem cells. The determination of an effective amount is within the abilities of those skilled in the art, especially under the inspiration of the disclosure provided herein.

According to the present invention, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention can be administered to a subject at any effective dose. Preferably, the pharmaceutical product (medicine, medicament or kit) or pharmaceutical composition of the present invention can be administered in multiple doses, for example, from about 2 to about 10 doses, more preferably from about 3-5 doses, most preferably About 3 doses. In a particularly preferred embodiment, during the administration process, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention is administered to the subject at a frequency of about once every two months, such as injection or infusion. Note. In a particularly preferred embodiment, the administration is the administration of cytokines and adult stem cells by intravenous infusion.

It should be understood that the pharmaceutical product (drug, medicament or kit) or pharmaceutical composition of the present invention can be formulated in any suitable manner for administration by any suitable route.

The dosage unit of the pharmaceutical product (medicine, medicament) or pharmaceutical composition of the present invention is based on routine administration to the subject. For example, the dosage unit can be administered more than once a day, once a week, once a month, etc. The dosage unit can be administered on a twice/week basis, that is, twice a week, for example, once every three days.

As used herein, "comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended, and does not exclude additional unstated elements or method steps. Any expression of the term "comprising" in this text, especially when describing the method, use or product of the present invention, should be understood to include essentially consisting of the component or element or step and consisting of the component or element or The products, methods, and uses of the steps. The present invention exemplarily described herein can suitably be practiced in the absence of any one or more elements, one or more limitations not specifically disclosed herein.

The pharmaceutical product (e.g., pharmaceutical composition or kit) of the present invention may include instructions related to the pharmaceutical product, and the instructions may contain the following contents: indications (e.g., ovarian cancer), dosage (e.g., the exemplified above ) And possible side effects, etc.

The terms and expressions that have been used herein are used as descriptive rather than restrictive terms, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or parts thereof, but various modifications should be recognized It is possible within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art can adopt modifications and variations of the concepts disclosed herein, and such modifications and variations are deemed to be as defined by the appended claims Defined within the scope of the invention.

In order to illustrate the present invention more clearly, a detailed description is now combined with the following embodiments, but these embodiments are merely exemplary descriptions of the present invention and cannot be construed as limiting the present application.

Detailed ways

The effective component in the pharmaceutical composition of the present invention is present in an effective amount. As used herein, the term "effective amount" refers to an amount that is sufficient to treat (therapeutically or prophylactically) this target disorder when it is administered in an appropriate dosage regimen. For example, an effective amount is sufficient to reduce or improve the severity, duration, or progression of the disorder being treated, prevent the advancement of the disorder being treated, cause the deterioration of the disorder being treated, or enhance or improve another therapy The preventive or therapeutic effect.

The term "treatment" includes both therapeutic treatment and preventive treatment (reducing the possibility of development). The term refers to reducing, inhibiting, attenuating, reducing, stopping or stabilizing the development or progression of a disease (such as the disease or condition described herein, male sperm motility deficiency), reducing the severity of the disease or improving the disease associated with the disease Symptoms.

In order to mobilize and recruit more adult stem cells from the subject, especially peripheral blood stem cells, it is preferable to administer the granulocyte colony stimulating factor to the subject for at least 5 days before collecting the peripheral blood stem cells from the subject (G-CSF), for example, about 5ug/kg/day to 10ug/kg/day; more preferably, the granulocyte colony stimulating factor is administered to the subject at a dose of about 6-8 μg/kg/day or equivalent, and Monitor white blood cells daily.

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Considering that the inventors of the present invention found in previous scientific research or clinical trials that the effect of reinfusing stem cells alone on asthenospermia is not excellent (there is a high failure rate), the inventors directly in this project trial The combination of the selected cytokines and stem cells is used to try to treat asthenospermia in order to find a better stem cell treatment plan. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

Example 1 Preparation of PBMC from peripheral blood stem cells

Daily intramuscular injection of mobilizer (recombinant human granulocyte stimulating factor GCSF) at a dose of 5-10ug/kg/day, continuous injection of mobilizer for 5 days, and 1ml of venous blood daily from the next day to monitor white blood cells, when white blood cells rise To 2.5×10 ⁹ /L, the amount of mobilizer does not exceed (5ug/kg/day). Five days after the injection of GCSF, PBMC can be collected when the white blood cell value reaches 2.5×10 ⁹ /L or more. In general, white blood cells can reach 4.5×10 ⁹ /L, which is the "ideal mobilization state", that is, GCSF successfully stimulates the bone marrow colony .

After successful mobilization, stem cells (PBMC) were collected with a blood separator. Specifically, puncture the veins of the elbow, drain the blood into the blood separator through a closed tube, and collect it by a machine with a centrifugal setting. The whole process takes about 3-5 hours, and 120-200ml PBMC can be collected. . The specific collection amount depends on actual needs, usually based on body weight. For example, taking 70kg as an example, 150ml is required.

The blood preservation solution was used in the collection process: the use ratio of 22.0g sodium citrate (C6H5Na307.2H2O) blood preservation solution to PBMC in 1000ml glucose was 1:10. The collected PBMCs are divided into bags for infusion.

Example 2 The composition improves male sperm motility

Sperm motility has a significant positive correlation with parameters such as VSL, VCL, and VAP. Sperm motility parameters such as VSL, VCL, and VAP are conventional indicators that reflect sperm motility. In this example, the effect of the composition of the present invention on improving sperm motility of healthy men with normal sexual life was investigated. The specific method is as follows. For 16 normal male volunteers aged 22-44 who were married, healthy and sexually active, before the start of the experiment, peripheral blood stem cells were prepared by the method of Example 1, and He Jiang et al. (Chinese Journal of Eugenics and Genetics, 2010, 18(5): 110) collected semen and measured VSL, VCL, VAP values and sperm motility values (a+b%). The results were expressed as the average value of 16 volunteers and recorded as the starting value; Then, the pharmaceutical composition of the present invention was administered to all subjects by infusion, that is, 1×10 ⁶ cells/kg body weight of peripheral blood stem cells + 0.3 mg MCP-1 + 0.3 mg GDNF were used to return the pharmaceutical composition of the present invention along the vein Infused into the patient, one dose.

The results showed that the initial values (mean ± SD) of sperm motility (a+b, %), VSL, VCL, and VAP were 40.5±5.6%, 32.6±1.8μm/s, 60.3±5.8μm/s, 36.5± At 4.3 μm/s, the values of sperm motility (a+b, %), VSL, VCL, and VAP increased by 24.5%, 28.7%, 29.2%, and 28.8% respectively 2 months after the first intravenous injection. The above results indicate that the composition of the present invention has an excellent effect of improving male sperm motility.

Example 3: The composition improves the effect of male asthenospermia

Mr. Wu, about 40 years old, weighs 70Kg and suffers from infertility. PBMC was collected according to Example 1, and the cytokine composition 0.3mgMCP-1+0.3mgGDNF+0.3mgOPG was added to 50ml PBMC, infused once every two months, a total of three reinfusions, a total of 150ml PBMC (which contains Stem cell number is about 6X10 ⁷ th), wherein the first recirculation date September 18, 2016; second recirculation date November 20, 2016; third recirculation date January 20, 2017. Successfully improved sperm motility rate from 1.80% (physical examination value before reinfusion, see Table 1, where standard value: ≥40%) to 17.6% of the second time (two months after the first reinfusion, see Table 2), the third time 70% (two months after the second return, see Table 3). Successfully improved the sperm forward rate from 1.58 (standard value: ≥32%, Table 1) to 14.81 for the second time (Table 2) and 48.2 for the third time (Table 3). The subject finally improved the asthenospermia and completed natural conception with his wife in April 2017.

Table 1: Various indexes of Mr. Wu's semen before treatment

Table 2: The semen indexes of Mr. Wu two months after the first infusion

Table 3: The semen indexes of Mr. Wu two months after the second infusion

It can be seen from the above examples that the product of the present invention has been clinically verified to effectively improve energy and vitality, so that the family of patients who have not conceived for many years can be successfully conceived without side effects and immune rejection, and is against the infertility currently faced in the field. The problem is very important.

For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description, and therefore it is intended to fall within the claims. All changes within the meaning and scope of the equivalent elements of are included in the present invention.

Claims (10)

1. A pharmaceutical composition or kit for improving a subject's low sperm motility, which comprises adult stem cells and one selected from the group consisting of monocyte chemotactic protein-1, glial cell-derived neurotrophic factor and osteoprotegerin, Two or three cytokines, and optionally a pharmaceutically acceptable carrier.
2. The pharmaceutical composition or kit according to claim 1, wherein the pharmaceutical composition or kit comprises adult stem cells, monocyte chemoattractant protein-1 and glial cell-derived neurotrophic factor, wherein relative to every 10 ^{6-10 8} adult stem cells, the content of the monocyte chemoattractant protein-1 is 0.01-10 mg, and the content of the glial cell-derived neurotrophic factor is 0.01-10 mg.
3. The pharmaceutical composition or kit according to claim 2, wherein the content of the monocyte chemoattractant protein-1 is 0.1-0.5 mg per 10 ⁶ -10 ⁸ adult stem cells, and the glia The content of cell-derived neurotrophic factor is 0.1-0.5mg.
4. The pharmaceutical composition or kit according to claim 1, wherein the pharmaceutical composition or kit comprises adult stem cells, monocyte chemoattractant protein-1, glial cell-derived neurotrophic factor and osteoprotegerin, wherein Relative to every 10 ⁶ -10 ⁸ adult stem cells, the content of the monocyte chemoattractant protein-1 is 0.01-10 mg, the content of the glial cell-derived neurotrophic factor is 0.01-10 mg, and the osteoprotegerin The content is 0.01-10mg.
5. The pharmaceutical composition or kit according to claim 4, wherein the content of the monocyte chemoattractant protein-1 is 0.1-0.5 mg per 10 ⁶ -10 ⁸ adult stem cells, and the glia The content of cell-derived neurotrophic factor is 0.1-0.5 mg, and the content of the osteoprotegerin is 0.1-0.5 mg.
6. The pharmaceutical composition or kit according to any one of claims 1-5, wherein the monocyte chemotactic protein-1, glial cell-derived neurotrophic factor and osteoprotegerin are recombinant proteins.
7. The pharmaceutical composition or kit according to claim 1, wherein the adult stem cells and cytokines in the pharmaceutical composition or the kit are stored separately or mixedly.
8. The pharmaceutical composition or kit according to claim 1, wherein the adult stem cells are autologous peripheral blood stem cells of a subject.
9. Use of adult stem cells in the preparation of the pharmaceutical composition or kit for improving sperm motility in a subject according to any one of claims 1-8.
10. One, two or three cytokines of MCP-1, GDNF and OPG are used in the preparation of the pharmaceutical composition or medicine for improving sperm motility in a subject according to any one of claims 1-8 Use in the box.