Application of sperm exosomes and / or fallopian tube cell exosomes in the preparation of therapeutic drugs for infertility-related diseases

Abstract

The present invention relates to the field of biomedical technology, and specifically to the use of sperm exosomes and / or fallopian tube cell exosomes in the preparation of drugs for the treatment of infertility-related diseases. The drug provided by the present invention can effectively reduce the degree of endometrial damage, promote the normal implantation of embryos, promote the vitality of endometrial cells, and reduce the damage of endometrial cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of sperm exosomes and / or fallopian tube cell exosomes in the preparation of drugs for treating infertility-related diseases. Background Art

[0002] Exosomes are membrane vesicles released into the extracellular matrix after the fusion of intracellular multivesicular bodies (MVB) with the cell membrane. Almost all types of cells can produce and release exosomes. It is a nanoscale lipid inclusion structure with a diameter of 30 to 100 nm, which contains substances such as proteins, mRNA and microRNA. Exosomes exist naturally in body fluids, including blood, saliva, urine and breast milk. It is closely related to diseases such as immune response, viral pathogenicity, pregnancy, and cardiovascular disease.

[0003] Due to its own characteristics, the embryo is easily rejected by the mother and can only be accepted by the mother's endometrial tissue within a specific developmental period. In existing research, little is known about the factors related to embryo implantation. Studies have shown that seminal plasma and uterine-related cells have the function of regulating endometrial receptivity, but the specific regulatory molecules and mechanisms are not yet clear. Summary of the invention

[0004] In order to solve the above problems, the present invention provides an application of sperm exosomes and / or fallopian tube cell exosomes in the preparation of drugs for the treatment of infertility-related diseases. Since exosomes are biological molecules with immune regulation and information transmission functions, if sperm exosomes and / or fallopian tube cell exosomes can be used to regulate the immune response between mother and fetus during embryo implantation and promote the formation of endometrial receptivity, the purpose of facilitating embryo implantation can be achieved.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides an application of sperm exosomes and / or fallopian tube cell exosomes in preparing a drug for treating infertility-related diseases.

[0007] The present invention also provides the use of sperm exosomes and / or fallopian tube cell exosomes in the preparation of a drug for increasing the probability of fertilized eggs and / or embryos implanting in the uterus.

[0008] The present invention also provides the use of sperm exosomes and / or fallopian tube cell exosomes in the preparation of a drug for treating endometrial growth disorder.

[0009] The present invention also provides the use of sperm exosomes and / or fallopian tube cell exosomes in the preparation of a drug for increasing the thickness of the endometrium.

[0010] The present invention also provides the use of sperm exosomes and / or fallopian tube cell exosomes in the preparation of a drug for promoting endometrial cell activity.

[0011] In the present invention, the dosage form of the drug includes gel and solution.

[0012] In the present invention, the method for preparing sperm exosomes comprises the following steps:

[0013] Cut the testis washed with PBS solution into pieces, place the cut testis into pieces in a culture medium without exosomes for culture, collect the culture supernatant in the culture medium, and centrifuge the culture supernatant to obtain sperm exosomes;

[0014] When the sperm exosomes are used, the sperm exosomes are resuspended in PBS buffer;

[0015] The specific operation is preferably as follows: healthy adult male BALB / c mice are selected, and the mice are killed by cervical dislocation after anesthesia, the mouse testis is taken out, washed with PBS solution for 2 to 3 times, cut into pieces, and placed in exosome-free culture medium for culture, the culture supernatant is collected, the sperm exosomes are collected by low-temperature freezing ultracentrifugation, and the sperm exosomes are resuspended with PBS buffer;

[0016] The method for preparing fallopian tube cell exosomes comprises the following steps:

[0017] The cleaned fallopian tubes are dispersed to obtain fallopian tube cells, the fallopian tube cells are cultured in a culture medium, the culture medium supernatant is collected, the culture medium is centrifuged at low temperature to obtain fallopian tube cell exosomes, and the fallopian tube cell exosomes are resuspended in PBS buffer and stored in a low temperature environment;

[0018] The specific operation is preferably as follows: healthy adult female BALB / c mice are selected, and after anesthesia, they are killed by cervical dislocation, and the uterus of the mice is taken. After opening the abdomen, the connection between the uterus and the fallopian tube is clamped with ophthalmic forceps, and the fallopian tube mesentery is cut with sharp scissors. Then, the connection structure between the ovary and the fallopian tube is cut first, and then the connection between the uterus and the fallopian tube is cut to obtain the fallopian tube part, and the fallopian tube is transferred to a surface dish containing PBS solution. The fat on the fallopian tube is removed with ophthalmic scissors, and then rinsed clean.

[0019] The fallopian tubes are dispersed to obtain fallopian tube cells, the fallopian tube cells are cultured in a culture medium, the culture medium supernatant is collected, and the culture medium is centrifuged at low temperature to obtain fallopian tube cell exosomes.

[0020] In the present invention, the dosage of sperm exosomes and / or fallopian tube cell exosomes in the drug is 1×10 8 ~1×10 13 units; further, in the present invention, the amount of the exosomes is preferably 5×10 12 ~6×1012 units. DETAILED DESCRIPTION

[0021] Example 1

[0022] 1. Preparation of sperm exosomes

[0023] Healthy adult male BALB / c mice were selected and killed by cervical dislocation after anesthesia. The mouse testis was taken and washed with PBS solution for 2-3 times, cut into pieces and placed in exosome-free culture medium. Sperm exosomes were collected by low-temperature freezing ultracentrifugation, resuspended in PBS buffer, and stored in a low-temperature environment (-80°C).

[0024] 2. Preparation of Fallopian Tube Exosomes

[0025] Healthy adult female BALB / c mice were selected and killed by cervical dislocation after anesthesia. The uterus of the mice was taken. After opening the abdomen, ophthalmic forceps were used to clamp the connection between the uterus and the fallopian tube, and sharp scissors were used to cut the fallopian tube mesentery. Then, the connecting structure between the ovary and the fallopian tube was cut first, and then the connection between the uterus and the fallopian tube was cut to obtain the fallopian tube part. The fallopian tube was transferred to a surface dish containing PBS solution. The fat on the fallopian tube was removed with ophthalmic scissors and rinsed clean.

[0026] The fallopian tubes were dispersed using trypsin to obtain fallopian tube cells, and the fallopian tube cells were cultured in culture medium (DMEM / F12 cell culture medium containing 10% calf serum, epidermal growth factor concentration of 0.1 mg / ml, penicillin concentration of 0.4%), and the culture medium supernatant was collected. The culture medium was centrifuged at low temperature to obtain fallopian tube cell exosomes, and the fallopian tube cell exosomes were resuspended in PBS buffer and stored in a low temperature environment (-80°C).

[0027] 3. Preparation of sperm exosomes and fallopian tube exosomes drugs

[0028] The active ingredients of the drug are sperm exosomes, fallopian tube exosomes or a mixture of the two in a certain ratio:

[0029] 1) Sperm exosomes (about 6×10 12 units / mL);

[0030] 2) Fallopian tube exosomes (about 5.5×10 12 units / mL);

[0031] 3) 50% sperm exosomes in step 1) + 50% fallopian tube exosomes in step 2), about 5.8×10 13 Units / mL.

[0032] Application Example 1

[0033] Sperm exosomes and fallopian tube exosomes drugs promote endometrial cell vitality and reduce endometrial cell damage.

[0034] After culturing non-pathological endometrial stromal cells (stroma cells) for 24 hours, the three groups of drugs provided in Example 1 were added respectively, and the final concentration of exosomes was 10 9 Particles / mL level. The same cells and culture medium were cultured without exosomes (blank control). After 48h, the cells were sampled for analysis. The viability of stromal cells was detected by the PrestoBlue method (ThermoFisher Scientific). The test results are shown in Table 1.

[0035] Table 1

[0036]

[0037]

[0038] It can be seen from the experimental results that the three groups of drugs provided in Example 1 of the present invention all have the ability to increase the viability of uterine stromal cells.

[0039] Application Example 2

[0040] Sperm exosomes and fallopian tube exosomes promote the expression of endometrial stromal cell markers

[0041] After culturing non-pathological endometrial stromal cells for 24 h, the three drugs provided in Example 1 (final concentration of 10 9 The control group did not contain exosomes. After culturing in an incubator at 37°C and 5% CO2 for 24 h, the ALDH positivity and Ki67 positivity of stromal cells were detected by flow cytometry. The results are shown in Table 2.

[0042] Table 2

[0043] Group ALDH positive rate% SD p-value Ki67 positive rate % SD p-value 1) 20.58 0.62 ＞0.05 40.35 0.32 ＞0.05 2) 22.78 0.98 ＜0.05 39.01 0.48 ＜0.05 3) 19.65 1.23 ＜0.05 42.61 0.39 ＜0.05 Blank group 5.99 0.36 - 14.22 0.19 -

[0044] The results show that the drug provided in Example 1 of the present invention has a very strong effect of maintaining the stemness of matrix cells and stimulating proliferation.

[0045] Application Example 3

[0046] Application of sperm exosomes and fallopian tube exosomes in the treatment of premature ovarian failure

[0047] Female Wistar rats aged 9 to 10 weeks were selected, and rats with regular estrus were selected. Some rats were selected for modeling, and 50 mg / kg of Tripterygium wilfordii polyglycosides tablet suspension was gavage daily for 14 days. After the modeling, the rats were tested multiple times, and the rats with significantly higher body weight, significantly lower serum E2 level, and significantly higher serum LH level than the control group were selected. They were considered to have successfully modeled premature ovarian failure. Six rats were randomly divided into two groups (control group and experimental group), and 3 rats were randomly selected as blank control group.

[0048] After the modeling was completed, the rats in the experimental group were injected with the exosome mixture in Example 3) through the tail vein, once every two days, for a total of 3 injections; the control group was injected with an equal amount of normal saline intravenously. The arterial blood of the rats was taken 1 day, 4 days and 7 days after the modeling was successful to detect the concentrations of progesterone, follicle-stimulating hormone and luteinizing hormone. The test results are shown in Table 3.

[0049] Table 3

[0050]

[0051] Application Example 4

[0052] 1. Preparation of gel drugs

[0053] The sperm exosomes and fallopian tube exosomes mixture provided in 3) of Example 1 was mixed with a thermosensitive gel to prepare a gel drug. The content of exosomes in the gel was 10 9 Particles / mL level.

[0054] 2. The application of gel drugs in improving the probability of pregnancy

[0055] Several sexually mature female BALB / c mice with regular estrus were selected to establish a thin endometrium mouse model.

[0056] The mice were selected for surgery during the estrus period. The mice were anesthetized by intraperitoneal injection of chloral hydrate. The laparotomy was performed until the abdominal cavity was entered. A pink V-shaped uterus was visible behind the bladder. The uterus was separated to expose the vaginal and ovarian ends of the uterus. The needle of the syringe entered the uterine cavity on one side, and 0.1 mL of anhydrous ethanol was slowly injected into the vaginal end of the uterus. After the anhydrous ethanol was retained for 4 to 5 minutes, the ethanol was slowly aspirated and rinsed with saline 1 to 2 times. The other uterus was treated in the same way.

[0057] Several mice with successful modeling were selected and randomly divided into three groups (experimental group, control group and blank control group), with 4 mice in each group and repeated twice.

[0058] After the estrus cycle was determined, the experimental group was treated with 0.5 mL of gel drug infused into the uterine cavity at a fixed time every day for three days before estrus, the control group was treated with 0.5 mL of normal saline in the uterine cavity, and the blank control group was not treated at all. All mice were artificially inseminated during the estrus period, and the mice were tested for pregnancy one week after the artificial insemination. The test results are shown in Table 4.

[0059] Table 4

[0060] Average number of pregnancies Blank control group 1.3 Control group 2.7 Experimental Group 3.7

[0061] As shown in Table 4, the gel drug provided by the present invention can effectively increase the probability of pregnancy in female mice.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that the inventor of the present invention can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of sperm exosomes and fallopian tube cell exosomes in the preparation of drugs for the treatment of premature ovarian failure.

2. The use according to claim 1, characterized in that: The dosage form of the drug includes gel and solution.

3. The use according to claim 1, characterized in that: The method for preparing sperm exosomes comprises the following steps: Cut the testis washed with PBS solution into pieces, place the cut testis into a culture medium without exosomes, centrifuge to obtain sperm exosomes, resuspend the sperm exosomes in PBS buffer and store them in a low-temperature environment; The method for preparing fallopian tube cell exosomes comprises the following steps: The cleaned fallopian tubes were dispersed to obtain fallopian tube cells, the fallopian tube cells were cultured in a culture medium, the culture medium supernatant was collected, the culture medium was centrifuged at low temperature to obtain fallopian tube cell exosomes, and the fallopian tube cell exosomes were resuspended in PBS buffer and stored in a low temperature environment.

4. The use according to claim 1, characterized in that: In the drug, the dosage of sperm exosomes and / or fallopian tube cell exosomes is 1×10 8 ~1×10 13 units.

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