CN110946878B - Application of uterine cavity fluid source exosome in preparation of medicine and adjuvant therapy agent for treating infertility related diseases - Google Patents

Abstract

The invention relates to the technical field of medicines, and provides application of uterine cavity fluid-derived exosomes in preparation of medicines and adjuvant therapeutic agents for treating infertility-related diseases. Experiments prove that the exosome realizes the treatment or alleviation of the endometrial growth disorder diseases by promoting the increase of the thickness of an endometrium, enhancing the activity of endometrial cells, reducing the damage of endometrial cells, promoting the expression of VEGF, maintaining the dryness of endometrial stromal cells and stimulating the proliferation; the method realizes the treatment or alleviation of the diseases related to the maternal-fetal immune tolerance disorder by playing the role of immune tolerance, treating spontaneous abortion and improving the level of T-helper lymphocytes, and provides a new theoretical basis for the treatment of infertility. In addition, the exosome of the invention can effectively improve the development rate of fertilized eggs in vivo and fertilized eggs in vitro in a multiple mode, simultaneously improve the implantation rate and the birth rate of transplantation of the fertilization rate in vitro, and play a positive auxiliary role in the treatment of infertility.

Description

Application of uterine cavity fluid source exosome in preparation of medicine and adjuvant therapy agent for treating infertility related diseases

Technical Field

The invention belongs to the field of biological medicine, and relates to application of a uterine cavity fluid source exosome in preparation of a medicine for treating infertility related diseases and an auxiliary therapeutic agent, a preparation method of the exosome and a pharmaceutical composition containing the exosome.

Background

The damage of endometrium is mainly damage of endometrium basal layer, the main reason of the damage is related to uterine curettage in pregnancy, and the endometrium basal layer in pregnancy is loose and is easier to be damaged compared with normal endometrium. Based on the national conditions and the current situation that the artificial abortion rate is gradually increased, the occurrence of damaged endometrium is not negligible. Damage to the basement membrane layer of the endometrium, possibly resulting in damage or loss of endometrial stem cells; meanwhile, local infection and aseptic inflammation of the damaged intima can destroy niche microenvironment of stem cells, so that epithelial and interstitial cell regeneration and repair are hindered, blood vessel formation is blocked, compact fibrous tissues are formed, and finally, the uterine cavity only covers a small amount of intima, even no intima exists, the gland is atrophied, and the normal form and function of the uterine cavity are lost. Further, disturbances of the immune status in the uterine cavity further mediate the risk of immunological abortion of the endometrium.

The exosome isA bilayer membrane vesicle with a diameter of 30-100nm can carry various bioactive substances, including proteins, lipids and genetic substances such as mRNA, micro RNA (miRNA) and long non-coding RNA (lncRNA). The exosome has extremely high stability and can be stored for years under various freezing, refrigerating and unfreezing conditions; the source is wide, and the natural source exists in biological body fluid such as blood, urine, saliva, breast milk, cell culture medium and the like; meanwhile, the content of the extract is very rich, and each milliliter of plasma contains 10^ s⁸-13 exosomes. These advantages give the possibility of exosomes as pharmaceutically active ingredients.

Uterine cavity fluid is a physiological fluid present in the uterine cavity, in which exosomes are also present. Patent application 201811552777.3 discloses a method for extracting uterine cavity fluid exosome from pig living body, but the application of the exosome is not clear, and the application research of the exosome is not seen in the prior art.

Disclosure of Invention

The invention is carried out to solve the problems and aims to provide the application and the preparation method of the exosome from uterine cavity fluid and the pharmaceutical composition containing the exosome.

The first aspect of the invention provides the application of uterine cavity fluid source exosome in preparing medicines and adjuvant treatment agents for treating diseases related to infertility.

The exosome from uterine cavity fluid has the following characteristics: has a diameter of about 30-150nm, has lipid membrane, and contains genetic material such as protein, mRNA, and microRNA.

The infertility related diseases include endometrium growth disorder diseases and maternal and fetal immune tolerance disorder related diseases. The former includes endometrium damage, premature ovarian failure, sex hormone disorder, polycystic ovary syndrome, pelvic inflammatory disease, decreased endometrial receptivity, endometritis, endometrial polyp, intrauterine adhesion, endometrial gland reduction, endometrial fibrosis, amenorrhea, abnormal uterine bleeding, adenomyosis and endometriosis, reproductive system infection, hysteromyoma, etc.; the latter includes recurrent spontaneous abortion, threatened abortion or failure of assisted reproductive technology treatment, etc.

Therefore, the medicine for treating the diseases related to infertility mainly comprises a medicine for treating the diseases related to the growth disorder of endometrium or a medicine for treating the diseases related to the immunological tolerance disorder of mothers and fetuses.

Specifically, the medicament for treating the endometrial growth disorder disease is any one or more of a medicament for promoting the increase of the thickness of endometrium, a medicament for enhancing the activity of endometrial cells, a medicament for reducing damage of endometrial cells, a medicament for promoting the expression of VEGF, a medicament for maintaining the dryness of endometrial stromal cells and a medicament for stimulating the proliferation.

The medicine for treating the disease related to the maternal-fetal immune tolerance disorder is any one or combination of medicines for playing the immune tolerance effect, treating spontaneous abortion and improving the level of T-helper lymphocytes.

The adjuvant therapeutic agent for diseases related to infertility is a reagent composition for improving the normal development rate of fertilized eggs, and the composition can be a reagent for promoting the development and maturation of the fertilized eggs, blastocysts or blastocysts, and can also be a culture medium of animal fertilized eggs containing the exosomes or a culture medium for in vitro fertilization.

Agent for promoting maturation of fertilized egg, blastocyst or blastocyst, it is preferable to use an agent in the medium of fertilized egg to increase the rate of development of fertilized egg. The agent is administered orally, sublingually, subcutaneously, intravenously, intramuscularly, nasally, via follicles, vaginally to the female human or non-human animal parent as the source of the ova. In the case of using the composition of the present invention, the normal development rate of fertilized eggs is significantly improved and the conception rate is also improved, as compared with the case of not using the composition for improving the development rate of the present invention. The composition may also be added to a medium for in vitro maturation of ova from ova, an egg cryopreservative, and the like.

The culture medium for animal fertilized eggs or the culture medium for in vitro fertilization containing the exosome is not particularly limited, and may be any medium as long as it can culture mammalian fertilized eggs, and examples thereof include HTF medium, m-HTF medium, Ham medium, HamF-10 medium, MEM medium, 199 medium, BME medium, CMRL1066 medium, McCoy-5A medium, Weymouth medium, Trowallt-8 medium, LeibovitzL-15 medium, NCTC medium, William-E medium, KaneandFoote medium, Brinster medium, m-Tyrode medium, WK W medium, WK Whitten medium, TYH medium, Hoppes & Pitts medium, m-KRB medium, BO medium, T6 medium, GPM medium, OM, HECM medium, and modified medium thereof. A commercially available medium for in vitro fertilization such as CARDMEDIUM mouse in vitro fertilization medium or porcine embryo development culture medium (PZM-5) may also be used.

In a second aspect of the present invention, there is provided a method for preparing the uterine cavity fluid-derived exosome, comprising the steps of:

filtering uterine cavity liquid with 0.45 μm filter membrane, centrifuging at 4 deg.C for 10min at 1000g, and collecting supernatant; centrifuging the collected supernatant at 4 ℃ for 20min at 2000g, and collecting the supernatant; centrifuging the collected supernatant at 4 ℃ for 30min at 10000g, and collecting the supernatant; centrifuging 110000g of collected supernatant for 100min, discarding the supernatant, and resuspending the precipitate by using a phosphate buffer solution; centrifuging at 110000g for 100min again, discarding the supernatant, resuspending the precipitate with a small amount of phosphate buffer solution, and filtering with 0.45 μm filter membrane to obtain exosome.

In a third aspect of the present invention, a pharmaceutical composition is provided, which comprises an exosome derived from uterine cavity fluid and a pharmaceutically acceptable excipient. The exosomes are their major or even sole active ingredients. The auxiliary materials help the exosome to exert curative effect more stably, and the preparations can ensure the conformation integrity of the exosome disclosed by the invention and protect the active functional group of the exosome to prevent the exosome from being degraded.

In general, liquid formulations can be stable for at least three months at 2 ℃ to 8 ℃ and lyophilized formulations can be stable for at least six months at-30 ℃.

In the form of medicine, the pharmaceutical composition comprises tablets, pills, powder, injection, tincture, solution, extract, ointment and the like which are commonly used in the pharmaceutical field; also comprises a uterine mucosa administration preparation, such as an intrauterine drug release system and the like of films, suppositories, tablets, effervescent tablets, gels, stents and the like; mucosal absorption enhancers such as surfactants, chelating agents, fatty acids, fatty alcohols, fatty acid esters, cyclodextrin derivatives, protease inhibitors, and the like may also be included.

Similar to the use of exosomes, the present invention also provides the use of exosomes derived from uterine cavity fluid for the preparation of a product for treating and preventing infertility-related diseases, which product employs exosomes as disclosed above as active ingredients, and an effective amount of exosomes and/or composition is administered to a subject (human or animal) having an infertility-related disease, and also an effective amount of exosomes and/or composition is administered prophylactically to a healthy subject at risk of infertility.

The invention provides an adjuvant therapeutic agent for infertility-related diseases, which consists of exosomes derived from uterine cavity fluid and pharmaceutically acceptable auxiliary materials, or is an animal fertilized egg culture medium or a culture medium for in vitro fertilization containing the exosomes.

Preferably, in the adjuvant therapeutic agent, the mass-to-volume ratio of the exosomes is 0.02% (exosome mass means the mass of protein contained in exosomes), and other values are not particularly limited as long as the effect of improving the normal development rate of fertilized eggs can be obtained.

In the fifth aspect of the present invention, there is provided a kit for in vitro development or in vivo development for increasing the normal development rate of fertilized eggs, which is not particularly limited as long as it contains the exosomes as an active ingredient, and a kit further containing one or more kinds of in vitro culture media of mammalian fertilized eggs may be used.

Action and Effect of the invention

The exosome, the composition and the application are provided, the preparation process is simple, experiments prove that the exosome has a good treatment effect on infertility diseases such as endometrium growth disorder diseases and maternal-fetal immune tolerance disorder related diseases, and the development potential of fertilized eggs can be enhanced. The compound can be used alone or in combination with other related disease drugs, or used as an auxiliary product for treatment, and has wide clinical application prospect.

Drawings

FIG. 1 shows electron microscopy of exosomes.

FIG. 2 shows the size distribution of exosomes detected by NTA.

Detailed Description

The present invention will be described in detail below with reference to examples and the accompanying drawings. The following examples should not be construed as limiting the scope of the invention.

Example 1 preparation of uterine cavity fluid exosomes

Filtering uterine cavity fluid from healthy volunteers with 0.45 μm filter membrane, centrifuging at 4 deg.C for 10min at 1000g, and collecting supernatant; centrifuging the collected supernatant at 4 ℃ for 20min at 2000g, and collecting the supernatant; centrifuging the collected supernatant at 4 ℃ for 30min at 10000g, and collecting the supernatant; centrifuging the collected supernatant at 110000g for 100min, discarding the supernatant, and resuspending the precipitate by using a phosphate buffer solution; centrifuging at 110000g for 100min again, discarding the supernatant, resuspending the precipitate with a small amount of phosphate buffer solution, and filtering with 0.45 μm filter membrane to obtain exosome. Total exosome protein was assayed by the Bradford method (Bio-RadProteinAssayReagen). Exosomes were cryopreserved at-80 ℃. Exosomes were extracted from the sera of the same healthy volunteers in the same manner as control exosomes.

Example 2 Electron microscopy of exosomes

Carrying out electron microscope detection on the exosome according to the following steps: thawing the uterine cavity fluid exosome obtained in example 1, and then resuspending the thawed uterine cavity fluid exosome with a buffer solution to obtain a sample; dropping the sample on a copper net with a supporting film; uniformly mixing the dye solution and the sample to obtain a uniformly mixed solution, dripping the uniformly mixed solution on a copper net after the suspension on the copper net is slightly dried, and naturally drying after negative dyeing for 2 minutes; and observing under a transmission electron microscope. Bilayer membrane vesicles that fit the morphological features of exosomes 30-150nm were observed, as shown in figure 1.

Example 3 exosome size analysis

The method for detecting the particle size of the uterine cavity fluid exosome obtained in example 1 by using a NanoFCM Flow NanoAnalyzer (nanometer particle size analyzer) specifically comprises the following steps:

and (3) particle size detection: sampling a particle size standard product, a blank control product and a sample to be detected (uterine cavity fluid) under the same detection condition (the laser power and the scattering channel attenuation coefficient are completely the same); all samples were tested at lower injection pressures (sampling no more than 1.0 kPa); detecting a particle size standard under a proper condition (a scattered light signal of a silicon sphere with the smallest particle size is completely separated from the background, a signal of the largest silicon sphere is not saturated, and a saturation value of a detector is 3.6 k); detecting a buffer solution for resuspending a sample to be detected, and using the buffer solution as a blank control for deducting background particles; detecting a sample to be detected, wherein if the signal intensity of more than 20% of particles in the sample to be detected in a scattering channel reaches saturation, the current particle size standard is not the optimal selection for the particle size characterization of the sample; the particle size of the sample detected by the steps is mostly concentrated to about 74nm, which is in accordance with the range of 30-150nm of the diameter of the exosome, and the detected sample is the exosome, as shown in fig. 2.

Statistical calculations in the examples described below calculate p-values based on different statistical models of the two-set comparison, the multiple-set comparison, and the different selection software SPSS 22.0 versions of the rate comparison.

Example 4 treatment of endometrial injury with exosomes

The thickness of the endometrium of the patient is less than 8mm due to factors such as artificial abortion, uterine curettage, infection and the like, and the clinical diagnosis is that the endometrium is thin. The administration to the patient is therapeutically ineffective against infection and the like. A composition comprising 1% by mass/volume of the exosome as the active ingredient prepared in example 1 was administered. One or more intrauterine infusions of 5ml of the composition are used to promote endometrial thickness increase.

Example 5 exosomes promote endometrial cell viability, reduce endometrial cell damage, increase VEGF expression

After culturing non-pathological endometrial stromal cells (stroma cells) for 24 hours, uterine cavity fluid exosomes and control exosomes as described in example 1 were added to a final concentration of 0.02% by mass/volume relative to the total volume of the culture medium. The same medium was cultured without exosomes (blank). After 48 hours of treatment, the media and cells were sampled for analysis.

Stromal cells were tested for cell viability by the Prestoblue method (Thermo Fisher Scientific). Measurements were made after 48 hours of treatment. Values are expressed as the mean value after normalization of the control (table 1).

TABLE 1 relative cell viability

Group of	Mean value of	SD	p value
				Blank control (Medium only)	100	5.61
Control exosomes as described in example 1 0.02% (w/v medium)	97.51	4.67	p＞0.05
				Uterine cavity fluid exosomes described in example 1 0.02% (w/v medium)	208.56	18.53	p＜0.05

And (4) conclusion: the exosome provided by the invention has the capacity of increasing the viability of uterine stromal cells, so that the exosome becomes an exosome for enhancing endometrial proliferation.

Cell damage at the stromal cell level was further assessed.

The cell damage was measured by colorimetry using a lactate dehydrogenase assay, enabling quantification of cell damage based on measurement of lactate dehydrogenase activity in damaged cells in the medium. The increase in cell membrane damage and cell lysis leads to an increase in lactate dehydrogenase activity, which is proportional to the number of lysed cells. Lactate dehydrogenase activity measurements were performed in the culture medium 48 hours after exosome treatment (table 2).

TABLE 2 relative cellular injury

Group of	Mean value of	SD	p value
				Blank control (Medium only)	100	11.64
Control exosomes as described in example 1 0.02% (w/v medium)	102.36	8.27	P＞0.05
				Uterine cavity fluid exosomes described in example 1 0.02% (w/v medium)	35.71	6.41	p＜0.05

And (4) conclusion: the uterine cavity fluid exosome has the capacity of reducing membrane damage, so that the uterine cavity fluid exosome becomes the exosome with activity on strengthening stromal cells.

The potential inhibitory effect of exosomes on endometrial stromal cell VEGF was further investigated. The results are presented in table 3. 48 hours after exosome treatment, ELISA measurements of VEGF concentrations were performed in culture medium.

Table 3: analysis of VEGF expression

Group of	VEGF(pg/ml)	SD	p value
				Blank control (Medium only)	356.51	41.64
Control exosomes as described in example 1 0.02% (w/v medium)	341.67	31.55	P＞0.05
				Uterine cavity fluid exosomes 0.02% (w/v medium) as described in example 1	845.22	101.71	p＜0.05

And (4) conclusion: the uterine cavity fluid exosome has the function of promoting VEGF expression and has the function of enhancing endometrial angiogenesis.

Example 6 exosomes promote endometrial stromal cell marker expression

After culturing non-pathological endometrial stromal cells (stromacell) for 24 hours, the uterine cavity fluid exosomes and control exosomes of example 1 as active ingredients were dissolved in a culture solution (final concentration 0.02% by mass/volume), the control group contained no exosomes and 5% CO at 37 ℃ in a controlled manner₂After culturing for 24 hours in the incubator under the conditions, the ALDH positive rate and Ki67 positive rate of the stromal cells were measured by flow cytometry, and the results are shown in tables 4 and 5.

Table 4: ALDH positive rate

Group of	ALDH positive rate%	SD	p value
				Blank control (Medium only)	7.35	0.45
Control exosomes as described in example 1 0.02% (w/v medium)	6.98	0.66	P＞0.05
				Uterine cavity fluid exosomes 0.02% (w/v medium) as described in example 1	21.21	1.02	p＜0.05

Table 5: ki67 positive rate

Group of	Ki67 Positive Rate%	SD	p value
				Blank control (Medium only)	12.57	0.22
Control exosomes as described in example 1 0.02% (w/v medium)	11.34	0.19	P＞0.05
				Uterine cavity fluid exosomes 0.02% (w/v medium) as described in example 1	35.14	0.41	p＜0.05

The results show that the uterine cavity fluid exosome has very strong effects of maintaining the dryness of stromal cells and stimulating proliferation.

Example 7 Effect of exosomes on decidua immune cells

Dendritic cell DC (CD1c positive) was isolated from decidua tissue terminating pregnancy for human non-medical reasons, and the isolation and screening methods were the same as those described in the publications (Guo P F, et al. blood,2010,116(12): 2061-. The results are shown in tables 6 and 7, showing that exosomes can significantly increase IL-10 secretion levels without increasing TNF α levels. These results demonstrate that uterine cavity fluid exosomes can exert an immune tolerizing effect by DCs.

Table 6: level of IL-10 secretion

Group of	IL-10pg/ml	SD	p value
				Blank control (Medium only)	87.56	10.55
Control exosomes as described in example 1 0.02% (w/v medium)	69.46	5.57	P＞0.05
				Uterine cavity fluid exosomes described in example 1 0.02% (w/v medium)	314.34	35.56	p＜0.05
LPS	290.16	30.66	p＜0.05

Table 7: TNF alpha secretion levels

Group of	TNFαpg/ml	SD	p value
				Blank control (Medium only))	18.25	3.12
Control exosomes as described in example 1 0.02% (w/v medium)	16.57	2.14	P＞0.05
				Uterine cavity fluid exosomes described in example 1 0.02% (w/v medium)	15.44	3.32	p＞0.05
LPS	1856.67	325.2	p＜0.05

Example 8 therapeutic Effect of exosomes on spontaneous abortion model

CBA/J female mice and DBA/2J male mice are used for establishing a stress abortion model which is a classic maternal-fetal immune tolerance disorder research model, and establishing methods, experimental methods and observation time point equivalent documents (Blois S M, et al. Nature Medicine,2007,13(12): 1450-. Before the CBA/J female mice are combined into a cage, the female mice are divided into a negative control group, a stress pressure + control exosome group and an exosome treatment group. 2mL of PBS containing 0.02% of the uterine cavity fluid exosome prepared in example 1 of the present invention was added into the uterine cavity, and the mixture was combined after 3 days. Mice were housed immediately after vaginal emboli were confirmed to be pregnant.

The experimental results show (table 8) that the abortion rate of the treatment group is significantly lower than that of the stress pressure abortion group, which indicates that the uterine cavity fluid exosome has good treatment effect.

Table 8: embryo absorption rate (fluid yield)

Example 9 Effect of exosomes on T helper cells

The periaortic lymph nodes of mice in the control group, stress-stressed group and exosome-treated group described in example 8 were isolated and tested for Foxp3 positive T helper lymphocyte levels. The isolation and detection methods are described in the literature (Kim B J, et al.. Proceedings of the National Academy of Sciences,2015,112(5): 1559-.

Table 9: foxp3⁺Helper T cell%

Group of	Foxp3+％	SD	p value
				Negative control group	25.4	2.31
Stress group	6.09	0.25	P＞0.05
				Stress group + control exosomes	5.14	0.16	P＞0.05
Stress pressure group + uterine cavity fluid exosome	19.9	1.26	p＜0.05

Example 10 preparation of uterine Cavity fluid exosome in healthy mice

A method for extracting uterine cavity fluid from a mouse (C57 mouse) living body comprises the following steps: carrying out pretreatment such as anesthesia on the mice; after the mouse is anesthetized, a longitudinal incision with the length of about 2cm in the middle of the abdomen is taken and enters the abdomen, a 0.5cm longitudinal incision is made at the 1/3 middle lower part of the uterus, a small opening is cut at the joint of the oviduct and the uterus by operation, one end of a embryo washing tube is inserted into the small opening and air is injected, so that the embryo washing tube is fixed in the uterine cavity; injecting a sterile buffer solution into the position of the cervix, which is far away from the end of the oviduct and is close to the uterine body, slightly squeezing the uterus by hands to enable the buffer solution to flow to the side of the oviduct so that a uterine flushing solution flows out from the opening of the embryo washing tube to obtain uterine cavity liquid; the uterus on the other side is treated in the same way to obtain the uterine cavity fluid. The collected uterine cavity fluid was stored at-80 ℃ or exosomes were enriched by centrifugation, and total exosome Protein amount was measured by Bradford Assay (Bio-Rad Protein Assay Reagent). Exosomes were lyophilized and stored at-80 ℃. The control exosomes were extracted from peripheral blood of C57 mice by centrifugation.

Example 11 mouse model of mouse uterine cavity fluid exosome treatment

Constructing an animal endometrium injury model (C57 mouse), dividing 8-week-old female mice into groups, and constructing the endometrium injury model by adopting a double (infection + mechanical) injury method for 10 mice in each group, namely after the mice are anesthetized, taking a longitudinal incision with the length of about 2cm in the middle of the lower abdomen into the abdomen, making a 0.5cm longitudinal incision at the position 1/3 in the middle and lower uterus, scratching the upper uterine cavity by adopting an endometrium spatula, stopping uterine curettage when the concave-convex feeling of the spatula entering and exiting the uterus disappears and the four walls feel rough, reserving lipopolysaccharide cotton threads in the uterine cavity after uterine curettage, suturing the abdominal incision, and taking out the lipopolysaccharide cotton threads after 48 hours. After completion of the modeling, 2mL of each of the exosomes-PBS containing 0.02% of the inventive sample 10 was injected into the uterine cavity. A blank control group (sham operation group) was set, and only a saline (model) group was injected; model + control exosome-treated group (control group); model + uterine cavity fluid exosomes (treatment group). Mice were mated with male mice 3 weeks after the mice were in estrus. Materials were taken 1 month later for evaluation of endometrial histology by HE staining and Masson staining, and mice were evaluated 3 months later for pregnancy results. As a result: tissue functional assessment at 1 month post-surgery showed a significant reduction in the degree of fibrosis in the exosome group compared to each control group; the number of exosome glands was higher than each control group. The pregnancy results evaluation showed that the pregnancy rate was higher in the exosome group than in the control group, and the results are shown in table 10.

TABLE 10 pregnancy results analysis in groups of mice

Group of	Rate of pregnancy	p value (vs. model group)
			Artificial operation group	100％
Model set	20％
			Model group + control exosomes	20％
Model group + uterine cavity exosome	80％	p＜0.05

Example 12 exosomes effect on fertilized eggs

Female mice of C57BL/6J line (21-27 weeks old, 20.0-24.5g in body weight) were used as female mice for collecting fertilized eggs. Male mice of the C57BL/6J line (32-38 weeks old, 31.0-35.5g in body weight) were used as male mice for the mating. According to a conventional method for inducing excessive ovulation, 5U (unit) of equine chorionic gonadotropin is intraperitoneally administered to each female mouse, and 45-48 hours later, 5U (unit) of human chorionic gonadotropin is intraperitoneally administered to each female mouse. Each female mouse was mated with each of the male mice described above immediately after administration of human chorionic gonadotropin.

The next day, it was determined whether or not the mated female mice had a milky resin-like vaginal plug, and the oviducts were collected from the female mice for which the vaginal plug was confirmed. The collected oviducts were left to stand in physiological saline (0.9% (w/v) NaCl) for about 15 minutes, and then the oviducts were transferred to M16 medium supplemented with about 300. mu.g/mL hyaluronidase (manufactured by Sigma Aldrich) to treat and remove cumulus cells. The oviduct is cut open, the fertilized egg is taken out,in CO₂After leaving the incubator at 37 ℃ for 5 to 10 minutes, fertilized eggs from which cumulus cells have been separated are collected and washed with M16 medium to which hyaluronidase has not been added, and hyaluronidase is removed. The resulting fertilized egg from which cumulus cells have been removed is left to stand at 37 ℃ in CO₂And (4) the heat preservation box.

Further, 200. mu.L of a drop of M16 medium was prepared in a 35mm petri dish, and mineral oil (manufactured by Sigma Aldrich) was laminated thereto, and the uterine cavity fluid-derived exosomes and the control exosomes described in example 1 were added to the resulting concentration of 0.02% by mass/volume. Standing at 37 deg.C in CO₂And (4) the heat preservation box. Transferring 40 fertilized eggs from which cumulus cells were removed to the drip in CO₂The in vitro culture was carried out in a incubator at 37 ℃. The blank control group did not add exosomes.

After 24 hours, 48 hours, 72 hours, and 96 hours from the start of in vitro culture in the spot (0 hour), the development stage of each embryo was determined by observation with a stereomicroscope, and the number of embryos that normally develop and the change in the development rate were calculated. Specifically, as embryos normally developing at each stage, the number of eggs at the 2-cell stage 24 hours after the start of culture, the number of eggs at the 3-cell stage, 4-cell stage and 8-cell stage 48 hours after the start of culture, the number of morula and blastocyst 72 hours after the start of culture, and the number of blastocyst 96 hours after the start of culture were calculated. The number of embryos at each stage is shown in Table 11, and the development rate at which the number of fertilized eggs (0 hour) is 100% is shown in Table 12. The evaluation of the development rate was performed by taking the number of fertilized eggs (0 hour) as 100%, and calculating the ratio of the number of embryos normally developing 24 hours later, 48 hours later, 72 hours later, and 96 hours later as the normal development rate. In addition, since natural mating is performed, the number of collected fertilized eggs (0 hour) includes some unfertilized eggs, and even if the colonies of the fertilized eggs (0 hour) collected in the same experiment are colonies of the fertilized eggs (0 hour), the content of unfertilized eggs may occasionally change in the colonies of the fertilized eggs (0 hour) distributed between the conditioned groups. The development rate calculated by taking the number of embryos at the 2-cell stage 24 hours after fertilization as 100% was calculated to exclude the influence of the content of unfertilized eggs which occasionally change among the condition groups. That is, in order to calculate the development rate based on the number of eggs for which embryo development was surely started, the number of eggs at the 2-cell stage after 24 hours was set to 100%, and the development rate in this case is shown in table 13. Statistical analysis of each result was performed by the chi-square test with statistically significant differences when p <0.05 (. sup.), p <0.01 (. sup. sup.). Since the control exosomes relatively reduced embryo development, the media groups without any exosomes were compared statistically.

TABLE 11 number of developing embryos of each group

Table 12 shows the developmental rate (%)

Table 13 shows the developmental rate (%)

(vs. media-only chi square assay: p <0.01, p <0.05)

The results showed that the development rate in the case of adding the uterine cavity fluid exosomes was improved in each group of exosomes when the number of fertilized eggs (0 hour) was 100%, compared to the blank group to which no exosomes were added.

Example 13 exosomes effect on fertilized eggs in vitro

5U (unit) of equine chorionic gonadotropin (Malus chorionic gonadotropin) was intraperitoneally administered to C57BL/6J female mice (3.9-4.0 weeks old) and 45-48 hours later, 5U (unit) of human chorionic gonadotropin (hCG) was intraperitoneally administered, and ASKA pharmaceutical corporation was usedBirth), inducing its excessive ovulation. After 15 hours of administration of human chorionic gonadotropin, the oviducts were recovered by laparotomy. In mineral oil, the enlarged part of the oviduct was dissected with a dissecting needle, and the ovum was recovered into drops of mHTF medium. Sperm were recovered from the tail of the epididymis of C57BL/6J male mice in mHTF medium at 37 deg.C with 5% CO₂Culturing for 40 min to 1 hr under the condition to capacitate the sperm. The collected ovum was dropped into a medium containing the collected ovum, and 2. mu.l to 4. mu.l of a sperm-containing mHTF medium was added thereto for insemination, and the resulting mixture was cultured at 37 ℃ under 5% CO 2. After 4 to 6 hours after fertilization, cumulus cells and sperm are removed by washing the fertilized eggs with KSOM medium. Temporarily cultured in KSOM or mWM at 37 deg.C and 5% CO₂Until the complete fertilized egg is recovered.

To each of the drops (laminated with mineral oil (manufactured by sigma aldrich) formed by adding no exosome or the uterine cavity fluid-derived exosome of example 1 and the control exosome and finally 100 μ l of the culture medium at a concentration of 0.02% by mass/volume, 25 fertilized eggs were transferred, and 200 fertilized eggs were counted for each treatment group. Thereafter, the culture was performed. The number of embryos at 2-cell stage 24 hours after the recovery of the ovum and the number of blastocysts 96 hours after the recovery of the ovum were measured. Furthermore, since the fertilized egg obtained from the in vitro fertilization develops slightly later, the number of blastocysts 120 hours after the egg is collected was also measured. The embryo development rate at each culture time was calculated based on the number of embryos at the 2-cell stage 24 hours after fertilization as 100%. The results are shown in tables 14 and 15.

TABLE 14 number of developing embryos of each group

Table 15 shows the developmental rate (%)

(vs. media-only chi square assay: p <0.01, p <0.05)

The results showed that the development rate of each group of exosomes was improved when the number of fertilized eggs (24 hours) was 100%, compared with the blank to which exosomes had not been added, when exosomes derived from uterine cavity fluid were added.

Example 14 Effect of exosomes on in vitro fertilized egg transplantation

Each group of embryos reaching blastocyst stage in example 12 was subjected to embryo transfer. The recipient mice were C57BL/6J female mice 6 to 10 weeks old, male mice of C57BL/6J female mice ligated with vas deferens on the day before the day of egg collection were used for the mating, and individuals of vaginal emboli were confirmed on the next day. General anesthesia was performed with pentobarbital (somnopentyl) anesthetic, and the uterus was exposed by incision of the back. The uterus was fixed with forceps, and the oviduct junction was opened with a 30G injection, and the embryo was transplanted into the uterus by inserting a glass capillary tube that has attracted the blastocyst. After implantation, the uterus was carefully returned to the body and the posterior peritoneal membrane and skin were sutured closed. At the time of transplantation, blastocysts obtained from each group of mice were individually transplanted into recipient mice. The second day of egg collection was set as the first day, and cesarean section was performed on the nineteenth day. Recipient mice were euthanized and opened abdominally, the uterus removed and the fetuses removed. The implantation rate was calculated by the number of implantation traces/number of embryo transfer (Table 16), and the litter size/number of embryo transfer was calculated by the number of litter size/number of embryo transfer (Table 17).

TABLE 16 implantation rates of the respective groups

Group of	Number of bed traces	Number of embryo transfer	The implantation rate%
				Culture medium only	41	80	51.25
Control exosomes	36	78	46.15
				Uterine cavity fluid exosomes described in example 1 0.02% (w/v medium)	119	136	87.50**

(vs. media-only chi square assay: p <0.01, p <0.05)

TABLE 17 farrowing rates for each group

(vs. media-only chi square assay: p <0.01, p <0.05)

The results showed that the implantation rate and farrowing rate were improved in each group of exosomes with addition of uterine cavity fluid exosomes compared to the control without addition of exosomes.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (5)

1. The application of the uterine cavity fluid source exosome in preparing medicines for treating infertility related diseases is characterized in that the medicines for treating the infertility related diseases are medicines for treating endometrial injury or medicines for treating maternal and fetal immune tolerance disorder related diseases.

2. Use of uterine cavity fluid-derived exosomes according to claim 1 in the preparation of a medicament for the treatment of infertility-related diseases, characterized in that:

wherein the medicament for treating the endometrial injury is any one or more of a combination of a medicament for promoting the increase of the thickness of endometrium, a medicament for enhancing the activity of endometrial cells, a medicament for reducing the injury of endometrial cells, a medicament for promoting the expression of VEGF, a medicament for maintaining the dryness of endometrial stromal cells and a medicament for stimulating the proliferation;

the medicine for treating the disease related to the maternal-fetal immune tolerance disorder is any one or combination of medicines for maintaining or enhancing immune tolerance, medicines for treating spontaneous abortion and medicines for improving the level of T-helper lymphocytes.

3. Use of uterine cavity fluid-derived exosomes according to any one of claims 1-2 in the preparation of a medicament for treating infertility-related diseases, characterized in that:

wherein the preparation method of the exosome comprises the following steps: filtering the uterine cavity liquid with 0.45 μm filter membrane, sequentially passing the liquid through

Centrifuging at 4 deg.C for 10min at 1000 g; centrifuging at 2000g for 20min at 4 ℃; centrifuging at 4 deg.C at 10000g for 30 min; centrifuging 110000g for 90min, discarding the supernatant, and resuspending the precipitate with phosphate buffer; and centrifuging the mixture for 90min at 110000g again, discarding the supernatant, resuspending the precipitate by using a small amount of phosphate buffer solution, and filtering the precipitate by using a 0.45-micrometer filter membrane to obtain the exosome.

4. Use of uterine cavity fluid-derived exosomes according to any one of claims 1-2 in the preparation of a medicament for treating infertility-related diseases, characterized in that:

wherein the drug is an exosome as the only active ingredient or a pharmaceutical composition comprising the exosome.

5. Use of uterine cavity fluid-derived exosomes according to claim 4 in the preparation of a medicament for treating infertility-related diseases, characterized in that:

wherein, the pharmaceutical composition is a solution.

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