Application of inducing mammary epithelial cell exosome in preparation of medicine for promoting lactation

Abstract

According to the application of the induced mammary epithelial cell exosome in preparation of the medicine for promoting lactation, induced mammary epithelial cells are obtained through a specific induction factor RepSox, the exosome with efficient lactation promoting activity can be secreted, and compared with a traditional lactation promoting method, the application has the advantages of being accurate in action target spot, high in safety and small in side effect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an application of inducing mammary epithelial cell exosomes in the preparation of a drug for promoting lactation. Background Art

[0002] In animal husbandry, increasing milk production in livestock, such as dairy cows, is crucial for the dairy industry. Meanwhile, during human lactation, some women also face insufficient milk production, impacting both maternal and infant health. Traditional methods for promoting lactation include improved nutrition, massage, and the use of hormones, but these methods have limitations, such as the potential for side effects. Exosomes, as crucial vehicles for intercellular communication, are increasingly attracting attention for their role in mammary gland physiology. However, research and application of specific exosome-induced mammary epithelial cell exosomes to promote lactation remain underdeveloped. Summary of the Invention

[0003] In view of this, the present invention aims to propose the use of induced mammary epithelial cell exosomes in the preparation of lactation-promoting drugs. By inducing mammary epithelial cell exosomes, lactation can be efficiently promoted, and it can be used to increase animal milk production and assist human lactation milk secretion.

[0004] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0005] Application of induced mammary epithelial cell exosomes in the preparation of lactation-promoting drugs.

[0006] Preferably, the dosage of the exosomes induced in mammary epithelial cells is 10 μg-1000 μg.

[0007] Preferably, the induced mammary epithelial cell exosomes are obtained by inducing fibroblasts with RepSox.

[0008] Preferably, it is prepared by the following method: using 10 μM RepSox to induce fibroblasts for 8 days to obtain induced mammary epithelial cells, collecting the cell supernatant of the induced mammary epithelial cells, centrifuging at 300 × g for 10 minutes at 4 ° C to remove large particles such as cell debris, and then taking the supernatant, and then centrifuging at 2000 × g for 10 minutes to further remove impurities; then aspirating the supernatant, performing high-speed centrifugation at 10000 × g for 30 minutes, and removing impurities again to obtain a relatively pure supernatant; the treated cell culture supernatant is subjected to 140000 × g ultracentrifugation for 90 minutes. After the centrifugation is completed, the supernatant is removed, and the obtained precipitate is the induced mammary epithelial cell exosomes R-CiMECs-Exo.

[0009] The obtained induced mammary epithelial cell exosomes R-CiMECs-Exo were washed with PBS buffer to remove residual impurities; a small amount of PBS buffer was added to resuspend the exosome pellet so that it was evenly dispersed in the solution and stored at -80°C.

[0010] Technical solution for verifying the effect of the present invention:

[0011] 1) Construction of postpartum hypogalactia mouse model: The postpartum hypogalactia mouse model was established by gavage of bromocriptine to female mice 3 days after delivery, and the gavage was continued for 10 days.

[0012] 2) Evaluation of the efficacy and safety of R-CiMECs-Exo (induced mammary epithelial cell exosomes) in restoring lactation in a postpartum hypogalactia model: PKH26-labeled R-CiMECs-Exo were injected into the mammary glands of dams on the same day of model establishment to facilitate subsequent experimental observations. The effectiveness of the hypogalactia model and the therapeutic recovery achieved with R-CiMECs-Exo were verified by measuring maternal weight loss, net litter weight gain, hourly milk production, and immunofluorescence analysis of mammary function antibody expression in each group. The establishment of the hypogalactia model with bromocriptine and the safety of R-CiMECs-Exo were evaluated by measuring key organ indices (liver, kidney, ovary, and uterus) and internal organ structure. Mammary gland tissue sections from dams in each group 14 days after treatment confirmed the persistence of R-CiMECs-Exo in mammary tissue. Dissemination of R-CiMECs-Exo to other organs was also assessed to verify its targeted and safety profile.

[0013] Compared with the prior art, the use of the induced mammary epithelial cell exosomes in the preparation of a drug for promoting lactation has the following advantages:

[0014] (1) The present invention induces mammary epithelial cells through a specific induction factor (RepSox (10 μM)), which can secrete exosomes with high-efficiency lactation-promoting activity. Compared with traditional lactation-promoting methods, it has the advantages of precise target action, high safety, and few side effects.

[0015] (2) The present invention can not only be applied to animal husbandry to increase livestock milk production and increase the output and quality of dairy products, but also provide a new and potential method for solving the problem of insufficient milk secretion in humans during lactation, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Statistical graph of weight loss of female mice in each group; * represents difference analysis compared with the blank control group, and Δ represents difference analysis compared with the sham operation group. (*p<0.05, **p<0.01, ***p<0.001; Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001);

[0017] Figure 2 This is a statistical chart of the hourly milk production of each group of female mice;

[0018] Figure 3 Statistical graphs of mammary gland weight and mammary gland index of each group of mice; * represents the difference analysis compared with the blank control group, and Δ represents the difference analysis compared with the sham operation group. (*p<0.05, **p<0.01, ***p<0.001; Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001);

[0019] Figure 4 The overall and subcutaneous appearance of the mammary glands of the female mice in each group is shown;

[0020] Figure 5 Shown are the mammary gland structures of the female mice in each group (HE staining);

[0021] Figure 6 Immunofluorescence images of EPCAM, a marker protein in the mammary gland of mammary glands of maternal rats; scale bar in the left column = 200 μm, scale bar in the local magnification image = 40 μm; scale bar in the right column = 200 μm, scale bar in the local magnification image = 10 μm;

[0022] Figure 7 Immunofluorescence images of mammary gland marker protein PRLR in mammary gland of mammary gland of maternal rats; scale bar in left column = 200 μm, scale bar in partial magnification image = 40 μm; scale bar in right column = 200 μm, scale bar in partial magnification image = 10 μm;

[0023] Figure 8 Statistical graphs of liver weight and liver index of female mice in each group; * represents the difference compared with the blank control group, and Δ represents the difference compared with the sham operation group. (*p<0.05, **p<0.01, ***p<0.001; Δp<0.05, ΔΔp<0.01, ΔΔΔp<0.001, blank represents no significant difference)

[0024] Figure 9 HE staining of the liver structure of female mice in each group;

[0025] Figure 10 Statistical graph of kidney weight and kidney index of female mice in each group (no significant difference);

[0026] Figure 11 HE staining of the kidney structure of female mice in each group; scale bar = 200 μm;

[0027] Figure 12 The statistical graphs of ovarian weight and ovarian index of female mice in each group (no significant differences were found);

[0028] Figure 13 HE staining of the ovarian structure of female mice in each group; scale bar = 200 μm;

[0029] Figure 14 The following are statistical graphs of uterine weight and uterine index of female mice in each group (no significant difference was found);

[0030] Figure 15 HE staining of the uterine structure of the female mice in each group; scale bar = 200 μm;

[0031] Figure 16 Verification of the retention of R-CiMECs-Exo. Scale bar = 200 μm, scale bar for the magnified image = 10 μm. DETAILED DESCRIPTION

[0032] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0033] The present invention will be described in detail below with reference to the embodiments.

[0034] Induced mammary epithelial cell exosomes were prepared by the following method: fibroblasts were induced with 10 μM RepSox for 8 days to obtain induced mammary epithelial cells, and the cell supernatant of the induced mammary epithelial cells was collected and centrifuged at 300×g for 10 minutes at 4°C to remove large particles such as cell debris. The supernatant was then taken and centrifuged at 2000×g for 10 minutes to further remove impurities. The supernatant was then aspirated and subjected to high-speed centrifugation at 10,000×g for 30 minutes to remove impurities again to obtain a relatively pure supernatant. The treated cell culture supernatant was subjected to ultracentrifugation at 140,000×g for 90 minutes. After the centrifugation, the supernatant was removed, and the obtained precipitate was the induced mammary epithelial cell exosomes R-CiMECs-Exo.

[0035] The obtained induced mammary epithelial cell exosomes R-CiMECs-Exo were washed with PBS buffer to remove residual impurities; a small amount of PBS buffer (e.g., 100 μL) was added to resuspend the exosome pellet so that it was evenly dispersed in the solution and stored at -80°C.

[0036] The RepSox-induced fibroblasts include the following steps: fibroblasts were cultured at a rate of 5×10 5The cells were evenly seeded into a 60 mm cell culture dish at a density of 100 μg / mL. After the cells settled to the bottom and were evenly distributed on the dish, they were placed in a constant temperature cell culture incubator at 37°C and 5% CO2. When the cells adhered to the wall and expanded into a typical fibroblast morphology, the culture medium was replaced with RepSox induction medium. The medium was changed every other day for eight consecutive days. The fibroblasts were goat ear fringe fibroblasts.

[0037] RepSox induction medium includes knockout DMEM / F12, KSR, N2, Neurobasal, B27, Glutamine (100×) and RepSox (R), wherein the volume ratio of knockout DMEM / F12, KSR, N2, Neurobasal, B27, and Glutamine (100×) is 40:20:0.4:40:0.8:1, and the concentration of RepSox (R) is 10 μM.

[0038] Construction of postpartum hypogalactia mouse model:

[0039] 1. Drug Dosage Testing in Postpartum Lactation Mouse Model

[0040] In this study, bromocriptine produced by the Hungarian Geri Pharmaceutical Company was used to establish a postpartum hypogalactia mouse model. 32 H 40 BrN5O5) is a dopamine receptor agonist that inhibits the secretion of prolactin to treat premenstrual syndrome caused by excessive prolactin, physiological excessive lactation and other related diseases.

[0041] The bromocriptine dose required for this model was 1.6 mg / kg. To minimize the influence of external experimental manipulations on the experimental results, mice in the blank control group were gavaged with the corresponding volume of normal saline. According to calculations, the required bromocriptine dose for each modeled mouse was 0.32 mg / ml. Bromocriptine tablets were dissolved in normal saline overnight at 4°C in the dark to obtain a bromocriptine stock solution, which was then stored at 4°C. Before the experiment began, the bromocriptine solution was dissolved in normal saline according to the required dose and prepared immediately. Before preparing the solution, the solution was thoroughly mixed using a vortex to avoid concentration deviations.

[0042] 2. Detection of the Estrous Cycle of Female Mice

[0043] To minimize the impact of large discrepancies in the breeding schedule of female mice, avoid external influences caused by large differences in the timing of their births, and improve the success rate of mating between male and female mice, the estrous cycle of female mice is tested using vaginal smears. Female mice undergoing subsequent experiments are tested for estrous cycle at 9:00 AM daily. After securing the female mice, the vaginal opening of the female mice to be tested is fully exposed. A cotton swab moistened with saline in advance is slowly inserted into the female's vagina. After gently rotating it for one week, the vaginal mucus is smeared on a glass slide and allowed to dry. After fixation with 4% PFA for 30 minutes, vaginal smears are stained with methylene blue. When a large number of anucleated keratinized epithelial cells (be careful to distinguish white blood cells) are observed under a microscope, the female mice are in estrus and can be co-housed with male mice for subsequent experiments.

[0044] 3. Breeding of experimental mice and determination of experimental groups

[0045] After the above-mentioned estrous cycle testing of female mice, select female mice in estrus at a ratio of 3:1 male mice and cage them together at 3:00 PM on the first day. At 9:00 AM on the second day, observe the female mice's vaginal opening for the presence of a pale yellow or milky white vaginal plug formed by coagulated male semen. Female mice that successfully mate are housed individually in a cage under standard breeding conditions to await calving. Female mice that fail to mate can be tested again and used later. After mating, male and female mice are housed separately. Female mice with a calving time difference of no more than 12 hours and similar weight are selected for experimental use. To consider factors such as pup survival rate and weight gain, the number of pups per female is adjusted to 8. The pup litter weight (8 pups) should be kept consistent across groups as much as possible. Grouping is performed using a random number table as shown in the table below.

[0046] Table 1 Experimental groups

[0047]

[0048] 4. Construction of Postpartum Lactation Mouse Model

[0049] (1) Preparation of bromocriptine solution: Prepare bromocriptine solution of experimental working concentration according to the method described in the drug dosage test section of the postpartum hypogalactia mouse model.

[0050] (2) Oral gavage of the mother mouse: Select an oral gavage needle that is suitable for the length of the mother mouse's esophagus. Carefully separate the mother mouse from the pups. Grasp the skin between the mother mouse's ears with your thumb and index finger. Do not press too tightly on the mother mouse's esophagus and trachea, which may affect the subsequent entry of the oral gavage needle and the mother mouse's normal breathing. Use your middle finger and thenar to fix the skin on the mother mouse's back so that the mother mouse's back is pressed against the palm of your hand. Use your little finger to fix the mother mouse's tail to facilitate the subsequent entry of the oral gavage needle. Try to ensure that the mother mouse's head and body trunk remain in the same straight line. Through the above method, the mother mouse is completely fixed in the palm of your hand and cannot move at will.

[0051] Keep the immobilized female mouse upright during gavage to facilitate smooth entry of the drug solution into the stomach and prevent backflow into the lungs, potentially causing injury or even death. Insert the gavage needle into the female mouse's mouth. Once contact with the hard palate is established, align the needle with the centerline of the female mouse's torso to ensure smooth passage from the esophagus into the stomach. Slowly administer the drug, observing the female mouse's response throughout the process.

[0052] Modeling Time: Record the time of parturition on the experimental record card, marking this time as Day 0. The insufficiency modeling experiment began on Day 3 after the female mice adapted to parturition. Bromocriptine was administered orally to the female mice in the corresponding experimental group starting at 9:00 AM daily for 10 consecutive days (the bromocriptine solution was prepared according to the above concentration and stored in a refrigerator at 4°C, protected from light. It must be rewarmed to room temperature for 40 minutes before use).

[0053] Evaluation of the effect of R-CiMECs-Exo on restoring lactation function in postpartum hypogalactia mice and its therapeutic safety:

[0054] 1. Screening and verification of R-CiMECs-Exo injection volume and concentration

[0055] In this experiment, the effective dose of R-CiMECs-Exo at the cellular level was used as the basis, and concentration screening tests were performed at the animal level (mice) using the original concentration (10 μg), 10-fold dose (100 μg), and 50-fold dose (500 μg). The optimal concentration at the animal level was finally determined through subsequent lactation-related indicators, mammary gland structure, and mammary epithelial cell marker proteins.

[0056] 2. R-CiMECs-Exo treatment of galactagogue model mice

[0057] At 9:00 a.m. each day, the female mice in the corresponding experimental groups were gavaged with bromocriptine to establish the model, and at 5:00 p.m., the female mice were injected with R-CiMECs-Exo for treatment. The injection steps were as follows:

[0058] (1) For the female mice that need to be injected, shave the hair on the lower abdomen in advance to ensure that the skin around the inguinal nipples (the fourth and fifth pairs) is completely exposed.

[0059] (2) Fixing the female mouse. Unlike the upright position of the gavage female mouse, the female mouse needs to lie on its back to expose the inguinal nipple for subsequent injection.

[0060] (3) Use straight forceps to pinch the skin around the fourth pair of nipples of the female mouse, insert an insulin needle loaded with R-CiMECs-Exo of the corresponding group concentration under the nipple, and slowly push the drug.

[0061] (4) To maintain a stable state during injection, you can fix the insulin needle with your hands.

[0062] The sham operation group used the same injection method and injected the same volume of PBS as that of R-CiMECs-Exo into the same location of the mammary gland at the same time to ensure good control with the experimental group.

[0063] 3. Collection and analysis of indicators for each group

[0064] (1) Maternal weight loss: The initial weight was obtained on the third day after delivery, i.e., the first day of modeling (also the start of treatment). The final weight was obtained on the last day of modeling (also the last day of treatment). Maternal weight loss = final weight - initial weight.

[0065] (2) Net increase in pup litter weight: The initial litter weight (8 pups) was calculated on the third day after the mother gave birth, which was also the first day of modeling (and treatment). The final litter weight (8 pups) was calculated on the last day of modeling (and treatment). Net increase in pup litter weight = final litter weight - initial litter weight.

[0066] (3) Hourly milk production of maternal mice: During the period of maternal feeding, in order to ensure that the maternal milk production is sufficient for the normal development and growth of each group of pups, it is not convenient to accurately measure the maternal milk production per hour. By fixing the number of pups per litter (8) and the initial weight of the maternal mice to be similar, the statistical data and calculation formula shown in the table below can be used to calculate the maternal milk production per hour.

[0067] Table 2 Statistics of milk production per hour

[0068]

[0069] Formula: Hourly milk production of female rats = W3-W2+(W1-W2) / 4

[0070] The hourly milk production directly reflects the success of the lactation model and the recovery of the treatment at the result level, and is an important indicator in the early stage of this study.

[0071] Table 3 Comparison of weight loss of female mice in each group

[0072]

[0073] like Figure 1 The statistical graph of maternal weight loss in each group reflects the dynamic changes in maternal weight after each treatment. This indicator provides a preliminary understanding of the therapeutic effects of different concentrations of R-CiMECs-Exo on the hypogalactia model mice. The graph shows that the 100μg and 500μg groups were similar to the positive control group, demonstrating a positive therapeutic effect.

[0074] Table 4 Comparison of milk production per hour in each group of female mice

[0075]

[0076] like Figure 2 As shown in the figure, the single-hour milk production statistics of each group of female mice show that the milk production of the 100μg and 500μg groups is similar to that of the positive and blank control groups, showing a good therapeutic effect.

[0077] (4) Tissue and organ index: To verify the changes in mammary gland tissue in the different treatment groups during the modeling process and whether damage to the major organs of the female mice occurs, the mammary glands, liver, kidneys, ovaries, and uterus were dissected and weighed after the treatment of each group. The tissue and organ index was used to preliminarily reflect the therapeutic effect of R-CiMECs-Exo and to assess its safety. Formula: Tissue and organ index = organ (tissue) weight / body weight;

[0078] Table 5 Comparison of mammary gland weight and mammary gland index of female mice in each group

[0079]

[0080] like Figure 3 As shown in the figure, the mammary gland weight and mammary gland index of each group are statistically analyzed. From this figure, we can see that the mammary gland index of the treatment group is similar to that of the positive control group, indicating that the treatment effect is good.

[0081] like Figure 4 Figure 2 shows supine top views of female mice in each group. The sham-operated female mice exhibited smaller nipples and a thinner body, demonstrating successful model establishment. The R-CiMECs-Exo (100 μg and 500 μg) groups exhibited fuller nipples and a plump body, indicating that lactation had been restored after treatment, similar to that of the positive control group.

[0082] Table 6 Comparison of liver weight and liver index of female mice in each group

[0083]

[0084] Table 7 Comparison of kidney weight and kidney index of female rats in each group

[0085]

[0086] Table 8 Comparison of ovarian weight and ovarian index of female mice in each group

[0087]

[0088] Table 9 Comparison of uterine weight and uterine index of female mice in each group

[0089]

[0090]

[0091] 4. Sampling record and preparation

[0092] After the modeling treatment ended (day 10), the final day of monitoring was completed. All groups were fasted for 12 hours. The next day, the female mice were weighed and sacrificed by cervical dislocation. Chest and abdomen photographs were taken before and after hair removal, and the female mice's body shape and nipple morphology were recorded.

[0093] The fourth pair of mammary glands, liver, kidneys, uterus and ovaries of the female mice were removed in turn as required, photographed and recorded with a ruler, and the above organs (tissues) were accurately weighed. The data of each indicator were calculated and statistically analyzed according to the above formula, and the organs were fixed with tissue fixative and stored at -80°C.

[0094] Paraffin sections and HE staining of various organs (tissues) before and after modeling treatment

[0095] In order to intuitively reflect the internal structural changes of breast tissue and major functional organs (liver, kidney, uterus and ovary) in each group after the establishment of the agalactia model and R-CiMECs-Exo treatment, the paraffin sections were sectioned and HE stained, and then observed under a microscope.

[0096] The general steps for paraffin sectioning are as follows:

[0097] (1)Acquisition of materials

[0098] The breast tissue, liver, kidney, uterus and ovary of the blank control group, sham operation group, R-CiMECs-Exo (10 μg), R-CiMECs-Exo (100 μg), R-CiMECs-Exo (500 μg) and positive control group were collected and placed in tissue fixative.

[0099] (2) Fixed

[0100] After adding tissue fixative, quickly transfer to -80°C to fix the current state of the organ (tissue) and terminate the internal metabolic process.

[0101] (3) Washing

[0102] Use running water to rinse off the fixative remaining in the organ (tissue).

[0103] (5) Dehydration

[0104] Table 10 Paraffin section dehydration steps

[0105]

[0106] (1) Transparency

[0107] In a constant temperature box at 55-60℃, paraffin is allowed to penetrate into the tissue and replace the alcohol in the tissue.

[0108] (2) Wax dipping and embedding

[0109] Infiltrate the transparent tissue with melted paraffin and place in a paraffin melting box to keep warm. Pour the melted paraffin into a paraffin melter and embed the soaked tissue in the paraffin melter to complete the sample embedding.

[0110] (3) Slicing

[0111] The above-mentioned embedded tissue wax block was fixed on an automatic microtome, cut into thin slices, and wrinkles were ironed out with hot water. The slices were placed on a glass slide and then dried in a 45°C oven.

[0112] (4) Dewaxing and dyeing

[0113] To facilitate subsequent HE staining, the sections were dewaxed with xylene and then HE staining experiments were performed.

[0114] The general steps of HE staining are as follows:

[0115] Table 11 HE staining steps

[0116]

[0117] Place the slides in a clean bench and air dry them, then seal them with a coverslip and take photos under a microscope.

[0118] like Figure 5 Figure 2 shows HE staining of the mammary glands of rats in each group. The mammary ducts of the R-CiMECs-Exo (100 μg and 500 μg) groups were plump and contained milk, indicating that lactation had been restored after treatment, similar to that of the positive control group. The mammary ducts of the sham-operated group were shrunken, indicating successful modeling.

[0119] 6. Frozen sections and immunofluorescence staining of various organs (tissues) before and after modeling and treatment

[0120] In order to more accurately evaluate the effect of R-CiMECs-Exo on the recovery of mammary gland lactation in the galactagogue model mice through immunofluorescence experiments, we took advantage of the frozen sectioning technique, which can better preserve the immune activity of multiple antigens. Immunofluorescence experiments were performed directly after frozen sections to detect the expression of mammary gland function marker proteins. The following is a general step for frozen sectioning:

[0121] (1) Frozen section sampling: Frozen section sampling is performed at the same time as the paraffin section sampling mentioned above.

[0122] (2) Sample quick freezing: Place the sample in a quick freezing box and add OCT embedding medium to cover the sample. Slowly place the quick freezing box containing the sample into liquid nitrogen. When the quick freezing box comes into contact with liquid nitrogen, it will quickly vaporize and boil. At this time, do not continue to extend the quick freezing box into the liquid nitrogen to prevent the sample from falling directly from the quick freezing box into the liquid nitrogen due to the violent boiling of liquid nitrogen. Keep the quick freezing box still. When the sample is frozen into a block in about 10-20 seconds, the sample quick freezing step is completed.

[0123] (3) Sample fixation: Place the quick-frozen sample on a sample holder that has been pre-coated with OCT embedding gel. To allow the OCT embedding gel to penetrate the tissue, place the sample in a 4°C refrigerator for 5-10 minutes. Quick-freeze the sample holder, place the sample on the quick-frozen sample holder, cover the sample with OCT gel, and quick-freeze for 30 minutes.

[0124] (4) Cryosectioning: Maintain the cryosectioning chamber at -15°C to -20°C, place the processed sample in a cryostat, and cut the sample into 5-10 μm slices.

[0125] After the frozen sections are completed, immunofluorescence experiments are performed to verify the expression of mammary epithelial cell functional markers EPCAM and PRLR. Tissue-level immunofluorescence experiments are different from cell-level immunofluorescence experiments. The general steps are as follows:

[0126] (1) Preparation of frozen sections: Prepare the sections according to the above steps and store them at -80°C until ready for use. When starting the experiment, rewarm them at room temperature for 15-30 minutes. In order to effectively remove the OCT embedding agent, soak them in PBS for 10 minutes.

[0127] (2) Tissue delineation: In order to save the use of antibodies, the antibody incubation is accurately carried out at the corresponding position of the tissue to improve the effectiveness of the antibody. The antibody incubation range is delineated using a special tissue chemistry pen.

[0128] (3) Sample permeabilization: Use 0.5% TritonX-100 solution and incubate at room temperature for 30 minutes to enhance the permeability of tissue samples and allow the breast function marker protein antibody to fully penetrate into the tissue.

[0129] (4) Blocking nonspecific sites: Use PBS-Tween solution (containing 0.3 M glycine and 1% bovine serum albumin) and incubate at room temperature for 2 h to block nonspecific binding sites and enhance the specific binding of the antibody.

[0130] (5) Primary antibody binding: Dilute the primary antibody with PBS-Tween according to the recommended concentration for immunofluorescence in the primary antibody instructions, completely immerse the tissue sections, and incubate at 4°C for 12 h.

[0131] (6) Washing: Wash the sections after primary antibody binding with PBS-Tween, and wash three times at room temperature, each time for 5 minutes.

[0132] (7) Secondary antibody binding: Dilute the secondary antibody according to the recommended concentration in PBS-Tween under light-proof conditions, add the nuclear dye Hoechst, and incubate at room temperature for 2 h.

[0133] (8) Sealing: Remove PBS-Tween and add 5-10 μL of anti-fluorescence quenching sealing agent under light-proof conditions, remove bubbles, and complete the sealing.

[0134] like Figure 6 Figure 2 shows immunofluorescence staining of mammary gland marker proteins in each group. The expression of mammary epithelial cell marker EPCAM (green fluorescence) in the R-CiMECs-Exo (100 μg and 500 μg) groups was similar to that in the positive control group, indicating a good treatment effect. R-CiMECs-Exo (red fluorescence) was also observed around the glandular ducts.

[0135] like Figure 7 Figure 2 shows immunofluorescence staining of mammary gland marker proteins in each group. The expression of PRLR (green fluorescence), a functional protein in mammary epithelial cells, in the R-CiMECs-Exo (100 μg and 500 μg) groups was similar to that in the positive control group, indicating a good treatment effect. R-CiMECs-Exo (red fluorescence) was also observed around the glandular ducts.

[0136] like Figure 8-15 Shown are: statistical graphs of liver, kidney, ovary and uterus weight index and HE staining images, indicating that the major organs of the female mice were not affected after treatment, indicating the safety of this treatment method.

[0137] like Figure 16 As shown in the figure, the distribution of R-CiMECs-Exo in the main organs of the mother mice 14 days after treatment. It can be seen from the figure that red fluorescence is distributed in the mammary gland, and red fluorescence is found in the other organs, indicating that R-CiMECs-Exo has a certain degree of targeting and will not spread to other organs to cause safety risks.

[0138] 7. Data Analysis and Data Visualization

[0139] The data were statistically analyzed and visualized using GraphPad Prism 9 and IBM SPSS Statistics 19, and significance was analyzed using T-test or one-way analysis of variance (*p<0.05, **p<0.01, ***p<0.001).

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of induced mammary epithelial cell exosomes in the preparation of lactation-promoting drugs.

2. The use according to claim 1, characterized in that: The dosage of the exosomes induced in mammary epithelial cells is 10 μg-1000 μg.

3. The use according to claim 1, characterized in that: Induction of mammary epithelial cell exosomes was obtained by inducing RepSox into fibroblasts.

4. The use according to claim 3, characterized in that: Induced mammary epithelial cell exosomes R-CiMECs-Exo were prepared by the following method: fibroblasts were induced with 10 μM RepSox for 8 days to obtain induced mammary epithelial cells, and the cell supernatant of the induced mammary epithelial cells was collected and centrifuged at 300×g for 10 minutes at 4°C to remove large particles such as cell debris. The supernatant was then taken and centrifuged at 2000×g for 10 minutes to further remove impurities. The supernatant was then aspirated and subjected to high-speed centrifugation at 10,000×g for 30 minutes to remove impurities again to obtain a relatively pure supernatant. The treated cell culture supernatant was subjected to ultracentrifugation at 140,000×g for 90 minutes. After the centrifugation, the supernatant was removed, and the obtained precipitate was the induced mammary epithelial cell exosomes R-CiMECs-Exo.

5. The use according to claim 4, characterized in that: The obtained induced mammary epithelial cell exosomes R-CiMECs-Exo were washed with PBS buffer to remove residual impurities; a small amount of PBS buffer was added to resuspend the exosome pellet so that it was evenly dispersed in the solution and stored at -80°C.

Images