Preparation method and application of engineered exosome derived from placenta mesenchymal stem cells

Abstract

The invention provides a preparation method and application of an engineered exosome derived from placenta mesenchymal stem cells, and belongs to the field of biological medicine. According to the invention, the placenta mesenchymal stem cells transfected with the miRNA virus vector are constructed, the engineered exosome is prepared in a culture system under an anoxic condition and stimulated by estrogen, and the invention also provides application of the placenta mesenchymal stem cells and the exosome in preparation of medicines for treating ovarian insufficiency, female climacteric syndrome and the like. The miRNA transfected placenta mesenchymal stem cells and exosomes provided by the invention have good effects in the aspects of treating ovarian insufficiency, female climacteric syndrome and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a preparation method and application of engineered exosomes derived from placental mesenchymal stem cells. Background Art

[0002] Premature ovarian insufficiency (POI) refers to ovarian dysfunction in women before the age of 40, mainly manifested by abnormal menstruation (amenorrhea, oligomenorrhea), follicle-stimulating hormone (FSH)>25U / L, and fluctuating estrogen levels. The incidence of POI is 1% to 4%, and the analysis of the global incidence of POI shows it is 3.7%. Ovarian insufficiency may lead to amenorrhea and infertility in women. It is also accompanied by a series of low estrogen symptoms such as hot flashes, sweating, depression or anxiety, sleep disorders, decreased attention or memory, etc., which seriously affect women's mental health and quality of life. In addition, ovarian insufficiency also increases women's risk of osteoporosis, cardiovascular disease and Alzheimer's disease.

[0003] Menopausal syndrome refers to a group of symptoms that occur before and after menopause (45-55 years old), which are caused by fluctuations or reductions in sex hormones due to ovarian dysfunction, leading to vasomotor and autonomic nervous system disorders and mental and neurological abnormalities, with menstrual disorders, hot flashes, sweating, insomnia, abnormal emotions, bone, joint and muscle pain as the main manifestations. It is estimated that by 2030, the number of menopausal women in my country will reach 210 million, accounting for about 1 / 7 of the total menopausal population in the world, and more than 85% of women may experience menopausal symptoms to varying degrees.

[0004] The most commonly used treatment for ovarian insufficiency is hormone replacement therapy (HRT). Although HRT therapy has a certain effect on alleviating clinical symptoms, it cannot fundamentally repair damaged ovaries and restore ovarian function, and it has no therapeutic effect on infertility. In addition, long-term HRT treatment may increase the risk of breast cancer and ovarian cancer. Stem cells and stem cell derivatives, including mesenchymal stem cell suspension injection therapy and stem cell exosomes, have made certain research progress in recent years, but the repair effects of different stem cells vary significantly. After single-cell suspension injection, cells are easily absorbed and diffused, and cannot achieve uniform distribution, resulting in low repair efficiency. Exosomes will change with changes in the tissue microenvironment, and the treatment effect is not stable. Therefore, there is an urgent need to develop a new drug for the treatment of ovarian insufficiency. Summary of the invention

[0005] In view of this, the present invention aims to overcome the defects in the prior art and provide a safe and efficient method for engineering mesenchymal stem cell exosomes.

[0006] The present invention also aims to provide an application of engineered exosomes derived from placental mesenchymal stem cells.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0008] The engineered exosomes are derived from placental mesenchymal stem cells, wherein the engineered exosomes are prepared from placental mesenchymal stem cells transfected with miRNA viral vectors.

[0009] The method for preparing exosomes comprises the following steps:

[0010] (1) Construction of miRNA viral vector;

[0011] (2) preparing placental mesenchymal stem cells transfected with miRNA viral vectors;

[0012] (3) Under hypoxic conditions and estrogen stimulation, engineered exosomes are prepared using the placental mesenchymal stem cells transfected in step (2).

[0013] Preferably, the miRNA viral vector described in step (1) is any one of miRNA-133b, miR-144-5p, miR-146, 132-3p, and miRNA-21.

[0014] Preferably, the step of preparing engineered exosomes in step (3) is:

[0015] a) P3 placental mesenchymal stem cells were obtained 48 hours after miRNA virus transfection and enzymatic digestion was performed. 6 The cells were seeded into a 10 cm culture dish, 8 ml of mesenchymal stem cell complete medium was added, and the dish was placed in a 37°C, 5% CO2 incubator to allow the cells to adhere to the wall;

[0016] b) the cells were cultured in a 37°C, 5% CO2 incubator until the cell confluence was no less than 80%, and the culture supernatant was collected;

[0017] c) washing with PBS three times, adding 8 mL of DMEM-high glucose basal medium without FBS, placing in an incubator at 37° C., containing 2% O2 and 5% CO2, and culturing for 48 hours under hypoxic conditions and estrogen stimulation to allow the transfected placental mesenchymal stem cells to fully release exosomes;

[0018] d) The cell supernatant was aspirated and placed in a 50 ml centrifuge tube for ultracentrifugation to obtain an exosome suspension, which was then stored at -80°C.

[0019] A drug for treating ovarian insufficiency, comprising any one or a combination of placental mesenchymal stem cells transfected by a miRNA viral vector and engineered exosomes as claimed in claim 1.

[0020] The application of the placental mesenchymal stem cells transfected with the miRNA viral vector in the preparation of a drug for ovarian insufficiency. The application of the engineered exosomes derived from the placental mesenchymal stem cells in the preparation of a drug for ovarian insufficiency.

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

[0022] The present invention provides a safe and efficient method for preparing engineered placental mesenchymal stem cell exosomes, and establishes a technical platform for the production of engineered exosomes.

[0023] The placental mesenchymal stem cells transfected with miRNA constructed by the present invention can express estrogen receptor (ER), while mesenchymal stem cells derived from bone marrow, fat and umbilical cord do not express estrogen receptor (ER). Therefore, compared with mesenchymal stem cells derived from bone marrow, fat and umbilical cord, placental mesenchymal stem cells can better respond to the stimulation of female estrogen.

[0024] The engineered exosomes prepared by the present invention can secrete a large amount of growth factors, have anti-inflammatory and immunomodulatory effects, can promote the recovery of ovarian function, and have better effects in treating ovarian insufficiency and female menopausal syndrome.

[0025] Mesenchymal stem cells (MSCs) are adult pluripotent stem cells with the potential for self-renewal and multidirectional differentiation. MSCs secrete a large number of extracellular vesicles such as exosomes through paracrine effects, which contain a large number of cytokines and growth factors, play an immunomodulatory role, and promote tissue regeneration. Mesenchymal stem cell exosomes (MSC-Exo) are natural nanoparticles with a diameter of 30-150nm, which have the advantages of high biocompatibility, good stability, strong homing ability, low toxicity, low immunogenicity, and the ability to cross the blood-brain barrier. MSC-Exo is rich in growth factors, nucleic acids (RNA and DNA) and lipids.

[0026] MicroRNA (miRNA) is a class of non-coding single-stranded RNA molecules with a length of about 18-24 nucleotides encoded by endogenous genes, which have multiple important regulatory effects in cells. MiRNA can target and bind to the base sequence of the 3'-untranslated region (UTR) of messenger RNA (mRNA) to regulate the cleavage or translation of target mRNA. miRNA-133b promotes estrogen synthesis. In the culture system of hypoxia and estrogen (E2) stimulation, it positively feedback stimulates placental MSCs, prepares a large number of engineered exosomes, and in the animal model of chemotherapy-induced ovarian insufficiency, it is observed that miR-133b-PD-MSC cells and PD-MSC-derived miR-133b exosomes are effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0028] Figure 1 . This is the result of flow cytometry detection of surface markers of placental mesenchymal stem cells;

[0029] Figure 2 . is a diagram of the tissue differentiation of placental mesenchymal stem cells; A is placental mesenchymal stem cells, B is adipogenic differentiation, the fat cells differentiated from placental mesenchymal stem cells are positively stained with Oil Red O, and oil droplets in the cytoplasm can be seen, C is osteogenic differentiation, the osteoblasts differentiated from placental mesenchymal stem cells are positively stained with Alizarin Red, D is chondrogenic differentiation, the chondrogenic proteoglycans in the chondrocytes differentiated from placental mesenchymal stem cells are stained with Safranin O, scale bar = 100 μm;

[0030] Figure 3 The figure shows the expression of estrogen receptors in mesenchymal stem cells from different tissues, where PD-MSC: placental mesenchymal stem cells, BM-MSC: bone marrow mesenchymal stem cells, AD-MSC: adipose mesenchymal stem cells, and UC-MSC: umbilical cord mesenchymal stem cells;

[0031] Figure 4 . Transmission electron microscopy image of miR-133b-PD-MSCs engineered exosomes;

[0032] Figure 5 . The particle size and distribution of miR-133b-PD-MSCs engineered exosomes; the left picture is the particle size-concentration distribution diagram, and the right picture is the particle size-average concentration distribution diagram;

[0033] Figure 6 .This is the expression diagram of exosome-specific markers membrane protein CD9 and membrane protein CD81 detected by flow cytometry. DETAILED DESCRIPTION

[0034] The following specific descriptions are exemplary and are intended to provide further explanation of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention.

[0035] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods. Some reagents used in the present invention are conventional in the art or can be easily purchased from commercial sources. For example, serum-free culture medium (Gibco) was purchased from ThermoFisher Scientific; human platelet lysate was purchased from Precicion BioMedicals; phosphate buffer (pH 6.8, PBS) was prepared by taking 250 ml of 0.2 mol / L potassium dihydrogen phosphate solution, adding 118 ml of 0.2 mol / L sodium hydroxide solution, diluting with water to 1000 ml, shaking well, and sterilizing at 121°C for 15 min; tissue cleaning solution (PRS-TCR-1) was purchased from Precicion. The MSC complete culture medium described in this article is a serum-free culture medium containing 2% human platelet lysate.

[0036] Example 1 Preparation and Detection of Placental Mesenchymal Stem Cells

[0037] 1.1 Isolation and culture of placental mesenchymal stem cells (PD-MSCs)

[0038] Remove the amniotic membrane, cut the surface membrane-like tissue of the placental lobule, wash it with physiological saline, process the surface membrane-like tissue of the placental lobule to a volume of about 0.2 cm^3, wash it with physiological saline, add digestion solution containing complex enzymes to the tissue, mix it thoroughly, and shake and digest it in a shaker at 37°C for 30 minutes, stop digestion, filter, wash, collect cells, resuspend the precipitated cells with DMEM / F12, take the placental mesenchymal stem cell suspension, and calculate the number and survival rate of mesenchymal stem cells by ADAM automatic cell counter (NanoEnTek, Korea). The viability of freshly prepared mesenchymal stem cells is not less than 95%.

[0039] Passaging and culture of PD-MSCs. Placental primary cells (P0 generation) were inoculated into T75 culture flasks and cultured in a constant temperature and humidity incubator at 37°C, DMEM / F12 medium, and 5% CO2. The medium was completely replaced on the 3rd to 4th day, several cell clones appeared on the 6th to 7th day, and there were fusiform cells growing in a vortex shape on the 10th to 11th day. When PD-MSCs reached 80% confluence, they could be passaged. Digest with 0.25% trypsin at 37°C for 2 minutes, terminate the digestion, and centrifuge at 10,000 / cm 2 The inoculation density was used for subculture.

[0040] 1.2 Flow cytometry detection of placental mesenchymal stem cell surface markers

[0041] According to the definition of mesenchymal stem cells published by the International Society for Cellular Therapy (ISCT) in 2006, MSC cells should meet the following requirements in marker detection:

[0042] Positive expression of markers CD73, CD90, and CD105

[0043] Negative expression of markers CD11b, CD34, CD45, CD19, HLA-DR

[0044] Possesses the potential to differentiate into bone, cartilage and adipose tissue

[0045] The results of the surface marker detection of placental mesenchymal stem cells from generation P0 to P5 were as follows: CD73, CD90, and CD105 were positive, and the positive rate was not less than 95%; CD45, CD34, CD11b, CD19, and HLA-DR were negative, and the negative rate was not higher than 2%, which was in line with the MSC definition standard recommended by the International Society for Cellular Therapy (ISCT) in 2006. Figure 1 shown.

[0046] 1.3 Detection of multidirectional differentiation ability of placental mesenchymal stem cells

[0047] PD-MSCs have the ability to differentiate into bone, cartilage and adipose tissue. Figure 2 shown.

[0048] Example 2 Expression of estrogen receptor (ER) in placental mesenchymal stem cells by Western blot

[0049] (1) The collected ovarian, umbilical cord, fat and bone marrow tissues were lysed with RIPA protein lysis buffer, protease inhibitors (Roche, Switzerland) and phosphorylated protease inhibitor cocktail (Roche), and placed on ice for protein lysis for 10 min. Centrifuged at 4°C, 14,000 rpm for 15 min to obtain a total protein solution, which was stored at -20°C;

[0050] (2) Detect protein concentration using BCA kit;

[0051] (3) an equal amount of 30 μg of protein sample was loaded and separated by 10% SDS-polyacrylamide (PAGE) gel electrophoresis;

[0052] (4) transferring the separated proteins to a PVDF membrane;

[0053] (5) Block with 5% skim milk for 1 h, then add ER antibody and β-actin antibody for incubation;

[0054] (6) Wash three times with Tris buffer containing 0.2% Tween 20 and incubate with IgG secondary antibody;

[0055] (7) Development and exposure, imaging was performed using the ImageQuant LAS 4000 Mini System (GE Healthcare, USA), see Figure 3 .

[0056] The results showed that placental mesenchymal stem cells expressed estrogen receptors, while mesenchymal stem cells derived from bone marrow, fat and umbilical cord did not express estrogen receptors.

[0057] Example 3. Preparation of engineered exosomes

[0058] 1. Construction of miRNA viral vector

[0059] 1.1 Take the lentiviral vector pLVX-has-miR-133b as an example.

[0060] The pLVX-shRNA2 lentiviral expression vector was purchased from Clontech. The polymerase chain reaction (PCR) was performed using DNA containing the has-miR-133b-5p sequence as a template. The PCR amplification product was purified using 1% agarose gel electrophoresis. The target fragment was double-digested with BamHI and EcoRI. The has-miR-133b-5p fragment was further purified. The pLVX-shRNA2 empty vector was double-digested with BamHI and EcoRI. The cells were connected overnight at 16°C in the presence of T4 DNA ligase. The ligation product was used to transform Escherichia coli DH5α competent cells. Some colonies were selected in LB medium. 1 μl of colonies was used as a template for PCR amplification. The PCR product was verified by agarose gel electrophoresis. Positive clones were cultured, plasmids were extracted and sequenced. The sequencing results confirmed that the LV-hsa-miR-133b-5p sequence was correct.

[0061] 1.2 Packaging and titer determination of miR-133b lentiviral vector

[0062] After 24 hours of passage of HEK 293T cells, take a 1.5ml EP tube, add OPTI-MED, add auxiliary plasmids RRE, REV, Vsvg, add pLVX-has-miR-133b plasmid expressing the target gene, mix well, add liposomes, mix well by pipetting; let it stand for 30 minutes, add it to HEK293T cells, and continue to incubate at 37℃ for 48 hours; collect the supernatant, which is the lentivirus. Filter with a 0.45μm PVDF filter; centrifuge at 4℃, 50000g for 2.5 hours, and store at -80℃ after packaging.

[0063] The steps of lentivirus titer detection are as follows:

[0064] (1) Take a 48-well plate one day before the titer determination and add 5 × 10 4 HEK293T cells were inoculated at a density of cells.

[0065] (2) Add polybrene to fresh complete culture medium (DMEM+10% FBS+P / S) to a final concentration of 8 ug / ml.

[0066] (3) Use complete medium containing polybrene to dilute the virus 10-fold in a gradient. The specific steps are as follows: Take a 48-well plate, each well contains 90 μL of medium, add 10 μL of the virus stock solution to the first well, mix well and pipette 10 μL of the virus mixture into the second well. The dilutions are 1 μL, 0.1 μL, 0.01 μL, 0.001 μL, and 0.0001 μL respectively, and continue to dilute to the fifth well (i.e., dilution 10). 5 times).

[0067] (4) Pipette 10 μL of virus dilution into the corresponding 48-well plate, incubate at 37°C, 5% CO2 overnight, and replace the medium with fresh complete culture medium (DMEM+10% FBS+P / S) after 16 to 24 hours.

[0068] (5) After culturing in a 37°C, 5% CO2 incubator for 48 hours, count the number of fluorescent cells using a fluorescence microscope. In general, if 5 (N<10) fluorescent cells appear in the wells with the highest dilution gradient of 10m, the virus titer is 5×10mTU / μL, that is, the virus titer is 5×10m+3TU / ml, where 10m is the dilution multiple.

[0069] Assay results: The number of miR-133b lentiviral particles with biological activity was detected to be 5×10 8 TU / ml, titrated to 1×10 8 TU / ml.

[0070] 1.3LV-has-miR-133b lentiviral vector transfection of placental mesenchymal stem cells

[0071] 1.3.1 Determination of MOI value of LV-has-miR-133b lentiviral vector transfection of placental mesenchymal stem cells PD-MSCs

[0072] MOI, or multiplicity of infection, is the ratio of the number of virus particles to the number of cells during infection, and refers to the number of viruses infected in each cell.

[0073] MOI value = virus titer (TU / mL) × virus volume (mL) / number of cells.

[0074] The determination process of MOI value is as follows: According to the literature, the MOI of lentivirus infection of MSCs is 20, and the MOI gradient is set to 10-20-40. The amount of virus to be added is calculated according to the formula. P3 generation PD-MSCs in good condition are inoculated into 24-well plates with a cell inoculation density of 1×10 4 / cm 2 , total number of cells 2×10 4 / well, and cultured overnight at 37℃. When virus infection was performed on the second day, the confluence of PD-MSCs cells was 50%-70%. LV-miR-133b lentiviral vector was injected at MOI values ​​of 10, 20 and 40, and the virus titer was 1×10 8 TU / ml of virus solution was added to 250μL culture medium at 2μL, 4μL and 8μL volumes for virus transfection. The volume was replenished to 500μL culture volume 4h after lentiviral transfection, and the medium was changed 24h after infection. At 72h after lentiviral transfection, the cell state and fluorescence intensity were observed under a fluorescence microscope to determine the optimal MOI. At MOI 10, the GFP fluorescence intensity was about 50%; at MOI 20, the GFP fluorescence intensity was about 80%; at MOI 40, the GFP fluorescence intensity was about 80%.

[0075] Results: After gradient infection, the MOI values ​​were determined to be 20 and 40, which were used as the MOI values ​​for the formal transfection experiment.

[0076] 1.3.2 LV-miR-133b lentiviral vector transfection of PD-MSCs

[0077] LV-miR-133b lentiviral vector was used to transfect placental mesenchymal stem cells at MOI values ​​of 20 and 40. P3 PD-MSCs in good condition were seeded into 10 cm culture dishes with a total number of 1×10 6 / 10cm culture dish, culture at 37℃ overnight.

[0078] When virus infection was performed on the second day, the virus titer was 1×10 8TU / ml LV-miR-133b lentiviral vector was added to 3 ml of culture medium at a volume of 200 μL and 400 μL, respectively, at an MOI of 20 and 40. 4 hours after LV-miR-133b lentiviral transfection, the volume was replenished to 6 ml of culture medium, and 24 hours after transfection, the medium was replaced with normal medium and cultured continuously.

[0079] After 72 hours of LV-miR-133b lentiviral transfection, the cell status and GFP fluorescence intensity were observed under a fluorescence microscope to determine the lentiviral transfection efficiency of PD-MSCs. Engineered miR-133b-PD-MSCs cells overexpressing miR-133b were obtained.

[0080] Table 1. Efficiency of miRNA-133b lentiviral vector transfection into PD-MSCs

[0081] Cell number MOI Value Virus titer Virus fluid volume Transfection efficiency 10cm Petri dish <![CDATA[1×10 6 ]]> 20 <![CDATA[1×10 8 TU / ml]]> 200μL / 3ml >80％ 10cm Petri dish <![CDATA[1×10 6 ]]> 40 <![CDATA[1×10 8 TU / ml]]> 400μL / 3ml >80％

[0082] Example 4. Hypoxia estrogen (E2) stimulates large-scale preparation of engineered exosomes miR-133b-Exo

[0083] 1.1 P3 PD-MSC cells were cultured at 1×10 4 The cells were inoculated at a density of 1 / well in a 24-well plate, with three wells in each group, and treated with 0, 10nm, 30nm, 50nm, and 100nm E2, respectively. One group was digested with trypsin every 24h, and the cells were counted and averaged for 7 days. The cells were observed using a TS2 inverted microscope (Olympus), with the culture time (h) as the horizontal axis and the number of cells as the vertical axis, and the cell growth curve was drawn and the cell viability was detected to verify the activity of the cells and their self-proliferation and differentiation capabilities.

[0084] The cell doubling time can usually be calculated using the following formula:

[0085] By cell doubling time formula D(T doubling time) = t × [lg2 / (lgNt-lgNo)]

[0086] No: theoretical initial value of cells in the logarithmic growth phase, Nt: the value of cells at one point in the logarithmic growth phase, t: the time taken by cells to increase from No to Nt (h);

[0087] The doubling time of P3 cells treated with 0, 10nm, 30nm, 50nm, and 100nm E2 can be obtained.

[0088] The experimental results showed that the time required for PD-MSCs to double decreased with the increase of E2 concentration. E2 50nm and 100nm were used to stimulate the PD-MSCs cell culture system.

[0089] 1.2 PD-MSCs of generation P3 were enzymatically digested 48 hours after virus transfection and the cells were divided into 1×10 6 The cells were seeded into 10 cm culture dishes, 8 ml of MSCs complete culture medium was added to each dish, and the dishes were placed in a 37° C., 5% CO 2 incubator to allow the cells to adhere to the wall.

[0090] The cells were cultured in a 37°C, 5% CO2 incubator until the cell confluence was ≥80%, and the culture supernatant was collected.

[0091] Wash with PBS three times, then replace with 8 mL of DMEM-high glucose basal medium without FBS to each culture dish, and place the miR-133b-PD-MSCs cells in a 37°C, 2% O2, 5% CO2 incubator, wash the culture dish with PBS three times, and add serum-free fresh medium containing E2 (100 nM), and culture for another 48 hours in a hypoxic environment to obtain E2-CM, so that miR-133b-PD-MSCs cells can fully release engineered exosomes overexpressing miR-133b.

[0092] Pipette the cell supernatant (40 ml, the total amount of supernatant from 5 culture dishes) into a 50 ml centrifuge tube and perform the following centrifugation treatment:

[0093] (1) Centrifuge at 300g and 4°C for 10 min to remove dead cells and larger cell debris, and transfer the supernatant to another 50 ml centrifuge tube;

[0094] (2) Centrifuge at 2000 g and 4 °C for 20 min to further remove cell debris and other impurities, and transfer the supernatant to another high-speed centrifuge tube;

[0095] (3) Centrifuge at 12000g and 4°C for 30 min to further remove smaller cell debris and impurities, and filter the supernatant with a 0.22 μm sterile filter (Millipore) and place it in another ultracentrifuge tube;

[0096] (4) Centrifuge at 100,000 g and 4°C for 90 min and discard the supernatant;

[0097] (5) Add 25 ml of sterile PBS to the centrifuge tube to resuspend the exosome pellet, centrifuge at 100,000 g and 4 °C for 90 min, discard the supernatant, and add 1 mL of sterile PBS to resuspend the exosomes to obtain 1 ml of exosome suspension, which can be stored at -80 °C and / or aliquoted for performance measurement.

[0098] 1.3 Transmission electron microscopy identification of the morphology of miR-133b-PD-MSCs engineered exosome particles

[0099] The extracted exosomes were observed under transmission electron microscopy. The specific operation was as follows:

[0100] (1) Take 50 μl of the isolated and purified exosome precipitate, add an equal volume of 2.5% glutaraldehyde, and place it in a 4°C refrigerator for fixation for 1 hour;

[0101] (2) 20 μl of the fixed exosome suspension was dripped onto the front of the copper mesh and allowed to stand for 20 min;

[0102] (3) Carefully absorb the excess solution with filter paper; then wash the copper mesh with ultrapure water 5 times, each time for 30 seconds, and dry it with filter paper;

[0103] (4) Add 1 drop of 2% uranyl acetate staining solution to the front of the copper mesh and stain for 1 min. Then use filter paper to absorb the excess stain along the edge of the copper mesh.

[0104] (5) The copper mesh was placed in air at room temperature and away from light to dry naturally. After drying, it was observed on a microscope and the particle size and distribution of the exosomes were calculated and counted.

[0105] (6) Finally, transmission electron microscopy was used to observe exosomes. The results are shown in Figure 4 .

[0106] 1.4 Detection of particle size of miR-133b-PD-MSCs engineered exosomes

[0107] The particle size was determined by nanoparticle tracking analysis (NTA) and analyzed by Finite Track Length Adjustment (FTLA). The exosome concentration was 4.14x10^10particles / mL. The exosome particle size had obvious peaks at 118nm and 199nm. The test results showed that the peak particle size was 118.4±3.0nm and the average particle size was 131.8±0.4nm, see Figure 5 .

[0108] 1.5 Flow cytometry detection of exosome-specific markers CD9 and CD81

[0109] The results of flow cytometry detection of the expression of exosome-specific markers membrane protein CD9 and membrane protein CD81 can be found in Figure 6 The results showed that the positive expression rate of miR-133b-PD-MSCs engineered exosomal membrane protein CD9 was approximately 85.29%, and the positive expression rate of exosomal membrane protein CD81 was approximately 96.76%.

[0110] Example 5. Application of miR-133b-PD-MSCs and miR-133b-Exo in ovarian insufficiency mouse model

[0111] 1.1 Establishment and grouping of ovarian insufficiency mouse model

[0112] Twenty-five adult C57BL / 6 female mice were randomly divided into five groups, with five mice in each group. They were control group, POI model group, PD-MSCs group, miR-133b-PD-MSCs group, and miR-133b-Exo group. Female C57BL / 6 mice were intraperitoneally injected with 50 mg / kg / day cyclophosphamide (CTX) for 14 consecutive days at the same time every day to establish a chemotherapy-induced POI mouse model. Ovarian function was evaluated by follicle number and hormone level indicators.

[0113] The mice in the control group were not injected, and the mice in the POI model group were injected with 200 μL of normal saline via the tail vein. The mice in the PD-MSCs group were injected with 1×10 6 PD-MSCs / 200 μL saline. The miR-133b-PD-MSCs group was injected with 1×10 6 miR-133b-PD-MSCs / 200μL saline. The miR-133b-Exo group was injected with 1×10 8 miR-133b-Exo / 200 μL saline. 14 days after transplantation, all mice were killed, and the therapeutic effects of POI after transplantation in the PD-MSCs group, miR-133b-PD-MSCs group, and miR-133b-Exo group were observed.

[0114] 1.2 Determination of ovarian follicle count

[0115] After 14 days of transplantation, the ovaries of both mice were harvested. One ovary was used to measure the ovarian weight, structure, follicle count, and histopathology was performed using HE staining. The number of primordial follicles, primary follicles, secondary follicles, antral follicles, and atretic follicles in the ovaries was counted under an optical microscope. The results are shown in Table 2.

[0116] The results showed that compared with the control group, the number of primordial follicles, primary follicles, secondary follicles and antral follicles in the POI group was significantly reduced, and the number of atretic follicles was significantly increased (P<0.01). Compared with the POI group, 14 days after transplantation, the number of primordial follicles, primary follicles, secondary follicles and antral follicles in the PD-MSCs group, miR-133b-PD-MSCs group and miR-133b-Exo group was significantly increased, and the number of atretic follicles was significantly decreased (P<0.01). 14 days after transplantation, compared with the PD-MSCs group, the number of primordial follicles, antral follicles and atretic follicles in the miR-133b-PD-MSCs group and miR-133b-Exo group was different (P<0.05).

[0117] Conclusion: PD-MSCs, miR-133b-PD-MSCs and miR-133b-Exo can improve the counts of primordial follicles, primary follicles, secondary follicles and antral follicles in POI mice and restore ovarian function. The effects of miR-133b-PD-MSCs and miR-133b-Exo are more significant than those of PD-MSCs.

[0118] Table 2 Follicle counting results

[0119]

[0120] 3. Effects on serum hormone levels

[0121] All mice were killed 14 days after transplantation. Blood samples were collected and centrifuged at 4000 r / min for 10 min to obtain mouse serum, which was stored at –80°C. ELISA kits were used to measure serum estradiol (E2) and follicle stimulating hormone (FSH) hormone levels. The results are shown in Table 3.

[0122] The results showed that compared with the control group, the E2 level in the POI group was significantly decreased, and the FSH level was significantly increased, P<0.01. After 14 days of transplantation, the E2 levels in the PD-MSCs group, miR-133b-PD-MSCs group, and miR-133b-Exo group were significantly higher than those in the model group, and the FSH level was significantly lower than that in the model group, P<0.01. The E2 levels in the miR-133b-PD-MSCs group and miR-133b-Exo group were higher than those in the PD-MSCs group, and the FSH levels were lower than those in the PD-MSCs group, with differences between the groups, P<0.05.

[0123] Conclusion: PD-MSCs, miR-133b-PD-MSCs and miR-133b-Exo all increased the E2 level and decreased the FSH level in POI mice. The effects of miR-133b-PD-MSCs and miR-133b-Exo were better than those of PD-MSCs.

[0124] Table 3 Hormone level test results

[0125] E2 (pg / ml) FSH (U / L) P-value Control group 90 5 POI group 20 16 Compared with the control group, P<0.01 PD-MSCs group 40 10 Compared with the POI group, P < 0.01 miR-133b-PD-MSCs 50 8 Compared with the POI group, P < 0.01 miR-133b-Exo 55 7 Compared with the POI group, P < 0.01

[0126] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Engineered exosomes derived from placental mesenchymal stem cells, characterized in that: The engineered exosomes are prepared from placental mesenchymal stem cells transfected with miRNA viral vectors.

2. The engineered exosomes derived from placental mesenchymal stem cells according to claim 1, characterized in that: The method for preparing exosomes comprises the following steps: (1) Construction of miRNA viral vector; (2) preparing placental mesenchymal stem cells transfected with miRNA viral vectors; (3) Under conditions of hypoxia and estrogen stimulation, engineered exosomes are prepared using the placental mesenchymal stem cells transfected in step (2).

3. The engineered exosomes derived from placental mesenchymal stem cells according to claim 1, characterized in that: The miRNA viral vector described in step (1) is any one of miRNA-133b, miR-144, miR-146, miRNA-21-5p, and miR-132.

4. The engineered exosomes derived from placental mesenchymal stem cells according to claim 3, characterized in that: The steps for preparing engineered exosomes in step (3) are: a) P3 placental mesenchymal stem cells were obtained 48 hours after miRNA virus transfection and enzymatic digestion was performed. 6 The cells were seeded into a 10 cm culture dish, 8 ml of mesenchymal stem cell complete medium was added, and the dish was placed in a 37°C, 5% CO2 incubator to allow the cells to adhere to the wall; b) The cells were cultured in a 37° C., 5% CO 2 incubator until the cell confluence was no less than 80%, and the culture supernatant was collected; c) washing with PBS for 3 times, adding 8 mL of DMEM-high glucose basal medium without FBS, placing in an incubator at 37°C, containing 2% O2 and 5% CO2, adding 50-100 nm of estrogen in an oxygen-deficient environment, and culturing for 48 hours to allow the transfected placental mesenchymal stem cells to fully release exosomes; d) The cell supernatant was aspirated and placed in a 50 ml centrifuge tube for ultracentrifugation to obtain an exosome suspension, which was then stored at -80°C.

5. A drug for treating ovarian insufficiency, characterized in that: The drug comprises any one or a combination of placental mesenchymal stem cells and engineered exosomes transfected by a miRNA viral vector as described in claim 1.

6. Use of placental mesenchymal stem cells transfected with the miRNA viral vector as claimed in claim 1 in the preparation of drugs for ovarian insufficiency and female menopausal syndrome.

7. Use of the engineered exosomes derived from placental mesenchymal stem cells as claimed in claim 1 in the preparation of drugs for ovarian insufficiency and female menopausal syndrome.

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