CN117653664A - Use of vesicles in the treatment of ovarian disease - Google Patents
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- CN117653664A CN117653664A CN202311517230.0A CN202311517230A CN117653664A CN 117653664 A CN117653664 A CN 117653664A CN 202311517230 A CN202311517230 A CN 202311517230A CN 117653664 A CN117653664 A CN 117653664A
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Abstract
The invention belongs to the field of biological medicine, and relates to application of vesicles in treatment of ovarian diseases. The invention provides application of an inducible vesicle in preparing a medicament for preventing or treating polycystic ovary syndrome. Administration of the inducible vesicles can rescue the PCOS phenotype and reduced birth rate.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to application of vesicles in treatment of ovarian diseases.
Background
Apoptosis is one of the modes of Programmed Cell Death (PCD), contributes to cell turnover in organisms, and plays an important role in development, physiological pathogenesis, and aging [1,2]. As apoptosis progresses, cells produce a number of bilayer membrane coated cell structures, previously known as apoptotic bodies (apobds); furthermore, there is a great deal of recent evidence that vesicle products called apoptotic vesicles (ApoV) are also produced during the process of apoptotic cell lysis [3,4]. According to its unique mechanism of production, apoV is capable of encapsulating various functional components of its parent cell, including nucleic acids, proteins and lipids, facilitating intercellular communication and maintaining systemic homeostasis [5,6]. With the continued confirmation of the unique beneficial properties of ApoV, a series of preclinical studies have shown that ApoV, particularly those from Mesenchymal Stem Cells (MSCs), has great therapeutic potential in wound repair, tissue regeneration and immunomodulation [7-11]. However, it is not known whether apoptosis and its metabolite ApoV regulate ovarian folliculogenesis.
Ovaries are recognized as important organs for female gametogenesis and sex hormone production. It has developed a unique and tightly controlled loop structure and regulatory mechanisms that respond to follicular growth, steroid production, oocyte maturation, ovulation and luteal formation [12]. From fetal development to adulthood, apoptosis has long been considered an essential physiological process of the female reproductive system, playing an important role in endogenous mechanisms of ovarian function execution including germ cell depletion, follicular locking and luteal degeneration [13, 14]. In the pathological context, numerous studies have demonstrated that ovarian localized apoptosis, particularly that which occurs in Granulosa Cells (GC) and oocytes, is widely involved in the progression of ovarian diseases such as polycystic ovary syndrome (PCOS), premature Ovarian Failure (POF), ovarian aging and ovarian cancer [15-17]. However, it is still unknown whether systemic apoptosis and circulating apoptotic vesicles contribute to ovarian homeostasis and pathological progression.
Disclosure of Invention
In some embodiments, the invention provides the use of an inducible vesicle in the preparation of a medicament for preventing or treating polycystic ovary syndrome.
In some embodiments, the vesicle is one that is produced by an external factor that induces apoptosis of a stem cell while it is in normal survival. In some embodiments, the method of inducing comprises adding staurosporine, ultraviolet irradiation, starvation, or thermal stress.
In some embodiments, the stem cell is a mesenchymal stem cell.
Regarding "polycystic ovary syndrome (Polycystic ovarian syndrome, PCOS)", the most common endocrine disease in human females, is characterized by hypersecretion of androgens and concomitant abnormal secretion of insulin, affecting 5% -10% of women of childbearing age. The main symptoms of PCOS are marked by anovulatory infertility, menstrual disorder, amenorrhea, obesity, hirsutism, etc. in females. Recent studies have shown that PCOS-induced related hormonal metabolic disorders will increase the risk of developing metabolic diseases such as Type 2diabetes (t 2 d) and cardiovascular disease. Aiming at the etiology of PCOS, the current clinical treatment means of PCOS mainly comprises regulating hormone level by steroid drugs such as dexamethasone, improving contraceptive drugs such as insulin resistance, estrogen and progestogen by utilizing metformin, and the like, and antiandrogen drugs such as diethylstilbestrol tablet, ethinyl estradiol-cyproterone tablet, compound estradiol valerate tablet, and the like [28].
In some embodiments, the invention adopts the induction vesicle to treat the ovarian syndrome, belongs to a new drug treatment mode, achieves a very good effect, and finds a better substitution therapy for the treatment of the polycystic ovarian syndrome. In some embodiments, the mesenchymal stem cell source comprises bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon, or peripheral blood.
In some embodiments, the mesenchymal stem cells are derived from a mammal. In some embodiments, the mammal is selected from a primate or a mouse. In some embodiments, the primate is a human.
In some embodiments, the vesicles have a diameter of 0.03 to 10 μm. In some embodiments, the vesicles have a diameter of 0.03 to 6 μm.
In some embodiments, the vesicles have a diameter of 0.03 to 4.5 μm. In some embodiments, the vesicles have a diameter of 0.03 to 1 μm.
In some embodiments, the concentration of staurosporine is 1-15000nM. In some embodiments, the concentration of staurosporine is 200-10000nM. In some embodiments, the concentration of staurosporine is 500-1000nM. In some embodiments, the concentration of staurosporine is 500-900nM. In some embodiments, the concentration of staurosporine is 500-800nM.
In some embodiments, the vesicle has the marker syncaxin 4. In some embodiments, the vesicle high-expression marker Syntaxin 4. In some embodiments, the vesicles express higher expression of the marker syncaxin 4 than the MSC or exosome of the same origin. In some embodiments, the markers further comprise one or more of Annexin V, flotillin-1, cadherin 11, or Intigrin alpha 5. In some embodiments, the vesicle high-expression marker Annexin V, flotillin-1, cadherin 11, or Intigrin alpha 5. In some embodiments, the inducible vesicle has a higher expression level of the markers Annexin V, flotillin-1, cadherin 11, intigrin alpha 5 than the homolog-derived MSC or exosome.
In some embodiments, the medicament is a topical injection formulation. In some embodiments, the local injection is subcutaneous, intramuscular, intra-articular, and intravenous injection. In some embodiments, any one or more of a pH adjuster, a buffer, a stabilizer, an isotonic agent, and a local anesthetic is added when used for the local injection. In some embodiments, the method of preparing the vesicle comprises the steps of: 1) Culturing mesenchymal stem cells; 2) Adding the mesenchymal stem cells of the step 1) into a serum-free medium containing 500-1000nM staurosporine, and collecting cell supernatant; 3) Subjecting the cell supernatant collected in step 2) to ultracentrifugation to obtain the vesicles. In some embodiments, the step of separating the vesicles by the ultracentrifugation method comprises: (a) Centrifuging the collected culture supernatant for the first time, and taking the supernatant; (b) Subjecting the supernatant collected in step (a) to a second centrifugation to obtain a supernatant; (c) Centrifuging the supernatant received in step (b) for a third time to obtain a precipitate; (d) Centrifuging the precipitate obtained in step (c) for the fourth time, and taking the precipitate.
In some embodiments, the first centrifugation is 500-1500g centrifugation for 5-30 minutes. In some embodiments, the first centrifugation is 500-1000g centrifugation for 5-20 minutes. In some embodiments, the first centrifugation is 500-900g centrifugation for 5-15 minutes. In some embodiments, the second centrifugation is from 1000 to 3000g centrifugation for 5 to 30 minutes. In some embodiments, the second centrifugation is from 1500 to 2500g centrifugation for 5 to 20 minutes. In some embodiments, the second centrifugation is from 1500 to 2200g centrifugation for 5 to 15 minutes. In some embodiments, the third centrifugation is 10000-30000g centrifugation for 15-60 minutes. In some embodiments, the third centrifugation is 12000-25000g centrifugation for 20-60 minutes. In some embodiments, the third centrifugation is 12000-20000g centrifugation for 20-40 minutes. In some embodiments, the fourth centrifugation is 10000-30000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is from 12000 g to 25000g for 20 to 60 minutes. In some embodiments, the fourth centrifugation is from 12000 to 20000g centrifugation for 20 to 40 minutes. In some embodiments, the invention provides a composition comprising an inducible vesicle and a medicament for treating polycystic ovary syndrome. In some embodiments, the vesicle is one that is produced by an external factor that induces apoptosis of a stem cell while it is in normal survival.
In some embodiments, the method of inducing comprises adding staurosporine, ultraviolet irradiation, starvation, or thermal stress.
In some embodiments, the stem cell is a mesenchymal stem cell. In some embodiments, the mesenchymal stem cell source comprises bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon, or peripheral blood.
In some embodiments, the mesenchymal stem cells are derived from a mammal. In some embodiments, the mammal is selected from a primate or a mouse. In some embodiments, the primate is a human.
In some embodiments, the vesicles have a diameter of 0.03 to 10 μm. In some embodiments, the vesicles have a diameter of 0.03 to 6 μm. In some embodiments, the vesicles have a diameter of 0.03 to 4.5 μm. In some embodiments, the vesicles have a diameter of 0.03 to 1 μm. In some embodiments, the concentration of staurosporine is 1-15000nM. In some embodiments, the concentration of staurosporine is 200-10000nM. In some embodiments, the concentration of staurosporine is 500-1000nM. In some embodiments, the concentration of staurosporine is 500-900nM. In some embodiments, the concentration of staurosporine is 500-800nM.
In some embodiments, the vesicle has the marker syncaxin 4. In some embodiments, the vesicle high-expression marker Syntaxin 4. In some embodiments, the vesicles express higher expression of the marker syncaxin 4 than the MSC or exosome of the same origin. In some embodiments, the markers further comprise one or more of Annexin V, flotillin-1, cadherin 11, or Intigrin alpha 5. In some embodiments, the vesicle high-expression marker Annexin V, flotillin-1, cadherin 11, or Intigrin alpha 5. In some embodiments, the inducible vesicle has a higher expression level of the markers Annexin V, flotillin-1, cadherin 11, intigrin alpha 5 than the homolog-derived MSC or exosome.
In some embodiments, the composition is a topical injection composition.
In some embodiments, the invention provides the use of the composition in the preparation of a medicament for preventing or treating polycystic ovary syndrome.
In some embodiments, the invention provides the use of an inducible vesicle in the preparation of a product that promotes, improves, restores, or restores fertility in a subject.
In some embodiments, the product comprises a food product, a nutraceutical, or a pharmaceutical product.
In some embodiments, the subject is an individual including elderly individuals. In some embodiments, the elderly individual refers to an individual older than 50 years of age.
In some embodiments, the invention provides an ovarian disease medication guidance system comprising the following components: (1) RNF43 level analysis means; for analyzing the deviation of the RNF43 level in the test inducible vesicle sample from the RNF43 level in the normal inducible vesicle sample; (2) a result judgment means; the method is used for judging a medication scheme for treating the ovarian diseases according to the RNF43 level analysis result, wherein the medication scheme is as follows: when the RNF43 level in the inducible vesicle sample to be tested is normal, directly administering the inducible vesicle to be tested for medication; when the RNF43 level is low, the inducer vesicles to be tested are not selected for administration.
In some embodiments, the ovarian disease comprises polycystic ovary syndrome.
Drawings
FIG. 1 shows that MRL/lpr mice exhibit ovarian dysfunction such as PCOS. (a) representative HE staining pattern of ovarian tissue. The lower panel is an enlarged view of the area within the box in the HE stained image described above. Black arrows indicate mature follicles (AF). The # symbol indicates a multi-saccular follicle. Scale bar: the low power is 500 μm and the high power is 100 μm. Number of Secondary Follicles (SF) and AF in ovaries. N=3. (C) appearance of meiotic second-phase (MII) oocytes. The # tag indicates an abnormal oocyte. Black arrows indicate high power field of view. The scale bar is 50 μm. (D) Normal MII oocyte/total oocyte ratio, n=3. (E and F) enzyme-linked immunosorbent assay (ELISA) for detecting estradiol in serum (E) 2 ) And testosterone levels, n=3. (G) appearance of WT and MRL/lpr mouse fetuses. # indicates fetal malformation. (H and I) fetal number and normal fetal/total fetal ratio. N=6, error bars represent mean ± SD. Data were analyzed for two sets of comparisons using a two-tailed Student's unpaired t-test. NS, has no meaning. * P (P)<0.05，**P<0.01，***P<0.001。
Figure 2 shows that mesenchymal stem cell-ApoV infusion can rescue ovarian dysfunction in MRL/lpr mice and GLD mice. (a) representative HE staining pattern of ovarian tissue. The lower panel is an enlarged view of the area within the box in the HE stained image described above. The black arrow indicates AF. The # symbol indicates a multi-saccular follicle. Scale bar: the low power is 500 μm and the high power is 100 μm. (B) number of SF and AF in ovaries. N=3. (C and D) ELISA analysis showed E in serum 2 And testosterone levels. N=6. (E) appearance of MII oocytes. The # tag indicates oocyte abnormality. Black arrows indicate high power field of view. Bar = 50 μm. (F) Normal MIIOocyte/total oocyte ratio. N=6. (G) fetal appearance. The scale bar is 0.5 cm. (H) appearance of fetal extremities and tail. The scale bar is 1 mm. (I) Alxin blue and alizarin red staining of the scaffold. Error bars represent mean ± SD. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey test. * P <0.01，***P<0.001。
FIG. 3 shows the apoptosis ratio of circulating ApoV in Wild Type (WT), fas mutant (MRL/lpr) and Fas ligand (FasL) mutant (GLD) mice by nano-flow cytometry analysis.
Figure 4 shows the characteristics of mesenchymal stem cells-ApoV. (A) Scheme for isolation of ApoV from apoptotic mesenchymal stem cells. (B) Mesenchymal stem cell morphology and morphology change after STS induction for 6 hours observed under microscope. The scale bar is 50 μm. (C) apoptosis was detected using TUNEL staining. The scale bar is 50 μm. (D) representative TEM images of ApoV. The scale bar is 500nm. (E and F) size and potential of ApoV in 1 XPBS buffer (pH 7.4). N=3. Figure 5 shows that mesenchymal stem cell-ApoV infusion can rescue ovarian dysfunction in MRL/lpr mice. (a) representative HE staining pattern of ovarian tissue. The lower panel is an enlarged view of the area within the box in the HE stained image described above. Black arrows indicate mature follicles (AF). The # symbol indicates a multivesicular follicle. The scale bar is 500 μm low and 100 μm high. Number of Secondary Follicles (SF) and AF in ovaries. N=3. (C) appearance of meiotic second-phase (MII) oocytes. Black arrows indicate high power field of view. The scale bar is 50 μm. (D) normal MII oocyte/total oocyte ratio. N=3. (E and F) ELISA detection of estradiol in serum (E 2 ) And testosterone levels. N=3. (G) Appearance of MRL/lpr mouse fetuses with or without ApoV injection. Sign # indicates fetal malformation. (H and I) fetal number and normal fetal/total fetal ratio. N=6, error bars represent mean ± SD. Data were analyzed for two sets of comparisons using a two-tailed Student's unpaired t-test. NS, has no meaning. * P (P)<0.05，**P<0.01，***P<0.001。
Fig. 6 shows TC is an MSC-like cell, preferentially taking up ApoV. (A and B) mesenchymal stem cells-ApoV (red) labeled with PKH26 and AIE genes in ovarian follicles were followed. The right image is an enlarged view of the left box area. The white dotted line represents the follicular membrane cell (TC) region. The scale bar is 50 μm low and 10 μm high. (C) Quantification of the ratio of the cellular uptake area of mesenchymal stem cells-ApoV to the total area of the whole ovary. N=3. (D) PKH26 labeled mesenchymal stem cells-ApoV were co-cultured with TCs for tracking the location of mesenchymal stem cells-ApoV in TCs in vitro. ApoV was shown to be predominantly enriched in perinuclear regions under high power microscopy. The scale bar is 10 μm. N=6. ApoV error bars represent mean ± SD. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey test. NS, not significant. * P <0.05, < P <0.01, < P <0.001.
Fig. 7 shows that the follicular membrane cells have the characteristics of mesenchymal stem cells. (A) Follicular membrane cells (TC) and Granulosa Cells (GC) morphology under a bright field microscope. The inset shows the high power field of view of the area within the box. The scale bar is 200 μm. (B) Immunofluorescent staining of Gli1-tdTomato mouse mesenchymal stem cell marker Gli 1. The right image is an enlarged view of the area within the left image box. White arrows indicate Gli1 positive regions. The scale bar is 200 μm. (C) The osteogenic differentiation capacity of TCs and GCs was examined by alizarin red staining. The scale bar is 200 μm. (D) Colony Forming Unit (CFU) assay of TCs and GCs. (E) Flow cytometry analysis of mesenchymal stem cell-specific markers, including negative markers CD34 and CD45; positive markers CD44, CD73, CD90, CD105 and Sca-1. (F to H) representative HE and immunofluorescence images of Wild Type (WT) mouse renal capsule engrafted with TCs and GCs. The right panel is an enlarged view of the left HE and the area within the box in the immunofluorescence image. The scale bar is 500 μm.
FIG. 8 shows that infusion of MSC-ApoV restores the NPPC/cGMP/PDE3A/cAMP cascade. (A) ELISA showed NPPC levels in MGCs. N=3. (B) ELISA showed cGMP levels in CC. (C and D) ELISA showed PDE3A and cAMP levels in the oocytes. Error bars represent mean ± SD. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey test. NS, not significant. * P <0.05, < P <0.01, < P <0.001.
Figure 9 shows that RNF43 contributes to the rescue of mesenchymal stem cell-ApoV mediated ovarian dysfunction. (A) Western Blot analysis showed mesenchymal stem cells (i.e., group UCs) and mesenchymal stem cellsExpression of activated RNF43, axin1 and GSK-3 beta in ApoV (i.e., apopVs). (B) Flow cytometry analysis showed mesenchymal stem cells and mesenchymal stem cell-ApoV and expression of RNF43 in circulating ApoV. (C) Immunofluorescent staining showed expression of RNF43 on MSC-ApoV surface. The scale bar is 500nm. (D) Western blot analysis showed in vitro expression levels of RNF43 in ovarian TCs co-cultured with mesenchymal stem cells-ApoV. (E) Representative HE staining of ovarian tissue showed that ApoV from RNF43 knockout (RNF 43 KD) -MSCs failed to rescue polycystic-like disorders in MRL/lpr mice. The black arrow indicates AF. The # shaped markers represent polycystic follicles. The scale bar is 500 μm low and 100 μm high. (F) number of SF and AF in ovaries. N=3. (G and H) ELISA showed that RNF43 KD-mesenchymal stem cell-derived ApoV failed to rescue E from MRL/lpr mice 2 And testosterone levels. N=3. (I) The appearance of MII oocytes showed that ApoV derived from RNF43 KD-mesenchymal stem cells failed to rescue abnormal oocytes from MRL/lpr mice. The # tag indicates an abnormal oocyte. Black arrows indicate high power field of view. The scale bar is 50 μm. Error bars represent mean ± SD. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey test. NS, not significant. * P (P) <0.05，**P<0.01，***P<0.001。
FIG. 10 shows Western Blot analysis demonstrating that mesenchymal stem cells and RNF43 gene knockouts (RNF 43 KD) -mesenchymal stem cells and RNF43 levels in ApoV and RNF 43-KDaoV.
Figure 11 shows that mesenchymal stem cell-ApoV infusion can rescue DHEA-induced polycystic ovary syndrome and ovarian aging injury. (A) Representative HE staining of ovarian tissue of WT mice, PCOS mice injected or not with ApoV. The # shaped markers represent polycystic follicles. Scale bar: the low power is 500 μm and the high power is 100 μm. (B) number of SF and AF in ovaries. N=3. (C) ELISA analysis showed testosterone levels in the serum. N=3. Error bars represent mean ± SD. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey test. NS, not significant. * P <0.05, < P <0.01, < P <0.001.
Fig. 12TUNEL staining shows apoptosis levels in ovarian Chocolate Cyst (CC) patients (as control group) and polycystic ovary syndrome patients in ovarian interstitial and GC areas. The scale bar is 100 μm low and 20 μm high.
Figure 13 shows that MSC-ApoV infusion improved fertility in aged mice. (A) Representative HE staining of ovarian tissue of WT mice and aged mice with or without ApoV injection. The scale bar is 100 μm. (B) Number of Primordial Follicles (PF), primary follicles (PRF), SF, and AF in ovaries. N=3. (C) generating the general appearance of a foaming (GV) oocyte. The # tag indicates an abnormal oocyte. Black arrows indicate high power field of view. The scale bar is 50 μm. (D) ratio of normal GV oocytes/total oocytes. N=4. (E) fetal number. N=10. (F) appearance of fetuses from WT mice and geriatric mice. Error bars represent mean ± SD. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey test. NS, not significant. * P <0.05, < P <0.01, < P <0.001.
Figure 14 shows that the effect of topical ApoV injection on treatment of ovarian defects in MRL/lpr mice was superior to topical MSC injection. (A) Representative HE staining of mouse ovarian tissue of MRL/lpr mice injected locally with ApoV or MSCs. The # shaped markers represent polycystic follicles. Scale bar: the low power is 500 μm and the high power is 100 μm. (B) Local injection of ApoV or MSC restored a comparison of MRL/lpr mice ovarian SF and AF numbers.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.
"comprising" or "including" is intended to mean that the compositions (e.g., media) and methods include the recited elements, but not exclude other elements. When used to define compositions and methods, "consisting essentially of … …" means excluding other elements that have any significance to the combination for the purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed application. "consisting of … …" means the process steps excluding trace elements and essential elements of other components. Embodiments defined by each of these transitional terms are within the scope of this application. The term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and/or" means that any one of the listed items can be used alone or any combination of two or more of the listed items can be used. For example, if a composition, combination, construction, etc. is described as comprising (or containing) components a, B, C, and/or D, the composition may contain a alone; comprising B alone; solely comprising C; separately comprising D; a combination comprising a and B; a combination comprising a and C; a combination comprising a and D; a combination comprising B and C; a combination comprising B and D; a combination comprising C and D; a combination comprising a, B and C; comprising a combination of a, B and D; a combination comprising a, C and D; comprising a combination of B, C and D; or A, B, C and D are used in combination. STS in the present invention is staurosporine.
Herein, "ApoV" is identical to "ApoVs" or "MSC-ApoV" or "mesenchymal stem cell-ApoV", and refers to "inducible vesicles", which may also be referred to as IEVs. The IEVs in the embodiments of the present invention are short for inducible vesicles, which may be referred to as inducible vesicles, and may be referred to as inducible extracellular vesicles (Induced extracellular vesicles, IEVs), and IEVs are the same as IEVs. An inducible extracellular vesicle refers to a class of subcellular products that are produced by precursor cells (e.g., stem cells) that are interfered with or induced to undergo apoptosis when they survive normally. Typically, this class of subcellular products has a membrane structure, expresses apoptotic markers, and contains in part the genetic material DNA. The inventors have found that inducible extracellular vesicles are a class of substances that are distinguished from cells and conventional extracellular vesicles (e.g., exosomes, etc.). In some embodiments, the cells that survive normally are, for example, non-apoptotic cells, non-senescent cells that proliferate arrested, cells that revive after non-cryopreservation, cells that do not become malignant and proliferate abnormally, or cells that do not become damaged, and the like. In some embodiments, the cells that survive normally are taken from cells that fuse 80-100% in contact during cell culture. In some embodiments, the cells that survive normally are obtained from log phase cells. In some embodiments, the normally viable cells are obtained from primary cultures of human or murine tissue origin and their subcultured cells. In some embodiments, the normally viable cells are taken from an established cell line or strain. In some embodiments, the precursor cells are taken from early cells.
In some embodiments, such as Zhang X, tang J, kou X, huang W, zhu Y, jiang Y, yang K, li C, hao M, qu Y, ma L, chen C, shi S, zhou Y.protein analysis of MSC-derived apoptotic vesicles identifies Fas inheritance to ameliorate haemophilia a via activating platelet functions.J excel Vehicles.2022Jun; 11 (7) e12240.doi:10.1002/jev2.12240.PMID:36856683; PMCID PMC9927920 the method described in the above paragraph identifies and characterizes the obtained surface protein marker of the induced vesicle, and suggests that the characteristic marker is consistent with the description in the above article. In some embodiments, the inducible vesicles are as disclosed in patent application 202110077486.9. Patent application 202110077486.9 is incorporated by reference in its entirety.
In the examples herein, the algebra used by the cells is from passage 5 to passage 10. SF indicates secondary follicles in the ovary, and AF indicates mature follicles in the ovary. Where not specifically indicated herein, tail vein injection (systemic injection) is 3.0-4.0X10 6 Cell-derived ApoV is treated. Intra-ovarian injection (local injection) is 1×10 6 Cell-derived ApoV is treated. In the examples herein, the cells of the examples were derived from umbilical cord mesenchymal stem cells, except that the MSCs of example 6 were derived from bone marrow mesenchymal stem cells, unless otherwise specified.
Example materials and methods herein
Animals
All animal experiments were in accordance with the university of Zhongshan's committee for animal management and use (SYSU-IACUC-2023-000098). Female C57BL/6J mice of 8 and 15 weeks old were purchased from laboratory animals at university of Zhongshan and Jizhikang (Nanjing, china)And (5) a heart. Female B6 MRL-Faslpr/J (MRL/lpr) mice, B6Smn.C3-Fasl gld A/J (GLD) mouse, gli1tm3 (cre/ERT 2) Alj/J and B6.Cg-Gt (ROSA) 26Sortm9 (CAG-tdTomato) Hze/J mouse were purchased from Jackson laboratories, USA. CYP17A1-Cre mice were purchased from Siro Biotech Inc. Axin1 fl/fl Mice were donated by the advanced technical institute of Shenzhen, national academy of sciences, human tissue and organ degeneration center. Age-matched 6 to 8 week old female mice with the same background were used in all experiments.
PCOS modeling and treatment
To establish the PCOS model, 8-week-old female mice were subcutaneously injected daily with 6mg/100g of dehydroepiandrosterone (DHEA, HY-14650, MCE) dissolved in 0.1ml of sesame oil (S27343, round leaf) for 21 days as a PCOS group; mice injected with an equal amount of sesame oil served as a control group [25 ]]. 3.0-4.0X10 6 Cell-derived ApoV was dissolved in filtered PBS, which was injected into PCOS mice by tail vein 24 hours after the last DHEA injection, once a week for two weeks. Control mice were injected with an equal amount of PBS.
Morphological observation and quantification of follicles
After mice were sacrificed, ovarian tissues were isolated and fixed in 4% Paraformaldehyde (PFA) (BL 539A, biosharp) overnight. The samples were then dehydrated through gradient ethanol and embedded in paraffin, producing serial sections of 5-8 μm (RM 2235, leica, germany). Sections were then stained with hematoxylin and eosin (H & E, G1080& G1100, solarbio).
Follicles were defined according to previous studies [26]. In short, follicles with more than one layer of granulosa cells without visible sinuses are defined as secondary follicles. A follicle with sinus space and a cumulus cell border is defined as a sinus follicle.
Separation of ovarian membrane cells, parietal granular cells, cumulus cells and oocytes
According to the previous report [20]Follicular membrane cells (TC) and parietal granulosa cells (MGCs) are isolated and cultured. Briefly, mouse ovarian tissue was gently isolated and placed in 60mm alpha-MEM containing culturesCulture dishes (430166, corning) of Medium (11380037, thermoFisher). After puncturing with an insulin needle, the cells were minced under a microscope and a single cell suspension of wall particles was obtained by passing the cells through a 70 μm filter (352350, falcon). The remaining ovarian tissue was digested with a solution containing 2mg/mL collagenase type I (LS 004197, biofiven) and 4mg/mL dispase II (4942078001-1, roche) in Phosphate Buffered Saline (PBS) at 37℃for 1 hour, and the digested ovarian tissue was then passed through a 70 μm filter to obtain a single cell suspension. Cumulus Cells (CCs) are produced by microscopic surgical excision of Cumulus Oocyte Complexes (COC). After 48 hours of administration of serum gonadotropins (PMSG, M2620, nanjing Abbe Biotechnology Co., ltd.) to pregnant horses, 10 UIMSG was intraperitoneally injected for 48 hours to obtain GV oocytes in the ovaries, and 10UI human chorionic gonadotropins (hCG, M2520, nanjing Abbe Biotechnology Co., ltd.) was intraperitoneally injected for 14 hours to obtain meiosis II (MII) oocytes from the oviducts. TC and MGCs, as well as CC, were inoculated into complete medium containing alpha-MEM supplemented with 20% fetal bovine serum (FBS, FSP500, excell Bio), 2mM L-glutamine (25030081, gibco), 100U mL-1 penicillin and 100 μg mL-1 streptomycin (15140163, invitrogen) in 60mM dishes, followed by 37℃and 5% CO 2 Initial incubation was performed.
Cell culture
The experiment used mainly two mesenchymal stem cells-umbilical cord mesenchymal stem cells (Umbilical cordmesenchymal stem cells, UMSCs) and bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMMSCs).
UMSC was purchased from science cell (7500) and cultured in complete medium containing alpha-MEM supplemented with 20% fetal bovine serum (FBS, FSP500, excell Bio), 2mM L-glutamine (25030081, gibco), 100U mL-1 penicillin and 100. Mu.g mL-1 streptomycin (15140163, invitrogen) followed by 37℃and 5% CO 2 Initial incubation was performed. MSCs were cultured to 80% confluence in 10cm dishes.
For BMMSCs, excess CO was used according to the guidelines of the animal ethics Committee 2 Killing mice, taking under aseptic conditionThe lower tibia and femur are peeled off, the muscles and connective tissues attached to the upper tibia and femur are further separated, the metaphysis is further separated, the bone marrow cavity is exposed, PBS with the volume fraction of 10% fetal calf serum is extracted by a 10mL sterile syringe, the bone marrow cavity is repeatedly flushed, a cell filter screen with the aperture of 70 mu m is used for filtering, 500g is centrifuged for 5min, the cell sediment at the bottom is collected after the supernatant is removed, PBS is resuspended, 500g is centrifuged for 5min again, and the final cell sediment is collected. The cells were then flow sorted, and bone marrow BMMSCs were sorted using CD 34-and CD90+ as sorting criteria. Finally, the cells were resuspended in Dex (-) medium and inoculated in 10cm diameter cell culture dishes at 37℃with 5% CO 2 Culturing. After 24h, the supernatant was aspirated off of non-adherent cells, and after PBS washing, dex (-) broth was added for continued culture. Equal amounts of Dex (+) medium were added after 1 week, and dense primary BMMSCs colonies were visible after another 1 week. The digested BMMSCs were incubated with trypsin at 37℃and passaged for expansion, after which the Dex (+) medium was changed every 3 days and passaged after confluence. Subsequent experiments were performed using P2 generation BMMSCs.
Wherein, the composition of the Dex (-) culture solution is shown in Table 1, and the composition of the Dex (+) culture solution is shown in Table 2:
TABLE 1Dex (-) culture composition table
TABLE 2Dex (+) culture fluid formulation table
Induction and detection of apoptosis
To induce apoptosis, MSCs were washed twice with 0.1 μm filtered PBS (C10010500 BT, thermosusher), and then added to a dish with STS (500 nM, ALX-380-014-M005, enzo life sciences) in alpha-MEM (5 mL) to induce apoptosis of MSCs for 6h. Apoptosis rate of MSCs was detected by terminal deoxynucleotidyl transferase mediated dUTP notch end-labeling (TUNEL, G3250, promega).
Wherein the alpha-MEM medium was supplemented with 20% fetal bovine serum (FBS, FSP500, excell Bio), 2mM L-glutamine (25030081, gibco), 100U mL-1 penicillin and 100 μg mL-1 streptomycin (15140163, invitrogen).
Isolation and characterization of ApoV
ApoV is isolated by continuous centrifugation according to the previously reported protocol [5, 11]. Briefly, apoptotic MSCs were collected and then cell debris was removed by centrifugation at 800g for 10 min at 4 ℃ and at 2000g for 10 min at 4 ℃, followed by collection of supernatant and centrifugation at 16,000g for 30 min at 4 ℃, suspension of pellet in sterile PBS, centrifugation at 16000g for 30 min, removal of supernatant, suspension of pellet in sterile PBS to obtain ApoV. Quantification of ApoV was performed using nanoparticle tracking analysis (NTA, zataview, particle metric) to detect size and Zata potential with NTA software (Zataview, particle metric).
Labelling of ApoV
ApoV is labeled with the previously reported method [10 ]]. To label ApoV with membrane dye, PKH26 (PKH 26PCL, sigma-Aldrich), cellMask was used according to manufacturer's instructions TM Deep Red (C10046, invitrogen) or membrane catenin V (640908, biolegand). In order to label ApoV with AIEgen, DCPY [19 ] was synthesized according to the protocol we set up]. DCPY was then added to serum-free cell culture medium at a final concentration of 5. Mu.M and incubated for 30 minutes followed by washing. Cells were then induced to undergo apoptosis by light irradiation.
Enzyme-linked immunosorbent assay (ELISA)
Determination of plasma estradiol (E) with mouse ELISA kit (CSB-E05109 m and CSB-E05101m, cusabio) 2 ) And testosterone (T) levels. Briefly, 50. Mu.L of standard solution at a concentration of 0, 50, 120, 250, 500, 1200 or 0, 1, 2.5, 5, 10, 15ng/ml, and E 2 Or T or diluted mouse plasma was added to antibody pre-coated microtiter wells. Then, 50. Mu. LHRP-conjugate was mixed with the solutionAdd to the microassay wells. Next, 96-Kong Hun was pooled and incubated at 37℃for one hour, then after three washes 50. Mu.L of substrate A and 50. Mu.L of substrate B were added to each well, and then allowed to stand for 15 minutes at 37℃and 50. Mu.L of stop solution was added to each well for 10 minutes using an microplate reader set at 450 nM. The concentration of Natriuretic Peptide Precursor C (NPPC), cGMP, phosphodiesterase 3A (PDE 3A) and cAMP was also determined using ELISA kits (JM-025M 2, JM-12746M2, JM-023048M 2, jinmei; abc55abcam) according to the manufacturer's instructions.
Mating test
Preparation of a total volume of 200. Mu.L of the composition from 1X 10 6 Solutions of ApoV of MSCs and injecting them into MRL/lpr and CYP17A1-cre; axin1 fl/fl The mice with the neutralization failure were administered via tail vein once a week for four weeks. The number of offspring per female is recorded.
Immunofluorescence (IF) staining
For IF staining, ovarian tissue was isolated and fixed in 4% pfa overnight. The samples were dehydrated in 30% sucrose solution and then embedded in an optimal cleavage temperature (OCT, 4583-12, sakura) compound. Tissue sections were incubated with primary antibodies to CYP17A1 (94004S, CST), FOXL2 (NB 100-1277, novus) and PPAR-gamma (SC 7273, santa Cruz) overnight at 4℃then washed in PBST (PBS+0.1% Triton X-100) and incubated with secondary antibodies and DAPI for 2 hours at room temperature, then sections were washed and blocked for confocal imaging. All confocal images were captured on an LSM 980 NLO/Zeiss.
Westernblot assay
The total protein of cells or ApoV was harvested and cleaved in RIPA (Sc-24948A,Santa Cruz) on ice for 30 min. Total protein concentration was measured by BCA protein assay kit (23225, invitrogen). Western blot analysis was performed using the following protocol. Briefly, total protein (20 μg) was loaded onto 4% -12% nupage TM Bis-Tris gels (NP 0321BOX, invitrogen) and transferred to 0.2 μm polyvinylidene difluoride (PVDF) membrane (ISEQ 00010, millipore). Blocking the membrane with 5% BSA for 1 hour at room temperature, howeverFollowed by overnight incubation with primary antibodies to AKT (9272 s, cst), axin1 (2087 s, cst), GSK-3β (12456 t, cst), RNF43 (bs-7007 r, bios), GAPDH (5174 s, cst) or β -actin (a 5441, sigma) at 4 ℃. After washing with Tris buffered saline containing 0.1% Tween-20 (TBST), the membranes were incubated with species-dependent HRP conjugated secondary antibody (Santa Cruz) at 1:10,000 dilution for 1 hour at room temperature. Using SuperSignal TM West Pico PLUS chemiluminescent substrate kit (34580,Thermo Scientific) and SuperSignal TM West Femto Maxi Sensitivity substrate kit (34095, invitrogen) visualizes immunoreactive proteins and passes through ChemiDoc TM MP imaging system (Bio-Rad, USA).
Flow cytometry analysis
To detect RNF43 expression, MSC and MSC-derived ApoV and circulating ApoV from organisms were treated with RNF43 primary antibody (bs-7007R, bios) in filtered PBS at a concentration of 1:200 at 4deg.C for 1 hour. After centrifugation and washing with PBS, MSC and MSC-derived ApoV as well as circulating ApoV were stained with FITC-conjugated donkey anti-rabbit secondary antibody (406403, biolegend) in filtered PBS at a concentration of 1:200 at 4℃for 1h. To detect surface marker expression of TC, TC was stained with PE anti-mouse CD34 (551387, BD), PE anti-mouse CD45 (12-0451-82, invitrogen), PE anti-mouse CD44 (553135, BD), PE anti-mouse CD73 (55741, BD), PE anti-mouse CD90 (554898, BD), PE anti-mouse CD105 (562759, BD) and PE anti-mouse sca-1 (108108, biolegand) in filtered PBS at a concentration of 1:200 for 1 hour at 4 ℃.
Nanoflow cytometry
Circulating ApoV stained with Annexin V in WT, MRL/lpr and GLD mice was analyzed using nanoflow (FlowNanoAnalyzer, nanoFCM company) according to the manufacturer's protocol [27]. Particle concentration and particle size distribution were calculated using NanoFCM software (NanoFCM specialty V1.0).
TUNEL assay
Apoptotic MSC and ovarian samples from PCOS patients (irb#) were fixed with 4% pfa and permeabilized. Cells were incubated with TUNEL reagent (G3250, promega) for 60 minutes at 37 ℃ according to the manufacturer's protocol. Positive cells were counted after counterstaining with DAPI under the microscope.
Kidney capsule implantation
Will be about 1X 10 6 Individual mice TC or GC are implanted under the kidney capsule of the recipient mouse. Mice were transplanted with TC or GC in the manner previously reported. Briefly, 1X 10 6 TC or GC were mixed and placed under the kidney capsule of C5757BL/6J mice. Mice were euthanized and harvested 2 months after implantation.
Systemic injection
Culturing MSCs to about 3.0-4.0X10 6 After resuspension of the individual MSCs-derived ApoV with 200. Mu.LPBS, 30. Mu.L heparin was added for use. Taking 8-week-old MRL/lpr mice or GLD mice, and taking about 3×10 mice 6 Cell-derived ApoV was infused into mice via the tail vein. Once a week for a total of four times. One week after the last ApoV infusion, mice were sacrificed and blood and ovarian samples were obtained for further experimental treatment.
Ovarian local injection
After anesthesia of MRL/lpr mice, an approximately 3 cm long port was opened at the dorsal kidney, the ovaries were gently pulled out with forceps, and the total length was approximately 1X 10 6 After resuspension of individual cell-derived ApoV with 20 μlpbs, the ovaries were infused with an insulin needle, gently replaced with forceps, and the muscles and skin were sutured. After one week, mice were sacrificed and blood and ovarian samples were obtained for further experimental treatment.
Statistical analysis
Data are expressed as mean ± SD of at least three replicates. Comparisons between two or more groups were analyzed using one-way ANOVA. P values less than 0.05 were considered statistically significant. Graph analysis was performed using GraphPad Prism 8.00 (GraphPad Software, USA).
Example 1 apoptosis deficiency impairing ovarian folliculogenesis
We used two mouse models, fas mutant (MRL/lpr) and Fas ligand (FasL) mutant (GLD) smallMouse [18 ]]When compared to wild-type (WT) mice, they showed excessive sinus follicle (AF) and polycystic ovary syndrome (PCOS) phenotypes (fig. 1A and 1B; fig. 2A and 2B), as well as significantly reduced circulating ApoV levels (fig. 3). Enzyme-linked immunosorbent assay (ELISA) detection showed that estradiol (E) 2 ) And elevated levels of testosterone (figures 1E and 1F; fig. 2C and 2D). The present study found that these mice met the Rotterdam criteria established in 2003, according to which PCOS could be diagnosed by the presence of polycystic histology and elevated testosterone levels. To investigate whether fertility of apoptosis-deficient mice was impaired, we evaluated oocyte quality and mating tests in MRL/lpr and GLD mice and found significant abnormalities in meiosis II (MII) oocytes after superovulation (fig. 1C and 1D and fig. 2E and 2F). In addition, the number of fetuses in MRL/lpr mice was significantly reduced (FIGS. 1G and 1H). Interestingly, the general appearance of MRL/lpr mouse fetuses showed extensive deformation, such as body size reduction, limb atrophy and tail bending, as well as bone reduction and skull loss (fig. 1G and 1I; fig. 2G to 2I).
EXAMPLE 2ApoV infusion rescue of ovarian injury in MRL/lpr and GLD mice
This example is a study of the recovery of impaired ovarian function in MRL/lpr and GLD mice with respect to systemic infusion of MSC-derived ApoV (MSC-ApoV). We obtained ApoV from staurosporine (STS) -induced MSCs by continuous centrifugation (fig. 4A). The morphology of MSCs showed a significant change after 8h of STS induction (fig. 4B). TUNEL assay results indicated a typical apoptotic response (fig. 4C). The size, zeta potential and surface marker distribution of ApoV (fig. 4D to 4G) [5, 10 ] as described previously were confirmed using Transmission Electron Microscopy (TEM), nanoparticle Tracer Analysis (NTA), cell count analysis and western blot]. Then we go from 3.0-4.0X10 6 ApoV was collected by each MSC and injected into MRL/lpr and GLD mice via the tail vein. We found that MSC-ApoV infusion effectively restored impaired ovarian function in MRL/lpr and GLD mice, particularly with respect to the polycystic phenotype and the number of SF and AF (fig. 5A and 5B and fig. 2A and 2B).
ELISA detection showed MRL/lpr after MSC-ApoV infusionAnd E in GLD mice 2 And testosterone levels were rescued (fig. 5E and 5F and fig. 2C and 2D). The overall appearance of MII oocytes suggests that injury to MII oocytes was rescued by infusion of MSC-ApoV in MRL/lpr and GLD mice (fig. 5C and 5D and fig. 2E and 2F). Furthermore, following MSC-ApoV infusion, the number of fetal and fetal abnormalities in MRL/lpr mice was significantly rescued (fig. 5G to 5I and fig. 2G to 2I). Taken together, these results demonstrate that MSC-ApoV infusion provided an effective therapeutic effect in MRL/lpr and GLD mice to rescue damaged ovarian function.
Example 3TC is an MSC-like cell, preferentially uptake ApoV
It was found that systemic infusion of PKH26 labeled MSC-ApoV was concentrated mainly in ovarian TC, not in Granulosa Cells (GC) or oocytes (fig. 6A). To further confirm the tracing of ApoV in the ovary, we used the chromophore DCPY (AIEgen) with aggregation-induced emission characteristics for mitochondrial targeting labelling of ApoV to avoid membrane adhesion [19]. Tracing of AIE-genApoV did show that systemically infused MSC-ApoV specifically homing to ovary TC (FIG. 6B). There was no significant difference in cellular uptake (%) measured using these two different labeling methods (fig. 6C). Then, we isolated TC from the MSC-ApoV infused mice as previously described (FIG. 7A) [20]. TC is the only cell population in the ovary that produces androgens, which specifically express the mesenchymal cell marker Gli1 (fig. 7B) [21, 22]. As previously reported, apoV tends to be enriched in MSCs to regulate local tissue homeostasis [10, 11]. In view of this, we studied the properties of TCs and found that they express MSC markers and have clonally proliferative and osteogenic capacity (fig. 7C to 7E). Notably, the kidney capsule implantation test showed that TC also had potent adipogenic capacity in vivo (fig. 7F to 7H). Next, we co-cultured PKH 26-tagged ApoV with TC, and found that MSC-ApoV was mainly enriched in perinuclear regions within TC (fig. 6D). These data indicate that TC may be an MSC-like cell that preferentially ingests ApoV.
EXAMPLE 4MSC-ApoV infusion rescue of injured oocytes by NPPC/cGMP/PDE3A/cAMP cascade
The NPPC/cGMP/PDE3A/cAMP cascade involves the natriuretic peptide precursor C of MGC (NPPC), which interacts with the receptor NPR2 on CC to enhance cGMP levels, after which cGMP diffuses into the oocyte to inhibit phosphodiesterase 3A (PDE 3A) activity and ultimately increase cAMP levels [23]. cAMP is a key molecule regulating oocyte maturation and high levels cause meiosis [24]. We found that MGCs' NPPC levels and CC cGMP levels were increased in MRL/lpr oocytes, both of which were rescued by MSC-ApoV infusion (fig. 8A and 8B). We further isolated oocytes to detect their PDE3A and cAMP levels, found that PDEA3 was reduced and cAMP was increased in MRL/lpr oocytes, both of which were recovered after MSC-ApoV infusion (fig. 8C and 8D). These results show that MSC-ApoV infusion rescues NPPC/cGMP/PDE3A/cAMP cascade and ovarian function.
EXAMPLE 5MSC-ApoV infusion to rescue ovarian injury by transferring RNF43 from MRL/lpr mice
We found that MSC-ApoV contained ring finger protein 43 (RNF 43) (FIG. 9A). Flow cytometry analysis confirmed the expression of RNF43 in MSC and MSC-ApoV and circulating ApoV (fig. 9B). The results of the Structured Illumination Microscope (SIM) further showed that MSC-ApoV showed surface expression of RNF43 (fig. 9C). After co-culture with MSC-ApoV, the level of RNF43 expression in MRL/lpr ovary TC was increased (FIG. 9D).
We further investigated whether RNF43 is indeed a key factor in mediating the therapeutic effects of MSC-ApoV in MRL/lpr mice by using RNF43 deficient ApoV derived from RNF43 siRNA knockout (RNF 43 KD) MSCs (fig. 10). We showed that RNF43KD-ApoV was unable to rescue excess SF and AF as well as PCOS phenotype in MRL/lpr mice (FIGS. 9E and 9F). ELISA analysis showed that RNF43KD-ApoV failed to rescue E in MRL/lpr mice 2 And testosterone levels (figures 9G and 9H). RNF43KD-ApoV also failed to rescue abnormal oocytes in MRL/lpr mice, as assessed by the overall appearance of MII oocytes (FIG. 9I). In brief, these findings indicate that RNF43 is required for ApoV-mediated rescue of impaired ovarian function in MRL/lpr mice.
EXAMPLE 6MSC-ApoV infusion to improve DHEA-induced PCOS and rescue and promote agingFertility of annual individuals
BMMSCs-derived ApoV was given DHEA by subcutaneous injection to induce PCOS phenotype [25 ]]Followed by infusion through the tail vein of 3.0-4.0X10 6 ApoVs derived from individual BMMSCs. Representative HE staining of ovarian tissue in DHEA-induced PCOS mice showed that BMMSC-ApoV infusion effectively rescued PCOS phenotypes, including polycystic changes, excessive SF and AF, and homotestosterone (fig. 11A-11C).
Interestingly, we also found that the rate of apoptosis was reduced in the ovarian stroma of human PCOS patients (fig. 12), and that the ovaries of polycystic ovary syndrome (PCOS) patients showed defects in apoptosis in the ovarian stroma.
We studied 15 month old mice (approximately 50 years of age in the corresponding human) with and without BMMSC-ApoV infusion [29]Ovarian status and fertility. Representative HE staining showed a decrease in the number of follicles in ovaries of untreated geriatric mice, whereas 3.0-4.0X10 6 ApoV infusion from individual BMMSC cells effectively increased the number of follicles in aged mice (fig. 13A and 13B). Rough examination of GV oocyte appearance showed reduced number and low quality of GV oocytes in aged mice, which were rescued after BMMSC-ApoV infusion (fig. 13C and 13D). Importantly, we assessed fetal amounts in senescent mice following BMMSC-ApoV infusion and examined any significant abnormalities in offspring. We found that BMMSC-ApoV treatment significantly improved fertility in senescent mice (fig. 13E and 13F).
Wherein, ctrl group in FIGS. 13A-13F refers to eight weeks of C57BL/6 mice.
Example 8 local injection of ApoV treatment of MRL/lpr mice ovarian defects was better than local injection of MSC
In the experiment, MSC and ApoV treatment effects are compared by locally injecting MSC and ApoV to one side of an ovary of an MRL/lpr mouse. As shown in fig. 14A, PCOS phenotype of the ovaries of mice injected locally with MSC or ApoV side MRL/lpr was significantly improved compared to ovaries not injected locally with MSC or ApoV. As shown in fig. 14B, ovarian follicular counting results showed that local injection of MSC reduced the number of MRL/lpr mice ovaries SF, but did not have significant differences compared to control ovaries, whereas local injection of ApoV significantly restored the number of MRL/lpr mice ovaries SF and AF.
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Claims (10)
1. use of an inducible vesicle in the preparation of a medicament for preventing or treating polycystic ovary syndrome.
2. The use of claim 1, wherein the vesicles are vesicles that are produced by inducing stem cells to apoptosis by an external agent when they are in normal survival;
preferably, the method of induction comprises addition of staurosporine, ultraviolet irradiation, starvation, or thermal stress;
preferably, the stem cells are mesenchymal stem cells;
preferably, the mesenchymal stem cell source comprises bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon or peripheral blood;
preferably, the mesenchymal stem cells are derived from a mammal;
preferably, the mammal is selected from primate or murine;
preferably, the primate is a human.
3. The use according to claim 1, wherein the vesicles have a diameter of 0.03-10 μm;
preferably, the vesicles have a diameter of 0.03-6 μm;
preferably, the vesicles have a diameter of 0.03-4.5 μm;
preferably, the vesicles have a diameter of 0.03-1 μm.
4. The use according to claim 1, wherein the concentration of staurosporine is 1-15000nM;
Preferably, the concentration of staurosporine is 200-10000nM;
preferably, the concentration of staurosporine is 500-1000nM;
preferably, the concentration of staurosporine is 500-900nM;
preferably, the concentration of staurosporine is 500-800nM.
5. Use according to claim 1, wherein the vesicle has the marker syncaxin 4;
preferably, the vesicle high expression marker Syntaxin 4;
preferably, the expression of the marker Syntaxin 4 by the vesicles is higher than that of the MSC or exosomes of homogeneous origin;
preferably, the markers further comprise one or more of Annexin V, flotillin-1, cadherin 11 or Intigrin alpha 5;
preferably, the vesicle high expression marker Annexin V, flotillin-1, cadherin 11 or Intigrin alpha 5;
preferably, the expression level of the inducible vesicle pair markers Annexin V, flotillin-1, cadherin 11, intigrin alpha 5 is higher than that of the MSC or exosome of the same origin;
preferably, the medicament is a topical injection medicament;
preferably, the topical injection is a subcutaneous, intramuscular, intra-articular, intravenous or intra-ovarian injection;
preferably, the topical injection drug is added with at least one of a pH adjuster, a buffer, a stabilizer, an isotonic agent, or a local anesthetic.
6. The use according to any one of claims 1 to 5, wherein the method of preparation of the vesicles comprises the steps of:
1) Culturing mesenchymal stem cells; 2) Adding the mesenchymal stem cells of the step 1) into a serum-free medium containing 500-1000nM staurosporine, and collecting cell supernatant;
3) Subjecting the cell supernatant collected in step 2) to ultracentrifugation to obtain the vesicles;
preferably, the step of separating the vesicles by the ultracentrifugation method comprises:
(a) Centrifuging the collected culture supernatant for the first time, and taking the supernatant;
(b) Subjecting the supernatant collected in step (a) to a second centrifugation to obtain a supernatant;
(c) Centrifuging the supernatant received in step (b) for a third time to obtain a precipitate;
(d) Centrifuging the precipitate received in step (c) for the fourth time, and taking the precipitate;
preferably, the first centrifugation is 500-1500g for 5-30 minutes;
preferably, the first centrifugation is 500-1000g centrifugation for 5-20 minutes;
preferably, the first centrifugation is 500-900g for 5-15 minutes;
preferably, the second centrifugation is between 1000 and 3000g centrifugation for 5 to 30 minutes;
preferably, the second centrifugation is from 1500 to 2500g centrifugation for 5 to 20 minutes;
Preferably, the second centrifugation is from 1500 to 2200g centrifugation for 5 to 15 minutes;
preferably, the third centrifugation is 10000-30000g centrifugation for 15-60 minutes;
preferably, the third centrifugation is 12000-25000g centrifugation for 20-60 minutes;
preferably, the third centrifugation is 12000-20000g centrifugation for 20-40 minutes;
preferably, the fourth centrifugation is 10000-30000g centrifugation for 15-60 minutes;
preferably, the fourth centrifugation is 12000-25000g centrifugation for 20-60 minutes;
preferably, the fourth centrifugation is between 12000 and 20000g centrifugation for 20 to 40 minutes.
7. A composition comprising an inducible vesicle and a drug for treating polycystic ovary syndrome;
preferably, the vesicle is a vesicle that is produced by inducing apoptosis of stem cells by an external factor when the stem cells are in normal survival;
preferably, the method of induction comprises addition of staurosporine, ultraviolet irradiation, starvation, or thermal stress;
preferably, the stem cells are mesenchymal stem cells;
preferably, the mesenchymal stem cell source comprises bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon or peripheral blood;
preferably, the mesenchymal stem cells are derived from a mammal;
Preferably, the mammal is selected from primate or murine;
preferably, the primate is a human;
preferably, the vesicles have a diameter of 0.03-10 μm;
preferably, the vesicles have a diameter of 0.03-6 μm;
preferably, the vesicles have a diameter of 0.03-4.5 μm;
preferably, the vesicles have a diameter of 0.03-1 μm;
preferably, the concentration of staurosporine is 1-15000nM;
preferably, the concentration of staurosporine is 200-10000nM;
preferably, the concentration of staurosporine is 500-1000nM;
preferably, the concentration of staurosporine is 500-900nM;
preferably, the concentration of staurosporine is 500-800nM;
preferably, the vesicle has the marker syncaxin 4;
preferably, the vesicle high expression marker Syntaxin 4;
preferably, the expression of the marker Syntaxin 4 by the vesicles is higher than that of the MSC or exosomes of homogeneous origin;
preferably, the markers further comprise one or more of Annexin V, flotillin-1, cadherin 11 or Intigrin alpha 5;
preferably, the vesicle high expression marker Annexin V, flotillin-1, cadherin 11 or Intigrin alpha 5;
preferably, the expression level of the inducible vesicle pair markers Annexin V, flotillin-1, cadherin 11, intigrin alpha 5 is higher than that of the MSC or exosome of the same origin;
Preferably, the composition is a topical injection composition.
8. Use of the composition of claim 7 for the preparation of a medicament for the prevention or treatment of polycystic ovary syndrome.
9. Use of an inducible vesicle in the preparation of a product that promotes, improves, restores or restores fertility in a subject;
preferably, the product comprises a food, a health product or a pharmaceutical product;
preferably, the subject is a subject including elderly individuals;
preferably, the elderly individual is an elderly individual older than 50 years of age;
preferably, the vesicle is one that is produced by inducing apoptosis of stem cells by an external factor when they are in normal survival;
preferably, the method of induction comprises addition of staurosporine, ultraviolet irradiation, starvation, or thermal stress;
preferably, the stem cells are mesenchymal stem cells;
preferably, the mesenchymal stem cell source comprises bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon or peripheral blood;
preferably, the mesenchymal stem cells are derived from a mammal;
preferably, the mammal is selected from primate or murine;
preferably, the primate is a human;
Preferably, the vesicles have a diameter of 0.03-10 μm;
preferably, the vesicles have a diameter of 0.03-6 μm;
preferably, the vesicles have a diameter of 0.03-4.5 μm;
preferably, the vesicles have a diameter of 0.03-1 μm.
Preferably, the concentration of staurosporine is 1-15000nM;
preferably, the concentration of staurosporine is 200-10000nM;
preferably, the concentration of staurosporine is 500-1000nM;
preferably, the concentration of staurosporine is 500-900nM;
preferably, the concentration of staurosporine is 500-800nM.
Preferably, the vesicle has the marker syncaxin 4;
preferably, the vesicle high expression marker Syntaxin 4;
preferably, the expression of the marker Syntaxin 4 by the vesicles is higher than that of the MSC or exosomes of homogeneous origin;
preferably, the markers further comprise one or more of Annexin V, flotillin-1, cadherin 11 or Intigrin alpha 5;
preferably, the vesicle high expression marker Annexin V, flotillin-1, cadherin 11 or Intigrin alpha 5;
preferably, the expression level of the inducible vesicle pair markers Annexin V, flotillin-1, cadherin 11, intigrin alpha 5 is higher than that of the MSC or exosome of the same origin.
10. An ovarian disease medication guidance system comprising the following components:
(1) RNF43 level analysis means; for analyzing the deviation of the RNF43 level in the test inducible vesicle sample from the RNF43 level in the normal inducible vesicle sample;
(2) A result judgment means; the method is used for judging a medication scheme for treating the ovarian diseases according to the RNF43 level analysis result, wherein the medication scheme is as follows: when the RNF43 level in the inducible vesicle sample to be tested is normal, directly administering the inducible vesicle to be tested for medication; when the RNF43 level is low, the inducer vesicles to be detected are not selected for administration;
preferably, the ovarian disease comprises polycystic ovary syndrome.
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| CN202311517230.0A CN117653664A (en) | 2023-11-14 | 2023-11-14 | Use of vesicles in the treatment of ovarian disease |
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| CN202311517230.0A CN117653664A (en) | 2023-11-14 | 2023-11-14 | Use of vesicles in the treatment of ovarian disease |
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