Amniotic epithelial cell-derived exosome for treating gestational hypertension disease and application of amniotic epithelial cell-derived exosome

Abstract

The invention discloses an amniotic epithelial cell-derived exosome for treating gestational hypertension and application thereof, and belongs to the technical field of biological medicines. The exosome (hAECs-exos) is obtained by secreting and extracting human amniotic epithelial cells (hAECs), the particle size distribution of the exosome (hAECs-exos) is 30-150 nm, and the exosome (hAECs-exos) expresses exosome specific markers CD9, CD63 and CD81. The invention also provides a preparation method of the exosome. The preparation method comprises the following steps: isolated culture of hAECs, and ultracentrifugal extraction and identification of the exosome. Animal experiments prove that when the exosome is applied to a gestational hypertension disease model animal in an intravenous injection mode, the blood pressure and urine protein level of the gestational hypertension disease model animal can be remarkably reduced, and the fetal rat dysplasia condition is improved. The invention provides a brand-new and effective biological preparation candidate scheme for the treatment of the gestational hypertension disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to amnion epithelial cell-derived exosomes for treating pregnancy-induced hypertension and application thereof. BACKGROUND

[0002] Pregnancy-induced hypertension is a common and serious complication during pregnancy, characterized by high blood pressure and proteinuria after 20 weeks of gestation, and can develop into multiple organ dysfunction if not controlled in time, which is one of the main causes of maternal and perinatal mortality. Pregnancy-induced hypertension not only increases the risk of maternal diabetes, cardiovascular and kidney diseases in the future, but also is an important risk factor for neonatal respiratory distress syndrome and pulmonary hypoplasia, seriously endangering the life and health of mother and child. However, the specific pathogenesis has not been fully elucidated so far. At present, it is believed that chronic hypertension history, diabetes, multiple pregnancy, immune system diseases, congenital heart disease, advanced primipara and obesity are high-risk factors for the disease. A large number of studies have shown that the occurrence of pregnancy-induced hypertension is mainly related to remodeling disorders of uterine spiral artery trophoblasts, leading to placental ischemia and hypoxia, oxidative stress, excessive activation of inflammatory immune response, vascular endothelial cell damage, angiogenesis and dysfunction, mitochondrial dysfunction, maternal cardiovascular dysfunction and maternal-fetal interface immune imbalance. At present, the clinical treatment of pregnancy-induced hypertension mainly includes two aspects: one is to give low-dose aspirin to high-risk patients with pregnancy-induced hypertension in the early stage of pregnancy for prevention; the other is to give symptomatic and supportive treatment such as antihypertensive, antispasmodic and sedative treatment to patients who have been definitely diagnosed, and timely termination of pregnancy is the only effective treatment measure if the condition still progresses. Therefore, it is of great importance to study the causes and molecular mechanisms of PE and find safe and effective treatment options for the maintenance of maternal and child health. SUMMARY

[0003] In view of the problems in the prior art, the application provides amnion epithelial cell-derived exosomes for treating pregnancy-induced hypertension and application thereof. The application discloses amnion epithelial cell-derived exosomes for treating pregnancy-induced hypertension and application thereof, which comprises the following steps: extracting human amnion epithelial cell-derived exosomes and identifying; transplanting the extracted human amnion epithelial cell-derived exosomes into the body of an animal with pregnancy-induced hypertension through intravenous input to prove the treatment effect. The application provides a new direction for the clinical treatment of pregnancy-induced hypertension.

[0004] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0005] The application discloses application of amnion epithelial cell-derived exosomes in preparation of a medicine for preventing and / or treating pregnancy-induced hypertension.

[0006] Further, the amniotic epithelial cell-derived exosomes have a diameter of 30-150 nm and express one or more of exosome markers CD9, CD63 and CD81 on the surface.

[0007] The application further discloses a preparation method of the amniotic epithelial cell-derived exosomes. (a) isolating and culturing human amniotic epithelial cells (hAECs); (b) collecting cell culture supernatant; (c) performing differential centrifugation and ultracentrifugation on the supernatant to separate and extract the exosomes.

[0008] Further, in step (a), the human amniotic epithelial cells positively express CD29, CD90, CD105 and SSEA-4, and do not express CD31, CD34 and HLA-DR.

[0009] The application further discloses application of the preparation method in preparation of the exosomes for use in a medicine for preventing and / or treating gestational hypertension.

[0010] The application further discloses a pharmaceutical composition, characterized by comprising the exosomes prepared by the preparation method and a pharmaceutically acceptable carrier.

[0011] Further, the pharmaceutical composition is in the form of an injection.

[0012] Further, the pharmaceutical composition is in the form of an injection.

[0013] Compared with the prior art, the application has the following beneficial effects.

[0014] The application overcomes the difficulty that the treatment effect on gestational hypertension is not obvious at present, and significantly inhibits the occurrence and development of gestational hypertension by intravenous transplantation of human amniotic epithelial cell-derived exosomes.

[0015] The application first proposes to use human amniotic epithelial cell-derived exosomes (hAECs-exos) to treat gestational hypertension, which provides a brand-new biological agent selection with etiological treatment potential for the current clinical scheme that can only rely on symptomatic treatment (such as antihypertensive and antispasmodic) and termination of pregnancy. Animal experiment results show that intravenous injection of hAECs-exos can significantly reduce the systolic pressure and 24h urine protein level of a gestational hypertension model rat, and effectively relieve the core clinical symptoms of the disease.

[0016] The invention discloses methods that not only improve maternal physiological indicators but also significantly reduce fetal growth retardation and placental weight loss caused by disease, lowering the incidence of fetal malformations and miscarriages, demonstrating a beneficial effect of dual protection for both mother and child. hAECs are derived from discarded amniotic membrane tissue after birth, making them widely available and ethically uncontroversial. Flow cytometry analysis shows that hAECs do not express HLA-DR immune rejection-related antigens, and the exosomes they secrete have low immunogenicity, indicating high safety for clinical application.

[0017] This invention provides a complete technical solution from the isolation and culture of hAECs to the extraction and identification of exosomes. The parameters of key steps (such as centrifugation force, culture medium composition, and identification standards) are clearly defined, the process flow is clear, and it has good reproducibility and standardization potential, laying the foundation for subsequent industrial production.

[0018] hAECs-exos naturally carry a variety of bioactive substances (such as proteins and RNA), which can exert their effects through multiple pathways, including regulating inflammatory responses, protecting vascular endothelial function, and promoting angiogenesis. This aligns with the core pathological mechanism of "placental ischemia-oxidative stress-inflammatory activation" in gestational hypertension, providing a scientific basis for their therapeutic efficacy. Attached Figure Description

[0019] Figure 1 Flow cytometry was used to detect the expression of surface markers in hAECs.

[0020] Figure 2 Identification of hAECs-exos. A: Morphology of hAECs-exos observed under transmission electron microscopy. Scale bar = 100 nm. B: NTA assay of hAECs-exos particle size distribution. C: Western blot analysis of the expression of hAECs-exos surface proteins CD9, CD63, and CD81.

[0021] Figure 3 Changes in systolic blood pressure in rats of each group. Note: * indicates P<0.05 compared with the NP group; # This indicates that compared with the PE group, P < 0.05.

[0022] Figure 4 Changes in 24-hour urinary protein in rats of each group. Note: * indicates P<0.05 compared with the NP group; # This indicates that compared with the PE group, P < 0.05.

[0023] Figure 5 Morphological images of fetal mice in each group. A: Abortion image of fetal mice in the PE group. B: Fetal mice in the NP group. C: Fetal mice in the PE group. D: Fetal mice in the hAECs-exos group. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0025] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0026] 1. Method.

[0027] 1.1 Isolation and culture of hAECs.

[0028] (1) Cut fresh human amniotic membrane tissue into evenly sized pieces and wash repeatedly with phosphate-buffered saline (PBS) or 0.9% sodium chloride injection to remove residual blood and mucus.

[0029] (2) Place the cleaned amniotic membrane tissue in PBS buffer containing 1000 U / mL gentamicin and 2.5 μg / mL amphotericin B and soak for 20 min.

[0030] (3) After 20 minutes, the amniotic tissue was transferred to a culture flask, and an appropriate amount of trypsin-EDTA solution was added to digest the tissue. The flask was placed in a constant temperature shaker at 37°C and shaken repeatedly for three to four times.

[0031] (4) Collect the digestive fluid and add KnockOut TM After digestion was terminated with serum substitute (KSR), the cells were passed through a 200-mesh cell sieve, centrifuged, and the supernatant was discarded.

[0032] (5) Collect the cell pellet and resuspend it in DMEM / F12 medium containing 10% KSR, 10 ng / mL epidermal growth factor (EGF), 1% glutathione (GlutaMAX) and 1% non-essential amino acids (NEAA).

[0033] (6) Count the cells using a cell counter, at a rate of 2.5 × 10⁻⁶. 9 Seeds were planted at a cell density of 1 / L in 25 cm² cells. 2 In the culture flask, incubate at 37°C, 5% CO2, and saturated humidity in an incubator, and replace with complete culture medium after 24 h.

[0034] (7) Under an inverted microscope, regularly observe the morphology and growth of the cells. When the cell confluence reaches 80-90%, add trypsin and incubate at 37°C for 5 minutes. Then stop the digestion, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add culture medium, mix well, count the cells, and divide according to 2.5 × 10⁻⁶. 9 Cells were seeded at a density of 10 cells / L and continuously expanded to the first generation (P1).

[0035] 1.2 Determination of hAECs stem cell markers.

[0036] (1) Take hAECs and adjust the cell density to 1×10⁻⁶. 6 / mL.

[0037] (2) Take 1 mL of cell suspension, wash the cells with cold PBS, and then resuspend the cells in 100 μL of PBS.

[0038] (3) Add 5 μL of monoclonal antibody and set up the group without antibody as the negative control group.

[0039] (4) Incubate at 4℃ in the dark for 30 min, then rinse 3 times with cold PBS.

[0040] (5) The expression of CD29, CD31, CD34, CD90, CD105, HLA-DR and SSEA-4 was detected by flow cytometry.

[0041] 1.3 hAECs-exos separation and extraction.

[0042] (1) Take hAECs and inoculate them into T75 culture flasks and culture them in a conventional manner until the confluence reaches 80%.

[0043] (2) After washing the cells with PBS 2-3 times, the cells were seeded in DMEM / F12 medium and cultured for 48 h in an incubator at 37°C, 5% CO2 and saturated humidity.

[0044] (3) Collect the supernatant and use a 4℃ centrifuge to repeatedly centrifuge to obtain the supernatant. Centrifuge at 300g for 5 min, 2000g for 15 min, and 13000g for 35 min in sequence.

[0045] (4) After centrifugation, filter the supernatant using a 0.22μm sterile filter to remove cell debris. Place the filtered supernatant into an ultrafiltration tube for ultrafiltration and discard the lower layer liquid.

[0046] (5) After resuspending in PBS, centrifuge at 150,000g for 3 hours at 4°C, discard the supernatant, take the precipitate, and obtain the exosomes hAECs-exos.

[0047] (6) Detect the hAECs-exos concentration, observe the morphology of exosomes under transmission electron microscopy, detect the particle size distribution of exosomes by NTA, and store at -80℃ for later use.

[0048] 1.4 Transmission electron microscopy.

[0049] (1) Turn on the instrument and adjust it to the ready-to-use state.

[0050] (2) Fix the 2mm aperture copper mesh onto the fixing device with the glossy film facing upward.

[0051] (3) Take 10 μL of the hAECs-exos suspension prepared in 1.3 and drop it onto the copper grid, and let it stand at room temperature for 5 min.

[0052] (4) Use filter paper to absorb the excess liquid from the surface of the copper mesh.

[0053] (5) Take 10 μL of 3% phosphotungstic acid solution and add it to the copper grid to negatively stain the sample. Stain at room temperature for 5 min.

[0054] (6) Use filter paper again to absorb the excess liquid from one side of the copper mesh.

[0055] (7) Dry the exosomes and use 80 KV~120 KV imaging to observe the morphology of the exosomes and take pictures.

[0056] 1.5 NTA.

[0057] (1) Thoroughly clean the sample pool with distilled water.

[0058] (2) The detection instrument was calibrated using polystyrene microspheres (100nm).

[0059] (3) Clean the sample pool again with PBS solution.

[0060] (4) The hAECs-exos prepared in 1.3 were diluted with 1×PBS, and samples were taken for detection. The results were recorded and analyzed.

[0061] 1.6 Western blot.

[0062] (1) Perform total protein extraction and protein concentration determination according to the kit instructions.

[0063] (2) SDS-PAGE electrophoresis: After preparing the separating gel and stacking gel, add the protein. After electrophoresis at 80V for 30 minutes, adjust the voltage to 120V and perform constant voltage electrophoresis to separate 30μg of protein.

[0064] (3) Transfer: Prepare the transfer buffer in advance and pre-cool it to 4°C. Place the transfer electrophoresis core into the transfer tank, add sufficient transfer buffer, set the transfer current to constant current of 200mA, and transfer the protein in the separating gel to the PVDF membrane.

[0065] (4) After the transfer is complete, remove the PVDF membrane and rinse the membrane surface with TBST.

[0066] (5) Sealing: At room temperature, the transferred membrane was incubated with 5% skim milk powder sealing solution for 2 hours.

[0067] (6) Primary antibody incubation: Cut the PVDF membrane according to the molecular weight of the protein, place it in the diluted primary antibody hybridization solution, and incubate overnight at 4°C.

[0068] (7) Wash the PVDF membrane 5 times with TBST, 5 minutes each time.

[0069] (8) Secondary antibody incubation: Add the horseradish peroxidase-labeled secondary antibody diluted with 5% BSA (1:10000), and incubate at room temperature for 1 hour.

[0070] (9) Place the PVDF membrane in TBST and wash 5 times, 5 minutes each time.

[0071] (10) Development: In a light-proof environment, prepare the ECL chemiluminescent reagent solution A: solution B = 1:1, mix well, pipette an appropriate amount of the ECL mixture and drop it on the PVDF membrane, and use a chemiluminescent imager to collect images.

[0072] 1.7 Experimental animals.

[0073] Twelve healthy SPF-grade male SD rats aged 8 - 10 weeks with a body weight of (250 ± 20) g and twenty-four female SD rats with a body weight of (220 ± 20) g were provided by Beijing Huafukang Biotechnology Co., Ltd. The animal quality certificate number was NO.110322230100948264, and the animal use license number was SCXK (Beijing) 2019 - 0008. They were adaptively fed for 1 week in an environment with a room temperature of 18 - 22°C, a relative humidity of 50% - 70%, and automatically regulated day and night of 12 hours each, without controlling the supply of feed and water. This experimental study has been approved by the Sub-committee of the Hospital Animal Medicine Research Ethics Committee (Ethical number: No. 2024 - 19).

[0074] 1.8 Establishment and grouping of the preeclampsia model.

[0075] After one week of acclimatization, rats were caged together at 17:00 with a female:male ratio of 2:1. The vaginal plugs were examined before 8:00 the following morning. If a milky white gelatinous substance was observed in the female rat's vagina, it was considered a vaginal plug, and pregnancy was considered to have occurred on day 0 of pregnancy (GD0). Pregnant rats were randomly divided into three groups: normal pregnancy group (NP group), preeclampsia group (PE group), and treatment group (hAECs-exos group), with eight rats in each group. On GD5, the PE group and hAECs-exos group received intraperitoneal injections of L-NAME 150 mg / kg 1 mL for 7 consecutive days, while the NP group received 1 mL of physiological saline. On GD12, the hAECs-exos group received a tail vein injection of hAECs-exos 1 mg / kg 1 mL 6 hours after L-NAME injection, for 7 consecutive days. Simultaneously, the NP and PE groups received 1 mL of physiological saline.

[0076] 1.9 Measurement of blood pressure in rats.

[0077] On the mornings of GD4, 8, 11, 15, and 18, the blood pressure of rats was measured using a non-invasive blood pressure monitor. The measurement was taken three times while the rats were at rest, and the average value was recorded.

[0078] 1.10 24-hour urinary protein determination in rats.

[0079] Urine was collected from rats in groups GD4, 8, 11, 15, and 18 between 8:00 AM and 8:00 AM the following day using metabolic cages. Urine volume was measured, and urinary protein concentration was detected using the Coomassie brilliant blue method.

[0080] 1.11 Specimen collection and processing.

[0081] GD19 terminated the pregnancy, the fetus was removed by cesarean section, and the fetal weight was recorded.

[0082] 1.12 Statistical analysis.

[0083] SPSS 26.0 statistical software was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation. One-way ANOVA was used to compare differences among multiple groups, and LSD-t test was used for pairwise comparisons between groups. P < 0.05 was considered statistically significant.

[0084] 2. Results.

[0085] 2.1 Identification results of hAECs.

[0086] Microscopic examination revealed that the primary amniotic epithelial cells exhibited a uniform morphology and a typical cobblestone-like arrangement. Flow cytometry analysis at passage 1 showed that hAECs positively expressed CD29, CD90, CD105, and SSEA-4, but did not express CD31, CD34, or HLA-DR (see [link to flow cytometry]). Figure 1 ).

[0087] 2.2 Identification results of hAECs-exos.

[0088] Transmission electron microscopy revealed that hAECs-exos are round or oval vesicles, resembling saucers (see...). Figure 2 A). NTA analysis showed that 97.3% of the samples had a diameter distribution of 121.3 ± 71.9 (FWHM) nm, consistent with the exosome size range (see [reference]). Figure 2 B). Western blot results showed that hAECs-exos expressed exosome-specific markers CD9, CD63, and CD81 (see B). Figure 2 C).

[0089] 2.3 Blood pressure measurement results of rats in each group.

[0090] As shown in Table 1, Figure 3 As shown, there was no significant difference in systolic blood pressure among the three groups of GD4 rats (P>0.05). After intraperitoneal injection of L-NAME, the systolic blood pressure of rats in GD8, GD11, PE, and hAECs-exos groups was significantly higher than that in the NP group (P<0.05), indicating that the PE rat model was successfully established. With prolonged gestation, the blood pressure of rats in the PE group continued to rise. After tail vein injection of hAECs-exos, the systolic blood pressure of rats in GD15, GD18, and hAECs-exos groups was significantly lower than that in the PE group (P<0.05).

[0091] Table 1. Measurement results of systolic blood pressure in rats at different gestation days (n=8, mean ± standard deviation, mmHg).

[0092] Note: * indicates that P < 0.05 compared to the NP group; # This indicates that compared with the PE group, P < 0.05.

[0093] 2.4 Results of 24-hour urinary protein measurement in rats of each group.

[0094] like Figure 4 As shown, there was no significant difference in 24-hour urinary protein among the three GD4 groups (P>0.05). After intraperitoneal injection of L-NAME, the 24-hour urinary protein levels in GD8, GD11, PE, and hAECs-exos groups were significantly increased compared to the NP group (P<0.05). With prolonged gestation, the urinary protein content in the PE group continued to rise. After tail vein injection of hAECs-exos, the 24-hour urinary protein levels in GD15, GD18, and hAECs-exos groups were significantly lower than those in the PE group (P<0.05).

[0095] 2.5 Development of fetal mice in each group.

[0096] Visually, all fetal mice in the NP group showed normal development; in the PE group, one pregnant mouse experienced abortion, and the embryo stopped developing (see...). Figure 5 A), and multiple fetal mice in the PE group showed limb deformities and congestion and edema (see A). Figure 5 C); Fetal mice in the hAECs-exos group were larger in morphology than those in the PE group, and limb deformities and congestion were significantly improved (see...). Figure 5 D). As shown in Table 2, compared with the NP group, the fetal mouse and placental weights in the PE group were significantly reduced (P<0.05); compared with the PE group, the fetal mouse and placental weights in the hAECs-exos group were increased (P<0.05).

[0097] Table 2. Fetal mouse development in each group (n=8, mean ± standard deviation).

[0098] Note: * indicates that compared with the NP group, P<0.05. Δ This indicates that compared with the PE group, P < 0.05.

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of an exosome derived from amniotic epithelial cells in the preparation of a medicament for the prevention and / or treatment of hypertensive disorders of pregnancy.

2. The application according to claim 1, characterized in that, The exosomes derived from amniotic epithelial cells have diameters ranging from 30 nm to 150 nm and express one or more of the exosome markers CD9, CD63, and CD81 on their surface.

3. A method for preparing exosomes derived from amniotic epithelial cells as described in any one of claims 1-2, characterized in that, Includes the following steps: (a) Isolation and culture of human amniotic epithelial cells (hAECs); (b) Collect the cell culture supernatant; (c) The supernatant is subjected to differential centrifugation and ultracentrifugation to separate and extract the exosomes.

4. The preparation method according to claim 3, characterized in that, In step (a), the human amniotic epithelial cells positively express CD29, CD90, CD105 and SSEA-4, but do not express CD31, CD34 and HLA-DR.

5. The use of the preparation method according to any one of claims 3-4 in a medicament for the prevention and / or treatment of gestational hypertension by preparing the exosomes.

6. A pharmaceutical composition, characterized in that, It includes exosomes prepared by the preparation method of any one of claims 3-4 and pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition is an injection.

8. Use of the pharmaceutical composition of any one of claims 6-7 in the preparation of a medicament for the prevention and / or treatment of hypertension during pregnancy.

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