Brain microvascular endothelial cell-derived exosome as well as preparation method and application thereof

Abstract

The invention relates to a brain microvascular endothelial cell-derived exosome and a preparation method and application thereof, cerebral vascular endothelial cells are pretreated by ischemia and hypoxia, and the exosome rich in miRNA with a nerve and blood vessel protection effect can be efficiently prepared. The invention discloses a preparation method of a preferable vascular endothelial cell-derived exosome, which can be used for promoting the repair of nerve and blood vessel units in ischemic cerebrovascular diseases and has a potential wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to brain microvascular endothelial cell-derived exosomes and a preparation method and application thereof. Background Art

[0002] Exosomes (Exos), also known as extracellular vesicles, are double-layer phospholipid particles with a diameter between 30-100nm. Cells can actively secrete them through exocytosis. They contain mRNAs, microRNAs, proteins, DNAs and other bioactive substances, which can be internalized by target cells. Exos are widely present in cerebrospinal fluid (CSF), serum, urine and other body fluids. They are key mediators of intercellular communication and regulate various physiological and pathological processes such as immune response, cell differentiation, angiogenesis and neurotransmission.

[0003] Stroke is a major disease that threatens human health and has extremely high disability and mortality rates. In recent years, the neurovascular unit (NVU) has played a key role in the occurrence, development and rehabilitation of stroke. The NVU is the basic unit for maintaining normal brain function. It is mainly composed of neural cells (neurons, glial cells) and vascular cells (endothelial cells and pericytes). The destruction of its structure and function is closely related to the occurrence and development of a variety of neurological diseases. As a structurally complete and functionally coordinated overall system, the NVU regulates neural metabolism and cerebral blood flow activity, and achieves a steady state of interaction and communication between nerves and blood vessels. In ischemic cerebrovascular diseases such as vascular cognitive dysfunction, dementia, and stroke, the NVU is damaged. How to target and protect the NVU and promote neural function recovery and angiogenesis is an important brain science issue worthy of study. However, the introduction of the concept of NVU provides a new target for neural repair and functional reconstruction in stroke. Summary of the invention

[0004] In view of the existing technical problems, the technical problem to be solved by the present invention is to provide brain microvascular endothelial cell-derived exosomes and a preparation method and application thereof.

[0005] The present invention provides brain microvascular endothelial cell-derived exosomes, wherein the brain microvascular endothelial cell-derived exosomes contain miR-23b-3p.

[0006] The present invention provides a method for preparing brain microvascular endothelial cell-derived exosomes, comprising:

[0007] (1) Cultivating brain microvascular endothelial cells and then subjecting them to hypoxia preconditioning;

[0008] (2) After continuing to culture the cells after hypoxia pretreatment, the cell supernatant was collected;

[0009] (3) Extracting and purifying the cell supernatant to obtain brain microvascular endothelial cell-derived exosomes.

[0010] Preferably, the brain microvascular endothelial cells in step (1) are bEnd.3 vascular endothelial cells;

[0011] The cell culture in step (1) includes culturing in a cell culture medium, changing the cell culture medium, and subculturing the cells.

[0012] Furthermore, the cells are cultured in a medium containing 10% fetal bovine serum, 5% FBS, 1% endothelial cell growth supplement and 1% penicillin / streptomycin solution, placed in an incubator at 37°C and 5% CO2, the cell growth state is observed under a light microscope for 1-2 days, and the medium is changed and passaged after 1-2 minutes of trypsin digestion with 0.25% ETDA.

[0013] Preferably, the hypoxic pretreatment in step (1) is to place the cells in a hypoxic culture container (Mic-101, AIPUINS, USA) for 1 to 8 hours; wherein the hypoxic culture container contains 0.5 to 1% O by volume. 2 2-6% CO 2 and 93-97% N 2 .

[0014] Furthermore, the cells are cells with a confluence of 70 to 80%.

[0015] Preferably, the continued culturing in step (2) is continued culturing in serum-free culture medium for 24-48 hours.

[0016] Preferably, the extraction in step (3) includes: first centrifuging at 300-350g for 5-10min, then centrifuging at 3000-3500g for 15-20min, and then centrifuging at 10000-12000g for 25-30min, collecting the supernatant, centrifuging at 100000-110000g for 2-2.5h to obtain precipitated exosomes Exos, and then resuspending in PBS and centrifuging at 12000-15000g for 2-5min, retaining the supernatant.

[0017] The centrifugation was performed at 4°C.

[0018] Preferably, the purification in step (3) is performed by adding an exosome purification column Exosomoe Purafication Filter (41201ES, Yeasen, China), centrifuging, and the liquid at the bottom of the tube is the purified exosome Exos particles, wherein the centrifugation is performed at 3000-3200g for 10-15 min.

[0019] The present invention provides use of the brain microvascular endothelial cell-derived exosomes or the brain microvascular endothelial cell-derived exosomes prepared by the method in preparing a drug for preventing or treating ischemic cerebrovascular disease.

[0020] The present invention provides use of the brain microvascular endothelial cell-derived exosomes or the brain microvascular endothelial cell-derived exosomes prepared by the method in preparing a drug for repairing a stroke neurovascular unit.

[0021] Beneficial Effects

[0022] The present invention uses ischemia-hypoxia pretreatment of cerebral vascular endothelial cells to efficiently prepare exosomes rich in miRNA with neurovascular protective effects.

[0023] The present invention discloses a method for preparing a preferred vascular endothelial cell-derived exosome, which can be applied to promote the repair of neurovascular units in ischemic cerebrovascular diseases, but is not limited thereto, and has potential broad application prospects.

[0024] The present invention adopts an immortalized mouse brain capillary endothelial cell line (bEnd.3) for preparation, and the cell culture is easy, which can make up for the shortcomings such as stem cell heterogeneity and high preparation cost; the present invention does not use exogenous viruses or infected cells, but uses ischemia and hypoxia pretreatment cells to simulate the cell protection mechanism under the ischemic state of the body, and autonomously expresses and secretes exosomes rich in miRNA; the present invention establishes a neurovascular unit model in vitro, simulates the disease state of cerebral ischemia in the body, uses exosomes to act on co-cultured neurons and vascular endothelial cells, and studies its effect on the neurovascular unit, which is of great significance to the repair of NVU in stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Neurovascular unit model: The establishment and identification of the in vitro neurovascular unit. Hippocampal neurons were identified by NSE fluorescence, and vascular endothelial cells were identified by vWF fluorescence;

[0026] Figure 2 :Identification of exosomes: (A) TEM detection of Exos; (B-C) NTA detection of Exos size and concentration; (D) Western blot detection of Exos positive markers TSG101, Alix and negative marker Calnexin;

[0027] Figure 3:The expression levels of miRNAs in endothelial cell-derived Exos at different times of hypoxic pretreatment (A) Comparison of significantly different miRNAs in endothelial cell RNA-seq between Control and OGD models, miR-23b-3p was significantly overexpressed (B) RT-PCR detection of the expression of endothelial cell-specific miRNAs at different times of OGD; (C) CCK-8 detection of cell viability, *p<0.05 compared with 0h, #p<0.05 compared with 1h and 2h;

[0028] Figure 4 :Exosome uptake by neurons and endothelial cells: Exos were labeled with PKH 26 (red), vWF and NeuN were labeled with brain microvascular endothelial cells and hippocampal neurons (green), respectively, and DAPI was used for background staining (blue). Immunofluorescence detection showed that Exos could be taken up by microvascular endothelial cells (A) and neurons (B);

[0029] Figure 5 :Exosomes promote OGD vascular endothelial cell lumen formation (24h), where (A) vascular endothelial cells in the Con group, OGD group and Exos group grew into tubes; (B) Statistical analysis of the number of cells forming tubes in each group, *p<0.05 compared with the Con group, #p<0.05 compared with the OGD group;

[0030] Figure 6 : Exosomes promote the survival of OGD neurons; (A) Immunofluorescence detection of NeuN expression in neurons of Con group, OGD group and Exos group; (B) Statistical analysis of the number of NeuN-positive cells in each group, *p<0.05 compared with Con group, #p<0.05 compared with OGD group. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0032] Example 1

[0033] 1. Establishment of in vitro neurovascular unit model

[0034] (1) Purchase mouse bEnd.3 vascular endothelial cells (SCSP-5267, Cell Bank of the Chinese Academy of Sciences) and HT22 hippocampal neurons (GNM47, Cell Bank of the Chinese Academy of Sciences);

[0035] (2) For routine cell culture, fresh culture medium containing 5% fetal bovine serum (G8003, Servicebio, Wuhan) was used to prepare a cell suspension, which was then placed in a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 3 min; remove the supernatant, add 3 mL of fresh culture medium containing 5% fetal bovine serum to the cell pellet, and mix by pipetting to obtain a diluted cell suspension;

[0036] (3) Transwell chamber (WG3415, COSTAR, USA), take the upper chamber, pipette 10-15 μL HT22 neuronal cell suspension, and slowly fill the bottom of the cover and the counting plate with the cell suspension from the edge of the bottom of the plate. Move slowly to prevent bubbles from forming;

[0037] (4) For the Transwell chamber, remove the chamber and pipette 10-15 μb of End.3 endothelial cell suspension. Slowly fill the bottom of the cover and the counting plate with the cell suspension from the edge of the bottom of the plate. Move slowly to prevent bubbles from forming.

[0038] (5) HT22 cell culture medium (NO.GZ20011-500M, Servicebio) containing 10% fetal bovine serum and 1% endothelial cell growth supplement was used. The cells were placed at 37°C and 5% CO 2 incubator.

[0039] (6) Observe the cell culture status under a microscope and change the medium after 2-3 days.

[0040] 2. Construction of hypoxia preconditioning vascular endothelial cell (bEnd3) model

[0041] (1) Purchase mouse bEnd.3 vascular endothelial cells;

[0042] (2) Cell culture: Advanced RPMI 1640 medium (12633012, Gibco) containing 10% fetal bovine serum, 5% FBS, 1% endothelial cell growth supplement and 1% penicillin / streptomycin solution were used. The cells were placed at 37°C and 5% CO 2 incubator.

[0043] (3) Cell medium replacement and cell subculturing: The cell growth status was observed under a light microscope for 1-2 days. The cells were digested with 0.25% ETDA trypsin (25200056, Gibco) for 1-2 min, and then the medium was replaced and subculturing was performed.

[0044] (4) Hypoxia pretreatment: Observe the cell culture status. When the cells in the culture flask reach 80% confluence, place the cells in a hypoxic incubator (1% O 2 ,5%CO 2 , and 94% N 2), the hypoxia pretreatment time was 0h, 1h, 2h, 4h and 8h respectively.

[0045] 3. Preparation and Extraction of Endothelial Cell-derived Exosomes

[0046] (1) bEnd.3 cells pretreated with hypoxia for 0 h, 1 h, 2 h, 4 h, and 8 h were cultured in serum-free medium for 48 h, and the cell supernatant was collected for exosome extraction;

[0047] (2) Remove cells by ultracentrifugation at 300 g for 10 min at 4°C;

[0048] (3) Centrifugation at 3000 g for 20 min at 4°C to remove dead cells;

[0049] (4) Centrifuge at 10,000 g for 30 min at 4 °C to remove cell debris;

[0050] (5) Collect the supernatant and centrifuge at 100,000 g for 2 h at 4 °C to precipitate Exos;

[0051] (6) Exos resuspension: The precipitate is Exos. Take an appropriate amount of PBS, blow the precipitate, resuspend it, and divide it into 1.5 mL centrifuge tubes and store it at -80°C.

[0052] (7) Harvesting Exos particles: Centrifuge the 1.5 mL centrifuge tube containing the resuspension at 12,000-15,000 g for 2-5 min at 4°C, and retain the supernatant, which is rich in exosome particles.

[0053] (8) Exos purification: At 4°C, the supernatant was added to Exosomoe Purification Filter (41201ES, Yeasen, China) and centrifuged at 3000 g for 10 min. The liquid at the bottom of the tube was the purified Exos particles.

[0054] 4. Detection of miRNAs expression and cell activity in bEnd3 cells treated with hypoxia

[0055] (1) Culture cells routinely. When the cells grow to 60-80% of the culture dish, place them in an anoxic incubator (1% O 2 ,5%CO 2 , and 94% N 2 ), the hypoxia pretreatment time was 0h, 1h, 2h, 4h and 8h respectively.

[0056] (2) Extraction of RNA: After conventional cell digestion, centrifuge at 1500 g for 3 min, add lysis buffer, and place the lysed sample or homogenate at room temperature for 5-10 min to completely separate the nuclear protein from the nucleic acid;

[0057] (3) Add 0.2 ml of chloroform, shake and place at room temperature for 5 min, then centrifuge at 2,000 rpm at 4°C for 10 min.

[0058] (4) After absorbing the supernatant, add an equal volume of isopropanol, mix well, and leave at room temperature for 20 min; then centrifuge at 4°C, 12,000 rpm for 10 min and discard the supernatant;

[0059] (5) Add 1 ml of 75% ethanol to wash the precipitate, then centrifuge at 12,000 rpm for 5 min at 4°C and leave to dry at room temperature for 20 min;

[0060] (6) Add appropriate amount of RNase-free ddH 2 O, prepare RNA stock solution and store in liquid nitrogen.

[0061] (7) Reverse transcription of cDNA: Add 0.5-5ug (5ul) total RNA, 181ul oligo(dT), and appropriate amount of ddH2O to a total volume of 12ul to a PCR tube, mix well, and then place in a PCR instrument at 70°C for 5 min; add 4ul 5× Buffer, 1ul RNAase inhibitor, and 2ul 10mM dNTP, and mix gently;

[0062] (8) PCR amplification: Place the transcription reaction mixture (DNA polymerase, DNA template, primers and nucleotides), denature at 94°C for 30 seconds, and cycle 30-35 times: denature at 94°C for 30 seconds, anneal at 55-60°C for 30 seconds, and extend at 72°C for 24 seconds; terminate the extension at 72°C for 5-10 minutes.

[0063] (9) CCK-8 experiment: CCK-8 test kit (Order No. E606335) was used to detect the activity of bEnd.3 vascular endothelial cells after hypoxia pretreatment for different time periods (0 h, 1 h, 2 h, 4 h, 8 h) according to the product instructions.

[0064] (10) Screening of hypoxic pretreatment time points: Based on the heat map of miRNAs with significant expression differences and the correlation analysis between the time course changes of miR-23b-3p expression and the survival rate of bEnd.3 vascular endothelial cells, Exos at appropriate hypoxic pretreatment time points were screened for subsequent experiments.

[0065] 5. Exosome Uptake by Neurovascular Unit Cells

[0066] (1) bEnd.3 cells pretreated with hypoxia for 0 h, 1 h, 2 h, 4 h, and 8 h were cultured in serum-free medium for 48 h, and the cell supernatant was collected for exosome extraction;

[0067] (2) Remove cells by ultracentrifugation at 300 g for 10 min at 4°C;

[0068] (3) Centrifugation at 3000 g for 20 min at 4°C to remove dead cells;

[0069] (4) Centrifuge at 10,000 g for 30 min at 4°C to remove cell debris;

[0070] (5) Collect the supernatant and centrifuge at 100,000 g for 2 h at 4 °C to precipitate Exos;

[0071] (6) After the exosomes were resuspended, PKH26 (NO.PKH26GL, Sigma-Aldrich) was added to label the Exos and incubated with the in vitro neurovascular unit model for 24 h.

[0072] (7) The upper chamber HT22 hippocampal neuronal cells were stained with NSE (1:200, NO.ab79757, Abcam), bEnd3 brain microvascular endothelial cells were stained with vWF (1:200, NO.ab287962, Abcam), and DAPI was used for contrast staining. The uptake of Exos by endothelial cells and neurons was observed under a laser confocal microscope.

[0073] 6. Microvascular Endothelial Cell Growth and Lumen Formation Experiment

[0074] (1) Grouping: normal mouse brain microvascular endothelial cell group (Con group), oxygen-glucose deprivation (OGD) group, OGD group + hypoxia preconditioning (4 h) bEnd.3 vascular endothelial cell-derived Exos intervention group (Exos group), 12 h after intervention;

[0075] (2) Preparation of cell suspension: Normally cultured cells were digested with trypsin, centrifuged, and 3 ml of culture medium containing 5% fetal bovine serum was added to prepare a cell suspension;

[0076] (3) Preparing a culture dish containing collagen coating: Add 5 ml of the coating solution containing collagen to the culture dish and place it at room temperature 25°C for 1 hour;

[0077] (4) Preparation of cell coating: Pipette 10-15 μb End.3 vascular endothelial cell suspension and slowly spread the cell suspension over the culture dish coating from the edge of the coating;

[0078] (5) Place at 37°C, 5% CO 2 incubator culture;

[0079] (6) Observe cell morphology and lumen formation under an optical microscope at 0, 12, and 24 h. Use the number or index of lumen formation to perform quantitative analysis and calculate the cell lumen forming ability.

[0080] like Figure 5 As shown: Figure 5 Under the A-light microscope, the endothelial cells in the normal group grew for 24 hours, and the endothelial cells formed cords and then formed a lumen. In the OGD group, the ability and number of endothelial cells to form cords decreased. However, after exosome treatment (Exos group), the lumen growth of endothelial cells was significantly improved, and the number was significantly increased compared with the OGD group ( Figure 5 B), indicating that Exos can significantly improve the growth of endothelial cells and promote the formation of vascular lumen under OGD state.

[0081] 7. NeuN fluorescence detection of neuronal survival

[0082] (1) Grouping: mouse hippocampal neuron HT22 cells (Con group), OGD group, and OGD group + hypoxia pretreatment (4 h) bEnd.3 vascular endothelial cell-derived Exos intervention group (Exos group), 48 h after intervention;

[0083] (2) Take a cell slide grown for 48 hours and fix the cells with 4% paraformaldehyde;

[0084] (3) After routine washing with PBS, add 10 μl of 10% normal fetal bovine serum to the slide using a micropipette and block at room temperature for 30 min;

[0085] (4) Add primary antibody for incubation: Add NeuN antibody (1:200, NO.ab177487, Abcam) with a pipette and place in a 4°C refrigerator overnight;

[0086] (5) Add fluorescent secondary antibody: After routine washing, add Alexa Fluor TM 594 secondary antibody, incubate at room temperature for 1 h in the dark.

[0087] (6) Add 1 ml of DAPI in a dark environment and continue incubation for 5-10 minutes.

[0088] (7) Photograph and analyze using a fluorescence microscope.

[0089] like Figure 6 As shown under a fluorescence microscope, OGD can induce HT22 hippocampal neuron damage and reduce the number of surviving cells. After exosome treatment (Exos group), the number of surviving HT22 hippocampal neurons increased significantly compared with the OGD group ( Figure 6 B), indicating that Exos can significantly reduce OGD-induced neuronal damage.

Claims

1. A brain microvascular endothelial cell-derived exosome, characterized in that: The brain microvascular endothelial cell-derived exosomes contain miR-23b-3p.

2. A method for preparing brain microvascular endothelial cell-derived exosomes, comprising: (1) Cultivating brain microvascular endothelial cells and then subjecting them to hypoxia preconditioning; (2) After continuing to culture the cells after hypoxia pretreatment, the cell supernatant was collected; (3) Extracting and purifying the cell supernatant to obtain brain microvascular endothelial cell-derived exosomes.

3. The preparation method according to claim 2, characterized in that: The brain microvascular endothelial cells in the step (1) are bEnd.3 mouse brain vascular endothelioma cells; the cell culture includes culturing in a cell culture medium, changing the cell medium, and subculturing.

4. The preparation method according to claim 2, characterized in that: The hypoxic pretreatment in step (1) is to place the cells in a hypoxic culture container for 1 to 8 hours; wherein the hypoxic culture container contains 0.5 to 1% O2, 2 to 6% CO2 and 93 to 97% N2 by volume.

5. The preparation method according to claim 4, characterized in that: The cells are cells with a confluence of 70 to 80%.

6. The preparation method according to claim 2, characterized in that: The continued culturing in step (2) is continued culturing in serum-free culture medium for 24-48 hours.

7. The preparation method according to claim 2, characterized in that: The extraction in step (3) includes: first centrifuging at 300-350g for 5-10min, then centrifuging at 3000-3500g for 15-20min, and then centrifuging at 10000-12000g for 25-30min, collecting the supernatant, centrifuging at 100000-110000g for 2-2.5h to obtain precipitated exosomes Exos, and then resuspending in PBS and centrifuging at 12000-15000g for 2-5min, and retaining the supernatant.

8. The preparation method according to claim 2, characterized in that: The purification in step (3) is performed by adding an exosome purification column and centrifuging.

9. Use of the brain microvascular endothelial cell-derived exosomes according to claim 1 or the brain microvascular endothelial cell-derived exosomes prepared by the method according to claim 2 in the preparation of a drug for preventing or treating ischemic cerebrovascular disease.

10. Use of the brain microvascular endothelial cell-derived exosomes according to claim 1 or the brain microvascular endothelial cell-derived exosomes prepared by the method according to claim 2 in preparing a drug for repairing neurovascular units in stroke.

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