CN117045676A - Extracellular vesicles for preventing and/or treating acute lung injury and preparation method and application thereof - Google Patents
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- CN117045676A CN117045676A CN202310932628.4A CN202310932628A CN117045676A CN 117045676 A CN117045676 A CN 117045676A CN 202310932628 A CN202310932628 A CN 202310932628A CN 117045676 A CN117045676 A CN 117045676A
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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Abstract
The invention belongs to the technical field of biological medicines, and relates to an extracellular vesicle for preventing and/or treating acute lung injury, and a preparation method and application thereof. The invention provides application of extracellular vesicles over-expressing OIT3 in preparing a medicament for preventing and/or treating acute lung injury caused by cardiac surgery extracorporeal circulation and/or a medicament for inhibiting activation of lung bronchogenic dermatitis small NLRP3 and secretion of inflammatory factors. The extracellular vesicles over-expressing OIT3 have therapeutic effects on acute lung injury caused by cardiac surgery extracorporeal circulation, and can inhibit activation of lung bronchogenic dermatitis small NLRP3 and secretion of inflammatory factors.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an extracellular vesicle for preventing and/or treating acute lung injury, and a preparation method and application thereof.
Background
Acute lung injury (Acute lung injury, ALI)/acute respiratory distress syndrome (Acute respiratory distress syndrome ARDS) is a common life-threatening complication following surgery for extracorporeal circulation of the heart (cardiopulmonary bypass, CPB). The risk of ALI occurrence in patients following excessive inflammatory response, ischemia reperfusion injury and coagulation dysfunction during CPB in cardiac surgery is significantly increased, with mortality rates of severe ALI/ARDS still up to 46%, mainly due to lack of specific predictable biomarkers and treatment methods for ALI/ARDS after cardiac surgery. Thus, there is a need for a solution to the problem of treating ALI/ARDS that is concurrent after CPB surgery.
Extracellular vesicles (Extracellular vesles, EVs) are secretory membrane vesicles with a diameter of 100-1000 nm, which transport molecules such as proteins, nucleic acids and lipids to target cells, perform their functions, and regulate organ communication. More and more studies have demonstrated that circulating extracellular vesicles are involved in regulating the development of body inflammation in physiological and disease states. Extracellular vesicles have the function of amplifying the effects of biomolecules and compounds, namely, the characteristics of low efficiency and high efficiency. Moreover, the extracellular vesicles are easily extracted and obtained in large quantities by means of differential centrifugation. Thus, the treatment of concurrent ALI/ARDS after CPB surgery using extracellular vesicles is a good way. However, there is currently no extracellular vesicle that enables concurrent ALI/ARDS treatment after CPB surgery.
Disclosure of Invention
The invention aims to provide an extracellular vesicle for preventing and/or treating acute lung injury, and a preparation method and application thereof. Extracellular vesicles over-expressing OIT3 have therapeutic effects on acute lung injury and can inhibit activation of lung bronchodermatitis exosome NLRP3 and inflammatory factor secretion.
The invention provides application of extracellular vesicles over-expressing OIT3 in preparing medicines for preventing and/or treating acute lung injury and/or medicines for inhibiting activation of lung bronchogenic dermatitis exosome NLRP3 and secretion of inflammatory factors.
Preferably, the acute lung injury comprises acute lung injury caused by cardiac surgery extracorporeal circulation.
Preferably, the acute lung injury comprises acute respiratory distress syndrome.
Preferably, the inflammatory factors include interleukin-1 beta, interleukin-6 and tumor necrosis factor alpha.
Preferably, the nucleotide sequence of OIT3 is shown in SEQ ID NO. 1.
Preferably, the extracellular vesicles are prepared by transfecting HEK-293T cell lines based on lentiviral vectors that overexpress OIT3.
The invention also provides an extracellular vesicle for preventing and/or treating acute lung injury, which extracellular vesicle is capable of overexpressing OIT3.
The invention also provides a preparation method of the extracellular vesicles, which comprises the following steps:
the lentiviral vector over-expressing OIT3 is transfected into a HEK-293T cell line to extract extracellular vesicles secreted by the HEK-293T cell line.
The invention also provides a medicine for preventing and/or treating acute lung injury, which comprises the extracellular vesicles and pharmaceutically acceptable auxiliary materials according to the technical scheme.
Preferably, the drug comprises an intravenous drug.
The invention provides an extracellular vesicle for preventing and/or treating acute lung injury. The extracellular vesicles over-expressing OIT3 are easy to construct and obtain in vitro, have a therapeutic effect on acute lung injury, and can inhibit activation of lung bronchogenic dermatitis small NLRP3 and secretion of inflammatory factors. The extracellular vesicles rich in OIT3 protein molecules can treat ALI/ARDS complicated with CPB operation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic representation of the extraction of OIT 3-enriched extracellular vesicles (OE-OIT 3 EV) secreted by HEK-293T cells provided by the present invention;
FIG. 2 is a graph of the nanoparticle trace analysis results of OE-OIT 3EV extracts provided by the present invention;
FIG. 3 is a Western blot of OE-OIT 3EV provided by the present invention;
FIG. 4 is a graph showing the successful results of over-expression of OIT3 by OE-OIT 3EV provided by the present invention;
FIG. 5 shows intravenous injection of a blank EV (EVs) in the course of the rat extracorporeal circulation model provided by the invention NC ) And OIT3EV (EVs) rich OE-OIT3 ) Is a lung tissue HE staining pattern;
FIG. 6 is a plasmid map of a lentiviral vector over-expressing OIT3.
Detailed Description
The invention provides application of Extracellular Vesicles (EV) over-expressing OIT3 in preparing medicines for preventing and/or treating acute lung injury and/or medicines for inhibiting activation and inflammatory factor secretion (release) of lung bronchogenic dermatitis small NLRP 3. In the invention, the nucleotide sequence of the OIT3 is shown in SEQ ID NO. 1: gacaaaggaacaccagtattaagaggattttccagtgtttctggcagttggtccagaaggatgcctccattcctgcttctcacctgcctcttcatcacaggcacctccgtgtcacccgtggccctagatccttgttctgcttacatcagcctgaatgagccctggaggaacactgaccaccagttggatgagtctcaaggtcctcctctatgtgacaaccatgtgaatggggagtggtaccacttcacgggcatggcgggagatgccatgcctaccttctgcataccagaaaaccactgtggaacccacgcacctgtctggctcaatggcagccaccccctagaaggcgacggcattgtgcaacgccaggcttgtgccagcttcaatgggaactgctgtctctggaacaccacggtggaagtcaaggcttgccctggaggctactatgtgtatcgtctgaccaagcccagcgtctgcttccacgtctactgtggtcatttttatgacatctgcgacgaggactgccatggcagctgctcagataccagcgagtgcacatgcgctccaggaactgtgctaggccctgacaggcagacatgctttgatgaaaatgaatgtgagcaaaacaacggtggctgcagtgagatctgtgtgaacctcaaaaactcctaccgctgtgagtgtggggttggccgtgtgctaagaagtgatggcaagacttgtgaagacgttgaaggatgccacaataacaatggtggctgcagccactcttgccttggatctgagaaaggctaccagtgtgaatgtccccggggcctggtgctgtctgaggataaccacacttgccaagtccctgtgttgtgcaaatcaaatgccattgaagtgaacatccccagggagctggttggtggcctggagctcttcctgaccaacacctcctgccgaggagtgtccaacggcacccatgtcaacatcctcttctctctcaagacatgtggtacagtggtcgatgtggtgaatgacaagattgtggccagcaacctcgtgacaggtctacccaagcagaccccggggagcagcggggacttcatcatccgaaccagcaagctgctgatcccggtgacctgcgagtttccacgcctgtacaccatttctgaaggatacgttcccaaccttcgaaactccccactggaaatcatgagccgaaatcatgggatcttcccattcactctggagatcttcaaggacaatgagtttgaagagccttaccgggaagctctgcccaccctcaagcttcgtgactccctctactttggcattgagcccgtggtgcacgtgagcggcttggaaagcttggtggagagctgctttgccacccccacctccaagatcgacgaggtcctgaaatactacctcatccgggatggctgtgtttcagatgactcggtaaagcagtacacatcccgggatcacctagcaaagcacttccaggtccctgtcttcaagtttgtgggcaaagaccacaaggaagtgtttctgcactgccgggttcttgtctgtggagtgttggacgagcgttcccgctgtgcccagggttgccaccggcgaatgcgtcgtggggcaggaggagaggactcagccggtctacagggccagacgctaacaggcggcccgatccgcatcgactgggaggactagttcgtagccatacctcgagtccctgcattggacggctctgctctttggagcttctccccccaccgccctctaagaacatctgccaacagctgggttcagacttcacactgtgagttcagactcccagcaccaactcactctgattctggtccattcagtgggcacaggtcacagcactgctgaacaatgtggcctgggtggggtttcatctttctagggttgaaaactaaactgtccacccagaaagacactcaccccatttccctcatttctttcctacacttaaatacctcgtgtatggtgcaatcagaccacaaaatcagaagctgggtataatatttcaagttacaaaccctagaaaaattaaacagttactgaaattatgacttaaatacccaatgactccttaaatatgtaaattatagttataccttgaaatttcaattcaaatgcagactaattatagggaatttggaagtgtatcaataaaacagtatataattttaaaagta. In the present invention, the average diameter of the extracellular vesicles is preferably 100 to 1000nm. Extracellular vesicles having diameters not between 100 and 1000nm are not included in the present invention.
In the present invention, the acute lung injury preferably includes acute lung injury caused by cardiac surgery extracorporeal Circulation (CPB). In the present invention, the acute lung injury preferably comprises acute respiratory distress syndrome. That is, the extracellular vesicles over-expressing OIT3 of the present invention have good preventive and/or therapeutic effects on both acute lung injury (Acute lung injury, ALI) and severe acute lung injury, acute respiratory distress syndrome (Acute respiratory distress syndrome ARDS). In the present invention, the inflammatory factors include interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). The extracellular vesicles have the effects of inhibiting activation of lung bronchogenic dermatitis small NLRP3 and secretion of inflammatory factors, and improving acute lung injury caused by cardiac surgery extracorporeal circulation. In the present invention, when verifying the effect of the extracellular vesicles on activation of lung bronchodermatitis minibody NLRP3 and secretion of inflammatory factors, it is preferable to use human lung epithelial cells for verification. In the present invention, the human lung epithelial cells are preferably BEAS-2B cell lines. The culture medium for BEAS-2B cell lines preferably comprises 1640 medium. In the present invention, the culture medium for the BEAS-2B cell line preferably further contains exosome Fetal Bovine Serum (FBS). In the present invention, the culture medium for BEAS-2B cell line is preferably obtained by mixing 50ml of exosome-free Fetal Bovine Serum (FBS) per 500ml of basal medium (1640 medium).
In the present invention, the extracellular vesicles are preferably prepared by transfecting HEK-293T cell line (human kidney epithelial cell line) based on lentiviral vector overexpressing OIT3. That is, the extracellular vesicles of the present invention are secreted by HEK-293T cells, and other extracellular vesicles of cellular origin are not included in the present invention. OIT3 protein-rich extracellular vesicles released by HEK-293T cells overexpressing OIT3 protein molecules can assist in the treatment of acute lung injury/acute respiratory distress syndrome following extracorporeal circulation surgery.
The invention also provides an extracellular vesicle for preventing and/or treating acute lung injury, which extracellular vesicle is capable of overexpressing OIT3. In the present invention, the average diameter of the extracellular vesicles is preferably 100 to 1000nm.
The invention also provides a preparation method of the extracellular vesicles, which comprises the following steps:
the lentiviral vector over-expressing OIT3 is transfected into a HEK-293T cell line to extract extracellular vesicles secreted by the HEK-293T cell line.
The method for obtaining the lentiviral vector over-expressing OIT3 is not particularly limited, and a conventional lentiviral vector construction method is adopted, or a company is entrusted to construct, such as Hantaan. The plasmid map of the lentiviral vector overexpressing OIT3 of the present invention is shown in FIG. 6, and is synthesized for Hantao by a second-generation three-plasmid system. After transfection, HEK-293T cells are preferably cultured using a medium according to the present invention. In the present invention, the medium preferably includes DMEM high sugar medium. In the present invention, the medium is more preferably DMEM high sugar medium containing FBS. Specifically, the culture medium is preferably prepared by uniformly mixing every 500ml of basic culture medium DMEM high-sugar culture medium with 50ml of exosome-removed Fetal Bovine Serum (FBS). In the present invention, it is preferable to change the medium 2 times after successful overexpression of 293T cells overexpressing OIT3 protein.
In the present invention, the extraction method preferably includes differential centrifugation. In the present invention, the conditions of the differential centrifugation are preferably 13000g,45min,4 ℃. Specifically, the present invention preferably collects culture supernatants of 293T cells that overexpress OIT3, removes cells or cell debris from the supernatants (i.e., removes sediment) by centrifugation at 2500g for 10min, and then extracts 13000g of the supernatant, which is centrifuged at 4℃for 45min to obtain EV sediment. After the precipitation of EV, the EV is preferably resuspended in an appropriate amount of physiological saline. The solvent for resuspension of the present invention preferably also includes water, PBS or HBSS.
The invention also provides a medicine for preventing and/or treating acute lung injury, which comprises the extracellular vesicles and pharmaceutically acceptable auxiliary materials according to the technical scheme. The medicament of the invention preferably comprises as active ingredient extracellular vesicles secreted from HEK-293T cell lines over-expressing OIT3 proteins. In the present invention, the drug preferably includes an intravenous drug.
When the medicine is applied, the concentration of extracellular vesicles is preferably 150-200 mug/kg. In the present invention, the means for quantifying extracellular vesicles is preferably BCA protein quantification. In the present invention, the drug is preferably administered 2 hours after the start of the cardiac surgery extracorporeal circulation operation.
For further explanation of the present invention, an extracellular vesicle for preventing and/or treating acute lung injury, a preparation method and application thereof, provided by the present invention, will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
HEK-293T cells were transfected with over-expressed OIT3 lentivirus constructed by Hantao (second generation three plasmid system) (plasmid map shown in FIG. 6). Following transfection, cells were cultured in medium (500 ml dmem medium plus 50ml of exosome Fetal Bovine Serum (FBS)) at 37 ℃ for 24 h. After the over-expression is successful, the culture medium is replaced for 2 times, and the possible residual lentivirus in the culture medium is removed.
Then extracting the OIT3 enriched EV: culture supernatants of 293T cells overexpressing OIT3 were collected, centrifuged at 2500g for 10min to remove cells or cell debris from the supernatants, followed by centrifugation at 13000g for 45min at 4℃to obtain EV pellet. The EV was resuspended in an appropriate amount of physiological saline.
The control EV was prepared by the same method as OIT 3-enriched EV, and lentivirus transfected with control virus. Control viruses were also purchased from hantao, in order to construct empty vectors without the OIT3 gene sequence for use in overexpressing OIT3 lentiviruses.
Performing transmission electron microscope observation on the resuspended EV, performing a nano particle size tracking analysis experiment, and verifying an extracellular vesicle marker in a western blotting mode; and performing a subsequent effect verification experiment.
All animal experiments were approved by the ethical review Committee and the animal research Committee of the university of Zhongshan (ethical review code SYSU-IACUC-2022-001417), and all animal studies were performed according to the National Institutes of Health (NIH) guidelines for laboratory animal care and use. The study was performed using wild Sprague-Dawley rats weighing 350-450 g purchased from the laboratory animal center in the east school district of the university of Zhongshan. The rats are routinely fed for 1 week after transfer and surgery is scheduled to exclude the transfer process or the new environment from causing the associated stress response.
Weigh the mark well, use 2.5% isoflurane in O 2 Anesthesia was induced in the mixed air (1:1 volume ratio), followed by 1.5% -2% inhalation of isoflurane to maintain anesthesia. Auxiliary ventilation of trachea cannula connected with breathing machine, tidal volume maintenance of 8ml/kg and respiratory frequency60 times/min. The hair is shaved quickly at the place where the neck and the rat leg are placed, and the iodophor is sterilized. The right neck was cut and the right jugular vein was isolated. The bilateral thighs were each cut to isolate a femoral artery of sufficient length to prepare for cannulation. The right jugular vein is placed in a self-made improved pipeline with a side hole 18F retaining needle and fixed. After the tube is successfully placed, 500IU/kg heparinized is given from the venous pipeline, and the clamping pipeline is connected with the extracorporeal circulation venous return end. The right femoral artery is fixed by using a 24F indwelling needle as an imbedding pipeline and clamped with the input end of the extracorporeal circulation artery. The left femoral artery is similarly placed in the pipeline with a 24F indwelling needle for monitoring the blood pressure during operation. The extracorporeal circulation comprises an oxygenator, a blood reservoir, an axial pressure pump and corresponding connecting lines. 10ml of hydroxyethyl starch solution was used as priming solution to prime the CPB line and circulate the diverted exhaust. During CPB diversion, 50 ml/(kg×min) was targeted for flow. The other femoral artery was used to monitor blood pressure. An extracorporeal circulation model was made to investigate acute respiratory distress syndrome that occurs after extracorporeal circulation.
Only cannulation was performed in the sham surgery sham group. The whole procedure was performed in the CPB group. CPB+EV NC And CPB+EV OE-OIT3 The group was subjected to the subsequent injection administration procedure.
The content of the extracted EVs protein was determined by BCA in advance. Resuspension of EVs (50 μg dose, 2h administration before the initiation of extracorporeal circulation operation) with physiological saline, injecting into rat via jugular vein cannula side hole, and CPB+EV NC Group injection over-expressed control EV, i.e., EV NC ；CPB+EV OE-OIT3 Group injection overexpresses OIT3EV, i.e., EV OE-OIT3 。
The results are shown in FIGS. 1 to 5.
FIG. 1 is a schematic representation of the extraction of OIT 3-enriched extracellular vesicles (OE-OIT 3 EV) secreted by HEK-293T cells; FIG. 2 is a graph of the results of nanoparticle trace analysis of OE-OIT 3EV extract, demonstrating diameters between 100-1000 nm; FIG. 3 is a Western blot of OE-OIT 3EV showing differential centrifugation extracted EV-expressed markers including Flotillin1 and Annexin a5; FIG. 4 is a graph of the results of successful OE-OIT 3EV over-expression of OIT3.
FIG. 5 is a ratIntravenous injection blank control EV (EVs) during extracorporeal circulation model NC ) And OIT3EV (EVs) rich OE -OIT3 ) Is a graph of lung tissue HE staining. According to under-lens H&E staining results show that the lung interstitium thickens and inflammatory cells infiltrate areas, and the lung injury is evaluated as grade 0-3 and classified as grade 0-3. The 0 is divided into: has no thickening of lung interstitium and infiltration of inflammatory cells, and the lung tissue is normal in morphology. 1 is divided into: thickening lung interstitium and inflammatory cell infiltration area<25%.2 is divided into: the lung interstitial thickening and inflammatory cell infiltration area is 25% -50%. The method comprises the following steps: thickening lung interstitium and inflammatory cell infiltration area>50%. The lung injury of rats CPB after intravenous injection of OE-OIT 3EV is significantly improved.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. Use of OIT3 overexpressing extracellular vesicles for the preparation of a medicament for the prevention and/or treatment of acute lung injury and/or for the inhibition of activation of pulmonary bronchogenic dermatitis exosomes NLRP3 and secretion of inflammatory factors.
2. The use according to claim 1, wherein the acute lung injury comprises acute lung injury caused by cardiac surgery extracorporeal circulation.
3. The use of claim 1, wherein the acute lung injury comprises acute respiratory distress syndrome.
4. The use according to claim 1, wherein the inflammatory factors comprise interleukin-1 β, interleukin-6 and tumor necrosis factor α.
5. The use according to any one of claims 1 to 4, wherein the nucleotide sequence of OIT3 is shown in SEQ ID No. 1.
6. The use according to any one of claims 1 to 4, wherein the extracellular vesicles are prepared by transfecting HEK-293T cell line based on lentiviral vectors overexpressing OIT3.
7. An extracellular vesicle for preventing and/or treating acute lung injury, wherein the extracellular vesicle is capable of over-expressing OIT3.
8. The method for preparing extracellular vesicles according to claim 7, comprising the steps of:
the lentiviral vector over-expressing OIT3 is transfected into a HEK-293T cell line to extract extracellular vesicles secreted by the HEK-293T cell line.
9. A medicament for preventing and/or treating acute lung injury, comprising the extracellular vesicles of claim 7 and pharmaceutically acceptable excipients.
10. The medicament of claim 9, wherein the medicament comprises an intravenous medicament.
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