CN113713114A - Chimeric membrane vesicle and preparation method and application thereof - Google Patents
Chimeric membrane vesicle and preparation method and application thereof Download PDFInfo
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- CN113713114A CN113713114A CN202111032205.4A CN202111032205A CN113713114A CN 113713114 A CN113713114 A CN 113713114A CN 202111032205 A CN202111032205 A CN 202111032205A CN 113713114 A CN113713114 A CN 113713114A
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Abstract
The invention provides a chimeric membrane vesicle and a preparation method and application thereof, relating to the technical field of biological medicine. The raw materials for preparing the Neu-Ex comprise hucMSC-Ex and Neu-NVs, and the hucMSC-Ex and the Neu-NVs are ultrasonically mixed and then extruded to obtain the Neu-Ex. The Neu-Ex can be stored for a long time at the temperature of minus 80 ℃ after being subpackaged, is easy to store and use, can be directly used for preventing and treating the acute kidney injury induced by the cisplatin, and has obvious effect; and the Neu-Ex is counted according to particles, the dosage is convenient to master, and the Neu-Ex can be used for preparing a medicinal preparation for preventing and treating acute kidney injury.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a chimeric membrane vesicle and a preparation method and application thereof.
Background
Acute Kidney Injury (AKI), a clinical syndrome that occurs as a result of rapid decline in renal function due to a variety of etiologies, is one of the most common clinical acute complications, and has the characteristics of high morbidity and mortality. It is mainly manifested by scanty or no urine, swelling, poor appetite, etc., and most patients need to be hospitalized. In hospitalized patients, nearly 20% suffer from AKI, with higher mortality and poorer outcome, with prognosis related to a number of risk factors including sepsis, trauma, diabetes, age, etc. There are currently no truly effective drugs for the treatment of AKI. A plurality of preclinical studies show that hucMSC-Ex has obvious effect on treating AKI, but the clinical application of the human is limited by the insufficient targeting of exosome to kidney injury parts.
In recent years, cell transplantation therapies represented by stem cells, particularly umbilical cord mesenchymal stem cells (hucMSCs), have attracted considerable attention in medical research for the prevention and treatment of acute and chronic renal injuries. Research shows that hucMSC can play a role in protecting the kidney by directly differentiating into damaged kidney cells or by the action mechanisms of paracrine action, immunoregulation and the like, and has wide clinical transformation and application prospects in prevention and treatment of acute and chronic kidney injury. However, after the MSC is implanted into the body, the safety problem of long-term application and the problems of difficult storage and transportation exist, the clinical application of the MSC is greatly limited, and the search for a biological material or a stem cell product which can replace the MSC and has the effect of repairing the tissue damage is very urgent.
Disclosure of Invention
In view of the above, the present invention aims to provide a chimeric membrane vesicle, and a preparation method and an application thereof, wherein the chimeric membrane vesicle has better stability, stronger targeting property, better effect of preventing and/or treating acute kidney injury, and better commercialization prospect.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a chimeric membrane vesicle, which is prepared from the following raw materials: the human blood neutrophil-derived nano vesicles and the human umbilical mesenchymal stem cell-derived exosomes;
the number ratio of the nano vesicles to the exosomes is (0.8-1.2): (0.8 to 1.2).
Preferably, the preparation method of the nanovesicle comprises the following steps: and (3) carrying out ice bath ultrasonic treatment on the neutral particle cell membrane protein for 2-5 min, and then sequentially extruding the neutral particle cell membrane protein to pass through polycarbonate membranes with the aperture of 400nm, 200nm and 100nm to obtain the nano vesicles.
Preferably, the power of the ultrasound is 100W, and the ultrasound is performed every 3s and every 10 s.
Preferably, the preparation method of the exosome comprises the step of separating and obtaining the exosome from supernatant of human umbilical cord mesenchymal stem cells by using a sucrose density gradient centrifugation method with the mass percentage concentration of 30%.
The invention also provides a preparation method of the chimeric membrane vesicle, which comprises the following steps: and ultrasonically mixing the nano vesicles and the exosome for 2-5 min in an ice bath, and sequentially extruding the mixture to pass through polycarbonate membranes with the pore diameters of 400nm, 200nm and 100nm to obtain the chimeric membrane vesicles.
Preferably, the power of the ultrasound is 100W, and the ultrasound is performed every 3s and every 10 s.
The invention also provides application of the chimeric membrane vesicle in preparation of a medicine for preventing and/or treating acute kidney injury.
The invention also provides a medicament for preventing and/or treating acute kidney injury, which comprises the chimeric membrane vesicle.
Has the advantages that: the invention provides a chimeric membrane vesicle (Neu-Ex), the diameter of the Neu-Ex is distributed in the range of 30-200 nm, and the membrane potential is higher than two groups of absolute values of a human umbilical cord mesenchymal stem cell exosome (hucMSC-Ex) and a human blood neutrophil-derived nano vesicle (Neu-NVs) before chimeric; the fused Neu-Ex has a typical spherical structure; Neu-Ex expresses a protein component common to hucMSC-Ex and Neu-NVs cells.
Animal model experiments prove that Neu-Exs plays a significant role in renal function protection in vivo and vitro cisplatin-induced animal model experiments. In vivo results show that the Neu-Ex can effectively enhance the targeting property of the hucMSC-Ex to the kidney injury part; following the Neu-Ex intervention, tubular necrosis and inflammatory cell infiltration in renal tissue are reduced, creatinine and urea nitrogen levels are reduced, and renal tissue cell proliferative capacity is enhanced; in vitro results show that the Neu-Ex stem prognosis, the renal tubular epithelial cell apoptosis is reduced, and the expression of inflammatory factors is reduced.
In conclusion, the exosome used in the invention is derived from hucMSC of a newborn, so that the exosome has no ethical problem, lower immunogenicity and higher safety; the Neu-Ex can be stored for a long time at the temperature of minus 80 ℃ after being subpackaged, is easy to store and use, can be directly used for preventing and treating the acute kidney injury induced by the cisplatin, and has obvious effect; and the Neu-Ex is counted according to particles, the dosage is convenient to master, and the Neu-Ex can be used for preparing a medicinal preparation for preventing and treating acute kidney injury.
Drawings
FIG. 1 shows the identification of human umbilical mesenchymal stem cells;
FIG. 2 is a schematic diagram of a method for separating and purifying human umbilical mesenchymal stem cell exosomes;
FIG. 3 shows the identification of isolated human umbilical mesenchymal stem cell exosomes;
FIG. 4 shows the identification of human blood neutrophils;
FIG. 5 is a process of preparing human neutrophil-derived nanovesicles;
FIG. 6 is the identification of human neutrophil-derived nanovesicles;
FIG. 7 is the identification of chimeric membrane vesicles fused with umbilical cord mesenchymal stem cell-derived exosomes from neutrophils nanovesicles;
FIG. 8 is the identification of chimeric membrane vesicles fused with umbilical cord mesenchymal stem cell-derived exosomes from neutrophils nanovesicles;
FIG. 9 is the development of a cisplatin-induced acute kidney injury model;
figure 10 is a graph of the intervention of chimeric membrane vesicles on cisplatin-induced acute kidney injury in vivo and in vitro.
Detailed Description
The invention provides a chimeric membrane vesicle, which is prepared from the following raw materials: human neutrophil-derived nanovesicles (Neu-NVs) and human umbilical mesenchymal stem cell-derived exosomes (hucMSC-Ex);
the number ratio of the nano vesicles to the exosomes is (0.8-1.2): (0.8 to 1.2).
The number ratio of the nanovesicles and exosomes according to the invention is preferably 1:1, and the number of each particle is preferably 4 × 1011And (4) respectively.
The preparation method of the nanovesicle of the present invention preferably includes: and (3) carrying out ice bath ultrasound on the neutral granulocyte cell membrane protein for 2min, and sequentially extruding the neutral granulocyte cell membrane protein to pass through polycarbonate membranes with the pore diameters of 400nm, 200nm and 100nm to obtain the nano vesicles. The power of the ultrasonic wave is 100W, and the ultrasonic wave is performed at an interval of 10s after every 3s of ultrasonic wave. The extrusion described herein is preferably performed using an Avanti micro extruder.
The invention preferably extracts neutrophilic granulocyte membrane protein from human neutrophilic granulocyte, the extraction method of the human neutrophilic granulocyte and the extraction method of the neutrophilic granulocyte membrane protein are not particularly limited, and the human neutrophilic granulocyte membrane protein is extracted from a fresh anticoagulated blood sample by utilizing the conventional extraction method in the field and then is extracted from the neutrophilic granulocyte.
The preparation method of the exosome preferably comprises the step of separating and obtaining the exosome from supernatant of human umbilical cord mesenchymal stem cells by using a sucrose density gradient centrifugation method with the mass percentage concentration of 30%. The present invention is preferably selected from the group consisting of isolated and cultured human umbilical cord mesenchymal stem cells (hucMSC, Qiao Chun et al, human mesenchymal stem cells isolated from the umbilical cord. cell Biol int.2008; 32(1):8-15.) from umbilical cord in neonatal umbilical cord, cultured to the third generation (P3) hucMSC for subsequent testing.
After obtaining primary hucMSCs, the method preferably further comprises the following steps: (1) separating and culturing umbilical cord mesenchymal stem cells until the P3 generation is reached, when the fusion degree of the P3 generation hucMSCs reaches 50-60%, washing the cells by using PBS buffer solution, placing the washed P3 generation hucMSCs in an alpha-MEM culture medium of a serum-free exosome to culture until the P6 generation is reached, and taking hucMSCs supernatant; the alpha-MEM culture medium also contains 10% of fetal calf serum by mass;
(2) centrifuging and sterilizing the hucMSC supernatant to obtain an umbilical cord mesenchymal stem cell exosome hucMSC-Ex;
the centrifugation comprises the steps of carrying out first centrifugation on the hucMSC supernatant, carrying out second centrifugation on the supernatant, carrying out first ultrafiltration centrifugation on the supernatant, carrying out third centrifugation on the supernatant of the membrane, diluting the precipitate of the third centrifugation by using PBS buffer solution, carrying out second ultrafiltration centrifugation, and collecting the supernatant of the membrane;
the centrifugal force of the first centrifugation is 2000g, and the centrifugation time is 10 min;
the centrifugal force of the second centrifugation is 10000g, and the centrifugation time is 30 min;
the first ultrafiltration centrifugation is 100kDa MWCO ultrafiltration centrifugation, the centrifugal force is 1000g, and the centrifugation time is 30 min;
the third centrifugation is carried out by putting the membrane supernatant of the first ultrafiltration centrifugation on a 30% sucrose/heavy water density pad, the centrifugal force of the third centrifugation is 100000g, and the centrifugation time is 3 h;
the second ultrafiltration centrifugation is 100kDa MWCO ultrafiltration centrifugation, the centrifugal force is 1000g, and the centrifugation time is 30 min.
The invention preferably separates umbilical cord mesenchymal stem cells (hucMSC) at 37 ℃ and 5% CO2Culturing in a saturated humidity incubator; performing multidirectional differentiation potential and flow identification on the P3-generation hucMSCs, more preferably selecting P3-generation hucMSCs mother plates with good growth state, adding a adipogenic and osteogenic induced differentiation culture medium, and culturing for corresponding time to perform oil red O staining and alizarin red staining identification; p3 generation hucMSC with good growth state is selected for surface marker staining and then flow cytometry detection analysis is carried out.
The invention preferably selects the obtained P3 hucMSC, when the fusion degree reaches 50-60%, after PBS is washed for 3 times, the culture medium is changed into 10% alpha-MEM culture medium without serum exosome and then is continuously cultured for 48h (low sugar DMEM culture medium containing 10% fetal calf serum), and the culture is stopped after supernatant from P3 generation to P6 generation is collected. The supernatant according to the present invention is preferably obtained by centrifugation, more preferably 300 Xg for 10min to remove floating viable cells, and the supernatant is collected for exosome isolation.
After the hucMSC supernatant is obtained, the invention centrifuges and sterilizes the hucMSC supernatant to obtain an umbilical cord mesenchymal stem cell exosome hucMSC-Ex; the centrifugation comprises the steps of carrying out first centrifugation on the hucMSC supernatant, carrying out second centrifugation on the supernatant, carrying out first ultrafiltration centrifugation on the supernatant, carrying out third centrifugation on the supernatant of the membrane, diluting the precipitate of the third centrifugation by using PBS buffer solution, carrying out second ultrafiltration centrifugation, and collecting the supernatant of the membrane; the centrifugal force of the first centrifugation is 2000g, and the centrifugation time is 10 min; the centrifugal force of the second centrifugation is 10000g, and the centrifugation time is 30 min; the first ultrafiltration centrifugation is 100kDa MWCO ultrafiltration centrifugation, the centrifugal force is 1000g, and the centrifugation time is 30 min; the third centrifugation is carried out by putting the membrane supernatant of the first ultrafiltration centrifugation on a 30% sucrose/heavy water density pad, the centrifugal force of the third centrifugation is 100000g, and the centrifugation time is 3 h; the second ultrafiltration centrifugation is 100kDa MWCO ultrafiltration centrifugation, the centrifugal force is 1000g, and the centrifugation time is 30 min.
The centrifugation according to the invention is preferably all centrifugation at low temperature, more preferably at 4 ℃, wherein the first centrifugation removes intact dead cells and cell debris; a second centrifugation can remove organelles; the first ultrafiltration centrifugation can be concentrated. The concentrate obtained after the first ultrafiltration centrifugation was slowly transferred to a 5ml 30% sucrose/heavy water density pad (rho 1.210 g/cm)3) The third centrifugation was performed, and the bottom 5ml sucrose/heavy water layer (containing exosomes) was collected, diluted with PBS and subjected to the second ultrafiltration centrifugation. In the present invention, it is preferable that the supernatant after the second ultrafiltration centrifugation is washed 3 times with PBS, sterilized by filtration with a sterile filter of 0.22 μm, stored at-80 ℃ after being dispensed, and quantitatively detected by BCA protein quantitative kit method.
The invention also provides a preparation method of the chimeric membrane vesicle, which comprises the following steps: and ultrasonically mixing the nano vesicles and the exosome for 2-5 min in an ice bath, and sequentially extruding the mixture to pass through polycarbonate membranes with the pore diameters of 400nm, 200nm and 100nm to obtain the chimeric membrane vesicles.
The power of the ultrasonic wave is 100W, and the ultrasonic wave is performed at an interval of 10s after every 3s of ultrasonic wave. The extrusion described herein is preferably performed using an Avanti micro extruder.
The invention also provides application of the chimeric membrane vesicle in preparation of a medicine for preventing and/or treating acute kidney injury.
According to the embodiment of the invention, an acute kidney injury animal model is used for verification, and Neu-Ex effectively enhances the targeting property of hucMSC-Ex to a kidney injury part; reduce tubular necrosis and inflammatory cell infiltration in kidney tissue induced by cisplatin, reduce creatinine and urea nitrogen level, and enhance cell proliferation ability of kidney tissue; reduce the apoptosis of the renal tubular epithelial cells, inhibit the expression of inflammatory factors thereof, prove that the Neu-Ex has obvious therapeutic action on the acute renal injury induced by the cisplatin, and therefore, the Neu-Ex can be used for preparing the medicine for preventing and/or treating the acute renal injury.
The following examples are provided to illustrate the chimeric membrane vesicles of the present invention and the preparation and application thereof in detail, but they should not be construed as limiting the scope of the present invention.
The reagents and apparatus used in the examples of the present invention are, if not necessarily, conventional commercial products in the art:
MSC culture reagent: low sugar alpha-MEM (BI), fetal bovine serum (Gibco), antibiotics (Sigma), carbon dioxide incubator (Forma corporation);
an upright microscope, an ultra-clean workbench, a table centrifuge and an ultra-centrifuge;
heavy water (D)2O, shanghai wouisco corporation), analytically pure sucrose (guangzhou chemical reagent factory), adipogenic, osteogenic induction medium, stem cell surface marker detection kit (guangzhou seiki biotechnology limited), CD9, CD63, CD81, TSG101, Calnexin antibody (CST), BCA protein quantification kit, horseradish peroxidase (HRP) -labeled goat anti-rabbit IgG secondary antibody (beijing kang is a century corporation), premixed HRP chemiluminescent substrate, 100kDa MWCO ultrafiltration centrifuge tube, 0.22 μm sterile filter membrane (Millipore corporation, usa); transmission electron microscope (FEI Tecnai 12, Philips); atomic force microscopy (brueck, germany) nanoparticle tracking analyzer (ZetaView, germany);
ICR mice (animal experimental center, university of jiangsu, approved by ethics committee, university of jiangsu);
immunohistochemical staining reagent (warrior, bosch de, operating according to kit instructions);
tubular endothelial cell line NRK52E cells (purchased from ATCC);
an inverted microscope, a confocal and ultra-high resolution microscope, an ultra-clean workbench and a desk centrifuge;
reagents related to the qRTPCR technique (Vazyme).
Example 1 isolation and purification of human umbilical mesenchymal stem cells and exosomes derived therefrom
(1) Isolated culture and characterization of HucMSC: the method of Qiao Chun was used to successfully isolate and culture and identify hucMSCs (Qiao Chun et al, human mesenchyme stem cells isolated from the lithium cardiac cord. cell Biol int.2008 Jan; 32(1):8-15), and the isolated hucMSCs were cultured at 37 ℃ with 5% CO2Culturing in a saturated humidity incubator; selecting a third-generation hucMSC seed plate with good growth state, adding a adipogenic and osteogenic induced differentiation culture medium, and culturing for corresponding time to carry out oil red O staining and alizarin red staining identification; and selecting the third generation hucMSC with good growth state for surface marker staining and then carrying out flow cytometry detection analysis.
The adipogenic induced differentiation results show that the induced hucMSC body presents typical oil drops (A right in figure 1) compared with the negative control group (A left in figure 1); the osteogenic induced differentiation results showed that the induced hucMSC cell bodies exhibited calcium nodules (right B in fig. 1) compared to the negative control group (left B in fig. 1); flow cytometry results showed that hucMSC positively expressed CD29, CD73, and CD105, and negatively expressed CD11b, CD14, and CD45 (C in fig. 1).
(2) Preparation of human umbilical cord mesenchymal stem cell supernatant (hucMSC-CM): selecting 3-6 generation hucMSCs with good growth state, firstly culturing with low-sugar DMEM medium containing 10% fetal calf serum, changing serum-free medium for culturing when the cells are fused to 50% -60%, collecting culture supernatant after 48h, and centrifuging for 10min at 300g to remove floating living cells for exosome separation.
Example 2 separation and purification of human umbilical cord mesenchymal Stem cell-derived exosomes
(1) The separation and purification of exosomes in umbilical cord mesenchymal stem cell supernatant was performed according to the procedure shown in fig. 2: the collected hucMSC supernatant was centrifuged at 2000g for 10min at 4 ℃ to remove cell debris; collecting supernatant, centrifuging at 4 deg.C and 10000g for 30min to remove organelles; transferring the supernatant to a 100kDa MWCO ultrafiltration centrifugal tube, centrifuging at 4 ℃ and 1000g for 30min, and concentrating; the concentrate was slowly transferred to 5ml 30% sucrose/heavy water density pad (ρ ═ 1.210 g/cm)3) Centrifuging at 100000g for 3h at 4 ℃; collecting 5ml of sucrose/heavy water layer (containing exosome) at the bottom, diluting with PBS, adding into a 100kDa MWCO ultrafiltration centrifugal tube, centrifuging at 4 ℃ for 30min at 1000g, and washing with PBS for 3 times; and finally, filtering and sterilizing by using a sterile filter membrane of 0.22 mu m, subpackaging, storing at the temperature of minus 80 ℃, and carrying out protein quantitative detection by using a BCA protein quantitative kit method, wherein the concentration of the separated hucMSC-Ex is 40 mg/ml.
(2) The basic morphology of exosomes was observed by transmission electron microscopy: and (2) taking 20 mu L of hucMSC-Ex, fully and uniformly mixing, dropwise adding the mixture on a sample-carrying copper net with the diameter of 2mm, standing at room temperature for 5min, slightly absorbing residual liquid at the edge of the copper net by using filter paper, then reversely covering the copper net on 30g/L phosphotungstic acid (pH 6.8) liquid drops, carrying out negative dyeing at room temperature for 5min, finally drying the copper net under an incandescent lamp, and placing the dried copper net under a transmission electron microscope for observation and photographing, wherein an exosome is of a typical cup-shaped structure as shown in A in figure 3.
(3) And (3) observing the height and the appearance of the exosome by using an atomic force microscope: 10 μ L of hucMSC-Ex was added dropwise to the cell slide, and after standing and drying at room temperature, rinsed with double distilled water to remove PBS salt crystals. After natural drying again, the exosomes were typically "cup-shaped" in structure, as shown in fig. 3B, and observed and photographed under an atomic force microscope.
(4) Westernblot detection of surface marker proteins of hucMSC-Ex: preparing 15% SDS-PAGE electrophoresis gel, fully cracking the above-mentioned extracted exosomes, adding 1/4 volume of 5 xSDS sample-loading buffer solution, boiling for 5min, loading sample according to 200. mu.g protein total quantity, transferring protein onto PVDF membrane by means of electrotransfer (350mA, 120min), sealing with TBS/T containing 50g/L skimmed milk at room temperature for 1h, respectively reacting with CD9, CD63, CD81, TSG101 and Calnexin antibody (1:500) at 4 deg.C overnight, washing membrane 3 times with daily TBS/0.5% Tween 20, incubating with HRP-labeled goat anti-rabbit IgG secondary antibody at 37 ℃ for 1h), washing the membrane for 3 times with TBS/0.5% Tween 20, adding a premixed HRP chemiluminescent substrate, and detecting by a chemiluminescence gel imaging system, as shown in figure 3C, the hucMSC-Ex protein specific markers of CD9, CD63, TSG101, Alix and HSP70 are positively expressed, and Calnexin is negatively expressed.
(4) Particle size, concentration and potential of the NTA assay hucMSC-Ex: mu.L of hucMSC-Ex was diluted (1:5000) and detected on NTA. As shown in D and E in FIG. 3, the hucMSC-Ex particle size distribution is 30-150 nm, the peak is about 123nm, and the hucMSC-Ex particle size distribution is negative potential.
Example 3 preparation and identification of human neutrophilic granulocyte membrane nanovesicles
(1) The separation and identification of human blood neutrophils: obtaining a fresh anticoagulated blood sample from a university Hospital of Jiangsu university after soliciting informed consent of healthy volunteers, slowly adding 5ml of fresh whole blood into a 15ml sterile centrifuge tube with 5ml of polymorph serum spread at the bottom along the tube wall in an ultra-clean workbench, slowly lifting and slowly centrifuging at 23 ℃ for 30min at 600g, and carefully sucking a middle white membrane layer into a serum-free 1640 medium by a pipettor for washing; centrifuging at 23 deg.C for 5min at 800g, carefully removing supernatant, adding freshly prepared liquid, carefully blowing, stirring, standing for 5-10min, and terminating with serum-free 1640 medium; centrifuging at 23 deg.C and 800g for 5min, collecting the neutrophilic granulocyte precipitate, washing with precooled PBS for three times, centrifuging at 4 deg.C and 800g for 5min, and collecting the cell precipitate; detecting the cell morphology by using an upright microscope; giemsa reyi staining detects nuclei and cytoplasm; flow cytometry detected the neutrophil-specific markers CD11b and CD 33.
As shown in FIG. 4, observation under an upright microscope revealed that neutrophils were typically spherical (A in FIG. 4); flow assay showed cell surface expression of specific molecules CD11B and CD33 (B in fig. 4); giemsa reyi staining showed that neutrophils present a typical rod-like or lobular nuclear structure (C in FIG. 4); confocal laser microscopy also revealed that neutrophils were isolated with intact membrane structures.
(2) The extraction and identification of human neutrophil membrane proteins were performed according to the procedure shown in fig. 5: resuspending the neutrophil precipitation with IB-1 separating medium containing protease and phosphatase inhibitor, repeatedly freezing and thawing for 3 times, and performing ice bath ultrasound for 5min to break the cells; centrifuging at 800g for 10min at 4 ℃ to remove completely dead cells and cell debris; centrifuging the supernatant at 4 deg.C and 10000g for 30min to remove organelle components; centrifuging the supernatant at 4 deg.C for 2h at 100000g to obtain membrane protein precipitate; centrifuging at 4 deg.C for 2h at 100000g, washing the enriched membrane protein precipitate for 2 times, and resuspending to-80 deg.C for use. Detecting the protein content of the neutrophil membrane by using a BCA method; coomassie brilliant blue staining and Western blotting technique were used to detect the total membrane protein component of neutrophils and some specific protein molecules, respectively.
As shown in fig. 6, the coomassie bright assay showed that the extracted neutrophil membranes predominantly expressed the same protein component of the neutrophils at around 70kDa (C in fig. 6); the results of Westernblot show that the extracted neutrophile cell membrane protein is enriched with a large amount of membrane surface protein components: CCL2, CXCR4, Fas, ICAM-1, integrin α V and integrin β 3 (D in FIG. 6).
(3) Preparing and identifying human blood neutrophilic granulocyte membrane nano vesicles: after the neutral granulocyte colony membrane protein is subjected to ice bath ultrasound for 2min, sequentially extruding membrane protein components through polycarbonate membranes (400nm, 200nm and 100nm) with different apertures by using an Avanti miniature extruder to form neutral granulocyte colony membrane nano vesicles (Neu-NVs); a nanoparticle analyzer (NTA) detects the particle size, concentration and potential of Neu-NVs; detecting the size and the morphology of Neu-NVs by a Transmission Electron Microscope (TEM); and detecting the height and the morphology of the Neu-NVs by an atomic force microscope (TEM).
As shown in fig. 6, atomic force microscopy and transmission electron microscopy showed that the nanovesicles formed after extrusion had a typical spherical structure (E and F in fig. 6); NTA detection results show that the nanocapsules formed after extrusion have negative potential (G and H in figure 6) of 30-200 nm; the corresponding vesicular structure was also detected by ultra high resolution microscopy (I in fig. 6).
Example 4 preparation and characterization of Neu-Ex
Preparing and identifying a chimeric membrane vesicle fused by Neu-NVs of a neutrophil source and hucMSC-Ex: and ultrasonically mixing Neu-NVs and hucMSC-Ex for 2min, and sequentially extruding the mixture to pass through polycarbonate membranes (400nm, 200nm and 100nm) with different pore diameters to form Neu-Ex. A nanoparticle analyzer (NTA) detects the particle size, concentration and potential of Neu-Ex; detecting the size and the morphology of Neu-Ex by a Transmission Electron Microscope (TEM); detecting the height and the morphology of Neu-Ex by an atomic force microscope (TEM); detecting Neu-Ex specific protein by using a protein immunoblotting technology; and detecting the Neu-Ex fusion condition by using an ultra-high resolution microscope and a fluorescence resonance energy transfer technology.
NTA detection results show that the fused Neu-Ex diameters are distributed in the range of 30-200 nm, and the membrane potential is higher than absolute values of other two groups (A and B in figure 7); the detection results of a Transmission Electron Microscope (TEM) and an Atomic Force Microscope (AFM) show that the fused Neu-Ex has a typical spherical structure (C and D in FIG. 7); coomassie brilliant blue detection and Western blot detection results show that the fused Neu-Ex expression cells have common protein components; the results of the ultra-high resolution microscopy showed that significant fusion occurred between green fluorescent dye-labeled Neu-NVs and red fluorescent dye-labeled hucMSC-Ex after extrusion (FIG. 8).
EXAMPLE 5 therapeutic Effect of Neu-Ex
(1) Constructing a cisplatin-induced acute kidney injury model: acute kidney injury is caused 72 hours after a single intraperitoneal injection of 10mg/kg cis-platinum (Sigma) into ICR mice with the weight of 20-25 g.
Cisplatin treatment is carried out for 72h, and serum biochemical detection results show that indexes such as creatinine, urea nitrogen and the like of the model group mice are remarkably reduced (A in figure 9). HE staining results showed that the kidney tissue vacuole degeneration of the model group was significant and the tissue structure was disordered (B in fig. 9); the success of the cisplatin-induced acute kidney injury model construction is shown.
(2) In vivo and in vitro therapeutic effects of chimeric membrane vesicles on acute kidney injury: after cisplatin is induced for 72 hours, acute kidney injury model mice are randomly divided into a PBS group, a neutrophil nano vesicle group and a chimeric membrane vesicle group; the same number of particles (8X 10) were injected into the tail vein of each mouse10) The vesicles of (3) were treated with PBS as negative control. Evaluating the repairing effect after 48 hours;
HE staining results showed that mouse kidney tissue vacuolar degeneration, decreased tissue inflammatory cell infiltration and relative intact tissue structure following Neu-Ex stem prognosis were reduced compared to PBS and Neu-NVs stem pretreatment alone (A in FIG. 10); immunohistochemical staining showed that the kidney tissue cells of mice with Neu-Ex stem prognosis showed stronger proliferative activity (B in FIG. 10). Serum biochemical detection results show that indexes such as creatinine, urea nitrogen and the like of the mice in the model group are remarkably reduced, and renal function damage is effectively relieved after Neu-Ex intervention (C in figure 10). The in vivo results show that the Neu-Ex has obvious therapeutic action on acute kidney injury induced by cisplatin.
In an in-vitro cisplatin-induced renal tubular endothelial cell (NRK52E) injury model, qRT-PCR detection results show that after Neu-Ex intervention, the expression levels of inflammatory factors TNF-alpha and IL-6 of cisplatin on NRK52E cells are obviously inhibited. This indicates that Neu-Ex has significant therapeutic effect on cisplatin-induced acute kidney injury in vitro and in vivo experiments (fig. 10, D).
TABLE 1 primer sequences for TNF-alpha and IL-6
20 μ l qRT-PCR System: SYBR Green Mix 10. mu.l, RNase free ddH2O7. mu.l, F/R primers 0.5. mu.l each, and cDNA 2. mu.l.
qRT-PCR procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles; further extension was carried out at 72 ℃ for 10 min.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (8)
1. A chimeric membrane vesicle, which is prepared from the following raw materials: the human blood neutrophil-derived nano vesicles and the human umbilical mesenchymal stem cell-derived exosomes;
the number ratio of the nano vesicles to the exosomes is (0.8-1.2): (0.8 to 1.2).
2. The chimeric membrane vesicle according to claim 1, wherein the preparation method of the nanovesicle comprises: and (3) carrying out ice bath ultrasonic treatment on the neutral particle cell membrane protein for 2-5 min, and then sequentially extruding the neutral particle cell membrane protein to pass through polycarbonate membranes with the aperture of 400nm, 200nm and 100nm to obtain the nano vesicles.
3. The chimeric membrane vesicle according to claim 2, wherein the power of the ultrasound is 100W, and the ultrasound is performed every 3s and every 10 s.
4. The chimeric membrane vesicle according to claim 1, wherein the exosome is prepared by a method comprising separating the exosome from the supernatant of human umbilical cord mesenchymal stem cells by sucrose density gradient centrifugation at a concentration of 30% by mass.
5. The method for producing a chimeric membrane vesicle according to any one of claims 1 to 4, comprising the steps of: and ultrasonically mixing the nano vesicles and the exosome for 2-5 min in an ice bath, and sequentially extruding the mixture to pass through polycarbonate membranes with the pore diameters of 400nm, 200nm and 100nm to obtain the chimeric membrane vesicles.
6. The method of claim 5, wherein the power of the ultrasound is 100W, and the ultrasound is performed every 3s and every 10 s.
7. Use of the chimeric membrane vesicle of any one of claims 1 to 4 for the preparation of a medicament for the prevention and/or treatment of acute kidney injury.
8. An agent for preventing and/or treating acute kidney injury, which comprises the chimeric membrane vesicle according to any one of claims 1 to 4.
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| CN110038032A (en) * | 2019-05-10 | 2019-07-23 | 江苏大学 | The biological agent and preparation method of the novel anti-kidney fibrosis of people's umbilical cord MSC excretion body |
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| CN110038032A (en) * | 2019-05-10 | 2019-07-23 | 江苏大学 | The biological agent and preparation method of the novel anti-kidney fibrosis of people's umbilical cord MSC excretion body |
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| Title |
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| HAOYUAN JIA等: "HucMSC exosomes-delivered 14-3-3ζ enhanced autophagy via modulation of ATG16L in preventing cisplatin-induced acute kidney injury", 《AM J TRANSL RES》, vol. 10, no. 1, pages 101 - 113 * |
| 肖瑶等: "中性粒细胞膜纳米囊泡对小鼠脓毒症致急性肾损伤的保护作用研究", 《军事医学》, vol. 45, no. 1, pages 25 - 29 * |
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