CN113713114B - Chimeric membrane vesicle and preparation method and application thereof - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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Abstract
The invention provides a chimeric membrane vesicle and a preparation method and application thereof, and relates to the technical field of biological medicines. The preparation raw materials of the Neu-Ex comprise hucMSC-Ex and Neu-NVs, and the hucMSC-Ex and the Neu-NVs are extruded after ultrasonic mixing to obtain the Neu-Ex. The Neu-Ex can be packaged and stored at-80 ℃ for a long time, is easy to store and use, can be directly used for preventing and treating acute kidney injury induced by cisplatin, and has remarkable effect; and the Neu-Ex is counted according to particles, the dosage is convenient to grasp, and the Neu-Ex can be used for preparing medicinal preparations 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 due to rapid decline of renal function caused by various etiologies, is one of the clinically extremely common acute complications, and has the characteristics of high morbidity and mortality. It is mainly manifested by oliguria or lack of oliguria, swelling, inappetence, etc., and most patients need hospitalization. Of the hospitalized patients, nearly 20% suffer from AKI, with higher mortality and poorer outcome, and their prognosis is associated with a number of risk factors such as sepsis, trauma, diabetes, age, etc. There is currently no truly effective drug for treating AKI. Several preclinical studies indicate that hucMSC-Ex has significant efficacy in treating AKI, but insufficient targeting of exosomes to kidney injury sites limits its clinical application.
In recent years, cell transplantation therapies typified by stem cells, particularly umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cell, hucMSC), have received attention in medical research for preventing and treating acute and chronic kidney injury. Research shows that hucMSC can play a role in protecting kidney through direct differentiation into damaged kidney cells or through paracrine action, immune regulation and other action mechanisms, and has wide clinical transformation and application prospects in prevention and treatment of acute and chronic kidney injury. However, the MSC has problems of long-term application safety, difficult storage and transportation, and the like after being implanted into the body, so that the clinical application of the MSC is greatly limited, and the search for biological materials or stem cell products capable of replacing the MSC to play a role in repairing tissue injury is urgent.
Disclosure of Invention
In view of the above, the invention aims to provide a chimeric membrane vesicle, a preparation method and application thereof, wherein the chimeric membrane vesicle has better stability, stronger targeting, better prevention and/or treatment effects on 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 comprises the following raw materials: human neutrophil-derived nanovesicles and human umbilical cord mesenchymal stem cell-derived exosomes;
the number ratio of the nano vesicle to the exosome is (0.8-1.2): (0.8-1.2).
Preferably, the preparation method of the nano vesicle comprises the following steps: and (3) sequentially extruding the neutral granulocyte membrane protein through a polycarbonate membrane with the pore diameters of 400nm, 200nm and 100nm after carrying out ice bath ultrasound for 2-5 min, so as to obtain the nano vesicle.
Preferably, the power of the ultrasonic wave is 100W, and the ultrasonic wave is further ultrasonic waves at intervals of 10s after each ultrasonic wave is performed for 3 s.
Preferably, the preparation method of the exosome comprises the step of separating the exosome from the supernatant of the human umbilical cord mesenchymal stem cells by utilizing 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 (3) uniformly mixing the nano vesicles and the exosome by ice bath ultrasound for 2-5 min, and sequentially extruding the mixture through a poly-polycarbonate membrane with the pore diameters of 400nm, 200nm and 100nm to obtain the chimeric membrane vesicles.
Preferably, the power of the ultrasonic wave is 100W, and the ultrasonic wave is further ultrasonic waves at intervals of 10s after each ultrasonic wave is performed for 3 s.
The invention also provides application of the chimeric membrane vesicle in preparing a medicament for preventing and/or treating acute kidney injury.
The invention also provides a medicine for preventing and/or treating acute kidney injury, which comprises the chimeric membrane vesicle.
The beneficial effects are that: the invention provides a chimeric membrane vesicle (Neu-Ex), wherein the diameter of the Neu-Ex is distributed at the size of 30-200 nm, and the membrane potential is higher than two groups of absolute values of human umbilical cord mesenchymal stem cell exosomes (hucMSC-Ex) and human neutrophil-derived nano vesicles (Neu-NVs) before being 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 remarkable role in protecting kidney function in-vivo and in-vitro cisplatin-induced animal model experiments. In vivo results show that the Neu-Ex can effectively enhance the targeting property of hucMSC-Ex to the kidney injury site; the Neu-Ex dry prognosis has reduced tubular necrosis and inflammatory cell infiltration in kidney tissue, reduced creatinine and urea nitrogen levels, and enhanced proliferation of kidney tissue cells; in vitro results showed reduced apoptosis of tubular epithelial cells and reduced inflammatory factor expression following the Neu-Ex intervention.
In conclusion, the exosomes used in the invention are derived from neonatal hucMSC, so that the method has no ethical problem, lower immunogenicity and higher safety; the Neu-Ex can be packaged and stored at-80 ℃ for a long time, is easy to store and use, can be directly used for preventing and treating acute kidney injury induced by cisplatin, and has remarkable effect; and the Neu-Ex is counted according to particles, the dosage is convenient to grasp, and the Neu-Ex can be used for preparing medicinal preparations for preventing and treating acute kidney injury.
Drawings
FIG. 1 is an identification of human umbilical cord mesenchymal stem cells;
FIG. 2 is a schematic diagram of a method for separating and purifying human umbilical cord mesenchymal stem cell exosomes;
FIG. 3 is an isolated identification of human umbilical cord mesenchymal stem cell exosomes;
FIG. 4 is a human blood neutrophil identification;
FIG. 5 is a process flow of preparation of human blood neutrophil-derived nanovesicles;
FIG. 6 is an identification of human blood neutrophil-derived nanovesicles;
FIG. 7 is an identification of chimeric membrane vesicles fused with umbilical cord mesenchymal stem cell-derived exosomes;
FIG. 8 is an identification of chimeric membrane vesicles fused with umbilical cord mesenchymal stem cell-derived exosomes;
FIG. 9 is the establishment of a cisplatin-induced acute kidney injury model;
figure 10 is a graph showing the intervention of cisplatin-induced acute kidney injury in vivo and in vitro with chimeric membrane vesicles.
Detailed Description
The invention provides a chimeric membrane vesicle, which comprises the following raw materials: human neutrophil-derived nanovesicles (Neu-NVs) and human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex);
the number ratio of the nano vesicle to the exosome is (0.8-1.2): (0.8-1.2).
The ratio of the number of the nano vesicles to the number of the exosomes is preferably 1:1, and the number of each particle is preferably 4×10 11 And each.
The preparation method of the nano vesicle preferably comprises the following steps: and (3) sequentially extruding the neutral granulocyte membrane protein through a polycarbonate membrane with the pore diameters of 400nm, 200nm and 100nm after carrying out ice bath ultrasound for 2min, so as to obtain the nano vesicle. The power of the ultrasonic wave is 100W, and the ultrasonic wave is further ultrasonic waves at intervals of 10s after each ultrasonic wave is performed for 3 s. The extrusion according to the invention is preferably carried out using an Avanti mini extruder.
The invention preferably extracts neutrophil membrane protein from human neutrophil, the method for extracting human neutrophil and the method for extracting neutrophil membrane protein are not particularly limited, and the method for extracting neutrophil membrane protein from fresh anticoagulated blood sample and then extracting human neutrophil membrane protein from neutrophil by using the conventional extraction method in the field.
The preparation method of the exosome preferably comprises the step of separating the exosome from the supernatant of the human umbilical cord mesenchymal stem cells by utilizing a sucrose density gradient centrifugation method with the mass percentage concentration of 30%. The present invention preferably uses the method of Qiao Chun et al to isolate and culture human umbilical cord mesenchymal stem cells in neonatal umbilical cord (hucMSCs, qiao Chun et al human mesenchymal stem cells isolated from the umbilical cord. Cell Biol int.2008;32 (1): 8-15.) to obtain primary hucMSCs, and cultures to third generation (P3) hucMSCs for subsequent detection experiments.
After the primary hucMSC is obtained, the method preferably further comprises the following steps: (1) Separating and culturing umbilical cord mesenchymal stem cells to P3 generation, washing with PBS buffer solution when the fusion degree of the P3 generation hucMC reaches 50% -60%, culturing the washed P3 generation hucMC in alpha-MEM culture medium without serum exosomes to P6 generation, and taking hucMC supernatant; the alpha-MEM culture medium is also provided with 10 mass percent of fetal bovine serum;
(2) Centrifuging and degerming the hucMSC supernatant to obtain 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 membrane supernatant, diluting sediment of the third centrifugation by using PBS buffer solution, carrying out second ultrafiltration centrifugation, and collecting the membrane supernatant;
the centrifugal force of the first centrifugation is 2000g, and the centrifugation time is 10min;
the centrifugal force of the second centrifugation is 10000g, and the centrifugation time is 30min;
the first ultrafiltration centrifugation is 100kDa MWCO ultrafiltration centrifugation, the centrifugal force is 1000g, and the centrifugation time is 30min;
the third centrifugation is carried out by placing the membrane supernatant of the first ultrafiltration centrifugation on a 30% sucrose/heavy water density pad, wherein the centrifugal force of the third centrifugation is 100000g, and the centrifugation time is 3h;
the second ultrafiltration centrifugation was 100kDa MWCO ultrafiltration centrifugation, the centrifugal force was 1000g, and the centrifugation time was 30min.
The invention preferably uses isolated umbilical cord mesenchymal stem cells (hucMSCs) at 37 ℃ and 5% CO 2 Culturing in a saturated humidity incubator; the hucMSC of the P3 generation is subjected to multidirectional differentiation potential and flow identification, and more preferably, the method comprises the steps of selecting a P3 generation hucMSC plate with good growth state, adding a adipogenic and osteogenic induction differentiation medium, and culturing until the corresponding time is reached, and carrying out oil red O staining and alizarin red staining identification; and selecting the P3 generation hucMSC with good growth state for surface marker staining and then performing flow cytometry detection analysis.
The invention preferably changes the obtained P3 generation hucMSC into the 10 percent alpha-MEM culture medium without serum exosomes after PBS is washed for 3 times when the fusion degree reaches 50 to 60 percent, and then continues to culture for 48 hours (the low-sugar DMEM culture medium containing 10 percent fetal calf serum), and collects the supernatant of the P3 generation to the P6 generation. The supernatant according to the present invention is preferably obtained by centrifugation, more preferably 300 Xg centrifugation for 10min to remove floating living cells, and the supernatant is collected for isolation of exosomes.
After hucMSC supernatant is obtained, the hucMSC supernatant is centrifuged and sterilized to obtain 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 membrane supernatant, diluting sediment of the third centrifugation by using PBS buffer solution, carrying out second ultrafiltration centrifugation, and collecting the membrane supernatant; the centrifugal force of the first centrifugation is 2000g, and the centrifugation time is 10min; the centrifugal force of the second centrifugation is 10000g, and the centrifugation time is 30min; the first ultrafiltration centrifugation is 100kDa MWCO ultrafiltration centrifugation, the centrifugal force is 1000g, and the centrifugation time is 30min; the third centrifugation is carried out by placing the membrane supernatant of the first ultrafiltration centrifugation on a 30% sucrose/heavy water density pad, wherein the centrifugal force of the third centrifugation is 100000g, and the centrifugation time is 3h; the second ultrafiltration centrifugation was a 100kDa MWCO ultrafiltration centrifugation, the centrifugal force was 1 g, and the centrifugation time was 30min.
The centrifugation according to the invention is preferably a centrifugation at low temperature, more preferably a centrifugation at 4 ℃, wherein the first centrifugation removes intact dead cells and cell debris; a second centrifugation removes organelles; the first ultrafiltration centrifugation may be concentrated. The invention slowly moves the concentrated solution obtained after the first ultrafiltration centrifugation to 5ml of 30% sucrose/heavy water density pad (ρ=1.210 g/cm) 3 ) The upper part was subjected to a third centrifugation, and the bottom 5ml of sucrose/heavy water layer (containing exosomes) was collected, diluted with PBS and subjected to a second ultrafiltration centrifugation. The membrane supernatant after the second ultrafiltration centrifugation is preferably washed 3 times by PBS, filtered and sterilized by a sterile filter membrane with the diameter of 0.22 mu m, sub-packaged and stored at the temperature of minus 80 ℃, and protein quantitative detection is carried out by a BCA protein quantitative kit method.
The invention also provides a preparation method of the chimeric membrane vesicle, which comprises the following steps: and (3) uniformly mixing the nano vesicles and the exosome by ice bath ultrasound for 2-5 min, and sequentially extruding the mixture through a poly-polycarbonate membrane 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 further ultrasonic waves at intervals of 10s after each ultrasonic wave is performed for 3 s. The extrusion according to the invention is preferably carried out using an Avanti mini extruder.
The invention also provides application of the chimeric membrane vesicle in preparing a medicament for preventing and/or treating acute kidney injury.
In the embodiment of the invention, an acute kidney injury animal model is utilized for verification, and Neu-Ex effectively enhances the targeting characteristic of hucMSC-Ex to kidney injury parts; reducing tubular necrosis and inflammatory cell infiltration in cisplatin-induced kidney tissues, reducing creatinine and urea nitrogen levels, and enhancing proliferation capacity of kidney tissue cells; reduce the apoptosis of tubular epithelial cells and inhibit the expression of inflammatory factors, and prove that Neu-Ex has obvious treatment effect on acute kidney injury induced by cisplatin, so that Neu-Ex can be used for preparing medicines for preventing and/or treating acute kidney injury.
The following describes a chimeric membrane vesicle, a method for preparing the same and applications thereof in detail with reference to examples, but they should not be construed as limiting the scope of the 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 (forta corporation);
a positive microscope, an ultra-clean workbench, a desk centrifuge and an ultra-centrifuge;
heavy water (D) 2 O, shanghai, ind.) analytically pure sucrose (Guangzhou chemical reagent works), adipogenic, osteogenic induction medium, stem cell surface marker detection kit (Guangzhou Sier Biotechnology Co., ltd.), CD9, CD63, CD81, TSG101, calnexin antibody (CST), BCA protein quantification kit, horseradish peroxidase (HRP) -labeled goat anti-rabbit IgG secondary antibody (Beijing kang century Co.), premix HRP chemiluminescent substrate, 100kDa MWCO ultrafiltration centrifuge tube, 0.22 μm sterile filter membrane (Millipore company, usa); transmission electron microscopy (FEI Tecnai 12, philips); atomic force microscopy (bruk, germany) nanoparticle tracking analyzer (ZetaView, germany);
ICR mice (university of Jiangsu animal center for experiments approved by the ethical committee of Jiangsu university);
immunohistochemical staining reagents (warrior doctor company, operating according to kit instructions);
tubular endothelial cell line NRK52E cells (purchased from ATCC);
inverted microscope, confocal and ultra-high resolution microscope, ultra-clean bench, table centrifuge;
qRTPCR technology related reagent (Vazyme).
EXAMPLE 1 human umbilical cord mesenchymal stem cells and isolation and purification of their derived exosomes
(1) Isolated culture and identification of HucMSC: the hucMSCs were successfully isolated and cultured and characterized by the method of Qiao Chun (Qiao Chun et al human mesenchymal stem cells isolated from the umbilical cord. Cell Biol Int.2008 Jan;32 (1): 8-15), and the isolated hucMSCs were cultured at 37℃and 5% CO 2 Culturing in a saturated humidity incubator; good selective growth stateAdding adipogenic and osteogenic induction differentiation culture medium into the third generation hucSC plate, culturing until the corresponding time, and carrying 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 performing flow cytometry detection analysis.
The adipogenic induced differentiation results showed that the induced hucMSC cell bodies exhibited typical oil droplets (right in fig. 1 a) compared to the negative control group (left in fig. 1 a); osteogenic induced differentiation results showed that induced hucMSC cell bodies exhibited calcium nodules (right in fig. 1B) compared to negative control group (left in fig. 1B); flow cytometry results showed that hucMSC expressed CD29, CD73 and CD105 positively, CD11b, CD14 and CD45 negatively (C in fig. 1).
(2) Preparation of human umbilical cord mesenchymal stem cell supernatant (hucMSC-CM): 3-6 generations of hucMSC with good growth state is selected to be firstly cultured by a low-sugar DMEM culture medium containing 10% of fetal calf serum, when cells are fused to 50% -60%, the culture medium is replaced by the serum-free culture medium to be cultured, after 48 hours, culture supernatant is collected, and 300g of culture supernatant is centrifuged for 10 minutes to remove floating living cells for separation of exosomes.
EXAMPLE 2 isolation and purification of human umbilical cord mesenchymal Stem cell-derived exosomes
(1) Separation and purification of exosomes in umbilical cord mesenchymal stem cell supernatants were performed according to the procedure shown in fig. 2: centrifuging the collected hucMSC supernatant at 4 ℃ for 10min at 2000g to remove cell debris; collecting supernatant, centrifuging at 4deg.C and 10000g for 30min to remove organelles; transferring the supernatant to a 100kDa MWCO ultrafiltration centrifuge tube, centrifuging at 4 ℃ for 30min at 1000g, and concentrating; the concentrate was slowly transferred to 5ml of a 30% sucrose/heavy water density pad (ρ=1.210 g/cm 3 ) Centrifuging with 100000g at 4deg.C for 3 hr; collecting bottom 5ml sucrose/heavy water layer (containing exosomes), diluting with PBS, adding into 100kDa MWCO ultrafiltration centrifuge tube, centrifuging at 4deg.C for 30min at 1000g, and washing with PBS for 3 times; finally, filtering and sterilizing the mixture by using a sterile filter membrane with the thickness of 0.22 mu m, subpackaging the mixture, preserving the mixture at the temperature of minus 80 ℃, and quantitatively detecting protein by using a BCA protein quantitative kit method, wherein the concentration of the separated hucMSC-Ex is 40mg/ml.
(2) Basic morphology of exosomes was observed by transmission electron microscopy: taking 20 mu L of hucMSC-Ex, fully and uniformly mixing, dripping the mixture on a sample-carrying copper mesh with the diameter of 2mm, standing the mixture at room temperature for 5min, slightly sucking residual liquid at the edge of the copper mesh by using filter paper, reversely buckling the copper mesh on 30g/L phosphotungstic acid (pH 6.8) liquid drops, carrying out negative dyeing at room temperature for 5min, finally drying the copper mesh under an incandescent lamp, observing and photographing the copper mesh under a transmission electron microscope, and wherein an exosome is of a typical cup-shaped structure as shown in A in fig. 3.
(3) The height and morphology of exosomes were observed with an atomic force microscope: mu.L of hucMSC-Ex was dropped onto a cell slide, and after standing and drying at room temperature, PBS salt crystals were removed by rinsing with double distilled water. After again naturally drying, the exosomes were observed under an atomic force microscope and photographed, as shown in fig. 3B, as typical "cup-shaped" structures.
(4) Westernblot detection of surface marker proteins of hucMSC-Ex: preparation of 15% SDS-PAGE gel, after the above extracted exosomes were fully lysed, 1/4 volume of 5 XSDS loading buffer was added, boiled for 5min, loaded with 200. Mu.g total protein, electrotransferred (350 mA,120 min) to PVDF membrane, blocked with 50g/L skim milk in TBS/T room temperature for 1h, reacted overnight with CD9, CD63, CD81, TSG101, calnexin antibodies (1:500) at 4℃respectively, after 3 washes of the membrane with daily TBS/0.5% Tween 20, incubated with HRP-labeled goat anti-rabbit IgG secondary antibody for 1h at 37℃and after 3 washes of TBS/0.5% Tween 20, premix HRP chemiluminescent substrate was added and detected by a chemiluminescent gel imaging system, as shown in FIG. 3C, CD9, CD63, TSG101, alix and HSP70 for the huSC-Ex protein specific markers expressed positively and negative.
(4) NTA detection hucMSC-Ex particle size, concentration and potential: mu.L of hucMSC-Ex was diluted (1:5000) and tested on NTA. As shown in FIGS. 3D and E, hucMSC-Ex has a particle size distribution of 30 to 150nm and a peak value of about 123nm, and exhibits a negative potential.
Example 3 preparation and identification of human neutrophil membrane nanovesicles
(1) Isolation and identification of human neutrophils: after soliciting the informed consent of healthy volunteers, fresh anticoagulated blood samples are obtained from Jiangsu university school hospitals, 5ml of fresh whole blood is slowly added into a 15ml sterile centrifuge tube with 5ml of polymorphhPrep separating liquid paved at the bottom along the tube wall in an ultra clean workbench, and after centrifugation for 30min at 23 ℃ and 600g in a slow-rise and slow-fall way, a pipette carefully sucks the middle white membrane layer into serum-free 1640 medium for washing; centrifuging at 23deg.C for 5min at 800g, carefully removing supernatant, adding fresh split red liquid, carefully blowing, mixing, standing for 5-10min, and stopping with serum-free 1640 culture medium; harvesting neutrophil sediment after centrifugation at 23 ℃ and 800g for 5min, washing with precooled PBS for three times, and obtaining the cell sediment after centrifugation at 4 ℃ and 800g for 5min; detecting cell morphology by a front microscope; staining the cell nucleus and cytoplasm by Giemsa ruishiba; the flow cytometer detects neutrophil specific markers CD11b and CD33.
As shown in fig. 4, neutrophils were shown to be typically round and spherical when viewed under a normal microscope (a in fig. 4); flow-through assays revealed cell surface expression of specific molecules CD11B and CD33 (B in fig. 4); giemsa staining of rayleigh showed that neutrophils were in a typical rod-like or split nuclear structure (C in fig. 4); confocal laser microscopy also showed that the isolated neutrophils had a complete membrane structure.
(2) Extraction and identification of human neutrophil membrane proteins were performed according to the procedure shown in fig. 5: re-suspending the neutrophil precipitation with IB-1 separating liquid containing protease and phosphatase inhibitor, repeating freezing and thawing for 3 times, and performing ice bath ultrasonic treatment for 5min to break the cells; centrifuging at 4deg.C for 10min at 800g to remove dead cells and cell debris completely; centrifuging the supernatant at 4deg.C and 10000g for 30min to remove organelle components; the supernatant is further centrifuged at 100000g for 2h at 4 ℃ to harvest membrane protein precipitate; centrifuging at 4 ℃ and 100000g for 2h, washing the enriched membrane protein precipitate for 2 times, and re-suspending to-80 ℃ for standby. Detecting the protein content of the neutrophil membrane by a BCA method; coomassie brilliant blue staining and Western immunoblotting techniques detected the whole membrane protein fraction of neutrophils and some specific protein molecules, respectively.
As shown in fig. 6, the coomassie-light detection results show that the extracted neutrophil membrane mainly expresses the same protein component of neutrophils at about 70kDa (C in fig. 6); the Westernblot results show that a great amount of membrane surface protein components are enriched in the extracted neutrophil membrane protein: CCL2, CXCR4, fas, ICAM-1, integrin αv and integrin β3 (D in fig. 6).
(3) Preparation and identification of human neutrophil membrane nanovesicles: after ice bath ultrasound of neutrophil membrane proteins for 2min, sequentially extruding membrane protein components through polycarbonate membranes (400 nm, 200nm and 100 nm) with different pore diameters by using an Avanti micro extruder to form neutrophil membrane nanovesicles (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); atomic force microscopy (TEM) detects the height and morphology of Neu-NVs.
As shown in fig. 6, atomic force microscopy shows and transmission electron microscopy shows that the nanovesicles formed after extrusion have a typical spherical structure (E and F in fig. 6); NTA detection results show that nanocapsules formed after extrusion are 30-200 nm and present negative potential (G and H in FIG. 6); the corresponding vesicle-like structure was also detected by ultra-high resolution microscopy (I in fig. 6).
Example 4 preparation and characterization of Neu-Ex
Preparation and identification of chimeric membrane vesicles fused with Neu-NVs and hucMSC-Ex, which are neutrophil sources: neu-NVs and hucMSC-Ex were sonicated for 2min and then sequentially extruded through the different pore size poly polycarbonate membranes (400 nm, 200nm, 100 nm) to form Neu-Ex. A nanoparticle analyzer (NTA) detects particle size, concentration and potential of Neu-Ex; detecting the size and morphology of Neu-Ex by a Transmission Electron Microscope (TEM); detecting the height and morphology of Neu-Ex by an atomic force microscope (TEM); detecting Neu-Ex specific proteins by a western immunoblotting technology; neu-Ex fusion was detected by ultra-high resolution microscopy and fluorescence resonance energy transfer techniques.
NTA detection results show that the diameter of the Neu-Ex after fusion is distributed at the size of 30-200 nm, and the membrane potential is higher than the absolute values of other two independent groups (A and B in FIG. 7); transmission Electron Microscope (TEM) and Atomic Force Microscope (AFM) measurements showed that the fused Neu-Ex had a typical spherical structure (C and D in fig. 7); the coomassie brilliant blue detection and Western blot detection results show that the fused Neu-Ex expression cells share the common protein component; ultra-high resolution microscopy showed that the green fluorescent dye-labeled Neu-NVs and the red fluorescent dye-labeled hucMSC-Ex were significantly fused after extrusion (fig. 8).
EXAMPLE 5 therapeutic Effect of Neu-Ex
(1) Cisplatin-induced acute kidney injury model construction: ICR mice weighing 20-25 g were given an acute kidney injury 72 hours after a single intraperitoneal injection of 10mg/kg cisplatin (Sigma).
Cisplatin treatment for 72 hours, serum biochemical detection results show that the indexes of creatinine, urea nitrogen and the like of the mice in the model group are obviously reduced (A in figure 9). HE staining results showed that model group kidney tissue vacuoles were significantly denatured and tissue structure was disordered (B in fig. 9); shows that the cisplatin-induced acute kidney injury model is successfully constructed.
(2) In vivo and in vitro therapeutic effects of chimeric membrane vesicles on acute kidney injury: randomly dividing model mice with acute kidney injury after cisplatin induction for 72 hours into PBS groups, neutrophil nano-vesicle groups and chimeric membrane vesicle groups; the same number of particles per mouse tail was given by intravenous injection (8X 10 10 ) With PBS as a negative control. After 48 hours, evaluating the repairing effect;
HE staining results showed reduced vacuolation of kidney tissue, reduced infiltration of inflammatory cells of tissue, and relatively intact tissue structure compared to PBS and Neu-NVs intervention group alone for Neu-Ex dry prognosis (fig. 10 a); immunohistochemical staining results showed that mouse kidney tissue cells with Neu-Ex dry prognosis showed stronger proliferation 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 obviously reduced, and kidney function damage is effectively relieved after Neu-Ex intervention (C in fig. 10). The above in vivo results show that Neu-Ex has significant therapeutic effects on cisplatin-induced acute kidney injury.
In vitro cisplatin-induced renal tubular endothelial cell (NRK 52E) injury model, qRT-PCR detection results show that after Neu-Ex intervention, the expression level of inflammatory factors TNF-alpha and IL-6 of the cisplatin on the NRK52E cells is obviously inhibited. This indicates that Neu-Ex has a significant therapeutic effect on cisplatin-induced acute kidney injury in vitro and in vivo experiments (D in fig. 10).
TABLE 1 primer sequences for TNF-alpha and IL-6
20 μl qRT-PCR system: SYBR Green Mix 10 μl, RNase free ddH 2 O7. Mu.l, 0.5. Mu.l each of F/R primers and 2. Mu.l each of cDNA.
qRT-PCR procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 30s,40 cycles; and extending at 72 ℃ for 10min.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
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Claims (2)
1. An application of chimeric membrane vesicles in preparing a medicament for preventing and/or treating acute kidney injury, which is characterized in that the preparation method of the chimeric membrane vesicles comprises the following steps: uniformly mixing human neutrophil-derived nano vesicles and human umbilical cord mesenchymal stem cell-derived exosomes in an ice bath for 2-5 min, and sequentially extruding the mixture through a poly-polycarbonate membrane with the pore diameter of 400nm, 200nm and 100nm to obtain chimeric membrane vesicles;
the number ratio of the nano vesicle to the exosome is (0.8-1.2): (0.8-1.2);
the power of the ultrasonic wave is 100W, and the ultrasonic wave is further ultrasonic waves at intervals of 10s after each ultrasonic wave is performed for 3 s;
the preparation method of the nano vesicle comprises the following steps: and (3) sequentially extruding the neutral granulocyte membrane protein through a polycarbonate membrane with the pore diameters of 400nm, 200nm and 100nm after carrying out ice bath ultrasound for 2-5 min, so as to obtain the nano vesicle.
2. A medicament for the prevention and/or treatment of acute kidney injury, characterized in that it comprises chimeric membrane vesicles for use according to claim 1.
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| CN101890050A (en) * | 2010-07-14 | 2010-11-24 | 江苏大学 | Human umbilical cordmesenchymal stem cell-derived exosome and application thereof |
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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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| HucMSC exosomes-delivered 14-3-3ζ enhanced autophagy via modulation of ATG16L in preventing cisplatin-induced acute kidney injury;Haoyuan Jia等;《Am J Transl Res》;第10卷(第1期);第101-113页 * |
| 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;肖瑶等;《军事医学》;第45卷(第1期);第25-29、63页，尤其是第25页摘要，第27页左栏第3段，第29页左栏第1段 * |
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