CN115634254B - Preparation method and application of allium chinense plant exosome - Google Patents
Preparation method and application of allium chinense plant exosome Download PDFInfo
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- CN115634254B CN115634254B CN202211233971.1A CN202211233971A CN115634254B CN 115634254 B CN115634254 B CN 115634254B CN 202211233971 A CN202211233971 A CN 202211233971A CN 115634254 B CN115634254 B CN 115634254B
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- Medicines Containing Plant Substances (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of exosome biological preparations, in particular to a preparation method and application of an allium chinense plant exosome, which comprises the steps of squeezing allium chinense to obtain juice, and filtering to obtain allium chinense stock solution; centrifugally extracting the allium chinense stock solution to obtain exosomes; purifying exosomes by using a sucrose cushion method to obtain allium chinense exosomes; according to the technical scheme, fresh allium chinense juice is used for obtaining allium chinense stock solution, and an exosome is obtained through a gradient ultracentrifugation method, and has the effects of effectively promoting healing of tendinous bones and promoting osteogenesis, so that the problem of lack of medicines with the function of promoting healing of tendinous bones and good biocompatibility is solved.
Description
Technical Field
The invention relates to the technical field of exosome biological preparations, in particular to a preparation method and application of allium chinense plant exosomes.
Background
The exosomes are nano-level vesicles secreted by cells, have phospholipid bilayer structures, contain substances such as DNA, small RNA, protein and the like, carry proteins and nucleic acids and participate in the communication among cells. The exosomes are used as natural intercellular information carriers, have smaller molecular structures and good biocompatibility, and have the potential of developing the exosomes into biological agents or medicines with therapeutic effects.
Exosomes were first found in sheep reticulocytes in 1983, and animal extracellular vesicles were separated into exosomes, microvesicles and apoptotic bodies by their specific protein markers and sources, and research into exosomes of animal origin is now mature. The present study found that plants secrete exosomes, while partial data was obtained on plant exosomes with a transmission electron microscope. The plant exosomes have the diameter of about 40-150 nm, are similar to the animal exosome morphological structure, have a phospholipid bilayer structure, are nanoscale vesicles secreted by most cells, are in a tea tray shape or cup shape, are composed of a large number of lipids including microRNAs and proteins, are relatively lack in the current research, and are still unclear in the related specific mechanism.
The tendon-bone junction is located between tendon and bone and is a key structure for mechanical transition, but the tendon-bone junction is difficult to recover to normal tissue structure and normal function after injury due to complex tissue structure. In particular, in the surgical treatment after injury of the anterior and posterior fork ligaments, tendons are often used to replace ligaments, and tendons and bones are connected by constructing a bone marrow duct to replace the ligaments, but healing between tendons and bone marrow ducts is difficult. Therefore, how to promote early tendon bone healing strength is a critical issue to be resolved urgently, and there is no drug with the above functions and good biocompatibility and bioavailability.
Disclosure of Invention
The invention aims to provide a preparation method and application of allium chinense plant exosome, and aims to solve the problem of lack of medicines with a function of promoting tendon bone healing and good biocompatibility.
In order to achieve the above object, in a first aspect, the present invention provides a preparation method of an allium chinense plant exosome, comprising the following steps:
squeezing Bulbus Allii Macrostemi, and filtering to obtain Bulbus Allii Macrostemi stock solution;
Centrifugally extracting the allium chinense stock solution to obtain exosomes;
Purifying the exosomes by using a sucrose cushion method to obtain allium chinense exosomes.
The specific mode for obtaining the allium chinense stock solution by squeezing the allium chinense and filtering the juice is as follows:
Cleaning and peeling 2.5 jin of allium chinense, and sterilizing with slicing instrument together with alcohol;
cutting the sterilized allium chinense into small blocks through the sterilized slicing instrument;
adding PBS into the small blocks of allium chinense, and squeezing to obtain juice;
Filtering the juice by using sterile gauze to obtain allium chinense stock solution.
The specific method for obtaining exosomes by carrying out centrifugal extraction on the allium chinense stock solution comprises the following steps:
Taking out the allium chinense stock solution for pretreatment, and then moving the allium chinense stock solution into a centrifuge tube for centrifugation for 10min to obtain a first centrifugate;
Transferring the supernatant of the first centrifugate into a new centrifuge tube, and centrifuging again for 30min to obtain a second centrifugate;
Taking out the supernatant of the second centrifugate, and filtering to obtain filtrate;
Transferring the filtrate into a new centrifuge tube, and centrifuging for 70min to obtain a third centrifugate;
removing the supernatant of the third centrifugate, and performing centrifugation for 70min after resuspension to obtain a fourth centrifugate;
And removing the supernatant of the fourth centrifugate, and re-suspending again to obtain exosomes.
The method for purifying the exosome by using the sucrose cushion method comprises the following specific steps of:
supplementing the volume of the exosome to 1mL by using precooled 1 XPBS to obtain a mixed solution;
adding the mixed solution into a sucrose cushion, and centrifuging for 70min;
Taking out 250 μl of 30% sucrose pad at the bottom, centrifuging for 70min again, removing supernatant precipitate, and re-suspending to obtain allium chinense exosome.
In a second aspect, an application of an allium chinense plant exosome is adopted in the preparation method of the allium chinense plant exosome in the claims 1-4,
The allium chinense exosome is applied to medicines for preventing and relieving osteoporosis and promoting healing of tendinous bones.
The preparation method and the application of the allium chinense plant exosome provided by the invention are that the allium chinense juice is squeezed and filtered to obtain allium chinense stock solution; centrifugally extracting the allium chinense stock solution to obtain exosomes; the method comprises the steps of purifying the exosome by using a sucrose cushion method to obtain the allium chinense exosome, squeezing fresh allium chinense to obtain allium chinense stock solution, and obtaining the exosome by using a gradient ultracentrifugation method, wherein the exosome has the effects of effectively promoting healing of tendinous bones and promoting osteogenesis, so that the problem of lack of medicines with the function of promoting healing of tendinous bones and good biocompatibility is solved.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the 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 flow chart of a preparation method of allium chinense plant exosomes.
FIG. 2 is an electron microscope picture of ChON-exos.
FIG. 3 is a size distribution diagram of ChON-exos.
FIG. 4 is a graph showing the results of an in vitro test cell alizarin red staining test control group.
FIG. 5 is a graph showing the results of an in vitro test cell alizarin red staining test group.
Fig. 6 is a schematic representation of tendon harvest in a mouse tendon bone healing model surgery.
Fig. 7 is a schematic representation of an implanted tendon in a mouse tendon bone healing model surgery.
FIG. 8 is a graph of the results of a MicroCT experiment.
Fig. 9 is a graph showing the osteogenic effect of a bone marrow canal model for tendon bone healing.
Fig. 10 is a schematic representation of safranin O fast green staining of the control group.
FIG. 11 is a schematic representation of the solid green staining of safranin O in the experimental group.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Referring to fig. 1-3, in a first aspect, the present invention provides a method for preparing and identifying an exosome of allium chinense plants, comprising the steps of:
S1, squeezing juice of allium chinense, and filtering to obtain an allium chinense stock solution;
s11, cleaning and peeling 2.5 jin of allium chinense, and sterilizing the allium chinense and a slicing device together by alcohol;
specifically, 2.5 jin of allium chinense is cleaned and peeled, and is sterilized with alcohol together with all the instruments.
S12, cutting the sterilized allium chinense into small blocks through the sterilized slicing instrument;
specifically, the process is carried out in an ultra clean bench, and the allium chinense is cut into small blocks.
S13, adding PBS into the allium chinense cut into small blocks, and squeezing juice to obtain squeezed juice;
specifically, a proper amount of PBS is added and then juice is obtained, so that about 500mL of juice is obtained.
S14, filtering the juice by using sterile gauze to obtain allium chinense stock solution.
Specifically, about 500mL of juice is squeezed, and the juice is filtered by sterile gauze to obtain allium chinense stock solution.
S2, carrying out centrifugal extraction on the allium chinense stock solution to obtain exosomes;
S21, taking out the allium chinense stock solution for pretreatment, and then moving the allium chinense stock solution into a centrifuge tube for centrifugation for 10min to obtain a first centrifugate;
Specifically, the samples were removed for pretreatment, and the samples were transferred to a new centrifuge tube, 2000 Xg, 4℃and centrifuged for 10 min.
S22, transferring the supernatant of the first centrifugate into a new centrifuge tube, and centrifuging again for 30min to obtain a second centrifugate;
specifically, the supernatant was carefully transferred to a new centrifuge tube, 10,000Xg, 4℃and centrifuged again for 30 min.
S23, taking out the supernatant of the second centrifugate, and filtering to obtain filtrate;
specifically, the supernatant was collected and filtered through a 0.45 μm filter membrane, and the filtrate was collected.
S24, transferring the filtrate into a new centrifuge tube, and centrifuging for 70min to obtain a third centrifugate;
Specifically, the filtrate was transferred to a new centrifuge tube, and the overspeed rotor was selected and centrifuged at 100,000Xg for 70min at 4 ℃.
S25, removing the supernatant of the third centrifugate, and performing resuspension and centrifugation for 70min to obtain a fourth centrifugate;
specifically, the supernatant was removed, and after resuspension with 10mL of pre-chilled 1 XPBS, an overspeed rotor was selected, and the supernatant was subjected to ultracentrifugation at 4℃again, 100,000Xg, for 70min, to obtain the fourth supernatant.
S26, removing the supernatant of the fourth centrifugate, and re-suspending again to obtain exosomes.
Specifically, the supernatant was removed and resuspended with 200. Mu.L of pre-chilled 1 XPBS to give the allium chinense exosomes.
S3, purifying the exosomes by using a sucrose cushion method to obtain allium chinense exosomes.
S31, supplementing the volume of the exosome to 1mL by using precooled 1 XPBS to obtain a mixed solution;
Specifically, the exosome volume was made up to 1mL with pre-chilled 1 XPBS to give the mixture.
S31, adding the mixed solution into a sucrose cushion, and centrifuging for 70min;
Specifically, 1mL of exosome sample was slowly added to 250. Mu.L of 30% sucrose pad (heavy water formulation), and centrifuged at 100,000Xg for 70min.
S32, taking out 250 mu L of the sucrose cushion positioned at the lower layer and 30 percent, centrifuging for 70 minutes again, removing the supernatant sediment, and then re-suspending to obtain the allium chinense exosome.
Specifically, 250. Mu.L of a 30% sucrose pad at the lower layer was taken out, diluted to 3mL with PBS, centrifuged at 100,000Xg for 70min, the supernatant was removed, the pellet was resuspended with 200. Mu.L of pre-chilled PBS, 20. Mu.L of electron microscopy was performed, 10. Mu.L of particle size was performed, 10. Mu.L of protein extraction was performed, 10. Mu.L of sterile detection was performed, and the remaining exosomes were stored at-80 ℃.
Referring to FIGS. 4-9, in a second aspect, an application of an allium chinense plant exosome is adopted in the preparation method of an allium chinense plant exosome as described in claims 1-4,
The allium chinense exosome is applied to medicines for preventing and relieving osteoporosis and promoting healing of tendinous bones.
Specifically, the proposal discovers that the allium chinense exosome has the effects of bone formation and tendon bone healing promotion for the first time. The exosome medicine has remarkable advantages over the traditional medicine, and is characterized in that: the exosomes are used as natural intercellular information carriers, and have small molecular structures and good biocompatibility; compared with the common medicine, the exosome has high bioavailability and good targeting property. The allium chinense exosome biological preparation for promoting tendon bone healing and promoting bone formation, which is developed by the scheme, has ideal application prospect.
The beneficial effects are that:
the method comprises the steps of squeezing fresh allium chinense to obtain allium chinense stock solution, and obtaining exosomes by a gradient ultracentrifugation method, wherein the exosomes have the effects of effectively promoting healing of tendinous bones and promoting osteogenesis. The exosome can be used as a biological agent for promoting healing of tendon bones and preventing osteoporosis in medical practice.
1. Construction of tendon bone healing model
Constructing a bone marrow duct: the mice were anesthetized with 0.5% sodium pentobarbital, then the skin in front of the hind limb achilles tendon was cut open, the plantar tendon was exposed and dissociated, and the removed plantar tendon was stored in allium chinense exosome solution. Then, a longitudinal incision is made in front of the knee of the hind limb, a 1mL syringe needle is used for drilling holes from the front inner side to the rear outer side of the proximal end of the tibial stem perpendicularly to the tibial shaft, a bone marrow canal is constructed, the pre-stripped plantar tendon is implanted into the bone marrow canal, and 50ul of allium chinense exosomes are injected into the bone marrow canal before implantation. Control groups replaced exosomes with PBS.
Drug treatment: 2 times per week of allium chinense exosomes are injected per mouse intraperitoneally, 500ug per week. The control group was injected with an equal amount of PBS solution.
1. Tissue section staining experiments
28 Days after molding, tibial samples were collected, fixed with 4% paraformaldehyde for 48 hours, then decalcified with EDTA decalcification solution for 10 days, and paraffin sections were made.
Safranine solid green staining: the area of the bone trabecula is observed through safranine solid-green staining, soaked for 3min with 0.1% safranine solution, then soaked for 10 seconds in 0.1% solid-green solution, then separated by 1% acetic acid solution, and finally sealed for observation after washing off the redundant solution.
MicroCT experiment
Tibia samples were collected 28 days after molding, fixed with 4% paraformaldehyde for 24 hours, and tibia MicroCT was photographed for correlation analysis.
The above disclosure is only illustrative of a preparation method and application of an allium chinense plant exosome of the present invention, but it is not limited thereto, and those skilled in the art will understand that all or part of the procedures for implementing the above embodiments are equivalent and still fall within the scope of the present invention.
Claims (2)
1. The preparation method of the allium chinense plant exosome is characterized by comprising the following steps of:
squeezing Bulbus Allii Macrostemi, and filtering to obtain Bulbus Allii Macrostemi stock solution;
Centrifugally extracting the allium chinense stock solution to obtain exosomes;
purifying the exosomes by using a sucrose cushion method to obtain allium chinense exosomes;
the method for purifying the exosomes by using the sucrose cushion method comprises the following specific modes of:
Supplementing the volume of the exosome to 1mL by using precooled 1 XPBS to obtain the mixed solution;
1mL of the exosome sample was slowly added to 250. Mu.L of 30% sucrose pad (heavy water formulation) and centrifuged at 100,000Xg for 70min;
Taking 250 μl of 30% sucrose pad at the bottom, diluting to 3mL with PBS, centrifuging at 100,000Xg for 70min, removing supernatant, resuspending the precipitate with 200 μl of precooled PBS, detecting with 20 μl of electron microscope, detecting with 10 μl of particle size, detecting with 10 μl of protein extract, detecting with 10 μl of sterile, and preserving the rest exosomes at-80deg.C;
The allium chinense stock solution is subjected to centrifugal extraction, and the specific mode for obtaining exosomes is as follows:
taking out the sample for pretreatment, moving the sample into a new centrifuge tube, centrifuging at 2000 Xg and 4 ℃ for 10 min;
carefully transfer the supernatant to a new centrifuge tube, 10,000Xg, 4℃and centrifuge again for 30 min;
filtering the supernatant with 0.45 μm filter membrane, and collecting filtrate;
transferring the filtrate into a new centrifuge tube, selecting an overspeed rotor, and centrifuging at 4 ℃ for 70min at 100×g;
Removing the supernatant, re-suspending with 10mL of pre-cooled 1 XPBS, selecting an overspeed rotor, and performing overspeed centrifugation at the temperature of 4 ℃ again for 70min at 100,000Xg to obtain the fourth centrifugate;
The supernatant was removed and resuspended with 200. Mu.L of pre-chilled 1 XPBS to give the allium macrostemon exosomes.
2. The method for preparing the allium chinense plant exosome according to claim 1, wherein,
The specific mode for obtaining the raw liquor of the allium chinense is as follows:
Cleaning and peeling 2.5 jin of allium chinense, and sterilizing with slicing instrument together with alcohol;
cutting the sterilized allium chinense into small blocks through the sterilized slicing instrument;
adding PBS into the small blocks of allium chinense, and squeezing to obtain juice;
Filtering the juice by using sterile gauze to obtain allium chinense stock solution.
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| CN113462632A (en) * | 2021-08-13 | 2021-10-01 | 徐州医科大学 | Bitter gourd exosome, extraction method and application in preparation of medicine for treating burns and scalds |
| CN113876798A (en) * | 2021-10-14 | 2022-01-04 | 中国人民解放军陆军军医大学第一附属医院 | Application of microRNA in preparation of medicine for promoting healing of tendon and bone |
| CN114015640A (en) * | 2021-10-19 | 2022-02-08 | 卡替睿舒(上海)医疗科技有限公司 | Pilot plant extraction method of plant-derived exosomes and application thereof |
| KR102391636B1 (en) * | 2021-12-02 | 2022-04-28 | 주식회사 에이바이오머티리얼즈 | Cosmetic composition for improving skin and hair condition containing Scutellaria baicalensis exosome, Houttuynia cordata exosome |
| KR102432749B1 (en) * | 2021-12-17 | 2022-08-16 | 주식회사 에이바이오머티리얼즈 | Cosmetic composition containing Pteridium aquilinum exosome |
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