CN115322950B - Bactrian camel milk exosome and application of preparation method thereof - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/20—Milk; Whey; Colostrum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention provides a bactrian camel milk exosome and a preparation method and application thereof. The preparation method of the bactrian camel milk exosome comprises the following steps: collecting milk secreted by Bactrian camels fed with lactobacillus, and extracting exosomes by differential centrifugation. The research finds that: lactic acid bacteria fed Bactrian camel milk exosomes (LAB-cExo) were found in which miRNAs could be targeted to many pathways directly or indirectly associated with the cardiovascular system, and thus were suspected to have potential for the treatment of cardiovascular disease. Subsequently, LAB-cExo can be successfully internalized by vascular endothelial cells (HUVECs), can effectively promote the proliferation of HUVECs and inhibit the apoptosis of HUVECs, and has the potential of treating cardiovascular diseases.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a preparation method and application of a bactrian camel milk exosome.
Background
Camels can thrive under extremely severe conditions such as drought, desert and the like. Camel milk is more known as "platinum" in desert. However, current studies indicate that: camel milk is rich in various amino acids and vitamins necessary for human body, and contains protective proteins such as lysozyme, lactoferrin, immunoglobulin and the like and various bioactive factors. Obviously, the development of effective components of camel milk is far from sufficient.
Disclosure of Invention
Based on this, it is necessary to provide a method for extracting the alpaca milk exosomes and application thereof, and intensive studies on the intrinsic components of the alpaca milk exosomes are performed. The phenotypic experiment result shows that the HUVECs can promote the proliferation of HUVECs and inhibit the aging of HUVECs, and are expected to be used for treating cardiovascular diseases.
The invention adopts the following technical scheme:
the invention provides a preparation method of a bactrian camel milk exosome, which comprises the following steps: collecting milk secreted by the Bactrian camel fed by lactic acid bacteria; separating the crude product from the milk by utilizing a differential centrifugation method to obtain a crude product of impurity removal and precipitation; and (3) re-suspending the precipitated crude product, placing the re-suspended product into the upper layer in a centrifuge tube containing the gradient iodixanol solution, and performing ultracentrifugation separation to form 12 layers to obtain a refined extract positioned in the middle layer.
In some of these embodiments, the step of separating the crude precipitate from the milk after primary removal of impurities using differential centrifugation comprises: centrifuging milk for 5-15 min at a low centrifugal speed of 1000g to obtain skim milk; centrifuging the skim milk at a medium centrifugal speed of 10000g to obtain emulsion for degreasing and removing large vesicles; continuously centrifuging the degreasing and de-vesicle emulsion for 1.5-2.5 h by using a high centrifugal speed of 100000g to obtain an enriched precipitate; suspending the enrichment sediment by using PBS buffer solution, further centrifuging and repeatedly cleaning the enrichment sediment by using a centrifugal speed of 2000g, centrifuging for 1.5-2.5 h under a high centrifugal speed condition of 100000g, and collecting a coarse impurity-removed sediment product.
In some of these embodiments, the gradient iodixanol solution has a concentration gradient of 40% iodixanol solution, 20% iodixanol solution, 10% iodixanol solution, and 5% iodixanol solution in that order.
In some of these embodiments, the process conditions for ultracentrifugation to form 12 layers are: centrifuging at a high centrifugal speed of 100000g for 2h to obtain a fine extract with 6-9 layers in the middle.
The invention also provides the bactrian camel milk exosome prepared and obtained by the preparation method of the bactrian camel milk exosome.
The invention also provides an application of the Bactrian camel milk exosome prepared by the preparation method of the Bactrian camel milk exosome in preparation of preparation products for promoting HUVECs proliferation.
The invention also provides a preparation product with the efficacy of promoting HUVECs proliferation, which comprises the Bactrian camel milk exosomes prepared by the preparation method of the Bactrian camel milk exosomes.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides an exosome which is prepared by extracting milk secreted by a Bactrian camel fed by lactic acid bacteria for the first time, and meanwhile, the exosome is verified to have the effects of promoting HUVECs proliferation and inhibiting apoptosis.
Drawings
FIG. 1 is a graph showing the particle size distribution of the alpaca milk secretion (LAB-cExo) body prepared in example 1.
FIG. 2 is a transmission electron microscope test chart of the alpaca milk exosomes (LAB-cExo) prepared in example 1.
FIG. 3 is a bar graph of the length distribution of small RNA tested against the alpaca milk exosomes (LAB-cExo) prepared in example 1 in example 2.
FIG. 4 is a graph showing the percentage of unique reads for each type of ncRNA tested against the alpaca milk exosomes (LAB-cExo) prepared in example 1 in example 2.
Fig. 5 is a bar graph of the expression levels of some of the mirnas tested against the alpaca milk exosomes (LAB-cero) prepared in example 1 in example 2.
Fig. 6 is the result of enrichment of miRNA target gene GO tested against the alpaca milk exosomes (LAB-cero) prepared in example 1 in example 2.
FIG. 7 is the results of enrichment of the miRNA target gene KEGG tested against the alpaca milk exosomes (LAB-cExo) prepared in example 1 in example 2.
FIG. 8 is a graph of the transfection of the alpaca milk exosomes (LAB-cExo) prepared in example 1 into HUVECs in example 3.
FIG. 9 is a statistical graph of the effect on cell activity of HUVECs cells treated in vitro against the alpaca milk exosomes (LAB-cExo) prepared in example 1 of example 3.
FIG. 10 is a statistical plot of the effect on apoptosis after in vitro treatment of HUVECs cells tested against the alpaca milk exosomes (LAB-cExo) prepared in example 1 in example 3.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
The key raw material sources are as follows:
bactrian camels, which grow in the arashan region of Xinjiang. Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from research domain biotechnology (Shanghai) limited. DMEM medium, available from sameidie technologies.
The preparation method of the PBS buffer solution comprises the following steps: weigh 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 And 0.24gKH 2 PO 4 Dissolving in 800mL distilled water, adjusting pH to 7.4 with HCl, adding distilled water to 1L, and sterilizing with high pressure steam.
Example 1
The embodiment provides a method for extracting a Bactrian camel milk exosome (LAB-cExo), which comprises the following steps:
(1) Feeding a dromedarion:
and selecting a female camel and earhole marker with the average age of 8-12 years, 3-4 fetuses and 2-3 months after delivery in Xinjiang. Lactic acid bacteria are mixed in feed for feeding the Bactrian camel. After continuous feeding for one month, milking and mixing evenly in the morning and in the evening, and obtaining camel milk samples.
(2) Preparation of exosomes:
transferring the camel milk sample into a new centrifuge tube, centrifuging at low speed (1000 g) at 4deg.C for 10min, and discarding precipitate to obtain skimmed milk.
Centrifuging skim milk at 4deg.C and 10000g for 45min to remove larger vesicles. Then selecting an overspeed rotor, centrifuging for 120 min at the temperature of 4 ℃ and the rotating speed of 100000g, removing the supernatant and collecting the sediment. After resuspension of the collected pellet with 20. Mu.L of pre-chilled 1 XPBS buffer, the pellet was removed by centrifugation at 2000g for 30 min at 4℃and repeated 2 times. Selecting an overspeed rotor, centrifuging the supernatant again at 4 ℃ and 100000g for 120 min, collecting precipitate, and re-suspending to form heavy suspension.
Iodixanol with different concentrations (40%, 20%, 10% and 5%) is prepared, the iodixanol with different concentrations is sequentially added into the super-separation tube along the tube wall according to the concentration from high to low, and finally 1 mL heavy suspension obtained in the above steps is added at the uppermost layer of the super-separation tube, and the rotating speed is centrifuged for 120 min under the condition of 4 ℃ and 100000 g. After centrifugation, the solution is divided into 12 layers, the liquid in the middle 6-9 layers is taken out, and the solution is centrifuged again for 120 min under the same conditions. The supernatant was removed, resuspended in 300. Mu.L of pre-chilled PBS, and the exosomes (LAB-cExo) were obtained and stored at-80 ℃.
PARTICLE size of camel milk exosomes (LAB-cero) was measured using a PARTICLE METRIX Nanoparticle Tracking Analyzer (NTA), the specific steps being: taking a frozen exosome (LAB-cExo) sample, thawing in a water bath at 25 ℃, and placing on ice; the exosome samples were diluted with PBS (exosome: pbs=1:1) and used directly for NTA detection of concentration and particle size of camel milk exosomes, and as a result, as shown in fig. 1, the average particle size was 85.20nm and the concentration was 1.11×10 12 Particles/mL。
The camel milk exosome pellet was fixed and examined by transmission electron microscopy using conventional procedures, and observed using a Hitachi transmission electron microscope, and the obtained transmission electron microscope micrograph is shown in fig. 2, while confirming the range of the above particle size distribution.
Example 2
This example was directed to sequencing and target gene prediction of the alpaca milk exosomes prepared in example 1.
After the data quality control and the length filtration are completed, the number and the duty ratio of the Reads with the length of 18-31 nt of each sample are counted, and the length distribution condition of the small RNA is obtained as shown in figure 3. mirnas are typically concentrated at 21 or 22nt. The figures show that the small RNA at lengths of 21nt and 22nt is at maximum, exceeding 41%.
Sequence miRBase 20.0 alignment of RNA after small RNA length screening, where small RNAs were annotated to different classifications, results are shown in figure 4, where small RNA species in the clean sequence include miRNA, rRNAs, snoRNAs, snRNAs, tRNA and other sRNAs. The total rRNA is used as a quality control index of a sample, and the proportion of the camel milk exosomes is lower than 40%, so that the research sample accords with the quality control index. According to the sequencing results, 233 mirnas were detected in total. The top 25 mirnas with the highest expression levels are shown in fig. 5.
And predicting target genes of the first 25 miRNAs with highest identified expression quantity, and performing functional enrichment analysis on the target genes. The results of GO enrichment indicate (fig. 6) that most target genes are mainly enriched in related pathways such as some angiogenesis (positive regulation of angiogenesis, negative regulation of angiogenesis), cardiac development (heart development), and some endothelial cell differentiation (epithelial cell differentiation). Most of the target genes are distributed mainly in some endosomes (early endosome, endosome membrane, early endosome membrane, late endosome, recycling endosome, late endosome membrane, recycling endosome membrane), actin scaffold (actin cytoskeleton) and extracellular matrix (extracellular matrix) parts. Many target genes have actin or microtubule binding capacity (actin binding, microtubule binding).
KEGG enrichment results (fig. 7) indicate that the target gene is mainly enriched in some cardiovascular disease-related pathways, such as Lipid and atherosclerosis, fluid shear stress and atherosclerosis, dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy.
EXAMPLE 3 investigation of the Effect of camel milk exosomes on Human Umbilical Vein Endothelial Cells (HUVECs)
(1) Absorption mark test
Camel milk exosomes (LAB-cero) extracted from example 1 were stained with PKH26 red fluorescent cell linker kit and unlabeled dye was removed with an exosome spin column. The dyed exosomes (2 mug/L multiplied by 10) 4 cells) or an equal volume of PBS (NC) was added to semi-miscible HUVECs. After 48h incubation, fixation was performed for 10min with 3.7% PFA wash. The nuclei of HUVECs were labeled with FITC-conjugated cyclopeptides and the labeled cells were incubated with labeled LAB-cExo to give two sets of cells (NC and LAB-cExo). The supernatant was removed and washed three more times with PBS to remove free exosomes. The exosome transfection effect of both groups of cells was measured with a fluorescence microscope, and the fluorescence microscope photograph obtained is shown in FIG. 8.
(2) Test of the Effect of camel milk exosomes on proliferation of HUVECs
As shown in FIG. 9, the survival of HUVECs cells transfected with LAB-cExo and PBS (NC) respectively was examined by CCK8 at 0, 24h, 48h and 72 h. CCK8 results indicate that LAB-cExo transfected cells have increased viability compared to control. This suggests that LAB-cExo can promote cell proliferation.
(3) Influence of camel milk exosomes on apoptosis of HUVECs
Flow cytometry was used to examine the percentage of apoptosis in two groups of HUVECs after transfection of LAB-cExo and PBS, respectively. As shown in fig. 10, the results demonstrate a significant decrease in the percentage of apoptotic cells in the LAB-cero group compared to the control group. This suggests that LAB-cExo can inhibit apoptosis.
In addition, the invention adopts a density gradient centrifugation method to extract and prepare the Bactrian camel milk exosome LAB-cExo from milk secreted by the Bactrian camel fed by lactic acid bacteria. In the method, the centrifugal times, the centrifugal time and the rotational speed are required to be strictly controlled, the time is too short or the rotational speed is too small, the outer vesicles cannot be fully extracted, the structure of the outer vesicles is damaged due to the excessive times or the excessive rotational speed, and the yield of the outer vesicles is reduced, so that the acquisition of the Bactrian camel milk exosome LAB-cExo is influenced.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. The application of the alpaca milk exosomes in preparing a preparation product for promoting the proliferation of HUVECs is characterized in that the preparation method of the alpaca milk exosomes comprises the following steps:
collecting milk secreted by the Bactrian camel fed by lactic acid bacteria;
separating the crude product from the milk by utilizing a differential centrifugation method to obtain a crude product of impurity removal and precipitation;
and (3) re-suspending the precipitated crude product, placing the re-suspended product into the upper layer in a centrifuge tube containing the gradient iodixanol solution, and performing ultracentrifugation separation to form 12 layers to obtain a refined extract positioned in the middle layer.
2. The use according to claim 1, wherein the step of separating the crude precipitate from the milk by differential centrifugation to obtain a preliminary impurity removal comprises:
centrifuging milk for 5-15 min at a low centrifugal speed of 1000g to obtain skim milk;
centrifuging the skim milk at a medium centrifugal speed of 10000g to obtain emulsion for degreasing and removing large vesicles;
continuously centrifuging the degreasing and de-vesicle emulsion for 1.5-2.5 h by using a high centrifugal speed of 100000g to obtain an enriched precipitate;
suspending the enrichment sediment by using PBS buffer solution, further centrifuging and repeatedly cleaning the enrichment sediment by using a centrifugal speed of 2000g, centrifuging for 1.5-2.5 h under a high centrifugal speed condition of 100000g, and collecting a coarse impurity-removed sediment product.
3. The use according to claim 1 or 2, wherein the gradient iodixanol solution has a concentration gradient of 40% iodixanol solution, 20% iodixanol solution, 10% iodixanol solution and 5% iodixanol solution in this order.
4. The use according to claim 3, wherein the process conditions for forming 12 layers by ultracentrifugation are: high centrifugation speed of 100000g was centrifuged for 2h.
5. The use according to claim 4, characterized in that a fine extract is obtained in the middle 6-9 layers.
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