CN115322950A - Bactrian camel milk exosome and application of preparation method thereof - Google Patents
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- 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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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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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 the milk secreted by Bactrian camel fed by lactobacillus, and extracting exosomes from the milk by differential centrifugation. The research finds that: lactic acid bacteria-fed bactrian camel milk exosomes (LAB-cExo) were found, in which mirnas could target many pathways directly or indirectly related to cardiovascular disease, and were hypothesized to have potential for treating cardiovascular disease. Subsequently, the LAB-cExo can be successfully internalized by vascular endothelial cells (HUVECs), can effectively promote the proliferation of the HUVECs and inhibit the apoptosis of the HUVECs, and has the potential of treating cardiovascular diseases.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to a preparation method and application of a bactrian camel milk exosome.
Background
Camels can thrive under extremely harsh conditions such as drought and desert. Camel milk is more known as platinum in desert. However, current research shows that: camel milk is rich in various amino acids and vitamins necessary for human body, and contains protective proteins such as lysozyme, lactoferrin and immunoglobulin and various bioactive factors. Obviously, the development of effective components of camel milk is far from enough.
Disclosure of Invention
Based on the above, there is a need to provide an extraction method of bactrian camel milk exosomes and application thereof, and the internal components of the bactrian camel milk exosomes are deeply researched. The results of phenotype experiments show that the compound can promote the proliferation of HUVECs and inhibit the senescence of HUVECs, and is 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 the milk secreted by the bactrian camel fed by the lactobacillus; separating the milk by using a differential centrifugation method to obtain a crude product of impurity-removed precipitate; and (3) resuspending the precipitated crude product, placing the crude product in the upper layer of a centrifugal tube containing a gradient iodixanol solution, and performing ultracentrifugation to form 12 layers to obtain a fine extract positioned in the middle layer.
In some embodiments, the step of separating the crude precipitate from the milk by differential centrifugation comprises: centrifuging the milk for 5-15 min at a low centrifugal speed of 1000g to obtain skim milk; centrifuging the skim milk at a medium-centrifugal rotation speed of 10000g to obtain a skim and large vesicle-removed emulsion; continuously centrifuging the degreased de-vesicular emulsion for 1.5-2.5 h by using 100000g of high centrifugal rotating speed to obtain an enrichment precipitate; suspending the enriched precipitate by PBS buffer solution, further centrifuging and repeatedly washing the enriched precipitate by using the centrifugal speed of 2000g, centrifuging for 1.5-2.5 h under the condition of high centrifugal speed of 100000g, and collecting the crude product of the impurity-removed precipitate.
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 ultracentrifugation process conditions to form 12 layers are: centrifuging at high centrifugation speed of 100000g for 2h to obtain refined extract in the middle 6-9 layers.
The invention also provides the bactrian camel milk exosome prepared by the preparation method of the bactrian camel milk exosome.
The invention also provides application of the bactrian camel milk exosome prepared by the preparation method of the bactrian camel milk exosome in preparation of a preparation product for promoting HUVECs proliferation.
The invention also provides application of the bactrian camel milk exosome prepared by the preparation method of the bactrian camel milk exosome in preparation of medicines, foods and health care products for treating cardiovascular diseases.
The invention also discloses a preparation product for promoting the proliferation of HUVECs or treating cardiovascular diseases, which comprises the bactrian camel milk exosome prepared by the preparation method of the bactrian camel milk exosome.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides an exosome extracted and prepared from milk secreted by bactrian camel fed by lactic acid bacteria for the first time, and simultaneously verifies that the exosome has the effects of promoting the proliferation of HUVECs and inhibiting apoptosis.
Drawings
FIG. 1 is a graph showing the results of the particle size distribution of the latex exosome (LAB-cExo) of the bactrian camel prepared in example 1.
FIG. 2 is a transmission electron microscopy assay of the bactrian camel milk exosome (LAB-cExo) prepared in example 1.
FIG. 3 is a histogram of the length distribution of small RNA tested in example 2 against the bactrian camel milk exosomes (LAB-cExo) prepared in example 1.
FIG. 4 is a graph of the percentage of unique reads of each type of ncRNA tested in example 2 for the bactrian camel milk exosomes (LAB-cExo) prepared in example 1.
FIG. 5 is a histogram of the expression levels of some of the miRNAs tested in example 2 against the Bactrian camel milk exosome (LAB-cExo) prepared in example 1.
FIG. 6 is the result of the GO enrichment of the miRNA target gene tested in example 2 for the Bactrian camel milk exosome (LAB-cExo) prepared in example 1.
FIG. 7 is the result of KEGG enrichment of miRNA target genes tested in example 2 for the bactrian camel milk exosome (LAB-cExo) prepared in example 1.
FIG. 8 is a graph of the test for the transfection of bactrian camel milk exosomes (LAB-cExo) prepared in example 1 into HUVECs in example 3.
FIG. 9 is a statistical chart showing the effect of in vitro treatment of HUVECs cells in example 3 on cell activity after bactrian camel milk exosomes (LAB-cExo) prepared in example 1.
FIG. 10 is a statistical chart of the effect on apoptosis of HUVECs treated in vitro in example 3 in testing bactrian camel milk exosomes (LAB-cExo) prepared in example 1.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.
The key raw material sources are as follows:
bactrian camel, growing in Alxa, xinjiang. Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from research biotechnology (Shanghai) Inc. DMEM medium, purchased from semer feishel technologies.
The preparation method of the PBS buffer solution comprises the following steps: weighing 8g of NaCl, 0.2g of KCl and 1.44g of 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 desired volume of 1L, and sterilizing with high pressure steam.
Example 1
This example provides a method for extracting a bactrian camel milk exosome (LAB-cExo), including the following steps:
(1) Feeding a humped camel:
selecting female camels with the average age of 8-12 years, 3-4 fetuses and 2-3 months after delivery in Xinjiang area, and marking earhole. Mixing lactobacillus in feed to feed Bactrian camel. After continuously feeding for one month, milking and mixing twice in the morning and evening to obtain camel milk samples.
(2) Preparing an exosome:
transferring the camel milk sample to a new centrifuge tube, centrifuging at low speed (1000 g) for 10min at 4 deg.C, and discarding the precipitate to obtain skimmed milk.
The skim milk was centrifuged at 10000g at 4 ℃ for 45min to remove larger vesicles. Selecting overspeed rotor, centrifuging at 4 deg.C and 100000g for 120min, removing supernatant, and collecting precipitate. The collected precipitate was resuspended in 20. Mu.L of precooled 1 XPBS buffer, centrifuged at 2000g for 30min at 4 ℃ to remove the precipitate, and the process was repeated 2 times. Selecting an overspeed rotor, centrifuging the supernatant again for 120min at the temperature of 4 ℃ and the speed of 100000g, collecting the precipitate, and carrying out heavy suspension to form a heavy suspension.
Preparing iodixanol with different concentrations (40%, 20%, 10% and 5%), sequentially adding iodixanol with different concentrations into a super separation tube along the tube wall according to the concentration from high to low, finally adding 1mL of heavy suspension obtained in the above step into the uppermost layer of the super separation tube, and centrifuging at the temperature of 4 ℃ and the rotation speed of 100000g for 120min. After centrifugation, the solution will be separated into 12 layers, and the liquid in the middle 6-9 layers is taken out and centrifuged again for 120min under the same conditions. The supernatant was removed and resuspended in 300. Mu.L of precooled PBS to obtain exosomes (LAB-cExo), which were stored at-80 ℃.
The PARTICLE size of camel milk exosome (LAB-cExo) is measured by using a PARTICLE METRIX Nanoparticle Tracking Analyzer (NTA), and the specific steps are as follows: taking a frozen exosome (LAB-cExo) sample, unfreezing in a water bath at 25 ℃, and placing on ice; the exosome sample is diluted by PBS (exosome: PBS = 1:1) and directly used for NTA to detect the concentration and the particle size of camel milk exosome, and the result is shown in figure 1, the average particle size is 85.20nm, and the concentration is 1.11X 10 12 Particles/mL。
The camel milk exosome particle sediment is fixed and examined by a transmission electron microscope using a conventional procedure, and observed by a Hitachi transmission electron microscope, and the obtained transmission electron microscope micrograph is shown in fig. 2, which also confirms the range of the particle size distribution.
Example 2
This example addresses sequencing and target gene prediction for the bactrian camel milk exosomes prepared in example 1.
After the data quality control and the length filtering are finished, counting the number of Reads with the length of 18-31 nt and the proportion of the Reads with the length of each sample to obtain the length distribution of small RNA shown in figure 3. mirnas are generally concentrated at 21 or 22nt. The figure shows that small RNA with the length of 21nt and 22nt accounts for the most, and exceeds 41 percent.
The RNAs were aligned by small RNA length screening sequence miRBase 20.0, where the small RNAs were annotated into different categories, with the results shown in fig. 4, and the small RNA species in the clean sequence included mirnas, rRNAs, snoRNAs, snRNAs, trnas, and other sRNAs. The total amount of rRNA is used as a quality control index of a sample, and the proportion of camel milk exosomes is lower than 40 percent, so that the research samples all accord with the quality control index. According to the sequencing results, a total of 233 mirnas were detected. The first 25 mirnas with the highest expression level are shown in fig. 5.
And (4) carrying out target gene prediction on the first 25 miRNAs with the highest expression quantity, and carrying out work energy enrichment analysis on the miRNAs. The GO enrichment results (FIG. 6) indicate that most target genes are mainly enriched in related pathways such as some angiogenesis, cardiac development and some endothelial cell differentiation. Most target genes are mainly distributed in some endosomes (early endosome, endosome membrane, early endosome, late endosome, regenerative endosome), actin skeleton (actin cytoskeleton) and extracellular matrix (extracellular matrix) parts. Many target genes have actin or microtubule binding ability.
The KEGG enrichment results (fig. 7) indicate that the target gene is mainly enriched in some cardiovascular disease-related pathways, such as Lipid and atherospermis, fluid front stress and atherospermis, divided cardio myopathiy, hypertrophhic cardio myopathiy, arrhytmogenic right vascular cardio myopathiy.
Example 3 Effect of camel milk exosomes on Human Umbilical Vein Endothelial Cells (HUVECs)
(1) Absorption labelling assay
The camel milk exosomes (LAB-cExo) extracted in example 1 were stained with a PKH26 red fluorescent cell ligase kit and unlabeled dye was removed with an exosome spin column. The stained exosomes (2. Mu.g/L.times.10) were assayed 4 cells) or equal volume of PBS (NC) was added to the semi-miscible HUVECs. After 48h incubation, the immobilization was washed with 3.7% PFA for 10min. The nuclei of HUVECs were then labeled with FITC-conjugated cyclic peptides 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 sets of cells was measured with a fluorescence microscope, and the resulting fluorescence microscope photograph is shown in fig. 8.
(2) Test of influence of camel milk exosomes on HUVECs proliferation
As shown in FIG. 9, the CCK8 method was used to examine the viability of HUVECs transfected with LAB-cExo and PBS (NC) at 0, 24h, 48h and 72 h. CCK8 results indicate that the survival rate of cells in the LAB-cExo transfected group is increased compared to the control group. This suggests that LAB-cExo can promote cell proliferation.
(3) Influence of camel milk exosomes on HUVECs apoptosis
Flow cytometry was used to determine the percentage of apoptosis in both groups of HUVECs after transfection into LAB-cExo and PBS, respectively. As shown in FIG. 10, the results showed a significant reduction in the percentage of apoptotic cells in the LAB-cExo group compared to the control group. This indicates that LAB-cExo is able to inhibit apoptosis.
In addition, the LAB-cExo of the bactrian camel milk exosome is extracted and prepared from the milk secreted by the bactrian camel fed by lactic acid bacteria by a density gradient centrifugation method. In the method, the centrifugation times, the centrifugation time and the rotation speed need to be strictly controlled, the outer vesicles cannot be fully extracted when the centrifugation times, the centrifugation time and the rotation speed are too short or too low, the structure of the outer vesicles is damaged when the centrifugation times or the rotation speed is too high, the yield of the outer vesicles is reduced, and the obtaining of the bimodal camel milk exosome LAB-cExo is influenced.
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further 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 protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A preparation method of bactrian camel milk exosomes is characterized by comprising the following steps:
collecting the milk secreted by the bactrian camel fed by the lactobacillus;
separating the milk by using a differential centrifugation method to obtain a crude product of impurity-removed precipitate;
and (3) resuspending the precipitated crude product, placing the crude product in the upper layer of a centrifugal tube containing a gradient iodixanol solution, and performing ultracentrifugation to form 12 layers to obtain a fine extract positioned in the middle layer.
2. The method for preparing the bactrian camel milk exosome according to claim 1, wherein the step of separating the crude precipitate after the primary impurity removal from the milk by using a differential centrifugation method comprises the following steps:
centrifuging the milk for 5-15 min at a low centrifugal speed of 1000g to obtain skim milk;
centrifuging the skim milk at a medium-centrifugal rotation speed of 10000g to obtain a skim and large vesicle-removed emulsion;
continuously centrifuging the degreased and de-vesiculated emulsion for 1.5-2.5 h by using 100000g of high centrifugal rotating speed to obtain enriched precipitate;
suspending the enriched precipitate by PBS buffer solution, further centrifuging and repeatedly washing the enriched precipitate by using the centrifugal speed of 2000g, centrifuging for 1.5-2.5 h under the condition of high centrifugal speed of 100000g, and collecting the crude product of the impurity-removed precipitate.
3. The method for preparing the bactrian camel milk exosome according to claim 1 or 2, wherein the concentration gradient of the gradient iodixanol solution is 40% iodixanol solution, 20% iodixanol solution, 10% iodixanol solution and 5% iodixanol solution in sequence.
4. The method for preparing the bactrian camel milk exosome according to claim 3, wherein the process conditions for forming 12 layers by ultracentrifugation are as follows: centrifuging at a high centrifugation speed of 100000g for 2h.
5. The method for preparing the bactrian camel milk exosome according to claim 4, wherein the fine extract is obtained in the middle 6-9 layers.
6. The bactrian camel milk exosome prepared by the method for preparing the bactrian camel milk exosome of any one of claims 1 to 5.
7. Use of the bactrian camel milk exosome prepared by the method for preparing the bactrian camel milk exosome according to any one of claims 1 to 5 in preparing a preparation product for promoting HUVECs proliferation.
8. The use of the bactrian camel milk exosome prepared by the method for preparing the bactrian camel milk exosome according to any one of claims 1 to 5 in the preparation of medicines, foods and health products for treating cardiovascular diseases.
9. A preparation product for promoting the proliferation of HUVECs or treating cardiovascular diseases, which comprises the bactrian camel milk exosome prepared by the preparation method of the bactrian camel milk exosome according to any one of claims 1 to 5.
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