CN116286376A

CN116286376A - Separation method of haematococcus pluvialis extracellular vesicles and application thereof

Info

Publication number: CN116286376A
Application number: CN202211709669.9A
Authority: CN
Inventors: 王潮岗; 胡群菊
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2022-12-29
Filing date: 2022-12-29
Publication date: 2023-06-23

Abstract

The invention provides a separation method and application of haematococcus pluvialis extracellular vesicles. According to the invention, the extracellular vesicles of the haematococcus pluvialis are separated from the haematococcus pluvialis culture solution by differential centrifugation, ultrafiltration concentration and other methods, so that the extracellular vesicles of the haematococcus pluvialis are clear in outline and complete in structure. The method provided by the invention can use cells from microalgae, and the extraction steps are simple to operate, low in cost and easy to control; the extracted extracellular vesicles of haematococcus pluvialis carry miRNA, protein, pigment and other components in haematococcus pluvialis, and can be taken up by haematococcus pluvialis cells and regulate a series of physiological activities of the haematococcus pluvialis cells. In addition, the isolated haematococcus pluvialis extracellular vesicles can be taken up by liver, kidney and skin cells, have obvious functions of resisting oxidization, resisting inflammation and regulating various signal paths, can be used as ideal choices for preparing therapeutic agents for treating eye, liver and kidney diseases and cosmetics for resisting skin aging, and have wide application prospects.

Description

Separation method of haematococcus pluvialis extracellular vesicles and application thereof

Technical Field

The invention relates to the technical field of molecular biology and biological medicine, in particular to a separation method of extracellular vesicles of haematococcus pluvialis and application thereof.

Background

Astaxanthin is a red fat-soluble substance, has extremely high tinting strength, oxidation resistance and other effects, and has potential application prospects in various fields of cosmetics, foods, medical health care, aquatic products and the like. The algae source astaxanthin can remove free radicals among cells of a human body, can also enter the cells through cell membranes, can remove the intracellular free radicals, can protect cells and DNA from being damaged by peroxidation, and can make the cells perform metabolism normally and more effectively, so that the algae source astaxanthin is considered to have wide application prospects in the aspects of protecting eyes, preventing and treating brain and central nervous system diseases, improving immunity, improving exercise capacity, protecting liver, preventing body kidney injury caused by diabetes, preventing and treating cardiovascular diseases, resisting aging, beautifying and other human medicines. However, because astaxanthin is a fat-soluble substance, the problems of poor solubility, low stability and the like severely limit the clinical application of astaxanthin.

Extracellular vesicles (Extracellular vesicle, EVs) are nanoscale-sized bilayer membrane organelles produced by cells and secreted outside the cells, representing the sum of cell secretory vesicles. EVs contain various biological components including mRNA, protein, lipid, metabolites and the like, and can participate in the material exchange among cells so as to influence the functions of the cells, and are considered to be key intermediate factors for the communication among the cells and participate in various physiological reaction processes of the cells and organisms. EVs can avoid rapid clearance in vivo due to biological endogenous and low immunogenicity, and can not cause immune response of organisms. The microalgae-derived EVs are free of animal pathogenic bacteria and immune response-inducing components, and can be used as human food or pharmaceutical source. Microalgae-derived EVs are naturally enriched in a variety of bioactive molecules, with their potential biomedical application potential including anti-inflammatory, anti-tumor, wound recovery, immunomodulation, and microbiota regulation. The unique morphological features and bioactive substance carrying functions of microalgae-derived EVs offer feasibility for their application as nano-carrier platforms for natural and synthetic drugs. In fact, microalgae-derived EVs have the advantages of sustainability, high stability, high biocompatibility, low toxicity, low immunogenicity, suitability for large-scale culture production, and the like, compared to milk-derived exosomes or artificial nanoparticles. In addition, the microalgae mass production process is mature, and the use of microalgae for producing EVs has economic feasibility and sustainability.

Haematococcus pluvialis is a biological resource with highest natural astaxanthin content known in the nature, and large-scale cultivation is realized at present. Extracellular vesicles derived from haematococcus pluvialis have good economic feasibility and stable sources, and have no worry about use safety and the like. Therefore, the extracellular vesicles of haematococcus pluvialis are novel natural source EVs which are economically feasible and sustainable to produce, and the transmission of astaxanthin in specific tissues (such as liver, kidney, eyes, dendrites or tumor cells and skin) of human bodies by using the extracellular vesicles of haematococcus pluvialis provides a good nano-carrier platform for promoting the application of astaxanthin in the fields of medicine, health care, cosmetics and the like, and can also promote the development of natural nano-drug carrier technology. However, due to the low density of microalgae extracellular vesicles, no efficient and simple method for separating extracellular vesicles from haematococcus pluvialis exists at present. At present, common methods for extracting extracellular vesicles from liquid, such as an ultracentrifugation method and a polymer precipitation method, have the defects of low separation efficiency, low purity, damaged denaturation of EVs, low biological activity and the like due to interference of various complex components in a sample. In addition, studies on extracellular vesicles of haematococcus pluvialis to deliver active ingredients between cells have not been reported.

Therefore, the prior art needs to be researched and improved, and the application development thereof needs to be further researched.

Disclosure of Invention

In view of the shortcomings of the prior art, the invention aims to provide a separation method of extracellular vesicles of haematococcus pluvialis and application thereof, and explores a more efficient method for separating extracellular vesicles from haematococcus pluvialis by combining an ultrafiltration method and an ultracentrifugation method, and aims to solve the problems that the extracellular vesicles obtained by the ultracentrifugation method only in the prior art are low in purity, poor in activity and the like.

The technical scheme of the invention is as follows:

a method for separating extracellular vesicles of haematococcus pluvialis, comprising the steps of:

collecting culture solution of haematococcus pluvialis;

centrifuging the culture solution under the first condition, and collecting to obtain a first supernatant;

centrifuging the supernatant I under the second condition, and collecting to obtain a supernatant II;

filtering the supernatant II, and collecting filtrate;

centrifuging and concentrating the filtrate under the third condition by using a ultrafilter tube, and collecting concentrated solution;

centrifuging the concentrated solution under a fourth condition, and collecting primary precipitate;

re-suspending the primary pellet with buffer, centrifuging again under condition four, and collecting the final pellet;

and re-suspending the final sediment by using a buffer solution to obtain the haematococcus pluvialis extracellular vesicles.

The method for separating extracellular vesicles of haematococcus pluvialis comprises the step of culturing the haematococcus pluvialis in a green swimming cell, a green motionless cell or a red cyst stage.

The separation method of the haematococcus pluvialis extracellular vesicles comprises the following steps of firstly, centrifuging at a temperature of 4 ℃ for 10-30 min at a centrifugal force of 2000 g; the second condition is that the centrifugal temperature is 4 ℃, the centrifugal force is 10000g, and the centrifugal time is 30-40 min; the third condition is that the centrifugal temperature is 4 ℃ and the centrifugal force is 6000g; and the fourth condition is that the centrifugal temperature is 4 ℃, the centrifugal force is 100500g, and the centrifugal time is 80-100 min.

The separation method of the haematococcus pluvialis extracellular vesicles comprises the steps of filtering the supernatant with a filter membrane with the aperture of 0.22 mu m, and collecting filtrate.

The separation method of the haematococcus pluvialis extracellular vesicles comprises the step of filtering the haematococcus pluvialis extracellular vesicles, wherein the ultrafiltration tube is a 100KD ultrafiltration tube.

The separation method of the haematococcus pluvialis extracellular vesicles comprises the step of mixing a solution of the haematococcus pluvialis extracellular vesicles with a buffer solution of PBS.

The separation method of the haematococcus pluvialis extracellular vesicles comprises the step of separating haematococcus pluvialis extracellular vesicles, wherein the particle size of the haematococcus pluvialis extracellular vesicles obtained through separation is 50-200 nm.

The separation method of the haematococcus pluvialis extracellular vesicles comprises the step of separating haematococcus pluvialis extracellular vesicles which contain astaxanthin.

The application of the haematococcus pluvialis extracellular vesicles separated by any one of the above methods in preparation of nano medicines, nutritional foods and cosmetics.

The application is that the nano-drug is used for treating eye, liver and kidney diseases, the nutritional food is microalgae food, and the cosmetic is anti-skin aging cosmetic.

The beneficial effects are that: the invention provides a separation method and application of haematococcus pluvialis extracellular vesicles. The invention separates and obtains the extracellular vesicles of haematococcus pluvialis from fresh haematococcus pluvialis culture solution by differential centrifugation, ultrafiltration concentration and other methods. The isolated haematococcus pluvialis extracellular vesicles have clear outline and complete structure, and are obtained by first disclosure. The separation method provided by the invention can use cells from microalgae, and the extraction steps are simple to operate, low in cost and easy to control; the extracted extracellular vesicles of haematococcus pluvialis carry active ingredients such as proteins, astaxanthin and the like in haematococcus pluvialis, and can be taken up by haematococcus pluvialis cells and regulate a series of physiological activities of the haematococcus pluvialis cells. The method for separating extracellular vesicles from microalgae culture solution provided by the invention comprises the following steps: 1) The operation is simple, and a large-volume culture solution sample can be treated; 2) The extraction is quick, the time consumption is short, and large-particle impurities can be removed efficiently; 3) The recovery rate of extracellular vesicles is high, the purity is high, and the like; 4) The extracellular vesicles have good activity, have the capability of carrying active ingredients among cells, can be used as ideal choices for preparing therapeutic agents for treating eye, liver and kidney diseases and cosmetics for resisting skin aging in the future, and have wide application prospects.

Drawings

Fig. 1 is a schematic diagram of EVs samples extracted from an algae cell culture solution of haematococcus pluvialis at different stages according to an embodiment of the present invention.

Fig. 2 is a schematic diagram of morphological structure of EVs extracted from an algal cell culture solution of haematococcus pluvialis at different stages according to an embodiment of the present invention, bar=200 nm.

Fig. 3 is a schematic diagram of quality control results of EVs proteins provided in the embodiment of the present invention.

FIG. 4 is a cluster heat map of the EVs protein expression level provided by the embodiment of the invention.

Fig. 5 is a schematic diagram of quality control results of total RNA of EVs samples at different stages according to an embodiment of the present invention.

Fig. 6 is a schematic diagram of the statistics of the expression level of EVs miRNA at different stages according to the embodiment of the present invention.

Fig. 7 is a schematic diagram showing the results of uptake of EVs by haematococcus pluvialis cells provided in the examples of the present invention.

Fig. 8 is a schematic diagram showing the effect of EVs on haematococcus pluvialis cell growth provided in the example of the present invention.

Fig. 9 is a schematic diagram showing the effect of EVs on haematococcus pluvialis cytochrome according to the embodiment of the present invention.

Detailed Description

The invention provides a separation method of haematococcus pluvialis extracellular vesicles and application thereof, and the invention is further described in detail below for the purpose, technical scheme and effect of the invention to be clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The embodiment of the invention provides a separation method of haematococcus pluvialis extracellular vesicles, which comprises the following steps:

s100, collecting a culture solution of haematococcus pluvialis;

s200, centrifuging the culture solution under a first condition, and collecting a supernatant I;

s300, centrifuging the supernatant I under a second condition, and collecting the supernatant II;

s400, filtering the supernatant II, and collecting filtrate;

s500, centrifugally concentrating the filtrate by using a ultrafiltration tube under a third condition, and collecting concentrated solution;

s600, centrifuging the concentrated solution under a fourth condition, and collecting primary precipitate;

s700, re-suspending the primary precipitate by using a buffer solution, centrifuging again under a fourth condition, and collecting a final precipitate;

s800, re-suspending the final sediment by using a buffer solution to obtain the haematococcus pluvialis extracellular vesicles.

In some embodiments, the culture medium comprises haematococcus pluvialis culture medium obtained by various culture methods, including haematococcus pluvialis culture medium of green motile cells, green motile cells or red cyst stage.

In some embodiments, in step S200, the first condition is a centrifugation temperature of 4 ℃, a centrifugal force of 2000g, and a centrifugation time of 10min to 30min. The culture solution is centrifuged for 10min to 30min at 4 ℃ and 2000 Xg, so as to remove algae cells.

In some embodiments, in step S300, the second condition is a centrifugal temperature of 4 ℃, a centrifugal force of 10000g, and a centrifugal time of 30min to 40min; the culture solution is centrifuged for 30 to 40 minutes at 4 ℃ and 10000 Xg, so as to remove cell debris.

In some embodiments, in step S400, the supernatant is filtered with a 0.22 μm pore size filter, and the filtrate is collected.

In some embodiments, in step S500, the third condition is a centrifugation temperature of 4 ℃, a centrifugal force of 6000g.

In some embodiments, in step S500, the ultrafiltration tube is a 100KD ultrafiltration tube.

Specifically, the Ultra-filtration tube is Ultra15 100KD Centrifugal Filter Units.

In the embodiment of the invention, the filtrate obtained in the step S400 is concentrated to about 20mL by using a 50mL ultrafiltration tube (100 KD) at 4 ℃ and 6000 Xg, and the extracellular vesicle concentrate of haematococcus pluvialis is collected.

The invention adopts ultrafiltration concentration method to extract, and the molecular weight cut-off and concentration condition of the ultrafiltration tube are required to be strictly controlled, and the quality and yield of obtaining haematococcus pluvialis extracellular vesicles are affected by too large or too small molecular weight cut-off of the ultrafiltration tube and too many centrifugation times or too large rotation speed during concentration.

In some embodiments, in the steps S600 and S700, the fourth condition is that the centrifugal temperature is 4 ℃, the centrifugal force is 100500g, and the centrifugal time is 80-100 min.

The primary pellet was resuspended in a large volume of buffer and then centrifuged again in order to wash the primary pellet to remove impurities.

In some embodiments, the buffer is a PBS buffer. But is not limited thereto, other conventional buffers may be used.

Specifically, the preparation method of the PBS buffer solution comprises the following steps: 8g of NaCl, 0.2g of KCl and 1.44g of Na are weighed ₂ HPO ₄ And 0.24g KH 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.

The invention adopts differential centrifugation and ultrafiltration to extract. In the differential centrifugation method, the centrifugation times, the centrifugation time and the rotation speed need to be strictly controlled, the time is too short or the rotation speed is too small, the outer vesicles cannot be fully extracted, the structure of the outer vesicles can be damaged due to too many times or too large rotation speed, and the yield of the outer vesicles is reduced. Finally, the invention separates and obtains the extracellular vesicles of the haematococcus pluvialis from the fresh haematococcus pluvialis culture solution by differential centrifugation, ultrafiltration concentration and other methods, and the separated extracellular vesicles of the haematococcus pluvialis have clear outline and complete structure.

In some embodiments, the isolated extracellular vesicles of haematococcus pluvialis have a particle size of 50 to 200nm.

The Extracellular Vesicles (EVs) of Haematococcus pluvialis isolated in the examples of the present invention were measured using a nanoparticle size detector (ZetaPlus, USA), and the particle size of the extracted EVs was found to be between 50nm and 200nm, with an average particle size of about 100nm.

Protein and RNA analysis shows that extracellular vesicles of haematococcus pluvialis extracted by the separation method provided by the embodiment of the invention carry miRNA, protein, astaxanthin and other active ingredients in haematococcus pluvialis. Meanwhile, the types and the contents of the protein and the miRNA have obvious differences in EVs samples at different stages, which means that the algae cells at different stages sort different contents such as the protein, the miRNA and the like into the EVs, then the EVs are released into a culture system by donor cells and are taken up by receptor cells, so that information transmission among cells is completed, and the process may be related to the synergic action of the algae cells in response to environmental changes.

Specifically, the isolated extracellular vesicles of haematococcus pluvialis contain carotenoids. More specifically, the haematococcus pluvialis extracellular vesicles contain active ingredients such as astaxanthin.

The invention provides a separation method which can use microalgae-derived extracellular vesicles, and the extraction steps are simple to operate, low in cost and easy to control; the extracted extracellular vesicles of haematococcus pluvialis carry miRNA, protein, astaxanthin and other active ingredients in haematococcus pluvialis, can be taken up by haematococcus pluvialis cells and regulate a series of physiological activities of the haematococcus pluvialis cells, and can be used as transport carriers for the active ingredients such as astaxanthin and the like entering liver cells, skin cells and the like in the future.

The embodiment of the invention also provides an application of the separation method of the haematococcus pluvialis extracellular vesicles, and the haematococcus pluvialis extracellular vesicles obtained by the separation method are applied to the preparation of nano medicines, nutritional foods and cosmetics.

Specifically, the haematococcus pluvialis extracellular vesicles are applied to the preparation of nano-drugs for treating eye, liver and kidney diseases, microalgae food and cosmetics for resisting skin aging.

Specifically, the prepared medicine for treating eye, liver and kidney diseases can be any one of granules, capsules, soft capsules, oral liquid preparations, injections and transdermal administration preparations.

The invention creatively extracts extracellular vesicles from haematococcus pluvialis culture solution, discovers that the extracellular vesicles contain various cell components of haematococcus pluvialis, and researches the functions of the haematococcus pluvialis culture solution to find that the haematococcus pluvialis culture solution can be taken up by algae cells and change the corresponding biological processes of the cells. In addition, extracellular vesicles derived from haematococcus pluvialis and extracellular vesicles derived from haematococcus pluvialis additionally loaded with astaxanthin can be taken up by relevant human cells, have the functions of resisting oxidization, resisting inflammation and regulating and controlling signal paths, prove that the extracellular vesicles have the potential of loading the astaxanthin for treating eye, liver and kidney diseases and resisting skin aging, and can be applied to preparing medicaments for treating the eye, liver and kidney diseases and cosmetics for resisting skin aging. Moreover, the EVs derived from microalgae can be used as an additional product of industries such as microalgae food development, biofuel production, bioremediation, high-value compound biosynthesis and the like, has a series of competitive advantages such as sustainability, expandability, operability, reproducibility, strong social acceptability and the like, and can be used in various industrial fields such as nano medicine, nutritional food, cosmetics and the like.

The method for separating extracellular vesicles of haematococcus pluvialis and the application thereof are further explained by the following specific examples.

Example 1

The material used in this example was Haematococcus pluvialis strain 192.80 algal cells (purchased from The Culture Collection of the University of)

Germany)。

The extraction method of the haematococcus pluvialis extracellular vesicles adopted in the embodiment comprises the following steps:

(1) Culturing haematococcus pluvialis based on a closed photo bioreactor by BBM, collecting algal cell culture solutions subjected to high-light high-salt treatment for 0h, 6h and 48h, and respectively obtaining fresh culture solutions of green motile cells, green motile cells and red cyst stages;

(2) Centrifuging at 1000g for 10min at 4deg.C for removing algae cells, collecting supernatant in a clean container;

(3) Centrifuging at 4deg.C for 30min at 10000g for removing cell debris, shedding vesicles and apoptotic bodies, collecting supernatant in a clean container;

(4) Filtering the collected supernatant with a filter membrane with a pore diameter of 0.22 μm, and collecting filtrate;

(5) Concentrating the filtrate collected in the previous step at about 6000g using a 50mL ultrafiltration tube (Ultra 15 100KD Centrifugal Filter Units) at 4deg.C to about 20mL, and collecting the concentrate;

(6) Collecting the concentrated solution in a special centrifuge tube of a differential ultra-high speed centrifuge, centrifuging for 80-100 minutes at 100500g at 4 ℃, and collecting extracellular vesicle sediment;

(7) After re-suspending the precipitate with precooled 1 XPBS buffer, centrifuging for 80-100 min at 4 ℃ with 100500g, washing extracellular vesicle precipitate, collecting extracellular vesicles of haematococcus pluvialis in a sterilized EP tube, and storing in a refrigerator at-80 ℃ for later use.

Example 2 characterization of extracellular vesicles of Haematococcus pluvialis

The morphology, particle size and content of extracellular vesicles derived from haematococcus pluvialis obtained in example 1 were characterized.

1. Morphological observation

The diameter of extracellular vesicles of haematococcus pluvialis is in the range of 20-500nm, and the extracellular vesicles can be clearly observed by combining a transmission electron microscope with negative staining.

The procedure for the observation of extracellular vesicles of haematococcus pluvialis was as follows: (1) Transferring a drop of 1 XPBS preservation solution containing EVs by using a liquid transferring gun, placing on a copper mesh with a polyvinyl acetate-carbon film attached to one surface (the sample is placed on one side of the carbon film, 2-3 copper mesh samples are prepared for each EVs sample), standing for 20min under dark drying condition, and allowing the carbon film to adsorb EVs; (2) Carefully sucking the EVs liquid drops on the side surface of the copper net by using water-absorbing paper, and airing for 2min; (3) A liquid transferring gun is used for taking 10 mu L of 1% phosphotungstic acid dye drops to a copper mesh, and the liquid is kept stand for 2min in a dark place; (4) Excess dye solution on the copper mesh was gently removed with a piece of absorbent paper, the copper mesh was placed on the piece of absorbent paper (with the film facing upward), and after drying for about 12 hours, it was observed on an 80kV electron microscope.

As a result, as shown in FIG. 1, EVs-1 in the green motile cell stage was green, EVs-2 in the green motile cell stage was yellowish green, and EVs-3 in the red sporangium stage was orange red.

FIG. 2 shows the morphology of EVs derived from Haematococcus pluvialis (H.pluvialis) cell culture solution extracted in example 1 under a transmission electron microscope, wherein the EVs are spherical or spheroid double-layer membrane vesicles, the diameters of the vesicles are distributed between 50 and 200nm, the outer layer is a dyed double-layer membrane structure, the membrane structure is clear, a deep dyed region is seen in the inner layer, and the EVs are presumably the contents of the vesicles.

2. Determination of HPEVs particle size Using a nanoparticle size Detector (ZetaPlus)

The particle size of the EVs from which the h.pluvialis algal cell culture fluid was derived was measured using a nano particle size tester (ZetaPlus, usa), and as shown in table 1, it was found that the particle size of the EVs extracted in example 1 was between 50nm and 200nm, consistent with the result of the transmission electron microscope, and the average particle size was about 100nm.

TABLE 1H particle size determination of algal cell culture fluid EVs at different stages of pluvialis

Example 3 quantitative proteomic analysis of extracellular vesicles of Haematococcus pluvialis

The method for quantifying the proteome by using the iTRAQ technology has the advantages of high sensitivity, strong separation capability, reliable result, high automation degree and the like.

The experimental process is as follows: (1) extraction and quality inspection of EVs proteins; (2) reductive alkylation and enzymatic hydrolysis of proteins; (3) iTRAQ-labeled and mixed in equal amounts; (4) pre-separating the mixed peptide fragments by a C18 reverse column; (5) liquid phase tandem mass spectrometry [ LC-MS/MS ] on-machine analysis; (6) outputting data by the database searching software; (7) data statistics and bioinformatics analysis.

The EVs protein extraction process comprises the following steps: (1) Taking out EVs sample from-80 deg.C, adding Urea and protease inhibitor with final concentration of 8M, and performing ultrasonic cleavage; (2) Centrifuging at 4deg.C for 10min at 12000g to remove fragments, transferring supernatant to a new centrifuge tube, and collecting supernatant as EVs protein solution; (3) protein concentration determination was performed using BCA kit.

The SDS-PAGE process of EVs proteins includes: (1) sample preparation: according to the protein concentration measurement result, taking an equal amount of protein from each sample into a centrifuge tube, adding 5 mu L of 4×loading buffer, and adding 2% SDS to make the volume 20 mu L; (2) sample loading: sequentially Loading 1 mu L of pre-dyed protein marker and 12 mu L of protein sample, and Loading 12 mu L of 1 xLoding buffer for closing the blank holes adjacent to the sample; (3) electrophoresis: concentrating gel 15mA/gel for about 15min until protein is concentrated into a line, separating gel 35mA to dye electrophoresis to gel bottom; (4) silver staining: after the gel is taken out, the gel is subjected to the steps of fixing, sensitization, silver staining and the like, and then is transferred into a color development liquid to be developed for about 10 minutes at room temperature, when the stripes are clear and the color development effect is moderate, the color development liquid is discarded, a stop solution is added, and the gel is photographed in a gel imager, and the result is shown in figure 3.

FIG. 3 shows the results of SDS-PAGE gel electrophoresis of the extracted EVs proteins for preliminary quality control testing. Wherein A: SDS-PAGE of proteins, 12. Mu.L per lane; b: quality control detection (peptide fragment length distribution) map of mass spectrometry data. The quality inspection results show that the protein bands of each EVs sample are clear and uniform, the proteins are not degraded, the parallelism of each lane in the group is good, and the electrophoresis behavior difference among groups is not obvious (figure 3A). In order to ensure the reliability of the experiment, the mass spectrum quality is detected among all proteomic related experiments. The vast majority of peptide lengths are distributed between 8-20 amino acid residues (fig. 3B), which conforms to the rules of pancreatin digestion of peptide, indicating that sample preparation meets the standards.

Bioinformatics analysis of proteins involves the following aspects: (1) protein annotation; (2) protein differential expression analysis; (3) Functional classification of differentially expressed proteins, including GO secondary annotation classification, subcellular structure localization classification, and COG/KOG functional classification; (4) Functional enrichment analysis of the differentially expressed proteins, including GO enrichment, KEGG pathway enrichment, and protein domain enrichment; (5) Functional enrichment clustering analysis, namely, using a hierarchical clustering method to gather related functions in different groups according to a Fisher's exact test p value obtained by enrichment analysis, and drawing a heat map; (6) Protein interaction network, i.e. the number or protein sequence of the differential protein database obtained by screening in different comparison groups, and the differential protein interaction relationship is obtained by comparing the protein interaction network with the STRING protein network interaction database and extracting according to the confidence score >0.7 (high confidence). EVs proteomic composition determination and trust analysis were carried out by commercial companies.

Finally, protein mass spectrometry (LC-MS) identified 2038 quantifiable proteins in six EVs samples from h.pluvialis algal cell culture broth (see fig. 4), indicating that EVs from h.pluvialis algal cell culture broth contain a large amount of protein.

EXAMPLE 4 Small RNA composition analysis of extracellular vesicles of Haematococcus pluvialis

And (3) extracting RNA of the EVs by using an RNA extraction kit, and completing sequencing of the small RNA by using an Illumina sequencing platform. And the statistics of base distribution and quality fluctuation is carried out on each cycle of all sequencing reads by using a statistical method, so that the library construction quality and sequencing quality of sequencing samples can be intuitively reflected from a macroscopic scale, and the base quality and base error rate of each sample are analyzed. In order to ensure the accuracy of subsequent biological information analysis, the original sequencing data is filtered, so that high-quality sequencing data (clean data) is obtained, after quality control, the reads length distribution of clean small RNA is counted, sequence comparison analysis is carried out, and the numbers and percentages of reads on comparison are counted. And comparing reads of the reference genome with the miRBase and Rfam databases, respectively obtaining known miRNA and ncRNA annotation information, and counting the numbers of the miRNAs and the ncRNA annotation information. And (3) performing base preference, base editing and family analysis on the obtained miRNA, and performing expression quantity and difference analysis on the miRNA. And further predicting target genes of all known miRNAs by using plant-specific target gene prediction software, and performing functional annotation on the target genes. And carrying out gene set analysis on the obtained miRNA or target genes according to certain screening conditions (such as functions, expression quantity, expression difference conditions and the like), wherein the gene set analysis comprises Venn analysis, cluster analysis, target gene function annotation, target gene function enrichment, target gene protein interaction analysis and the like. Small RNA composition measurement and functional analysis were carried out by commercial companies.

After total RNA extraction from EVs, agilent 2100 quality inspection of RNA was performed, and the results are shown in FIG. 5. The results show that 6 samples of H.pluvialis EVs total RNA have differences in sequence length, and each sample contains miRNA with an effective length of 20-24 nt. Meanwhile, the RNA contained in the EVs at the red cyst stage shows a characteristic of short sequence, and the lengths of the RNA fragments at the EVs-1 and EVs-2 stages show a characteristic of approximately normal distribution.

The total identified h.pluvialis EVs of this example are known and the new mirnas totalling 163, with 70 new mature mirnas predicted. The types of 6 sample mirnas of h.pluvialis EVs were counted separately, and the results are shown in table 2, and evs_1_1, evs_1_2, evs_2_1, evs_2_2, evs_3_1 and evs_3_2 contain 49, 40, 52, 39, 54 and 38 mirnas, respectively. The 6 samples are divided into three groups, namely EVs_1, EVs_2 and EVs_3, and the statistical results of the obtained expression quantity of the mature miRNA and the obtained expression quantity of the precursor miRNA are shown in FIG. 6. The results show that the types and the expression amounts of miRNA contained in EVs in the algae culture solution at different stages are different to a certain extent.

TABLE 2 statistics of EVs miRNAs

Example 5 verification of extracellular vesicles of Haematococcus pluvialis taken up by algal cells

This example was mainly validated using the cell membrane staining kit (PKH 67, shanghai vanda). The fluorescent dye PKH67 is a novel dye capable of carrying out fluorescent labeling on living cells, can label living cells, is used for labeling cells by combining with lipid molecules with a membrane structure, and is also a dye for carrying out cell uptake observation by commonly used labeled EVs. This example uses a laser confocal microscope (Confocol, LSM 710 NLO) for detection of PKH fluorescence.

The PKH67 staining and observation method is as follows: (1) Taking out PKH67 reagent from refrigerator, standing for several minutes to room temperature, centrifugingA tube for containing PKH67, so that PKH67 reagent falls into the bottom of the tube fully; (2) Packaging PKH67 reagent according to requirement to prevent repeated freezing and thawing; (3) Diluting PKH reagent 10 times with diluent to prepare PKH67 mother liquor; (4) Since EVs were dissolved in PBS, the working staining of EVs was performed directly at a ratio of 25:1 (EVs solution: PKH67 mother liquor); (5) incubating in an incubator (refrigerator) at 2-8 ℃ for 30min; (6) The dyed EVs were ultrafiltered in a 100KD ultrafilter tube at 4deg.C, washed 2 times with 10mL of PBS to remove free PKH67 dye; (7) The washed EVs were resuspended in approximately 100. Mu.L of PBS and a subsequent H.pluvialis cell uptake experiment was performed; (8) Centrifuging H.pluvialis cells in logarithmic phase at 1500g.times.5 min at 22deg.C, washing with fresh BBM medium at 150g.times.5 min at 22deg.C for 3 times, and re-suspending in fresh BBM medium to give algae cells with a number of 106 cells.ml ^-1 The method comprises the steps of carrying out a first treatment on the surface of the (9) All PKH67 stained and washed three-stage EVs were added to 1mL of resuspended algal cells, respectively, at 22℃and 25mmol photons.m ^-2 ·s ^-1 Continuously illuminating, standing and culturing for 24 hours, and shaking for 2-3 times during the period; (10) stained algal cells were observed with Confocol; (11) PKH-labeled EVs fluoresce green with a fluorescence wavelength γex=490 nm, γem=502 nm.

The results of observing the uptake of EVs by h.pluvialis algal cells by confocal laser microscopy are shown in fig. 7. Wherein, red is shown as chlorophyll fluorescence of algae cells, and green is PKH67 fluorescence. A: control group, PBS; b: evs_1 is taken up by h.pluvialis algal cells; c: evs_2 is taken up by h.pluvialis algal cells; d: EVs_3 is taken up by H.pluvialis algal cells. As can be seen, the EVs in the supernatant of the H.pluvialis culture medium can be taken into H.pluvialis algal cells at three cell stages, and the EVs stained by fluorescence are accumulated at the cell membrane/wall (FIG. 7).

Example 6 verification of extracellular vesicle function of Haematococcus pluvialis

Culturing to obtain H.pluvialis algae liquid in logarithmic growth stage, centrifuging at 2000 Xg for 10min at normal temperature, washing algae cell mass with fresh BBM culture medium for 3 times, and re-suspending in fresh culture medium to obtain algae cells in logarithmic growth stage.

Extracting and separating to obtain a sufficient amountThe EVs of different cell stages of the strain are resuspended in sterile 1 XPBS solution, and the protein concentration of the EVs is measured and stored at-80 ℃ for standby; centrifuging H.pluvialis cells in logarithmic phase at 1500g.times.5 min at 22deg.C, washing with fresh BBM medium at 1500g.times.5 min at 22deg.C for 3 times, and re-suspending in fresh BBM medium to give algae cells with number of about 10 ⁶ cells·ml ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The three EVs were used as targets, and the EVs were added to the resuspended algal cells, and the amount of EVs used in this example was all 10. Mu.g.L ^-1 Culturing for 48 hours under non-stress condition, and taking PBS group as control group; after the culture, 1mL of algal cells were taken and observed under a microscope, and the remaining algal cells were collected, and a part of the algal cells were subjected to a molecular biological experiment, a part of fatty acid components were analyzed, and a part of the algal cells were subjected to analysis of pigment components.

The expression amount analysis of 5 genes related to astaxanthin biosynthesis [ Phytoene Synthase (PSY), phytoene Dehydrogenase (PDS), lycopene beta cyclase (LCY), beta-carotene hydroxylase (crtR_b) and carotenoid hydroxylase (BKT) ], 4 genes related to fatty acid biosynthesis [ Biotin Carboxylase (BC), acyl Carrier Protein (ACP), SAD (stearoyl ACP desaturase) and omega-3 Fatty Acid Desaturase (FAD) ],5 genes related to cell wall biosynthesis [ endoglucanase (CW 738), cellulose synthase (CW 898), cellulose hydrolase (CW 207), mannitol dehydrogenase (CW 188) and mannose synthase (CW 778) ] and 3 genes related to algal cell cycle control [ cyclin A (CYCA), cyclin B (CYCB) and cell division cycle control protein 45 (CDC 45) ], and 17 genes in total was performed by qRT-PCR. The primer sequences used for the h.pluvialis beta-actin as reference gene are shown in table 3.

TABLE 3 fluorescent quantitative qRT-PCR primer sequences

This example explores the effect of EVs on h.pluvialis algal cells in the logarithmic growth phase. The results showed that EVs-1 and EVs-2 promoted the increase in algal cell number to some extent during the 2 day incubation time, but the three-stage EVs did not have a significantly different effect on the growth of h.pluvialis algal cells from the control PBS (as shown in fig. 8). Meanwhile, the EVs of the three stages and the PBS of the control group had an effect on the content of h.pluvialis algae cytochromes (including chlorophyll a, chlorophyll b and carotenoids) (as shown in fig. 9). At the same time, the algae cells were cultured under four treatment conditions for 2 days, and the total fatty acid content thereof was varied (as shown in Table 4).

TABLE 4 influence of EVs on fatty acid content of H.pluvialis algal cells

This example further explores the effect of EVs on the expression levels of 17 genes of H.pluvialis. As a result, as shown in table 5, it was found that EVs exhibited no significant difference in expression of these four genes PDS, BKT, ACP and CDC 45. EVs_3 significantly increased the expression levels of the PSY, LCY, crtR-b, SAD, FAD, CW738, CW898, and CW207 genes, while PBS, EVs_1, and EVs_2 showed no significant differences in the expression of these genes. EVs_2 and EVs_3 significantly increased the expression level of the BC gene, whereas EVs_1 did not show significant differences in the BC expression level. EVs_3 significantly increased the expression levels of CW118 and CW778 genes relative to EVs_1, while EVs_1 and EVs_2 did not significantly differ from the expression levels of both genes in the algae cells. In addition, evs_3 significantly increased the expression levels of the CYCA and CYCB genes, whereas evs_1 significantly decreased the expression levels of both genes relative to evs_2, whereas evs_1 did not exhibit significant differences in the expression levels of the CYCA and CYCB genes relative to the control group.

TABLE 5 influence of EVs on expression level of H.pluvialis 17 genes

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In conclusion, the invention separates and extracts Extracellular Vesicles (EVs) in the culture solution of three cell stages of H.pluvialis green motile cells, green motile cells and red cysts; the particle size of EVs is measured to be between 50nm and 200nm, and the average particle size is about 100nm; spherical or spheroidal bilayer membrane structures with diameter sizes distributed over EVs between 50-200nm were seen using transmission microscopy. Further, proteomic and miRNA histologic analyses were performed on the EVs content, which indicated that 2038 proteins could be quantified and 163 mirnas identified in the EVs of h.pluvialis. The types and amounts of proteins and mirnas have significant group-to-group differences in the EVs samples at different stages, indicating that the algal cells at different stages sort different inclusions into the EVs. In addition, EVs from three stages can be taken up by H.pluvialis algae cells, and participate in regulating and controlling gene expression of synthesis related paths of cell walls, astaxanthin, fatty acid and the like, thereby playing biological functions.

The EVs derived from microalgae can be used as an additional product of industries such as microalgae food development, biofuel production, bioremediation, high-value compound biosynthesis and the like, has a series of competitive advantages such as sustainability, expandability, controllability, reproducibility, strong social acceptability and the like, and can be used in various industrial fields such as nano medicine, nutritional food, cosmetics and the like.

In summary, the invention provides a separation method and application of haematococcus pluvialis extracellular vesicles. The invention separates and obtains the extracellular vesicles of haematococcus pluvialis from fresh haematococcus pluvialis culture solution by differential centrifugation, ultrafiltration concentration and other methods. The isolated haematococcus pluvialis extracellular vesicles have clear outline and complete structure, and are obtained by first disclosure. The separation method provided by the invention can use cells from microalgae, and the extraction steps are simple to operate, low in cost and easy to control; the extracted extracellular vesicles of haematococcus pluvialis carry miRNA, protein, pigment and other components in haematococcus pluvialis, and can be taken up by haematococcus pluvialis cells and regulate a series of physiological activities of the haematococcus pluvialis cells. The method for separating extracellular vesicles from microalgae culture solution provided by the invention comprises the following steps: 1) The operation is simple, and a large-volume culture solution sample can be treated; 2) The extraction is quick, the time consumption is short, and large-particle impurities can be removed efficiently; 3) The recovery rate of extracellular vesicles is high, the purity is high, and the like; 4) The extracellular vesicles have good activity, have the capability of carrying active ingredients among cells, can be used as ideal choices for preparing therapeutic agents for treating eye, liver and kidney diseases and cosmetics for resisting skin aging in the future, and have wide application prospects.

It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.

Claims

1. A method for separating extracellular vesicles of haematococcus pluvialis, which is characterized by comprising the following steps:

collecting culture solution of haematococcus pluvialis;

filtering the supernatant II, and collecting filtrate;

2. The method for isolating extracellular vesicles from haematococcus pluvialis according to claim 1, wherein the culture solution comprises a culture solution of haematococcus pluvialis in the stage of green motile cells, green motile cells or red cysts.

3. The method for separating extracellular vesicles from haematococcus pluvialis according to claim 1, wherein the first condition is a centrifugation temperature of 4 ℃, a centrifugal force of 2000g and a centrifugation time of 10-30 min; the second condition is that the centrifugal temperature is 4 ℃, the centrifugal force is 10000g, and the centrifugal time is 30-40 min; the third condition is that the centrifugal temperature is 4 ℃ and the centrifugal force is 6000g; and the fourth condition is that the centrifugal temperature is 4 ℃, the centrifugal force is 100500g, and the centrifugal time is 80-100 min.

4. The method for separating extracellular vesicles from haematococcus pluvialis according to claim 1, wherein the supernatant is filtered with a filter membrane having a pore size of 0.22 μm, and the filtrate is collected.

5. The method for separating extracellular vesicles from haematococcus pluvialis according to claim 1, wherein the ultrafiltration tube is a 100KD ultrafiltration tube.

6. The method for isolating extracellular vesicles from haematococcus pluvialis according to claim 1, wherein the buffer is PBS buffer.

7. The method for separating extracellular vesicles from haematococcus pluvialis according to claim 1 wherein the particle size of the extracellular vesicles from haematococcus pluvialis obtained by separation is 50 to 200nm.

8. The method for separating extracellular vesicles from haematococcus pluvialis according to claim 1, wherein the separated extracellular vesicles from haematococcus pluvialis contain astaxanthin.

9. Use of extracellular vesicles of haematococcus pluvialis isolated by the method according to any one of claims 1 to 8 for the preparation of nano-pharmaceuticals, nutraceuticals and cosmetics.

10. The use according to claim 9, wherein the nano-drug is a nano-drug for the treatment of eye, liver and kidney diseases, the nutritional food is a microalgae food, and the cosmetic is an anti-skin aging cosmetic.