Aloe acetylmannan milk fat ball fluorescent marker as well as preparation method and application thereof

Abstract

The invention provides an aloe acetylmannan milk fat ball fluorescent marker as well as a preparation method and application thereof. According to the aloe acetylmannan milk fat ball fluorescent marker, aloe acetylmannan and water are mixed and homogenized to obtain emulsion, then the emulsion is mixed with silicone oil, and secondary homogenization is performed to obtain aloe acetylmannan milk fat balls; the aloe acetylmannan milk fat ball is obtained by sequentially adopting rhodamine DHPE (docosahexaenoic acid) and fluorescently-labeled lectin to label and incubating in a dark place. The preparation method of the marker is simple to operate, chemical reagents are not used, the original space structure of the acetylmannan milk fat ball is not destroyed, the biological activity and experimental accuracy of acetylmannan are not influenced, and the prepared fluorescent marker milk fat ball is stable in structure, polysaccharide-lipid kernel dual-marking, high in visibility, good in experimental accuracy and suitable for large-scale popularization and application. The method is suitable for preparing a cell fluorescence labeling reagent and cell membrane labeling for non-diagnosis and non-treatment purposes.

Description

Technical Field

[0001] The present invention belongs to the technical field of polysaccharide markers, and particularly relates to an aloe acetyl mannan milk fat globule fluorescent marker, a preparation method thereof and an application thereof. Background Art

[0002] Aloe polysaccharide is a macromolecular compound formed by connecting multiple monosaccharide molecules with the same or different structures through glycosidic bonds, and mainly exists in the fresh leaf gel of aloe. Since aloe polysaccharide can be taken up by cells and trigger specific physiological reactions, and has high safety and low toxicity, it has attracted much attention in the biomedical field. Among them, aloe acetyl mannan, as the main component of aloe polysaccharide, has various biological activities such as anti-inflammatory, antioxidant and promoting tissue repair. In addition, aloe acetyl mannan has a complex spatial structure and amphiphilic properties of water and oil, is a natural macromolecular active polymer material, can be prepared into a milk fat globule structure, and shows broad application prospects in the field of drug delivery.

[0003] According to domestic and foreign literature, many studies have used radioactive isotopes to label polysaccharides to trace their biological activities and study their molecular mechanisms. However, radioactive isotopes have adverse effects on organisms and are not suitable for biological research. For this reason, the polysaccharide fluorescence labeling technology has emerged. This technology is based on the characteristic that polysaccharides have multiple reducing ends, and fluorescent substances are connected to the sugar chains by chemical synthesis methods. In the specific implementation process, polysaccharides need to be activated first to introduce reaction groups, and common activators include hydrogen bromide, isocyanate, etc.; then, under appropriate conditions, the polysaccharides are mixed and reacted with fluorescent dyes and catalysts, so that the fluorescent dyes form chemical bonds with groups such as the reducing end amino group, carboxyl group, and hydroxyl group of the polysaccharides; after the reaction is completed, a terminator is added to stop the reaction, and column chromatography, solvent precipitation and other methods are used to remove unreacted fluorescent dyes and other impurities, so as to purify the labeled polysaccharides. However, the above polysaccharide fluorescence labeling process takes a long time, has complex procedures, and there are problems of chemical reagent residues and reduced biological activity of the polysaccharides in the purified fluorescently labeled polysaccharides, which affect the accuracy of cell biology and biochemistry experiments.

[0004] In addition, fluorescein isothiocyanate (FITC) chemical labeling is a common method for fluorescent labeling of mannan, but there are few relevant research reports on whether this method is also applicable to aloe acetyl mannan milk fat globules. Some literature points out that aloe acetyl mannan has a large molecular weight, is rich in a large number of acetyl groups in its structure, and has a complex spatial conformation, making it difficult for the reducing end to be exposed, resulting in a low fluorescence labeling rate. In actual operation, researchers found that compared with aloe acetyl mannan without FITC chemical labeling, after aloe acetyl mannan was chemically labeled with FITC and prepared into milk fat globules, the emulsion stability decreased; when co-cultured with cells, the fluorescence of the milk fat globules was lost, and the tracing effect could not meet the experimental requirements. Therefore, it is of great significance to develop a new fluorescent labeling method for aloe acetyl mannan. Summary of the Invention

[0005] To solve the above problems, the present invention provides a fluorescent label for aloe acetyl mannan milk fat globules, a preparation method thereof, and an application thereof. The preparation method of the present invention is simple to operate, does not use chemical reagents, does not destroy the original spatial structure of the polysaccharide milk fat globules, does not affect the biological activity of the polysaccharide and the experimental accuracy, and the prepared fluorescent labeled milk fat globules have a stable structure, double labeling of the polysaccharide-lipid core, strong visibility, good experimental accuracy, and are suitable for fluorescence microscopy observation of cells.

[0006] A preparation method of a fluorescent label for aloe acetyl mannan milk fat globules, comprising the following steps: (1) Mix aloe acetyl mannan with water, homogenize once to obtain an emulsion, mix the emulsion with silicone oil, homogenize twice, let stand, and take the lower layer to obtain aloe acetyl mannan milk fat globules; (2) Mix the aloe acetyl mannan milk fat globules obtained in step (1) with rhodamine DHPE to obtain substance A, and then mix with a fluorescently labeled lectin, and incubate in the dark to obtain a fluorescent label for aloe acetyl mannan milk fat globules.

[0007] Preferably, the volume ratio of the aloe acetyl mannan to water in step (1) is 1:2 - 10.

[0008] Preferably, the volume ratio of the emulsion to silicone oil in step (1) is 1:2 - 10.

[0009] Preferably, the first homogenization in step (1) is carried out at 5000 - 20000 r / min for 10 - 30 min.

[0010] Preferably, the second homogenization in step (1) is carried out at 5000 - 20000 r / min for 10 - 30 min.

[0011] Preferably, the standing time in step (1) is 10 - 60 min.

[0012] Preferably, the volume ratio of the aloe acetyl mannan milk fat globules to rhodamine B in step (2) is 50 - 200:1.

[0013] Preferably, the rhodamine DHPE in step (2) is prepared with DMSO and the concentration is 0.5 - 2.5 mg / mL.

[0014] Preferably, the volume ratio of the substance A to the fluorescently labeled lectin in step (2) is 100 - 800:1.

[0015] Preferably, the fluorescently labeled lectin in step (2) is prepared with PBS and the concentration is 5 - 40 mg / mL.

[0016] Preferably, the light - avoiding incubation in step (2) is 10 - 60 min.

[0017] Preferably, the aloe acetyl mannan milk fat globule fluorescent label in step (2) is mixed with the uncoagulated agar, cooled, to obtain the fixed aloe acetyl mannan milk fat globule fluorescent label, which is more convenient for observation.

[0018] Preferably, the volume ratio of the uncoagulated agar to the aloe acetyl mannan milk fat globules is 1 - 5:1.

[0019] Preferably, the concentration of the uncoagulated agar is 1.5% - 2.0% in mass - volume ratio.

[0020] Preferably, the temperature of the uncoagulated agar is 45℃ - 50℃.

[0021] The aloe acetyl mannan in step (1) can be prepared by the following method: Cut off both ends of fresh aloe leaves, soak in pure water for 24 - 48 h, wash with water, remove the epidermis, beat into pulp at 1450 r / min for 15 - 20 min to obtain an aloe polysaccharide liquid material, stir at a constant temperature of 4 - 15℃ for more than 24 h under light with an illuminance of not less than 800 lumens, and evaporate at a constant temperature of 75 - 95℃ to obtain an aloe polysaccharide extract rich in aloe acetyl mannan (Patent ZL202410397753.4, Method for promoting the formation of an aloe polysaccharide pervaporation membrane). The components of the aloe polysaccharide extract are detected to be aloe acetyl mannan, and the structural formula is shown as follows: .

[0022] After the incubation of the aloe acetyl mannan milk fat globule fluorescent marker of the present invention in the dark is completed, a washing step can be added to solve the problem of excessive dye and too high background fluorescence in some cases. The washing step is as follows: Add 1×PBS buffer in an equal volume, gently pipette and mix well, let it stand in the dark at room temperature for 10 - 20 min, and take the upper liquid, which is the aloe acetyl mannan milk fat globule fluorescent marker with reduced dye after washing.

[0023] An aloe acetyl mannan milk fat globule fluorescent marker is prepared by the above preparation method.

[0024] The application of the above aloe acetyl mannan milk fat globule fluorescent marker in the preparation of a cell fluorescent labeling reagent.

[0025] The application of the above aloe acetyl mannan milk fat globule fluorescent marker in the labeling of cell membranes for non-diagnostic and non-therapeutic purposes.

[0026] A method for non-diagnostic and non-therapeutic cell fluorescence imaging includes the following steps: co-culture cells with a culture medium containing the aloe acetyl mannan milk fat globule fluorescent marker, and then perform cell fluorescence imaging.

[0027] Preferably, the dosage of the aloe acetyl mannan milk fat globule fluorescent marker in the culture medium is 2.5% - 10% by volume.

[0028] Compared with the prior art, the present invention has the following beneficial effects: The preparation method of the aloe acetyl mannan milk fat globule fluorescent marker of the present invention is simple to operate, does not use chemical reagents, does not destroy the original spatial structure of the acetyl mannan milk fat globule, does not affect the biological activity of acetyl mannan and the experimental accuracy. The prepared fluorescent marker has a stable milk fat globule structure, double labeling of polysaccharide-lipid core, strong visibility, good experimental accuracy, and is suitable for cell fluorescence microscopy observation. Description of the Drawings

[0029] Figure 1 It is a micrograph of the aloe acetyl mannan milk fat globule R5 prepared in Example 1.

[0030] Figure 2 It is a fluorescence micrograph of the fixed aloe acetyl mannan milk fat globule fluorescent marker WGA-DOPH-R5 prepared in Example 1; among them, A is the fluorescence imaging diagram of R5 in the green fluorescence channel, and the green fluorescence labels the aloe acetyl mannan in R5; B is the fluorescence imaging diagram of R5 in the red fluorescence channel, and the red fluorescence labels the lipid core in R5; C is the overlapping imaging diagram of R5 in the red and green fluorescence channels.

[0031] Figure 3Fluorescence microscope photograph of the fixed aloe acetylmannan milk fat globule fluorescent marker WGA-DOPH-R5 prepared in Example 1 after being stored in the dark at 4 °C for 48 h; among them, A is the fluorescence imaging diagram of R5 under the green fluorescence channel, and the green fluorescence labels the aloe acetylmannan in R5; B is the fluorescence imaging diagram of R5 under the red fluorescence channel, and the red fluorescence labels the lipid core in R5; C is the overlapping imaging diagram of R5 under the red and green fluorescence channels.

[0032] Figure 4 Co-culture result diagram of rat hair follicle stem cells and the aloe acetylmannan milk fat globule fluorescent marker prepared in Example 1; among them, A is the fluorescence imaging diagram of R5 under the green fluorescence channel, and the green fluorescence labels the aloe acetylmannan in R5; B is the fluorescence imaging diagram of R5 under the red fluorescence channel, and the red fluorescence labels the lipid core in R5; C is the overlapping imaging diagram of R5 under the red and green fluorescence channels.

[0033] Figure 5 Fluorescence microscope photograph of the FITC-labeled aloe acetylmannan milk fat globule FITC-R5 prepared in Comparative Example 1; among them, A is the fluorescence imaging diagram of R5 labeled with FITC under the green fluorescence channel; B is the bright-field image corresponding to A; C is the fluorescence imaging diagram of R5 not labeled with FITC under the green fluorescence channel; D is the bright-field image corresponding to C.

[0034] Figure 6 Fluorescence microscope photograph of the fast green FCF-labeled aloe acetylmannan milk fat globule prepared in Comparative Example 2; among them, A is the fluorescence imaging diagram of R5 labeled with FCF under the green fluorescence channel in an environment of pH 2.3; B is the fluorescence imaging diagram of R5 labeled with FCF under the green fluorescence channel in an environment of pH 7.4; C is the fluorescence imaging diagram of R5 labeled with FCF under the green fluorescence channel in an environment of pH 8.5.

[0035] Figure 7 Fluorescence microscope photograph of the Nile Red-labeled aloe acetylmannan milk fat globule prepared in Comparative Example 3; among them, A is the fluorescence imaging diagram of R5 labeled with Nile Red under the red fluorescence channel; B is the bright-field image corresponding to A.

[0036] Figure 8 Fluorescence microscope photograph of the Nile Red-labeled aloe acetylmannan milk fat globule prepared in Comparative Example 3; A is the fluorescence imaging diagram of R5 at the observation time of 0 s, and B is the fluorescence imaging diagram of R5 when observed for 15 s.

[0037] Figure 9Fluorescence microscopy photograph of AF488-WGA-labeled aloe acetylmannan milk fat globules prepared for Comparative Example 4; wherein, A is the fluorescence imaging of R5 labeled with AF488-WGA under the green fluorescence channel; B is the bright field image corresponding to A.

[0038] Figure 10 Fluorescence microscopy photograph of rhodamine DHPE-labeled aloe acetylmannan milk fat globules prepared for Comparative Example 5; wherein, A is the fluorescence imaging of R5 labeled with rhodamine DHPE under the red fluorescence channel; B is the bright field image corresponding to A.

[0039] Figure 11 TMRE, ROS, and ATP detection results of R5, FITC-R5, and WGA-DOPH-R5; wherein, A is the ATP detection result; B is the TMRE detection result; C is the ROS detection result.

[0040] Figure 12 Fluorescence microscopy images after co-culturing FITC-aloe acetylmannan and FITC-R5 with cells; wherein, A is the fluorescence microscopy image after co-culturing FITC-aloe acetylmannan with cells; B is the fluorescence microscopy image after co-culturing FITC-R5 with cells. Detailed implementation mode

[0041] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are not specified otherwise. For the experimental methods without specific conditions indicated in the embodiments, they are usually carried out under conventional conditions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without specific instructions.

[0042] Example 1 (1) Preparation of aloe acetylmannan: Both ends of fresh aloe leaves were removed, soaked in pure water for 24 h, washed with water, the epidermis was removed, and pulped at 1450 r / min for 20 min to obtain an aloe polysaccharide liquid. It was stirred at a constant temperature for 28 h under light with an illuminance of 800 lumens, and evaporated at a constant temperature of 80 °C to obtain an upper film-like substance, that is, enriched aloe polysaccharide (ABPA2). After component detection, the main component of aloe polysaccharide (ABPA2) is acetylmannan, and the structural formula is shown as follows: .

[0043] (2) Preparation of aloe acetyl mannan milk fat globules: Mix the aloe acetyl mannan prepared in step (1) with water at a volume ratio of 1:5, and perform high-shear homogenization at 10,000 r / min for 20 min until the two are fully fused to obtain an emulsion. Mix the emulsion with silicone oil (CAS: 63148-62-9) at a volume ratio of 1:4, and perform high-shear homogenization at 10,000 r / min for 20 min until the emulsion and silicone oil are fully mixed. Let it stand for 30 min, and remove the upper layer of silicone oil to obtain the lower-layer aloe acetyl mannan milk fat globules (R5). As observed under a microscope as Figure 1 shown, the milk fat globule structure can be clearly shown, including the outer polysaccharide and the internal lipid.

[0044] (3) Preparation of fluorescently labeled aloe acetyl mannan milk fat globules: Use rhodamine DHPE (prepared in DMSO, concentration 1 mg / mL) to fluorescently label the lipids in the aloe acetyl mannan milk fat globules, and mix the milk fat globules and rhodamine DHPE at a volume ratio of 100:1; then use Alexa Fluor 488-labeled wheat germ agglutinin AF488-WGA (prepared in 1×PBS, concentration 10 mg / mL) to label aloe acetyl mannan, and the volume ratio of aloe acetyl mannan milk fat globules to AF488-WGA is 200:1, and mix well; incubate in the dark at room temperature for 30 min to obtain fluorescently labeled aloe acetyl mannan milk fat globules (WGA-DOPH-R5).

[0045] (4) The fluorescently labeled aloe acetyl mannan milk fat globules are an emulsion and are in a flowing state under a microscope. They can be fixed with agar for easier observation. The specific method is as follows: Take 100 μL of the fluorescently labeled aloe acetyl mannan milk fat globules and mix them with 200 μL of agar (mass-volume ratio 2.0%) that has been completely melted by boiling and cooled to 45 °C. Quickly take 20 μL of the mixture and drop it onto a glass slide, cover it with a coverslip, gently press it, and then let it cool naturally to fix it to obtain fixed fluorescently labeled aloe acetyl mannan milk fat globules. As observed under a fluorescence microscope as Figure 2 shown, where A is the fluorescence imaging diagram of R5 in the green fluorescence channel, and the green fluorescence labels the aloe acetyl mannan in R5; B is the fluorescence imaging diagram of R5 in the red fluorescence channel, and the red fluorescence labels the lipid core in R5; C is the overlapping imaging diagram of R5 in the red and green fluorescence channels. The fluorescently labeled milk fat globules still have a clear structure after being stored in the dark at 4 °C for 48 h, as Figure 3 shown.

[0046] Example 2 (1) Preparation of aloe acetyl mannan: Remove both ends of fresh aloe leaves, soak in pure water for 24 h, wash with water, remove the epidermis, beat into pulp at 1450 r / min for 20 min to obtain aloe polysaccharide liquid, stir at a constant temperature for 28 h under light with an illuminance of 800 lumens, and evaporate at a constant temperature of 80 °C to obtain the upper film-like substance, namely aloe acetyl mannan.

[0047] (2) Preparation of aloe acetyl mannan milk fat globules: Mix the aloe acetyl mannan prepared in step (1) with water at a volume ratio of 1:2, and perform high-shear homogenization at 20000 r / min for 10 min until the two are fully fused to obtain an emulsion. Mix the emulsion with silicone oil (CAS: 63148-62-9) at a volume ratio of 1:2, and perform high-shear homogenization at 20000 r / min for 10 min until the emulsion and silicone oil are fully mixed. Let it stand for 60 min, remove the upper silicone oil, and obtain the lower-layer aloe acetyl mannan milk fat globules (R5). Observe under a microscope as Figure 1 .

[0048] (3) Preparation of fluorescently labeled aloe acetyl mannan milk fat globules: Use rhodamine DHPE (configured with DMSO, concentration 2 mg / mL) to fluorescently label the lipids in aloe acetyl mannan milk fat globules, and mix the milk fat globules and rhodamine DHPE at a volume ratio of 200:1; then use Alexa Fluor 488-labeled wheat germ agglutinin AF488-WGA (configured with 1×PBS, concentration 30 mg / mL) to label aloe acetyl mannan, and the volume ratio of aloe acetyl mannan milk fat globules to AF488-WGA is 600:1, and mix well; incubate in the dark at room temperature for 60 min to obtain fluorescently labeled aloe acetyl mannan milk fat globules (WGA-DOPH-R5).

[0049] (4) The fluorescently labeled aloe acetyl mannan milk fat globules are fixed with agar, and the specific method is the same as that in Example 1 to obtain fixed fluorescently labeled aloe acetyl mannan milk fat globules. Observe under a fluorescence microscope as Figure 2 and Figure 3 .

[0050] Example 3 (1) Preparation of aloe acetyl mannan: Remove both ends of fresh aloe leaves, soak in pure water for 24 h, wash with water, remove the epidermis, beat into pulp at 1450 r / min for 20 min to obtain aloe polysaccharide liquid, stir at a constant temperature for 28 h under light with an illuminance of 800 lumens, and evaporate at a constant temperature of 80 °C to obtain the upper film-like substance, namely aloe acetyl mannan.

[0051] (2) Preparation of aloe acetyl mannan milk fat globules: Mix the aloe acetyl mannan prepared in step (1) with water at a volume ratio of 1:10, shear and homogenize at 5000 r / min for 30 min until the two are fully fused to obtain an emulsion. Mix the emulsion with silicone oil (CAS: 63148-62-9) at a volume ratio of 1:10, shear and homogenize at 5000 r / min for 30 min until the emulsion and silicone oil are fully mixed, let stand for 10 min, remove the upper layer of silicone oil, and obtain the lower layer of aloe acetyl mannan milk fat globules (R5). Observe under a microscope as follows Figure 1 .

[0052] (3) Preparation of fluorescently labeled aloe acetyl mannan milk fat globules: Use rhodamine DHPE (prepared in DMSO, concentration 0.5 mg / mL) to fluorescently label the lipids in the aloe acetyl mannan milk fat globules, and mix the milk fat globules and rhodamine DHPE at a volume ratio of 200:1; then use Alexa Fluor 488-labeled wheat germ agglutinin AF488-WGA (prepared in 1×PBS, concentration 5 mg / mL) to label aloe acetyl mannan, and the volume ratio of aloe acetyl mannan milk fat globules to AF488-WGA is 100:1, and mix well; incubate in the dark at room temperature for 20 min to obtain fluorescently labeled aloe acetyl mannan milk fat globules (WGA-DOPH-R5).

[0053] (4) The fluorescently labeled aloe acetyl mannan milk fat globules are fixed with agar, and the specific method is the same as that in Example 1 to obtain fixed fluorescently labeled aloe acetyl mannan milk fat globules. Observe under a fluorescence microscope as follows Figure 2 and Figure 3 .

[0054] Application Example Seed 150,000 rat hair follicle stem cells HFSC (Saibakang, product number iCell-0098a) per well in a 12-well plate and culture overnight at 37°C; dilute the fluorescently labeled aloe acetyl mannan milk fat globules prepared in Example 1 50-fold with a special cell culture medium for hair follicle stem cells (Saibakang, product number iCell-0098a-001b), replace the cell culture medium of rat hair follicle stem cells HFSC (the concentration of the label in the culture medium is 5% by volume), co-culture at 37°C for 12 h, remove the upper layer of the culture medium, and wash the cells 2 times with pre-warmed 1×PBS at 37°C; add 500 μL of 1×PBS, and then observe under a fluorescence microscope. The fluorescence signal of the fluorescently labeled aloe acetyl mannan milk fat globules appears inside the cells and is marked with a yellow arrow, as shown in Figure 4 (The application effects of Example 2 and Example 3 are the same, and the attached drawings are not provided repeatedly).

[0055] Comparative Example 1 Labeling of the glycoprotein component in FITC-labeled aloe acetyl mannan milk fat globules (1) The isothiocyanate group can react with the amino terminus or primary amine of glycoprotein. Aloe acetyl mannan milk fat globules cannot be labeled using the traditional chemical synthesis labeling method of fluorescein isothiocyanate ester (FITC) (the reaction process will destroy the structure of milk fat globules). Therefore, in this example, only FITC (CAS: 3326-32-7) was directly added to the aloe acetyl mannan milk fat globules prepared in step (2) of the example, and mixed evenly to label the aloe acetyl mannan milk fat globules, obtaining a light yellow liquid. The labeling results are as Figure 5 shown. A small amount of milk fat globules were specifically labeled, but the vast majority of milk fat globules had no positive label. It was found during actual use that compared with the aloe acetyl mannan milk fat globules (R5) prepared in step (2) of Example 1, the stability of the FITC-labeled aloe acetyl mannan milk fat globules decreased, and oil-water separation occurred more easily.

[0056] (2) Additionally, try to use the aloe acetyl mannan after FITC labeling to prepare milk fat globules through the steps of Example (2) to achieve the purpose of glycoprotein component (FITC-R5) in the FITC-labeled aloe acetyl mannan milk fat globules. The specific preparation method of FITC-labeled aloe acetyl mannan (FITC-aloe acetyl mannan) is as follows: 1) Prepare phosphate buffer (pH 8.0). First, prepare two stock solutions of 0.2 M Na2HPO4 (disodium hydrogen phosphate) and 0.2 M NaH2PO4 (sodium dihydrogen phosphate); 0.2 M Na2HPO4: Take 71.6 g of Na2HPO4·12H2O, dissolve it in deionized water, and add water to 1000 mL; 0.2 M NaH2PO4: Take 31.2 g of NaH2PO4·2H2O, dissolve it in deionized water, and add water to 1000 mL; Mix 5.3 mL of 0.2 M NaH2PO4 and 94.7 mL of 0.2 M Na2HPO4 to obtain a phosphate buffer with pH 8.0; 2) Dissolve 400 mg of aloe acetyl mannan in 15 mL of the phosphate buffer (pH 8.0) prepared in step 1), add 400 mg of tyramine, and react for 24 h. Add 150 mg of sodium cyanoborohydride, react at 37 °C for 96 h, shake intermittently, centrifuge to remove the supernatant, and obtain ammoniated aloe acetyl mannan. Dissolve 200 mg of the ammoniated aloe acetyl mannan in water, adjust the pH to 8.0 with 0.5 mol / L NaHCO3, add 50 mg of FITC, stir and react in the dark at room temperature for 24 h, filter the reaction solution, add absolute ethanol to the filtrate until the final concentration of ethanol is 75% (V / V), and a large amount of bright yellow-green precipitate will precipitate. Centrifuge to discard the supernatant, dissolve the precipitate in water, precipitate with ethanol again, repeat 3 times under the same conditions, and obtain a bright yellow precipitate. Dissolve the obtained bright yellow precipitate in a small amount of water, and further purify it through a 10KD ultrafiltration column until the filtrate basically does not contain FITC to obtain FITC-labeled aloe acetyl mannan.

[0057] Comparative Example 2 Fast Green FCF-labeled glycoprotein component in aloe acetyl mannan milk fat globules Fast Green FCF is an acidic fluorescent dye with hydrophilic and lipophilic groups. It can bind to basic components in cells or tissues, including DNA, RNA, basic proteins, and acidic lipids. Utilizing the amphoteric nature (acid-base property) of proteins and their ability to form salts with acidic and basic dye ions, at a pH of around 2.2, i.e., below the isoelectric point of all proteins, all proteins can be stained by Fast Green. At a pH of around 8.5, basic proteins are stained by Fast Green. The inventors tried different pH environments. They dissolved 0.1% Fast Green (CAS 2353-45-9) in 1×PBS, measured the pH value with a pH meter, and adjusted the pH value to 2.3, 7.4, and 8.5 respectively using 0.1 mol / L hydrochloric acid and 0.1 mol / L sodium hydroxide. Then, an equal volume of the Fast Green dye was added to the aloe acetylmannan milk fat globules prepared in step (2) of Example 1, and they were mixed evenly for 2 h to label the aloe acetylmannan milk fat globules. The results are as Figure 6 shown. The aloe acetylmannan milk fat globules were not labeled, and the flocculent substances in the figure are residual aloe residues. Therefore, Fast Green is not suitable for labeling aloe acetylmannan milk fat globules.

[0058] Comparative Example 3: Labeling of neutral lipid components of aloe acetylmannan milk fat globules with Nile Red As an ideal fluorescent dye for staining lipids, Nile Red is mainly used to show fatty degeneration of tissues and organs, abnormal deposition of lipids, and the study of the directional differentiation of stem cells into adipocytes. The inventors used Nile Red to label the neutral lipid components of aloe acetylmannan milk fat globules. The specific labeling process is as follows: The powder of Nile Red (Macklin, product number: N861501) was dissolved in DMSO at a concentration of 1 mM / mL. The Nile Red dye was added to the aloe acetylmannan milk fat globules R5 prepared in step (2) of Example 1 at a volume ratio of 200:1, and after mixing evenly, they were reacted in the dark for 30 min to label the aloe acetylmannan milk fat globules.

[0059] The results are as Figure 7 shown. The lipid core of R5 was marked in red. The edge of the R5 sphere was clear, the orange-red fluorescence was bright, and the background was clean, indicating that the labeling effect of Nile Red was good. However, the fluorescence quenching of Nile Red-labeled was relatively fast. As Figure 8 shown, where A is the fluorescence imaging diagram of R5 at the observation time of 0 s, and B is the fluorescence of R5 observed until 15 s.

[0060] Comparative Example 4: Labeling of polysaccharide and glycoprotein components in aloe acetylmannan milk fat globules with AF488-WGA Take the aloe acetyl mannan milk fat globule R5 prepared in step (2) of Example 1, and use Alexa Fluor 488-labeled wheat germ agglutinin AF488-WGA (prepared with 1×PBS, concentration 10 mg / mL) to label the aloe acetyl mannan. The volume ratio of the aloe acetyl mannan milk fat globule to AF488-WGA is 200:1. Mix well and incubate in the dark at room temperature for 30 min. The results are as Figure 9 shown. The spherical structure of R5 is clear and complete, and the outer structure of the polysaccharide is clear, but the internal components and structure of R5 cannot be revealed.

[0061] Comparative Example 5 Rhodamine DHPE labels the polar lipid components in aloe acetyl mannan milk fat globules Take the aloe acetyl mannan milk fat globule R5 prepared in step (2) of Example 1, and use rhodamine DHPE (prepared with DMSO, concentration 1 mg / mL) to fluorescently label the lipids in the aloe acetyl mannan milk fat globule. Mix the milk fat globule and rhodamine DHPE with a volume ratio of 100:1, and incubate in the dark at room temperature for 30 min. The labeling results are as Figure 10 shown. The spherical structure of R5 is clear and complete, the lipid core is complete, the fluorescence is bright, and the background is clean, but the distribution of aloe acetyl mannan in R5 cannot be reflected, and the multi-layer milk fat globule structure of R5 cannot be fully reflected.

[0062] Co-culture the aloe acetyl mannan milk fat globule R5 prepared in step (2) of Example 1, the FITC-labeled aloe acetyl mannan milk fat globule (FITC-R5) prepared in Comparative Example 1, the fluorescent label of the aloe acetyl mannan milk fat globule prepared in Example 1 (WGA-DOPH-R5), and the control group (blank control group, without adding R5) with hair follicle stem cells HFSC (Cellapy, product number iCell-0098a)) for 24 h, collect the cells, and detect cell ATP, mitochondrial membrane potential TMRE and reactive oxygen species ROS. For ATP detection, use an ATP detection kit (Beyotime, product number S0026); for ROS detection, use a reactive oxygen species detection kit (Solarbio, product number CA1410); for TMRE detection, use a mitochondrial membrane potential detection kit (Beyotime, product number C2001S).

[0063] The results are as Figure 11 shown. Compared with the control group, both the R5 group and the WGA-DOPH-R5 group can significantly increase cell ATP, TMRE and ROS; compared with the R5 group, the ATP and TMRE of the FITC-R5 group decreased (with statistical differences), and the ROS increased slightly (without statistical differences), indicating that the biological activity of the aloe acetyl mannan after FITC labeling was damaged.

[0064] The rat hair follicle stem cells HFSC (Cellapy, product number iCell-0098a) were seeded at 150,000 cells per well in a 12-well plate and cultured overnight at 37°C; the FITC-labeled aloe acetyl mannan and FITC-labeled aloe acetyl mannan milk fat globule (FITC-R5) prepared in Comparative Example 1 were each diluted 20-fold with the special cell culture medium for hair follicle stem cells (Cellapy, product number iCell-0098a-001b), and then the cell culture media of rat hair follicle stem cells HFSC were replaced respectively (the concentration of the label in the culture medium was 5% by volume), and co-cultured at 37°C for 12 h. The upper layer of the culture medium was removed, and the cells were washed 2 times with pre-warmed 1×PBS at 37°C; 500 μL of 1×PBS was added, and then the cells were observed using a fluorescence microscope. The results showed that the FITC-labeled aloe acetyl mannan could be observed under the fluorescence microscope ( Figure 12 indicated by the white arrow in A), but after co-culturing FITC-R5 with the cells, the target was lost under the fluorescence microscope ( Figure 12 B).

[0065] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Preparation method of aloe acetyl mannan milk fat globule fluorescent marker, characterized in that, It includes the following steps: (1) Mix aloe acetyl mannan with water and homogenize it once to obtain an emulsion. Mix the emulsion with silicone oil, homogenize it twice, let it stand, and take the lower layer to obtain aloe acetyl mannan milk fat globules; (2) Mix the aloe acetyl mannan milk fat globules described in step (1) with rhodamine DHPE evenly to obtain substance A, and then mix it with fluorescently labeled lectin and incubate it in the dark to obtain aloe acetyl mannan milk fat globule fluorescent label.

2. The preparation method of the aloe acetyl mannan milk fat globule fluorescence marker according to claim 1, characterized in that, It includes at least one of the following items (a)-(f): (a) The volume ratio of the aloe acetyl mannan described in step (1) to water is 1:2-10; (b) The volume ratio of the emulsion described in step (1) to silicone oil is 1:2-1; (c) The volume ratio of the aloe acetyl mannan milk fat globules described in step (2) to rhodamine B is 50-200:1; (d) The rhodamine DHPE described in step (2) is prepared with DMSO, and the concentration is 0.5-2.5 mg / mL; (e) The volume ratio of substance A described in step (2) to fluorescently labeled lectin is 100-800:1; (f) The fluorescently labeled lectin described in step (2) is prepared with PBS, and the concentration is 5-40 mg / mL.

3. The preparation method of the aloe acetyl mannan milk fat globule fluorescence marker according to claim 1, wherein, The aloe acetyl mannan described in step (1) can be prepared by the following method: Remove the two ends of fresh aloe leaves, soak them in pure water for 24-48 h, wash them with water, remove the epidermis, beat them into pulp at 1450 r / min for 15-20 min to obtain aloe polysaccharide liquid, stir at a constant temperature of 4-15 °C for more than 24 h under light with an illuminance of not less than 800 lumens, and evaporate at a constant temperature of 75-95 °C to obtain aloe polysaccharide extract rich in aloe acetyl mannan.

4. The preparation method of the aloe acetyl mannan milk fat globule fluorescent marker according to claim 1, characterized in that, Mix the aloe acetyl mannan milk fat globule fluorescent label described in step (2) with uncoagulated agar, cool it to obtain a fixed aloe acetyl mannan milk fat globule fluorescent label, which is more convenient for observation.

5. The preparation method of the aloe acetyl mannan milk fat globule fluorescent marker according to claim 4, wherein, It includes at least one of the following items (a)-(c): (a) The volume ratio of the uncoagulated agar to the aloe acetyl mannan milk fat globules is 1-5:1; (b) The concentration of the uncoagulated agar is 1.5%-2.0% in mass-volume ratio; (c) The temperature of the uncoagulated agar is 45 °C-50 °C.

6. A kind of aloe acetyl mannan milk fat globule fluorescent label, which is prepared by the preparation method described in any one of claims 1-5.

7. Application of the aloe acetyl mannan milk fat globule fluorescent label described in claim 6 in the preparation of cell fluorescent labeling reagent.

8. Application of the aloe acetyl mannan milk fat globule fluorescent label described in claim 6 in the labeling of cell membrane for non-diagnostic and non-therapeutic purposes.

9. A method for cell fluorescence imaging for non-diagnostic and non-therapeutic purposes, characterized in that, It includes the following steps: Co-culture cells with a culture medium containing the aloe acetyl mannan milk fat globule fluorescent label described in claim 6, and then perform cell fluorescence imaging.

10. The method for cell fluorescence imaging for non-diagnostic and non-therapeutic purposes according to claim 9, wherein The dosage of the aloe acetyl mannan milk fat globule fluorescent label in the culture medium is 2.5%-10% in volume ratio.

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