Aloe acemannan milk fat globule fluorescent marker and its preparation method and application

Abstract

The present invention provides an aloe acemannan milk fat globule fluorescent marker, a preparation method thereof, and an application thereof. The aloe acemannan milk fat globule fluorescent marker of the present invention is prepared by mixing aloe acemannan with water, homogenizing to obtain an emulsion, and then mixing with silicone oil, and homogenizing for a second time to obtain aloe acemannan milk fat globule; the aloe acemannan milk fat globule is sequentially labeled with rhodamine DHPE and a fluorescently labeled lectin, and incubated in the dark to obtain. The above-mentioned marker preparation method is simple to operate, does not use chemical reagents, does not destroy the original spatial structure of the acemannan milk fat globule, does not affect the biological activity of acemannan and the experimental accuracy, and the prepared fluorescent marker milk fat globule has a stable structure, a polysaccharide-lipid core dual labeling, strong visibility, and good experimental accuracy. It is suitable for preparing cell fluorescent labeling reagents and cell membrane markers for non-diagnostic and non-therapeutic purposes.

Description

Technical Field

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

[0002] Aloe polysaccharides are macromolecular compounds composed of multiple monosaccharide molecules of the same or different structures linked by glycosidic bonds. They are primarily found in the gel of fresh aloe leaves. Due to their ability to be taken up by cells and trigger specific physiological responses, coupled with their high safety and low toxicity, aloe polysaccharides have attracted considerable attention in the biomedical field. Among them, aloe acetomannan, as the main component of aloe polysaccharides, possesses multiple biological activities, including anti-inflammatory, antioxidant, and tissue repair. Furthermore, aloe acetomannan, with its complex spatial structure and water- and oil-amphipatic properties, is a natural macromolecular active polymer material that can be prepared to form milk fat globule structures, showing 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 track their biological activity and study their molecular mechanisms. However, radioactive isotopes have adverse effects on organisms and are not suitable for biological research. For this reason, polysaccharide fluorescent labeling technology has emerged. This technology is based on the characteristics of polysaccharides with multiple reducing ends, and fluorescent substances are attached to sugar chains through chemical synthesis. In the specific implementation process, the polysaccharide must first be activated to introduce reactive groups. Commonly used activators include hydrogen bromide, isocyanate, etc.; then, under suitable conditions, the polysaccharide is mixed with a fluorescent dye and a catalyst to react so that the fluorescent dye forms a chemical bond with the amino, carboxyl, hydroxyl and other groups of the polysaccharide's reducing end; 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, thereby purifying the labeled polysaccharide. However, the above-mentioned polysaccharide fluorescent labeling process is time-consuming and complex, and the purified fluorescent labeled polysaccharide has problems such as chemical reagent residues and reduced polysaccharide biological activity, which affects the accuracy of cell biology and biochemistry experiments.

[0004] Additionally, fluorescein isothiocyanate (FITC) chemical labeling is a common method for fluorescently labeling mannans, but few studies have examined whether this method is also applicable to aloe acemannan milk fat globules. Literature indicates that aloe acemannan has a large molecular weight, is rich in acetyl groups, and possesses a complex spatial conformation, making it difficult to expose its reducing end, resulting in a low fluorescence labeling rate. In practice, researchers have found that compared with unlabeled aloe acemannan, FITC-labeled aloe acemannan exhibits reduced emulsion stability after preparation into milk fat globules. Furthermore, when co-cultured with cells, the milk fat globules lose fluorescence, and the tracing effect fails to meet experimental requirements. Therefore, developing novel fluorescent labeling methods for aloe acemannan is of great significance. Summary of the Invention

[0005] To address the above-mentioned issues, the present invention provides an aloe acemannan milk fat globule fluorescent marker, a preparation method, and applications thereof. The preparation method is simple to operate, does not use chemical reagents, does not destroy the original spatial structure of the polysaccharide milk fat globules, and does not affect the polysaccharide's biological activity or experimental accuracy. The resulting fluorescently labeled milk fat globules have a stable structure, dual polysaccharide-lipid core labeling, strong visibility, and good experimental accuracy, making them suitable for cell fluorescence microscopy observation.

[0006] A method for preparing an aloe acemannan milk fat globule fluorescent marker comprises the following steps:

[0007] (1) Aloe acemannan is mixed with water and homogenized once to obtain an emulsion, the emulsion is mixed with silicone oil, homogenized a second time, allowed to stand, and the lower layer is removed to obtain aloe acemannan milk fat globules;

[0008] (2) The aloe acetyl mannan milk fat globules described in step (1) are mixed with rhodamine DHPE to obtain substance A, and then mixed with fluorescently labeled lectin, and incubated in the dark to obtain aloe acetyl mannan milk fat globules fluorescent marker.

[0009] Preferably, the volume ratio of the aloe acetomannan to water in step (1) is 1:2-10.

[0010] Preferably, the volume ratio of the emulsion to the silicone oil in step (1) is 1:2-10.

[0011] Preferably, the primary homogenization in step (1) is performed at 5000-20000 r / min for 10-30 min.

[0012] Preferably, the secondary homogenization in step (1) is 5000-20000 r / min for 10-30 min.

[0013] Preferably, the standing time in step (1) is 10-60 min.

[0014] Preferably, the volume ratio of the aloe acetyl mannan milk fat globules to rhodamine B in step (2) is 50-200:1.

[0015] Preferably, the rhodamine DHPE in step (2) is prepared using DMSO at a concentration of 0.5-2.5 mg / mL.

[0016] Preferably, the volume ratio of the substance A to the fluorescently labeled lectin in step (2) is 100-800:1.

[0017] Preferably, the fluorescently labeled lectin in step (2) is prepared in PBS at a concentration of 5-40 mg / mL.

[0018] Preferably, the dark-proof incubation in step (2) is 10-60 min.

[0019] Preferably, the aloe acetyl mannan milk fat globule fluorescent marker in step (2) is mixed with unsolidified agar and cooled to obtain fixed aloe acetyl mannan milk fat globule fluorescent marker, which is more convenient for observation.

[0020] Preferably, the volume ratio of the unsolidified agar to the aloe acemannan milk fat globules is 1-5:1.

[0021] Preferably, the concentration of the unsolidified agar is 1.5%-2.0% by mass volume.

[0022] Preferably, the temperature of the unsolidified agar is 45°C-50°C.

[0023] The aloe acetyl mannan described in step (1) can be prepared by the following method: remove both ends of fresh aloe leaves, soak in pure water for 24-48 hours, wash with water, remove the epidermis, and beat at 1450 r / min for 15-20 minutes to obtain an aloe polysaccharide liquid, stir at a constant temperature of 4-15°C for more than 24 hours under light with an illumination of not less than 800 lumens, and evaporate at a constant temperature of 75-95°C to obtain an aloe polysaccharide extract rich in aloe acetyl mannan (Patent ZL202410397753.4, a method for promoting aloe polysaccharide to form a pervaporation membrane). The components of the aloe polysaccharide extract were tested and found to be aloe acetyl mannan, with the structural formula shown below:

[0024] .

[0025] The aloe acemannan milk fat globule fluorescent marker of the present invention can be subjected to an additional washing step after the dark-proof incubation to solve the problem of excessive dye and high background fluorescence in some cases. The washing step is as follows: an equal volume of 1×PBS buffer is added, and the mixture is gently pipetted and mixed using a pipette. The mixture is allowed to stand at room temperature in the dark for 10-20 minutes, and the upper layer of liquid is collected, which is the aloe acemannan milk fat globule fluorescent marker with reduced dye after washing.

[0026] An aloe acemannan milk fat globule fluorescent marker is prepared by the above preparation method.

[0027] Application of the aloe acemannan milk fat globule fluorescent marker in the preparation of cell fluorescent labeling reagents.

[0028] Application of the aloe acemannan milk fat globule fluorescent marker in cell membrane labeling for non-diagnostic and non-therapeutic purposes.

[0029] A method for cell fluorescence imaging for non-diagnostic and non-therapeutic purposes comprises the following steps: co-culturing cells with a culture medium containing the aloe acemannan milk fat globule fluorescent marker, and then performing cell fluorescence imaging.

[0030] Preferably, the amount of the aloe acemannan milk fat globule fluorescent marker in the culture medium is 2.5%-10% by volume.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The method for preparing the aloe acemannan 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 acemannan milk fat globule, does not affect the biological activity of the acemannan and the experimental accuracy, and the prepared fluorescent marker milk fat globule has a stable structure, a polysaccharide-lipid core dual labeling, strong visibility, good experimental accuracy, and is suitable for cell fluorescence microscopy observation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a microscope photograph of the aloe acemannan milk fat globule R5 prepared in Example 1.

[0034] Figure 2 These are fluorescence microscope photos of the fixed aloe acetomannan milk fat globule fluorescent marker WGA-DOPH-R5 prepared in Example 1; wherein A is a fluorescence imaging image of R5 under the green fluorescence channel, with green fluorescence marking the aloe acetomannan in R5; B is a fluorescence imaging image of R5 under the red fluorescence channel, with red fluorescence marking the lipid core in R5; C is an overlapping imaging image of R5 under the red and green fluorescence channels.

[0035] Figure 3These are fluorescence microscope photographs of the fixed aloe acetomannan milk fat globule fluorescent marker WGA-DOPH-R5 prepared in Example 1 after storage at 4°C in the dark for 48 h; wherein A is a fluorescence imaging image of R5 in the green fluorescence channel, with green fluorescence marking the aloe acetomannan in R5; B is a fluorescence imaging image of R5 in the red fluorescence channel, with red fluorescence marking the lipid core in R5; and C is an overlapping imaging image of R5 in the red and green fluorescence channels.

[0036] Figure 4 Figures 1 and 2 show the co-culture results of rat hair follicle stem cells and the aloe acemannan milk fat globule fluorescent marker prepared in Example 1; Figure A is a fluorescence imaging image of R5 in the green fluorescence channel, where green fluorescence marks the aloe acemannan in R5; Figure B is a fluorescence imaging image of R5 in the red fluorescence channel, where red fluorescence marks the lipid core in R5; and Figure C is an overlapping imaging image of R5 in the red and green fluorescence channels.

[0037] Figure 5 These are fluorescence microscope photos of FITC-labeled aloe acemannan milk fat globules FITC-R5 prepared in Comparative Example 1; wherein A is the fluorescence imaging of FITC-labeled R5 under the green fluorescence channel; B is the bright field image corresponding to A; C is the fluorescence imaging of FITC-unlabeled R5 under the green fluorescence channel; D is the bright field image corresponding to C.

[0038] Figure 6 These are fluorescence microscope photos of the Fast Green FCF-labeled aloe acemannan milk fat globules prepared in Comparative Example 2; wherein, A is the fluorescence imaging of FCF-labeled R5 under the green fluorescence channel at pH 2.3; B is the fluorescence imaging of FCF-labeled R5 under the green fluorescence channel at pH 7.4; C is the fluorescence imaging of FCF-labeled R5 under the green fluorescence channel at pH 8.5.

[0039] Figure 7 These are fluorescence microscope photos of Nile-Red labeled aloe acemannan milk fat globules prepared in Comparative Example 3; wherein A is the fluorescence imaging of R5 labeled with Nile-Red under the red fluorescence channel; and B is the bright field image corresponding to A.

[0040] Figure 8 These are fluorescence microscope photos of Nile-Red labeled aloe acemannan milk fat globules prepared in Comparative Example 3; A is the fluorescence imaging of R5 when the observation time is 0s, and B is the fluorescence imaging of R5 when the observation time is 15s.

[0041] Figure 9These are fluorescence microscope photos of AF488-WGA-labeled aloe acemannan milk fat globules prepared in Comparative Example 4; wherein, A is the fluorescence imaging of R5 labeled with AF488-WGA under the green fluorescence channel; and B is the bright field image corresponding to A.

[0042] Figure 10 These are fluorescence microscope photos of rhodamine DHPE-labeled aloe acetylmannan milk fat globules prepared in Comparative Example 5; wherein, A is the fluorescence imaging of R5 labeled with rhodamine DHPE under the red fluorescence channel; and B is the bright field image corresponding to A.

[0043] Figure 11 Figure 3 is the TMRE, ROS, and ATP detection results of R5, FITC-R5, and WGA-DOPH-R5; A is the ATP detection result; B is the TMRE detection result; and C is the ROS detection result.

[0044] Figure 12 Figures 1 and 2 are fluorescence microscopy images of FITC-aloe acemannan and FITC-R5 after co-culture with cells; Figure 2 is a fluorescence microscopy image of FITC-aloe acemannan and cells after co-culture; Figure 2 is a fluorescence microscopy image of FITC-R5 and cells after co-culture. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0046] Example 1

[0047] (1) Preparation of Aloe Acetylmannan: Remove the ends of fresh aloe leaves, soak in pure water for 24 hours, wash with water, remove the epidermis, and beat at 1450 r / min for 20 minutes to obtain aloe polysaccharide liquid. Stir at a constant temperature for 28 hours under a light intensity of 800 lumens, and evaporate at a constant temperature of 80°C to obtain the upper film, which is the enriched aloe polysaccharide (ABPA2). After component analysis, the main component of aloe polysaccharide (ABPA2) is acetylmannan, and the structural formula is as follows:

[0048] .

[0049] (2) Preparation of aloe acetyl mannan milk fat globules: The aloe acetyl mannan prepared in step (1) was mixed with water in a volume ratio of 1:5, and high shear homogenization was performed at 10,000 r / min for 20 min until the two were fully fused to obtain an emulsion. The emulsion was mixed with silicone oil (CAS: 63148-62-9) in a volume ratio of 1:4, and high shear homogenization was performed at 10,000 r / min for 20 min until the emulsion and silicone oil were fully mixed. The mixture was allowed to stand for 30 min, and the upper layer of silicone oil was removed to obtain the aloe acetyl mannan milk fat globules (R5) in the lower layer. The emulsion was observed under a microscope as follows: Figure 1 As shown, the structure of milk fat globules can be clearly seen, including the outer polysaccharide layer and the inner lipids.

[0050] (3) Preparation of aloe acetyl mannan milk fat globule fluorescent marker: Use rhodamine DHPE (DMSO preparation, concentration 1 mg / mL) to fluorescently label the lipids in aloe acetyl mannan milk fat globule, and mix the milk fat globule and rhodamine DHPE at a volume ratio of 100:1; then use Alexa Fluor 488-labeled wheat germ agglutinin AF488-WGA (1×PBS preparation, concentration 10 mg / mL) to label aloe acetyl mannan, and the volume ratio of aloe acetyl mannan milk fat globule and AF488-WGA is 200:1, and mix; incubate at room temperature in the dark for 30 min to obtain aloe acetyl mannan milk fat globule fluorescent marker (WGA-DOPH-R5).

[0051] (4) The aloe acetyl mannan milk fat globule fluorescent marker is an emulsion and is in a flowing state under a microscope. It can be fixed with agar for easier observation. The specific method is as follows: take 100 μL of aloe acetyl mannan milk fat globule fluorescent marker and mix it with 200 μL of agar (mass volume ratio 2.0%) that is completely boiled and melted and then cooled to 45°C. Quickly take 20 μL of the mixture and drop it onto a glass slide. Cover it with a coverslip, press it lightly, and then cool it naturally to fix it. The fixed aloe acetyl mannan milk fat globule fluorescent marker is obtained. Observe it under a fluorescence microscope. Figure 2 As shown, A is the fluorescence imaging image of R5 under the green fluorescence channel, green fluorescence labels aloe acemannan in R5; B is the fluorescence imaging image of R5 under the red fluorescence channel, red fluorescence labels the lipid core in R5; C is the overlapping imaging image of R5 under the red and green fluorescence channels. The milk fat globule fluorescent marker still has a clear structure after being stored in the dark at 4℃ for 48 hours, as shown in Figure 2. Figure 3 shown.

[0052] Example 2

[0053] (1) Preparation of aloe acetyl mannan: remove the ends of fresh aloe leaves, soak in pure water for 24 h, wash with water, remove the epidermis, and beat at 1450 r / min for 20 min to obtain aloe polysaccharide liquid. Stir at a constant temperature for 28 h under a light intensity of 800 lumens, and evaporate at a constant temperature of 80 °C to obtain the upper film, i.e., aloe acetyl mannan.

[0054] (2) Preparation of aloe acetyl mannan milk fat globules: The aloe acetyl mannan prepared in step (1) was mixed with water in a volume ratio of 1:2, and high shear homogenization was performed at 20,000 r / min for 10 minutes until the two were fully fused to obtain an emulsion. The emulsion was mixed with silicone oil (CAS: 63148-62-9) in a volume ratio of 1:2, and high shear homogenization was performed at 20,000 r / min for 10 minutes until the emulsion and silicone oil were fully mixed. The mixture was allowed to stand for 60 minutes, and the upper layer of silicone oil was removed to obtain the aloe acetyl mannan milk fat globules (R5) in the lower layer. The same was observed under a microscope. Figure 1 .

[0055] (3) Preparation of aloe acetyl mannan milk fat globule fluorescent marker: Use rhodamine DHPE (DMSO preparation, concentration 2 mg / mL) to fluorescently label the lipids in aloe acetyl mannan milk fat globule, and mix the milk fat globule and rhodamine DHPE at a volume ratio of 200:1; then use Alexa Fluor 488-labeled wheat germ agglutinin AF488-WGA (1×PBS preparation, concentration 30 mg / mL) to label aloe acetyl mannan, and the volume ratio of aloe acetyl mannan milk fat globule and AF488-WGA is 600:1, and mix; incubate at room temperature in the dark for 60 min to obtain aloe acetyl mannan milk fat globule fluorescent marker (WGA-DOPH-R5).

[0056] (4) The aloe acetyl mannan milk fat globule fluorescent marker is fixed with agar, and the specific method is the same as in Example 1 to obtain the fixed aloe acetyl mannan milk fat globule fluorescent marker, and the same Figure 2 and Figure 3 .

[0057] Example 3

[0058] (1) Preparation of aloe acetyl mannan: remove the ends of fresh aloe leaves, soak in pure water for 24 h, wash with water, remove the epidermis, and beat at 1450 r / min for 20 min to obtain aloe polysaccharide liquid. Stir at a constant temperature for 28 h under a light intensity of 800 lumens, and evaporate at a constant temperature of 80 °C to obtain the upper film, i.e., aloe acetyl mannan.

[0059] (2) Preparation of aloe acetyl mannan milk fat globules: the aloe acetyl mannan prepared in step (1) was mixed with water in a volume ratio of 1:10, and sheared and homogenized at 5000 r / min for 30 min until the two were fully fused to obtain an emulsion. The emulsion was mixed with silicone oil (CAS: 63148-62-9) in a volume ratio of 1:10, and sheared and homogenized at 5000 r / min for 30 min until the emulsion and silicone oil were fully mixed. The mixture was allowed to stand for 10 min, and the upper layer of silicone oil was removed to obtain the aloe acetyl mannan milk fat globules (R5) in the lower layer. The same was observed under a microscope. Figure 1 .

[0060] (3) Preparation of aloe acetyl mannan milk fat globule fluorescent marker: Use rhodamine DHPE (DMSO preparation, concentration 0.5 mg / mL) to fluorescently label the lipids in aloe acetyl mannan milk fat globule, and mix the milk fat globule and rhodamine DHPE at a volume ratio of 200:1; then use Alexa Fluor 488-labeled wheat germ agglutinin AF488-WGA (1×PBS preparation, concentration 5 mg / mL) to label aloe acetyl mannan, and the volume ratio of aloe acetyl mannan milk fat globule and AF488-WGA is 100:1, and mix; incubate at room temperature in the dark for 20 min to obtain aloe acetyl mannan milk fat globule fluorescent marker (WGA-DOPH-R5).

[0061] (4) The aloe acetyl mannan milk fat globule fluorescent marker is fixed with agar, and the specific method is the same as in Example 1 to obtain the fixed aloe acetyl mannan milk fat globule fluorescent marker, and the same Figure 2 and Figure 3 .

[0062] Application Examples

[0063] 150,000 rat hair follicle stem cells (HFSC) (Probio, Catalog No. iCell-0098a) were seeded into a 12-well plate at a rate of 120,000 per well and cultured overnight at 37°C. The aloe acemannan milk fat globule fluorescent marker prepared in Example 1 was diluted 50-fold with a hair follicle stem cell-specific cell culture medium (Probio, Catalog No. iCell-0098a-001b), and the culture medium of rat hair follicle stem cells (HFSC) was replaced (the concentration of the marker in the culture medium was 5% by volume). The cells were co-cultured at 37°C for 12 h, the upper culture medium was removed, and the cells were washed twice with 1×PBS preheated at 37°C. 500 μL of 1×PBS was added, and the cells were observed under a fluorescence microscope. The fluorescent signal of the aloe acemannan milk fat globule fluorescent marker appeared inside the cells, as marked by yellow arrows. Figure 4 As shown (the application effects of Examples 2 and 3 are the same, and the drawings are not provided again).

[0064] Comparative Example 1 FITC-labeled glycoprotein components in aloe acemannan milk fat globules

[0065] (1) Isothiocyanate groups can react with the amino terminal or primary amine of glycoproteins. Aloe acetyl mannan milk fat globules cannot be labeled using the traditional fluorescein isothiocyanate (FITC) chemical synthesis labeling method (the reaction process will destroy the milk fat globule structure). Therefore, in this example, only FITC (CAS: 3326-32-7) is directly added to the aloe acetyl mannan milk fat globules prepared in step (2) of the example, and mixed to label the aloe acetyl mannan milk fat globules to obtain a light yellow liquid. The labeling results are shown in Figure 2. Figure 5 As shown, a small number of milk fat globules were specifically labeled, but the vast majority of milk fat globules were not positively labeled. In actual use, it was found that the FITC-labeled aloe acetyl mannan milk fat globules were less stable than the aloe acetyl mannan milk fat globules (R5) prepared in step (2) of Example 1, and were more prone to oil-water separation.

[0066] (2) In addition, an attempt was made to use FITC-labeled aloe acetylmannan to prepare milk fat globules through step (2) of the example, thereby achieving the purpose of FITC-labeling the glycoprotein component (FITC-R5) in the aloe acetylmannan 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 Na2HPO4·12H2O, dissolve it in deionized water, and add water to 1000 mL; 0.2 M NaH2PO4: take 31.2 g 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 pH 8.0 phosphate buffer; 2) dissolve 400 mg of aloe acetyl mannan in 15 mL of phosphate buffer (pH 8.0) prepared in step 1), add 400 mg tyramine, react for 24 h. Add 150 mg sodium cyanoborohydride, react at 37°C for 96 h, shaking occasionally, and centrifuge to remove the supernatant to obtain ammoniated aloe acetomannan. Dissolve 200 mg of ammoniated aloe acetomannan in water, adjust the pH to 8.0 with 0.5 mol / L NaHCO₃, add 50 mg of FITC, and react with stirring at room temperature in the dark for 24 h. Filter the reaction solution, and add anhydrous ethanol to the filtrate to a final ethanol concentration of 75% (v / v). A large amount of bright yellow-green precipitate will precipitate. Centrifuge and discard the supernatant, re-dissolve the precipitate in water, and reprecipitate with ethanol three times under the same conditions as above to obtain a bright yellow precipitate. The resulting bright yellow precipitate is dissolved in a small amount of water and further purified on a 10KD ultrafiltration column until the filtrate is essentially FITC-free, obtaining FITC-labeled aloe acetomannan.

[0067] Comparative Example 2 Fast Green FCF Labeling of Glycoprotein Components in Aloe Acetylmannan Milk Fat Globule

[0068] Fast Green FCF is an acidic fluorescent dye with hydrophilic and lipophilic groups. It can bind to alkaline components in cells or tissues, including DNA, RNA, alkaline proteins, and acidic lipids. By utilizing the duality (acidity and alkalinity) of proteins and their ability to form salts with acidic and alkaline dye ions, all proteins can be stained with Fast Green at a pH of about 2.2, that is, below the isoelectric point of all proteins. At a pH of about 8.5, alkaline proteins are stained with Fast Green. The inventors tried different pH environments, dissolving 0.1% Fast Green (CAS 2353-45-9) in 1×PBS, measuring the pH value with a pH meter, and adjusting the pH value to 2.3, 7.4, and 8.5 using 0.1 mol / L hydrochloric acid and 0.1 mol / L sodium hydroxide, respectively. An equal volume of Fast Green dye was added to the aloe acetomannan milk fat globules prepared in step (2) of Example 1, and mixed for 2 hours to label the aloe acetomannan milk fat globules. The results are shown in FIG. Figure 6 As shown, aloe acemannan milk fat globules are not labeled, and the flocs in the figure are residual aloe residues. Therefore, Fast Green is not suitable for labeling aloe acemannan milk fat globules.

[0069] Comparative Example 3: Nile-Red Labeled Aloe Acetylmannan Milk Fat Globule Neutral Lipid Component

[0070] Nile red is an ideal fluorescent dye for lipid staining. It is mainly used to show fatty degeneration and abnormal lipid deposition in tissues and organs, as well as 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: Nile-Red (McLean, Cat. No.: N861501) powder is dissolved in DMSO at a concentration of 1 mM / mL. Nile-Red dye is added to the aloe acetylmannan milk fat globules R5 prepared in step (2) of Example 1 at a volume ratio of 200:1. After mixing, the mixture is reacted in the dark for 30 minutes to label the aloe acetylmannan milk fat globules.

[0071] The results are as follows Figure 7 As shown in Figure 3, the lipid core of R5 is marked in red, the edge of the R5 sphere is clear, the orange-red fluorescence is bright, and the background is clean, indicating that the Nile red labeling effect is good; however, the fluorescence quenching of the Nile red labeling is fast, as shown in Figure 3. Figure 8 As shown, A is the fluorescence imaging of R5 when the observation time is 0s, and B is the fluorescence of R5 when the observation time is 15s.

[0072] Comparative Example 4 AF488-WGA labeling of polysaccharide and glycoprotein components in aloe acetylmannan milk fat globules

[0073] Take the aloe acemannan milk fat globules R5 prepared in step (2) of Example 1, and use Alexa Fluor 488-labeled wheat germ agglutinin AF488-WGA (prepared in 1×PBS, concentration 10 mg / mL) to label the aloe acemannan. The volume ratio of aloe acemannan milk fat globules and AF488-WGA is 200:1. Mix well and incubate at room temperature in the dark for 30 minutes. The results are as follows. Figure 9 As 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 are not visible.

[0074] Comparative Example 5: Labeling of polar lipid components in aloe acetylmannan milk fat globules with rhodamine DHPE

[0075] Take the aloe acetyl mannan milk fat globules R5 prepared in step (2) of Example 1, and use rhodamine DHPE (DMSO preparation, concentration 1 mg / mL) to fluorescently label the lipids in the aloe acetyl mannan milk fat globules. The milk fat globules and rhodamine DHPE at a volume ratio of 100:1 are mixed and incubated at room temperature in the dark for 30 minutes. The labeling results are shown in Figure 2. Figure 10 As shown in the figure, the spherical structure of R5 is clear and complete, the lipid core is intact, the fluorescence is bright, and the background is clean, but the distribution of aloe acemannan in R5 is not reflected, and the multi-layered milk fat globule structure of R5 cannot be fully reflected.

[0076] Aloe vera acetylmannan milk fat globules R5 prepared in step (2) of Example 1, FITC-labeled aloe vera acetylmannan milk fat globules (FITC-R5) prepared in Comparative Example 1, aloe vera acetylmannan milk fat globules fluorescently labeled (WGA-DOPH-R5) prepared in Example 1, and a control group (blank control group, without R5) were co-cultured with hair follicle stem cells HFSC (Cellbio, Catalog No. iCell-0098a) for 24 h. The cells were collected and assayed for cellular ATP, mitochondrial membrane potential (TMRE), and reactive oxygen species (ROS). ATP was assayed using an ATP assay kit (Biyuntian, Catalog No. S0026); ROS was assayed using a reactive oxygen species assay kit (Solarbio, Catalog No. CA1410); and TMRE was assayed using a mitochondrial membrane potential assay kit (Biyuntian, Catalog No. C2001S).

[0077] The results are as follows Figure 11 As shown in the figure, compared with the control group, both the R5 group and the WGA-DOPH-R5 group could significantly increase the cellular ATP, TMRE and ROS; compared with the R5 group, the ATP and TMRE of the FITC-R5 group decreased (with statistical difference), and ROS increased slightly (without statistical difference), indicating that the biological activity of aloe acetomannan labeled with FITC was damaged.

[0078] 150,000 rat hair follicle stem cells (HFSC) (Probio, Catalog No. iCell-0098a) were seeded into a 12-well plate at a rate of 120,000 per well and cultured overnight at 37°C. FITC-labeled aloe acetyl mannan and FITC-labeled aloe acetyl mannan milk fat globules (FITC-R5) prepared in Comparative Example 1 were diluted 20-fold using a hair follicle stem cell-specific cell culture medium (Probio, Catalog No. iCell-0098a-001b). The culture medium was then replaced with the rat hair follicle stem cell HFSC culture medium (the concentration of the marker in the culture medium was 5% by volume). The cells were cultured at 37°C for 12 h, the upper culture medium was removed, and the cells were washed twice with 1×PBS preheated at 37°C. 500 μL of 1×PBS was added, and the cells were then observed using a fluorescence microscope. The results showed that FITC-labeled aloe acetyl mannan could be observed under a fluorescence microscope ( Figure 12 Indicated by the white arrow in A), but after FITC-R5 was co-cultured with cells, the target was lost under the fluorescence microscope ( Figure 12 B).

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing aloe acemannan milk fat globule fluorescent marker, characterized in that: The steps include: (1) Aloe acemannan is mixed with water and homogenized once to obtain an emulsion, the emulsion is mixed with silicone oil, homogenized a second time, allowed to stand, and the lower layer is removed to obtain aloe acemannan milk fat globules; (2) The aloe acetyl mannan milk fat globules described in step (1) are mixed with rhodamine DHPE to obtain substance A, and then mixed with fluorescently labeled lectin, and incubated in the dark to obtain aloe acetyl mannan milk fat globules fluorescent marker.

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

3. The method for preparing the aloe acemannan milk fat globule fluorescent marker according to claim 1, characterized in that: The aloe acetyl mannan in step (1) is prepared by the following method: removing both ends of fresh aloe leaves, soaking in pure water for 24-48 hours, washing with water, removing the epidermis, beating at 1450 r / min for 15-20 minutes to obtain aloe polysaccharide liquid, stirring at a constant temperature of 4-15°C for more than 24 hours under light with an illumination of not less than 800 lumens, and evaporating at a constant temperature of 75-95°C to obtain an aloe polysaccharide extract rich in aloe acetyl mannan.

4. The method for preparing the aloe acemannan milk fat globule fluorescent marker according to claim 1, characterized in that: In step (2), the aloe acetyl mannan milk fat globule fluorescent marker is mixed with the unsolidified agar and cooled to obtain a fixed aloe acetyl mannan milk fat globule fluorescent marker, which is more convenient for observation.

5. The method for preparing the aloe acemannan milk fat globule fluorescent marker according to claim 4, characterized in that: Include at least one of the following (a)-(c): (a) the volume ratio of the unsolidified agar to the aloe acemannan milk fat globules is 1-5:1; (b) the concentration of the unsolidified agar is 1.5% to 2.0% by mass volume; (c) The temperature of the unsolidified agar is 45°C-50°C.

6. An aloe acemannan milk fat globule fluorescent marker, prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the aloe acemannan milk fat globule fluorescent marker according to claim 6 in the preparation of a cell fluorescent labeling reagent.

8. Use of the aloe acemannan milk fat globule fluorescent marker according to claim 6 in cell membrane labeling for non-diagnostic and non-therapeutic purposes.

9. A method for cell fluorescence imaging for non-diagnostic and non-therapeutic purposes, characterized in that: The method comprises the following steps: co-culturing cells with a culture medium containing the aloe acemannan milk fat globule fluorescent marker according to claim 6, and then performing cell fluorescence imaging.

10. The method for cell fluorescence imaging for non-diagnostic and non-therapeutic purposes according to claim 9, characterized in that: The amount of the aloe acemannan milk fat globule fluorescent marker in the culture medium is 2.5%-10% by volume.

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