Extraction method of milk-derived exosome

Abstract

The invention relates to the technical field of exosome extraction, and discloses a milk-derived exosome extraction method. The extraction method comprises the following steps: carrying out differential centrifugation and microporous membrane filtration pretreatment on a milk source to obtain a milk-derived exosome filtrate; the milk-derived exosome filtrate is subjected to column purification enrichment and concentration, and the milk-derived exosome is obtained. Wherein the differential centrifugation comprises the following steps: first-stage centrifugation is carried out, and the centrifugal force is 2500-3500 * g; second-stage centrifugation is carried out, and the centrifugal force is 1000-1500 * g; and carrying out three-stage centrifugation, wherein the centrifugal force is 10000-15000 * g. According to the extraction method provided by the invention, ultracentrifugal precipitation and resuspension of extracellular vesicle particles are not carried out before enrichment of the exosome, and only low-medium speed centrifugation and membrane filtration are carried out, so that the method is relatively mild, mechanical damage of ultracentrifugal exosome is reduced, and the integrity of the structure and function of the exosome is maintained; the method is simple to operate, low in cost, high in extraction efficiency and high in product purity.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome extraction, and in particular to a method for extracting milk-derived exosomes. Background Art

[0002] Exosomes are a subtype of extracellular vesicles (EVs) found in a variety of biological fluids, including urine, plasma, saliva, and breast milk. Exosomes contain components such as genomic DNA, RNA, proteins, and lipids, and have been extensively studied in medicine, playing diverse physiological and therapeutic roles in cell proliferation, inflammation, immune regulation, and cancer.

[0003] Human breast milk (HBM) is rich in various nutrients and plays an irreplaceable role in supporting early human growth and development. Human breast milk is composed of approximately 87%-88% water, with the remaining essential components being macronutrients (carbohydrates, protein, and fat). Macronutrients provide essential nutritional support for infant growth and development. In addition, human breast milk contains a variety of vitamins and micronutrients, as well as non-nutritional bioactive components that profoundly impact infant survival and health, such as immune proteins (lysozyme, lactoferrin), various cytokines (tumor necrosis factor α, interferon γ), and derived microorganisms such as lactic acid bacteria and Staphylococcus aureus.

[0004] Studies have shown that breast milk contains functional vesicles encapsulated in protective lipid droplets—milk-derived exosomes (MDEs). Milk-derived exosomes are a subtype of extracellular vesicles with diameters ranging from 30-120 nm and have been shown to be present in the milk of humans, cows, goats, pigs, rodents, and marsupials. Secreted by mammary epithelial cells and released from milk fat globules during lactation, milk-derived exosomes contain proteins and siRNAs and play a variety of physiological and therapeutic roles in cell proliferation, inflammation, immune regulation, and cancer. They have been developed as drug carriers for delivering small molecules and siRNAs to tumors.

[0005] Milk-derived exosomes have a wide range of applications in the medical field, but their extraction methods still have many deficiencies. Existing methods for extracting exosomes from biological fluids (urine, plasma, saliva and milk) mainly include ultracentrifugation, density gradient ultracentrifugation, precipitation-based, immunoaffinity, ultrafiltration or size exclusion chromatography, etc. Among them, ultracentrifugation is simple and easy to operate, but the centrifugation time is long, and long-term ultracentrifugation can easily cause damage to exosomes. Although size exclusion chromatography can better maintain the intact structure of exosomes, its purity, recovery rate and insufficient processing capacity greatly limit its use. Therefore, it is urgent to develop a simple, rapid, efficient, high-purity, low-cost milk-derived exosome extraction method that does not damage the exosome structure to promote basic and clinical application research on milk-derived exosomes. Summary of the Invention

[0006] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, one of the purposes of the present invention is to provide a method for extracting milk-derived exosomes.

[0007] A second object of the present invention is to provide an application of milk-derived exosomes obtained by this extraction method.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A first aspect of the present invention provides a method for extracting milk-derived exosomes, comprising: performing differential centrifugation and microporous membrane filtration pretreatment on milk to obtain a milk-derived exosome filtrate; performing column purification, enrichment, and concentration on the milk-derived exosome filtrate to obtain the milk-derived exosomes; wherein the differential centrifugation comprises: a first-stage centrifugation with a centrifugal force of 2500-3500×g; a second-stage centrifugation with a centrifugal force of 1000-1500×g; and a third-stage centrifugation with a centrifugal force of 10,000-15,000×g.

[0010] In some embodiments of the present invention, the milk source comprises human breast milk.

[0011] In some embodiments of the present invention, the temperature of the differential centrifugation is 2-6°C.

[0012] In some preferred embodiments of the present invention, the temperature of the differential centrifugation is 3-5°C.

[0013] In some embodiments of the present invention, the primary centrifugation comprises: centrifuging the milk source at a centrifugal force of 2500-3500×g for 2-3 times, with a single centrifugation time of 8-15 minutes, to obtain a first supernatant.

[0014] In some preferred embodiments of the present invention, the primary centrifugation comprises: centrifuging the milk source twice at a centrifugal force of 2800-3200×g, with a single centrifugation time of 8-12 minutes, to obtain a first supernatant.

[0015] In some embodiments of the present invention, the secondary centrifugation comprises: centrifuging the first supernatant at a centrifugal force of 1000-1500×g for 1-2 times, with a single centrifugation time of 15-25 min, to obtain a second supernatant.

[0016] In some preferred embodiments of the present invention, the secondary centrifugation comprises: centrifuging the first supernatant once at a centrifugal force of 1100-1300×g for 18-22 min to obtain a second supernatant.

[0017] In some embodiments of the present invention, the three-stage centrifugation includes: first centrifuging the second supernatant at a centrifugal force of 10,000-15,000 × g for 2-3 times, with a single centrifugation time of 25-35 minutes, taking the supernatant, and then centrifuging it at a centrifugal force of 10,000-15,000 × g for 1-2 times, with a single centrifugation time of 50-70 minutes, to obtain a third supernatant.

[0018] In some preferred embodiments of the present invention, the three-stage centrifugation includes: first centrifuging the second supernatant twice at a centrifugal force of 12000-14000×g, with a single centrifugation time of 28-32 minutes, taking the supernatant, and then centrifuging once at a centrifugal force of 12000-14000×g, with a single centrifugation time of 55-65 minutes, to obtain a third supernatant.

[0019] In some embodiments of the present invention, the microporous membrane filtration includes sequentially performing primary filtration and secondary filtration on the third supernatant; the filtration accuracy of the primary filtration is 0.4-0.6 μm; the filtration accuracy of the secondary filtration is 0.1-0.3 μm.

[0020] In some preferred embodiments of the present invention, the filtration accuracy of the primary filtration is 0.4-0.5 μm; the filtration accuracy of the secondary filtration is 0.2-0.3 μm.

[0021] In some embodiments of the present invention, after the microporous membrane filtration, a step of adding a buffer solution to the filtrate is further included, and the volume of the milk-derived exosome filtrate after the addition of the buffer solution is 1.5-2 times the initial volume of the milk source.

[0022] In some embodiments of the invention, the buffer comprises phosphate buffered saline (PBS).

[0023] In some embodiments of the present invention, the column purification and enrichment comprises adding the milk-derived exosome filtrate to an exosome purification column, eluting, and collecting 4-6 fractions after the void volume to obtain an exosome enriched solution.

[0024] In some embodiments of the present invention, the elution reagent comprises phosphate buffered saline.

[0025] In some embodiments of the present invention, the concentration comprises placing the exosome-enriched solution in an ultrafiltration tube with a molecular weight cut-off of 80-150 KD, and centrifuging at a centrifugal force of 3500-4500×g for 20-40 min.

[0026] In some preferred embodiments of the present invention, the concentration comprises placing the exosome-enriched solution in an ultrafiltration tube with a molecular weight cut-off of 80-120 KD, and centrifuging at a centrifugal force of 3800-4200×g for 25-35 min.

[0027] In some embodiments of the present invention, the concentration is 8-12 times.

[0028] The basic principles of the present invention are described as follows:

[0029] 1) The method for extracting milk-derived exosomes provided by the present invention first performs differential centrifugation on the milk source, and then gradually increases the centrifugal force to utilize the differences in size, density and sedimentation velocity of different components (such as fat, cell debris, and exosomes) to achieve fractional separation. Specifically, the following steps are taken: ① The fat globules (diameter 1-10 μm) in the milk source have a low density (<1 g / cm 3 ), which will quickly float up to form a cream layer under low-speed centrifugation. The first-stage centrifugation (centrifugal force 2500-3500×g) is sufficient to float the fat globules, but is not enough to precipitate smaller exosomes. Therefore, most of the fat (3%-5% of the volume of breast milk) is removed by the first-stage centrifugation to avoid fat interfering with exosome purification in subsequent steps; ② After reducing the centrifugal force in the second-stage centrifugation (centrifugal force 1000-1500×g), smaller fat globules (0.5-2μm) and cell debris (such as desquamated epithelial cells) will slowly settle, while exosomes are still retained in the supernatant due to their small particle size. Therefore, the second-stage centrifugation can further remove the remaining tiny fat globules and cell debris, reducing the impurity load of subsequent high-speed centrifugation. This step is a transitional stage, and it is necessary to balance the centrifugal force and time to avoid exosome loss; ③ Casein (diameter 50-500nm) is the main protein aggregate in breast milk and has a high density (about 1.3g / cm 3 ), under the medium-speed centrifugal force (10000-15000×g) of the three-stage centrifugation, casein will gradually precipitate, while exosomes (density about 1.1-1.2g / cm 3) is still suspended, and 2-3 short centrifugations (25-35 min) followed by 1-2 long centrifugations (50-70 min) are used to improve the efficiency of casein removal. Through three-stage centrifugation, casein (accounting for 80% of breast milk protein) is completely removed, thereby preventing it from co-precipitating with exosomes or clogging subsequent purification columns;

[0030] 2) In microporous membrane filtration, the remaining cell debris and protein aggregates (>220 nm) in the final filtrate of differential centrifugation are retained by filters with gradually decreasing pore sizes, while exosomes (30-150 nm) can pass through the microporous membrane and be retained. Microporous membrane filtration further removes tiny particles that are not completely removed by differential centrifugation, ensuring the purity of the milk-derived exosome filtrate;

[0031] 3) During purification, enrichment, and concentration, exosomes are selectively captured using an exosome purification column. Unretained macromolecular impurities (void volume) are first eluted, and then the delayed eluted exosomes are collected to obtain a purified exosome enrichment solution. The exosomes are then trapped by an ultrafiltration tube, and the eluate and small molecular impurities are filtered out to obtain milk-derived exosomes.

[0032] The second aspect of the present invention provides the use of milk-derived exosomes obtained by the extraction method described in the first aspect of the present invention in the preparation of a drug for treating neonatal small intestinal necrotizing colitis.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1) The present invention provides a method for extracting milk-derived exosomes. Pretreatment with differential centrifugation and microporous membrane filtration effectively removes impurities such as fat globules, cell debris, and casein. The milk-derived exosome filtrate is then subjected to column purification, enrichment, and concentration to obtain high-purity milk-derived exosomes. This method does not involve ultracentrifugation to precipitate and resuspend extracellular vesicle particles before exosome enrichment. Instead, it involves low- to medium-speed centrifugation and membrane filtration. This method is relatively gentle, reduces mechanical damage to exosomes caused by ultracentrifugation, and maintains the integrity of the exosome structure and function. The method is simple to operate, low in cost, and has high extraction efficiency and high product purity.

[0035] 2) The milk-derived exosome extraction method provided by the present invention has a complete exosome structure and high purity, and can be used to prepare drugs for the treatment of neonatal small intestinal necrotizing colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the BCA standard curve;

[0037] Figure 2 This is a particle size distribution diagram of milk-derived exosomes in the examples;

[0038] Figure 3This is a graph showing the concentration of milk-derived exosomes in the examples;

[0039] Figure 4 This is a transmission electron micrograph of milk-derived exosomes in the examples;

[0040] Figure 5 This is an immunoblot image for the identification of milk-derived exosome proteins in the examples. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below by way of specific examples. Unless otherwise specified, the raw materials, reagents, or devices used in the examples are all commercially available or can be obtained by conventional methods. Unless otherwise specified, all experiments or testing methods are conventional methods in the art.

[0042] 1. The human breast milk used in the examples was collected from 5 healthy postpartum women, with a sample volume of 2 mL / person. The samples were frozen at -80°C and thawed overnight before use.

[0043] 2. The PBS buffer used in the examples was purchased from Corning, USA; the BCA protein assay kit was purchased from Wuhan Solebao Company; CD63, TSG101, and Calnexin antibodies were purchased from Abcam, USA; Exosupur columns were purchased from Enze Kangtai Biotechnology Co., Ltd.; and Millipore centrifugal ultrafiltration tubes (molecular weight cutoff 100 kD) were purchased from Merck Millipore China Co., Ltd.

[0044] Example

[0045] This embodiment provides a method for extracting human breast milk exosomes, and the steps are as follows:

[0046] S1. Centrifuge five 2 mL human breast milk samples twice at 3000 × g at 4°C for 10 min each time, and filter to remove the cream layer formed by fat globules to obtain the first supernatant;

[0047] S2, centrifuging the first supernatant at 1200×g for 20 min at 4°C, filtering to remove residual fat globules and cell debris, and obtaining a second supernatant;

[0048] S3. Centrifuge the second supernatant twice at 13,000 × g at 4°C for 30 min each time, and then centrifuge again at 13,000 × g at 4°C for 60 min to precipitate and remove casein, thereby obtaining a third supernatant.

[0049] S4. Filter the third supernatant through 0.45 μm and 0.22 μm syringe filters respectively to eliminate cell debris, and then add PBS buffer to make the filtrate volume up to 2 mL to obtain milk-derived exosome filtrate;

[0050] S5. Add the milk-derived exosome filtrate to the Exosupur column and add 4 mL of PBS buffer for elution. The first four fractions are the void volume without exosome components. After the void volume, add 5 mL of PBS buffer to continue elution and collect 5 mL of exosome enrichment solution respectively.

[0051] S6. The collected exosome enrichment solution was concentrated using an ultrafiltration tube with a molecular weight cutoff of 100 kD. 2.5 mL of the exosome fraction was centrifuged at 4000 × g for 30 min to concentrate it to 250 μL. Five milk-derived exosome concentrates were obtained and stored at -80°C.

[0052] 1. Perform BCA protein detection on the milk-derived exosomes prepared in the example:

[0053] 1) Preparation of BCA working solution: Add 1 volume of Cu reagent to 50 volumes of BCA reagent and mix thoroughly to obtain BCA working solution. BCA working solution is stable at room temperature for 24 hours.

[0054] 2) Dilute the standard: Take 10 μL of BSA standard (bovine serum albumin) and dilute it to 100 μL with PBS buffer to a final concentration of 0.5 mg / mL;

[0055] 3) Protein concentration detection: BSA standard (0.5 mg / mL) was added to the protein standard wells of a 96-well plate at 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, 12 μL, 16 μL, and 20 μL, and an appropriate amount of PBS buffer was added to make the volume up to 20 μL, respectively, to obtain BSA standards with final concentrations of 0 mg / mL, 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.30 mg / mL, 0.40 mg / mL, and 0.50 mg / mL. Then, 20 μL of the milk-derived exosome concentrated solution in the example was added, with 3 replicate wells for each sample, and then 200 μL was added to each well. The BCA working solution was incubated at 37°C for 30 minutes. After incubation, the absorbance (OD value) at a wavelength of A570 nm was measured using a microplate reader. Finally, the BCA protein concentration of the milk-derived exosome concentrated solution was calculated based on the standard curve and the sample volume used.

[0056] Table 1 shows the absorbance corresponding to different concentrations of BSA standards. It can be seen from Table 1 that as the BSA concentration increases, the absorbance generally shows an upward trend, which is consistent with the basic principle of the BCA method.

[0057] Table 1 Absorbance corresponding to different concentrations of BSA standards

[0058] BSA standard concentration (mg / mL) 0.00 0.05 0.10 0.15 0.20 0.30 0.40 0.50 Absorbance (OD value) 0.127 0.138 0.141 0.149 0.144 0.157 0.162 0.175

[0059] Figure 1 is the BCA standard curve, Figure 1 It can be seen that the linear equation of the BCA standard curve is y=0.0844x+0.1312, R 2 =0.9496, R 2 The value is close to 0.95, indicating a reasonable linear relationship.

[0060] Table 2 shows the absorbance and BCA protein concentration of the concentrated milk-derived exosome solutions in the examples. As shown in Table 2, the absorbance of the samples in accessory wells 1 and 3 is close (with a small standard deviation), indicating that the experimental operation is stable. The BCA protein concentration of each sample varies, which is presumably related to individual differences in breast milk. The BCA protein concentration of most samples is greater than 1 mg / mL, which is consistent with the range of breast milk exosome protein content reported in the literature.

[0061] Table 2 Absorbance and BCA protein concentration of milk-derived exosome concentrated solution in Example

[0062]

[0063] 2. Exosome identification of the milk-derived exosomes prepared in the examples:

[0064] 1) Particle size detection: 50 μL of the concentrated exosome solution of sample 1 was taken and the particle size, distribution, and particle concentration of the exosomes were detected using a Flow NanoAnalyzer.

[0065] Figure 2 is the particle size distribution diagram of milk-derived exosomes in the embodiment, Figure 2 It can be seen that the average particle size of the milk-derived exosomes in the embodiment is 82.36nm, which is consistent with the exosome literature report (30-150nm), indicating that the extract in the embodiment is exosomes rather than cell debris or other vesicles. The particle size of the milk-derived exosomes is unimodal, indicating that the exosome particle size is uniform and there is no significant aggregate or impurity interference. It can be seen that the method provided by the present invention effectively removes impurities such as fat globules and cell debris through low-to-medium speed differential centrifugation and microporous membrane filtration pretreatment, without destroying the natural size of the exosomes.

[0066] Figure 3 is the concentration information diagram of milk-derived exosomes in the embodiment, Figure 3 It can be seen that the particle concentration of milk-derived exosomes in the embodiment is 1.27×10 11Particles / mL. Based on the initial volume of breast milk and the concentration multiple of the exosome solution, the average exosome particle concentration of the sample can be calculated to be 3.18×10 11 Particles / mL, which is consistent with the range reported in the literature on breast milk exosomes (usually 10 9 -10 11 particles / mL), indicating that the method provided by the present invention has a high extraction efficiency of exosomes.

[0067] 2) Electron Microscopy: 10 μL of the concentrated exosome solution from sample 1 was dropped onto a copper grid. The grid was incubated at room temperature for 10 min, then rinsed with sterile distilled water and the excess liquid was absorbed with absorbent paper. 10 μL of 2 wt% uranyl acetate was then dropped onto the grid for negative staining for 1 min. The supernatant was removed with filter paper and dried under an incandescent lamp for 2 min. The grid was then observed under a transmission electron microscope at 80 kV for imaging.

[0068] Figure 4 The transmission electron micrograph of the milk-derived exosomes in the embodiment is shown in FIG. Figure 4 (a) and (b) are transmission electron microscope images under different observations. Figure 4 It can be seen that the milk-derived exosomes in the examples are round or cup-shaped vesicles with intact membrane structures and no ruptures. Their diameters are consistent with the nanoflow cytometry results (about 80-100 nm), and the background in the transmission electron microscopy image is clean, without free protein aggregation or cell debris contamination, which once again confirms that the extracts in the examples are exosomes rather than cell debris or other vesicles, and that impurities such as cell debris have been removed, and the product is highly pure. The extracts in the examples show a double-layer membrane structure under an electron microscope, which is consistent with the morphology of exosomes extracted by ultracentrifugation, and the exosomes are not wrinkled or deformed, indicating that the extraction process does not damage the membrane structure. That is, the method provided by the present invention is relatively mild and can keep the exosomes in an intact morphology.

[0069] 3) Protein Identification: To further verify that the extracts in the examples are genuine exosomes, exosome-specific protein markers were detected by immunoblotting. The detection targets include positive markers CD63 and TSG101, and a negative control Calnexin. CD63 is a member of the tetraspanin family and is enriched on the exosome membrane surface (>90% exosome expression). TSG101 is an ESCRT complex protein involved in exosome biogenesis (intracellular multivesicular body formation). Calnexin is an endoplasmic reticulum-resident protein that should not be present in exosomes and is used to exclude contamination by cell debris.

[0070] The specific steps are as follows: calculate the protein loading volume according to the BCA protein concentration of the sample, add 5× loading buffer (final concentration is 1×), then place in a metal bath at 100°C for 10 minutes, place on ice, and centrifuge instantly; after the sample is loaded, perform electrophoresis at a voltage of 90V, adjust the voltage to 120V when the band runs to the separation gel, and stop electrophoresis until the band runs to the bottom; then perform membrane transfer, transfer the band on the gel to a polyvinylidene fluoride membrane (PVDF membrane) soaked in pre-cooled methanol, and transfer the membrane in an ice water bath at a voltage of 100V for 50 minutes; after transfer, wash the membrane three times with TBST buffer and pour into 5wt% skim milk on a shaker, block for 1 hour, then pour out the milk, wash three times with TBST buffer and label the target protein; finally, prepare the developing luminescent solution, incubate in the dark for 1 hour, and then develop on the machine to observe the clarity of the CD63, TSG101, and Calnexin bands.

[0071] Figure 5 The immunoblot images of the milk-derived exosome protein identification in the embodiment are shown in FIG. Figure 5 As can be seen, CD63 and TSG101 showed clear specific bands in the immunoblot, confirming that the extract was rich in exosomes and retained intact membrane structure and intracellular protein components. Calnexin bands were not detected in the immunoblot, indicating that the sample was free of significant cellular debris or endoplasmic reticulum contamination. This demonstrates that the method provided by the present invention can effectively extract highly pure human breast milk exosomes while maintaining the integrity of exosomal proteins. This will further enhance the functional integrity of human breast milk exosomes and their application in the preparation of therapeutic drugs for neonatal necrotizing colitis.

Claims

1. A method for extracting milk-derived exosomes, characterized in that: include: The milk source is pretreated by differential centrifugation and microporous membrane filtration to obtain milk-derived exosome filtrate; The milk-derived exosome filtrate is subjected to column purification, enrichment and concentration to obtain the milk-derived exosomes; wherein the differential centrifugation includes: first-stage centrifugation, centrifugal force 2500-3500×g; second-stage centrifugation, centrifugal force 1000-1500×g; and third-stage centrifugation, centrifugal force 10000-15000×g.

2. The extraction method according to claim 1, wherein The milk source includes human breast milk.

3. The extraction method according to claim 1, wherein The temperature of the differential centrifugation is 2-6°C.

4. The extraction method according to claim 3, characterized in that The first-stage centrifugation comprises: centrifuging the milk source at a centrifugal force of 2500-3500×g for 2-3 times, with a single centrifugation time of 8-15 minutes, to obtain a first supernatant.

5. The extraction method according to claim 4, characterized in that The secondary centrifugation comprises: centrifuging the first supernatant at a centrifugal force of 1000-1500×g for 1-2 times, with a single centrifugation time of 15-25 min, to obtain a second supernatant.

6. The extraction method according to claim 5, characterized in that The three-stage centrifugation includes: first centrifuging the second supernatant at 10,000-15,000×g for 2-3 times, with a single centrifugation time of 25-35 minutes, taking the supernatant, and then centrifuging it at 10,000-15,000×g for 1-2 times, with a single centrifugation time of 50-70 minutes, to obtain a third supernatant.

7. The extraction method according to claim 6, characterized in that The microporous membrane filtration includes sequentially performing primary filtration and secondary filtration on the third supernatant; the filtration accuracy of the primary filtration is 0.4-0.6 μm; the filtration accuracy of the secondary filtration is 0.1-0.3 μm.

8. The extraction method according to claim 1, characterized in that The column purification and enrichment comprises adding the milk-derived exosome filtrate to an exosome purification column, eluting, and collecting 4-6 fractions after the void volume to obtain an exosome enrichment solution.

9. The extraction method according to claim 8, characterized in that The concentration includes placing the exosome enriched solution in an ultrafiltration tube with a molecular weight cut-off of 80-150 KD, and centrifuging at a centrifugal force of 3500-4500×g for 20-40 minutes.

10. Use of milk-derived exosomes obtained by the extraction method according to any one of claims 1 to 9 in the preparation of a drug for treating neonatal small intestinal necrotizing colitis.

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