Composition for promoting differentiation of intestinal immune cells as well as preparation method and application of composition

Abstract

The invention provides a composition for promoting differentiation of intestinal immune cells as well as a preparation method and application of the composition. According to the composition, 1, 3-dioleic acid-2-palmitic acid triglyceride and breast milk oligosaccharide are used in a combined manner, so that monocytes can be effectively promoted to differentiate into intestinal macrophages, and inflammation is reduced to a certain extent. After the monocytes are differentiated into the intestinal macrophages, the intestinal immune homeostasis and intestinal microbiota inflammatory response can be regulated, so that the mucosal immune homeostasis and intestinal epithelial cell barrier function are maintained. Therefore, the composition provided by the invention can improve the intestinal immune barrier by promoting the monocytes to differentiate into the intestinal macrophages, and provides a new thought for improving the early intestinal barrier of infants through nutritional supplement.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to a composition for promoting differentiation of intestinal immune cells, and a preparation method and application thereof. BACKGROUND

[0002] The intestine is the largest independent immune system in the body, and intestinal macrophages (IMs) are key regulators of intestinal immune homeostasis and intestinal microbiota inflammatory response, and are crucial for maintaining mucosal immune homeostasis and intestinal epithelial cell barrier function. The natural immune response induced by intestinal macrophages is the first line of defense against pathogen infection in the intestine. Intestinal macrophages are distributed throughout the intestinal mucosa, mainly in the intestinal lamina propria, and secondarily in the intestinal wall smooth muscle layer. These intestinal macrophages can regulate the inflammatory response caused by pathogenic bacterial infection or antigen destruction of the epithelium, protect the mucosa from pathogens, and clean up necrotic debris of tissues and cells at the site of injury.

[0003] Studies have shown that the macrophage pool in the adult intestine comes from classical hematopoietic cells, rather than from yolk sac (YS)-derived self-renewing precursor cells, and this process needs to continue throughout adult life to maintain normal macrophage numbers. Therefore, how to promote the differentiation of monocytes derived from hematopoietic stem cells into intestinal macrophages to perfect the intestinal immune barrier is a problem that those skilled in the art are eager to solve. SUMMARY

[0004] The present application provides a composition which can effectively promote the differentiation of monocytes into intestinal macrophages and reduce inflammation to a certain extent.

[0005] The present application provides a nutritional composition which can perfect the intestinal immune barrier by promoting the differentiation of intestinal monocytes into intestinal macrophages, and provides a new idea for perfecting the intestinal barrier of infants and young children in early life through nutritional supplementation.

[0006] The present application also provides the use of the above-mentioned composition and / or the above-mentioned nutritional composition in promoting the differentiation of intestinal immune cells, in the preparation of a medicine for preventing, treating or adjuvant treatment of diseases related to intestinal immune function, and in the preparation of a food, health product, health food, functional food or special medical purpose food for improving intestinal immune function.

[0007] The present application provides a composition, wherein the composition comprises 1,3-dioleic acid-2-palmitic acid triglyceride and human milk oligosaccharide.

[0008] The composition described above, wherein the mass ratio of 1,3-dioleic acid-2-palmitic acid triglyceride and human milk oligosaccharide is (2-25): 1.

[0009] The composition as described above, wherein the human milk oligosaccharide comprises 2'-fucosyllactose.

[0010] The composition as described above, wherein the mass ratio of 1,3-dioleoyl-2-palmitoyl triglyceride and 2'-fucosyllactose is (2-25):1.

[0011] The composition as described above, wherein the human milk oligosaccharide further comprises lacto-N-neotetraose.

[0012] The present application provides a nutritional composition, wherein the nutritional composition comprises the composition described above and at least one of breast milk, skim milk, lactose, whey powder, edible vegetable blend oil, docosahexaenoic acid oil powder, eicosapentaenoic acid oil powder.

[0013] The present application provides an application of the composition described above and / or the nutritional composition described above in promoting differentiation of intestinal immune cells.

[0014] The present application provides an application of the composition described above and / or the nutritional composition described above in preparing an intestinal immune cell differentiation agonist.

[0015] The present application provides an application of the composition described above and / or the nutritional composition described above in preparing a medicine for preventing, treating or adjuvant treating a disease related to intestinal immune function.

[0016] The present application provides an application of the composition described above and / or the nutritional composition described above in preparing a food, health product, health food, functional food or special medical purpose food for improving intestinal immune function.

[0017] The present application provides a composition, by jointly using 1,3-dioleoyl-2-palmitoyl triglyceride and human milk oligosaccharide, monocytes can be effectively promoted to differentiate into intestinal macrophages, and inflammation can be reduced to a certain extent. After the differentiation of monocytes into intestinal macrophages, the intestinal immune homeostasis and the intestinal microbiota inflammatory response can be adjusted, so as to maintain the mucosal immune homeostasis and the intestinal epithelial cell barrier function. Therefore, the composition provided by the present application can improve the intestinal immune barrier by promoting the differentiation of monocytes into intestinal macrophages, and provides a new idea for improving the intestinal barrier of infants in early stage through nutritional supplementation. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Cell proliferation chart of the control group (con), component A (OPO), component B (2'-FL) and component C (LNnT) in Example 2;

[0019] Figure 2Cell proliferation graph for control (con), A+B=2:1, B+C=1:1, A+(B+C)=2:1 in Example 2;

[0020] Figure 3 Cell proliferation graph for control (con), A+B=10:1, B+C=2:1, A+(B+C)=10:1 in Example 2;

[0021] Figure 4 Cell proliferation graph for control (con), A+B=25:1, B+C=5:1, A+(B+C)=25:1 in Example 2;

[0022] Figure 5 qPCR amplification curve graph in Example 3;

[0023] Figure 6 qPCR melting curve graph in Example 3;

[0024] Figure 7 mRNA relative expression amount graph of CX3CR1 in Example 3;

[0025] Figure 8 mRNA relative expression amount graph of F4 / 80 in Example 3;

[0026] Figure 9 mRNA relative expression amount graph of Ly6C in Example 3;

[0027] Figure 10 mRNA relative expression amount graph of CD121b. DETAILED DESCRIPTION

[0028] In order for those skilled in the art to better understand the solutions of the present application, the present application is further described in detail below. The following specific embodiments are only used to describe the principles and characteristics of the present application, and the examples are only used to explain the present application, and do not limit the scope of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0029] In order to promote the differentiation of monocytes into intestinal macrophages to perfect the intestinal immune barrier, the present application provides a composition comprising 1,3-dioleic acid-2-palmitic acid triglyceride and breast milk oligosaccharide in the first aspect.

[0030] Among them, 1,3-dioleic acid-2-palmitic acid triglyceride (OPO structured fat) is a fatty acid similar to the glyceride structure type in breast milk, generally using plant oil as raw material, using enzyme ester exchange or acid hydrolysis reaction to esterify palmitate in ordinary plant oil to the Sn-2 position of the glycerol backbone, so that the proportion of 2-position palmitic acid is as high as 40% or more. With the unique structure of 2-position palmitic acid, OPO structured fat is not easy to form calcium soap after being digested and absorbed, is not easy to cause infantile constipation, is easier to digest and absorb fatty acids and calcium, and is often used in formula milk powder.

[0031] Human milk oligosaccharides (HMOs) are the third most abundant solid component in breast milk (next to fat and lactose), not only have the effects of prebiotics, anti-adhesion, antibacterial and prevention of infant infection, but also can regulate the response of intestinal epithelial cells and immune cells, reduce excessive mucosal leukocyte infiltration and activation, reduce the risk of neonatal necrotizing enterocolitis, and provide sialic acid as a potential essential nutrient for infant brain development and cognition.

[0032] Experiments have found that it is difficult to promote the transdifferentiation of monocytes to intestinal macrophages by using 1,3-dioleic acid-2-palmitic acid triglyceride or human milk oligosaccharides alone; only by using 1,3-dioleic acid-2-palmitic acid triglyceride and human milk oligosaccharides in combination can the differentiation of monocytes to intestinal macrophages be effectively promoted, and inflammation can be reduced to a certain extent. Since monocytes differentiated into intestinal macrophages can regulate intestinal immune homeostasis and intestinal microbiota inflammatory response, thereby maintaining mucosal immune homeostasis and intestinal epithelial cell barrier function. Therefore, the composition provided by the present application can perfect the intestinal immune barrier by promoting the differentiation of monocytes to intestinal macrophages, and provide a new idea for perfecting the early intestinal barrier of infants and young children through nutritional supplementation.

[0033] In a specific embodiment, applying 1,3-dioleic acid-2-palmitic acid triglyceride and human milk oligosaccharides to monocytes can effectively promote the high expression of CX3C chemokine receptor 1 (CX3CR1), adhesion G protein-coupled receptor E1 (F4 / 80) and interleukin-1 receptor type II (CD121b), and reduce the expression of lymphocyte antigen 6 complex (Ly6C) to a certain extent. Existing researches show that the macrophages in the intestinal lamina propria of mice are marked by the expression of Ly6C, CX3CR1, F4 / 80 and CD121b, and the higher the expression amount of Ly6C on intestinal macrophages, the higher the level of inflammation. Therefore, the composition provided by the present application can promote the high expression of CX3CR1, F4 / 80 and CD121b and reduce the expression of Ly6C, which proves that it can promote the differentiation of monocytes to intestinal macrophages and reduce inflammation to a certain extent.

[0034] Further, the mass ratio of 1,3-dioleoyl-2-palmitoyl triglyceride and human milk oligosaccharide is (2-25): 1.

[0035] Experiments show that when the composition provided by the present application is used, the degree of promoting the differentiation of monocytes into intestinal macrophages and the degree of reducing inflammation can be controlled by regulating the mass ratio of 1,3-dioleoyl-2-palmitoyl triglyceride and human milk oligosaccharide. Specifically, when the mass ratio of 1,3-dioleoyl-2-palmitoyl triglyceride and human milk oligosaccharide is (2-25): 1, the differentiation-promoting and anti-inflammatory effect is better. For example, the mass ratio of 1,3-dioleoyl-2-palmitoyl triglyceride and human milk oligosaccharide can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, etc.

[0036] Among them, when the ratio of 1,3-dioleoyl-2-palmitoyl triglyceride and human milk oligosaccharide is (10-25): 1, the composition provided by the present application has more obvious effect on promoting the differentiation of monocytes into intestinal macrophages and reducing inflammation.

[0037] In a specific embodiment, the human milk oligosaccharide includes 2'-fucosyllactose.

[0038] 2'-fucosyllactose (2'-FL) is a non-reducing trisaccharide composed of lactose and L-fucose, and its content in human milk oligosaccharide accounts for 31%. It has obvious effect on immunity improvement and intelligence development of infants.

[0039] Experiments show that the use of 2'-fucosyllactose in combination with 1,3-dioleoyl-2-palmitoyl triglyceride can further promote the differentiation of monocytes into intestinal macrophages.

[0040] Further, the mass ratio of 1,3-dioleoyl-2-palmitoyl triglyceride and 2'-fucosyllactose is (2-25): 1.

[0041] It is found through experiments that the degree of promoting the differentiation of monocytes into intestinal macrophages and the degree of reducing inflammation can be controlled by regulating the mass ratio of 1,3-dioleoyl-2-palmitoyl triglyceride and 2'-fucosyllactose when the composition provided by the application is used. Specifically, when the mass ratio of 1,3-dioleoyl-2-palmitoyl triglyceride and 2'-fucosyllactose is (2-25):1, the differentiation-promoting and anti-inflammatory effect is better. For example, the mass ratio of 1,3-dioleoyl-2-palmitoyl triglyceride and 2'-fucosyllactose can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, and the like.

[0042] When the ratio of 1,3-dioleoyl-2-palmitoyl triglyceride and 2'-fucosyllactose is (5-15):1, the composition provided by the application has a more obvious effect on promoting the differentiation of monocytes into intestinal macrophages and reducing inflammation.

[0043] In the above technical solution, the breast milk oligosaccharide further includes lactose-N-neotetraose.

[0044] Lactose-N-neotetraose (LNnT) is composed of galactose, N-acetylglucosamine and glucose, and is the core structure of breast milk oligosaccharides naturally existing in human milk, which has the effects of prebiotics, immune regulation, anti-inflammatory, intestinal cell response regulation, antibacterial activity and antiviral activity.

[0045] It is found through experiments that the use of 2'-fucosyllactose and lactose-N-neotetraose in combination with 1,3-dioleoyl-2-palmitoyl triglyceride can not only further promote the differentiation of monocytes into intestinal macrophages, but also effectively reduce inflammation.

[0046] In an alternative embodiment, the mass ratio of 1,3-dioleoyl-2-palmitoyl triglyceride, 2'-fucosyllactose and lactose-N-neotetraose is (6-75):2:1.

[0047] It is found through experiments that the degree of promoting monocytes to differentiate into intestinal macrophages and the degree of reducing inflammation can be controlled by regulating the mass ratio of 1,3-dioleic acid-2-palmitic acid triglyceride, 2'-fucosyllactose and lacto-N-neotetraose when the composition provided by the present application is used. Specifically, the degree of promoting differentiation and anti-inflammatory effect is better when the mass ratio of 1,3-dioleic acid-2-palmitic acid triglyceride, 2'-fucosyllactose and lacto-N-neotetraose is (6-75):2:1. For example, the mass ratio can be 6:2:1, 10:2:1, 15:2:1, 20:2:1, 25:2:1, 30:2:1, 35:2:1, 40:2:1, 45:2:1, 50:2:1, 55:2:1, 60:2:1, 65:2:1, 70:2:1, 75:2:1, etc.

[0048] When the mass ratio of 1,3-dioleic acid-2-palmitic acid triglyceride, 2'-fucosyllactose and lacto-N-neotetraose is (30-75):2:1, the composition provided by the present application has more obvious effects on promoting monocytes to differentiate into intestinal macrophages and reducing inflammation.

[0049] In an alternative embodiment, the mass ratio of 2'-fucosyllactose and lacto-N-neotetraose is (1-5):1.

[0050] It is found through experiments that the mass ratio of 2'-fucosyllactose and lacto-N-neotetraose is (1-5):1 when 2'-fucosyllactose, lacto-N-neotetraose and 1,3-dioleic acid-2-palmitic acid triglyceride are used at the same time, which has more obvious effects on promoting monocytes to differentiate into intestinal macrophages and reducing inflammation. For example, the mass ratio of 2'-fucosyllactose and lacto-N-neotetraose can be 1:1, 2:1, 3:1, 4:1, 5:1, etc.

[0051] When the mass ratio of 2'-fucosyllactose and lacto-N-neotetraose is 2:1, the composition provided by the present application has the most obvious effects on promoting monocytes to differentiate into intestinal macrophages and reducing inflammation.

[0052] The second aspect of the present application provides a nutritional composition, which comprises the above-mentioned composition, and at least one of raw milk, skim milk, lactose, whey powder, edible vegetable blend oil, docosahexaenoic acid oil powder, eicosapentaenoic acid oil powder.

[0053] Further, the nutritional composition further comprises a carrier and / or a physiologically acceptable adjuvant;

[0054] The carrier includes at least one of microcapsules, microspheres, nanoparticles, liposomes; and the physiologically acceptable adjuvant includes at least one of fillers, flavoring agents, diluents, wetting agents, dispersants, binders, disintegrants, lubricants, color, flavor and odor adjusting agents, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH adjusting agents, isotonic or tonicity adjusting agents.

[0055] The nutritional composition can improve the intestinal immune barrier by promoting the differentiation of monocytes into intestinal macrophages, and provides a new idea for improving the early intestinal barrier of infants and young children through nutritional supplementation.

[0056] According to the research results, the composition provided by the first aspect of the present application or the nutritional composition provided by the second aspect of the present application can promote the high expression of CX3CR1, F4 / 80 and CD121b and reduce the expression of Ly6C, promote the differentiation of monocytes into intestinal macrophages and reduce inflammation, and can be applied to promote the differentiation of intestinal immune cells, or be applied to prepare an intestinal immune cell differentiation agonist.

[0057] Therefore, the third aspect of the present application provides the use of the above-mentioned composition and / or the above-mentioned nutritional composition in promoting the differentiation of intestinal immune cells. The fourth aspect of the present application provides the use of the above-mentioned composition and / or the above-mentioned nutritional composition in preparing an intestinal immune cell differentiation agonist.

[0058] Specifically, the above-mentioned composition and / or the above-mentioned nutritional composition can be applied to promote the differentiation of intestinal monocytes or be applied to prepare an intestinal monocyte differentiation agonist.

[0059] According to the research results, the composition provided by the first aspect of the present application or the nutritional composition provided by the second aspect of the present application can promote the high expression of CX3CR1, F4 / 80 and CD121b and reduce the expression of Ly6C, promote the differentiation of monocytes into intestinal macrophages and reduce inflammation, and can be applied to prepare a drug for preventing, treating or adjuvant treating diseases related to intestinal immune function, for example, inflammatory bowel disease, irritable bowel syndrome, infectious enteritis and other intestinal immune function related diseases.

[0060] Therefore, the fifth aspect of the present application provides the use of the above-mentioned composition and / or the above-mentioned nutritional composition in preparing a drug for preventing, treating or adjuvant treating diseases related to intestinal immune function.

[0061] In addition to the ability to promote monocyte differentiation into intestinal macrophages and reduce inflammation, the composition provided in the first aspect of the present application or the nutritional composition provided in the second aspect of the present application comprises 1,3-dioleic acid-2-palmitic acid triglyceride and breast milk oligosaccharide, has the advantages of safe ingredients and rich nutrition, and can be used in food or health products for improving intestinal immune function.

[0062] Therefore, the sixth aspect of the present application provides the use of the above-mentioned composition and / or the above-mentioned nutritional composition in the preparation of food, health products, health foods, functional foods or special medical purpose foods for improving intestinal immune function.

[0063] The food can be infant food, child food, adolescent food or adult food; the adult food can include at least one of pregnant woman food, lying-in woman food and elderly food. Specifically, the food can be at least one of infant milk powder, child milk powder, adolescent milk powder, adult milk powder, dietary supplement, snack, complementary food, nutritional product, liquid milk, non-alcoholic beverage and special medical purpose food.

[0064] The health product can be at least one of functional beverage, functional granule, functional capsule and functional powder for perfecting intestinal immune barrier, regulating intestinal flora or improving intestinal immune health, or improving intestinal inflammation.

[0065] Hereinafter, the technical solutions of the present application will be further explained and described in combination with specific examples. The experimental methods not specified in the following examples are usually carried out under conventional conditions or under the conditions recommended by the manufacturers. The reagents used, if not specifically stated, are commercially available or can be obtained from public channels.

[0066] Example 1: Preparation of experimental samples

[0067] A commercially available food nutrient fortifier 1,3-dioleic acid-2-palmitic acid triglyceride (OPO structured lipid) was selected as component A, wherein the content of OPO structured lipid was ≥40wt%.

[0068] A commercially available food nutrient fortifier 2'-fucosyllactose (2'-FL) was selected as component B, wherein the content of 2'-FL was ≥94wt% on a dry basis.

[0069] A commercially available food nutrient fortifier lacto-N-neotetraose (LNnT) was selected as component C, wherein the content of LNnT was ≥92wt% on a dry basis.

[0070] The experimental samples were obtained by compounding component A, component B or component C, and the specific preparation can be seen from Table 1.

[0071] Table 1

[0072]

[0073] Example 2: Cell activity experiment

[0074] To detect the effect of the above experimental samples on the activity of monocytes, a cell activity experiment was performed using mouse monocyte leukemia cells (Raw264.7), including the following steps:

[0075] Preheat ddH2O to 37°C, then take the cryopreservation tube containing Raw264.7 cells (containing 1 mL of cell mixture) from the liquid nitrogen tank and immediately put it into the 37°C ddH2O, and shake the cryopreservation tube quickly to dissolve it within 1 min. After complete dissolution, disinfect the outer wall of the cryopreservation tube with 75% alcohol and bring it into the clean bench. In the clean bench, add 10 mL of freshly prepared culture medium (containing 10% fetal bovine serum, 1% double-antibiotic DMEM culture medium (Thermo Fisher)) to a 15 mL sterile centrifuge tube, open the cryopreservation tube, and add all the thawed cell suspension to the centrifuge tube. Centrifuge at 1000 rpm for 3 min at room temperature (22-25°C), and aspirate the supernatant. Add 1 mL of fresh culture medium to resuspend the cell pellet, mix gently, and then add it to a T25 cell culture flask. Add fresh culture medium to 5 mL, and place it in a 37°C, 5% CO2 incubator. Replace the medium after 48 h, and subculture when the cell fusion reaches 80%.

[0076] After stable subculture, take the logarithmic growth phase cells and digest them with 0.25% trypsin for cell counting. Seed the cells into a 96-well plate at a density of 5000 cells / well, and continue to culture for 16 h. Add the experimental samples prepared in Example 1 to the 96-well plate, so that the final concentration of the experimental samples in each well is 0.02 mg / mL, and continue to culture for 0 h, 24 h, 48 h, and 72 h. Add 10% (v / v) CCK8 solution to each well, and incubate for 1 h. Select the 450 nm wavelength and measure the absorbance of each well on an enzyme-linked immunosorbent detector. See Figure 1 、 Figure 2 、 Figure 3 and Figure 4 .

[0077] Figure 1 The cell proliferation graph of the control group (con), component A (OPO), component B (2'-FL), and component C (LNnT) is Figure 2 The cell proliferation graph of the control group (con), A+B=2:1, B+C=1:1, and A+(B+C)=2:1 is Figure 3Cell proliferation graph for control (con), A+B=10:1, B+C=2:1, A+(B+C)=10:1, Figure 4 Cell proliferation graph for control (con), A+B=25:1, B+C=5:1, A+(B+C)=25:1. It can be found that neither OPO, 2'-FL, LNnT alone nor OPO, 2'-FL, LNnT in combination has a promoting effect on cell proliferation after 48 h of application. In general, when cells are in the proliferation cycle, it is not possible to be in the differentiation cycle at the same time, and therefore the composition provided by the present application has no promoting effect on the proliferation of Raw264.7 cells, indicating that it can have a promoting effect on the differentiation of Raw264.7 cells.

[0078] Example 3: Reverse transcription quantitative polymerase chain reaction

[0079] Since CX3C chemokine receptor 1 (CX3CR1), lymphocyte antigen 6 complex (Ly6C), adhesion G protein-coupled receptor E1 (F4 / 80), and interleukin-1 receptor type II (CD121b) are markers of intestinal macrophages, and the higher the expression of Ly6C on intestinal macrophages, the higher the level of inflammation. Therefore, if the mRNA expression of CX3CR1, Ly6C, F4 / 80, and CD121b is detected after the experimental sample is applied, and the mRNA relative expression of CX3CR1, F4 / 80, and CD121b is up-regulated, and the mRNA relative expression of Ly6C is down-regulated, it can be proved that the experimental sample has the ability to induce monocytes to transform into intestinal macrophages and can reduce inflammation. Among them, CX3CR1 and Ly6C are the most critical marker genes for determining the transformation of monocytes into intestinal macrophages.

[0080] According to the above research, in order to detect the influence of the experimental sample on the differentiation of intestinal immune cells, this embodiment uses a human colorectal adenocarcinoma cell (Caco-2) / Raw264.7 cell co-culture system to perform reverse transcription quantitative polymerase chain reaction (RT-qPCR) to detect the mRNA expression level of CX3CR1, Ly6C, F4 / 80, and CD121b, including the following steps:

[0081] (1) Cell treatment: add the experimental sample prepared in Example 1 to the Caco-2 / Raw264.7 cell co-culture system to make the final concentration of the experimental sample in the system 0.02 mg / mL, and continue to culture for 48 h; wherein the Caco-2 / Raw264.7 cell co-culture system uses a separated co-culture system, and Raw264.7 is placed in the lower layer of the co-culture system.

[0082] (2) RNA extraction: After the end of culture, the Raw264.7 cells in the lower layer of the co-culture system were scraped off with a cell scraper and placed in a 1.5 mL EP tube, and Trizol reagent was added for cell lysis, then 200 μL of chloroform was added, shaken and mixed, and then stood for 5 min, and centrifuged at 12000 rpm at 4°C for 10 min. The 1.5 mL EP tube was taken out of the centrifuge, and at this time the sample in the EP tube was divided into three layers, the lower layer was the organic phase layer, the middle layer and the upper layer were the aqueous phase layer, and the RNA was in the upper layer of the aqueous phase. The colorless and transparent aqueous phase layer in the upper layer was sucked into another 1.5 mL EP tube, and an equal volume of isopropanol was added, and then mixed by gently inverting up and down, and then stood for 10 min, and then centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was discarded, and 1 mL of 75% ethanol was used to wash the RNA precipitate, and then centrifuged at 4°C at 7000 rpm for 5 min, and then the supernatant was removed, and then dried at room temperature (25°C) for 5-10 min, and then 25 μL of diethyl pyrocarbonate treated water (DEPC H2O) was added, and then the RNA was fully dissolved by blowing with a gun head, and then the RNA concentration was detected using a nucleic acid protein detector, and the specific values are shown in Table 2. The obtained RNA sample was stored at -80°C.

[0083] Table 2

[0084]

[0085] (3) RT-PCR: 3 μg of RNA sample, 2.5 μL of OligodT Primer were mixed, and diethyl pyrocarbonate treated water was added to a total volume of 12.5 μL; 70°C for 5 min; then 4 μL of RT Buffer, 2 μL of dNTPs, 1 μL of RT Enzyme, and 0.5 μL of RI Enzyme were added to obtain a reaction system with a total volume of 20 μL, which was placed at 42°C for 60 min, and then at 70°C for 10 min, to obtain a cDNA sample.

[0086] (4) qPCR: The qPCR reaction system includes: 0.2 μL of cDNA sample, 5 μL of SYBR Green I mix (2X), 0.3 μL of upstream primer (m-CX3CR1-162-F, m-F4 / 80-181-F, m-Ly6C-236-F, m-CD121b-352-F or GAPDH-128F (Marker)), 0.3 μL of downstream primer (m-CX3CR1-162-R, m-F4 / 80-181-R, m-Ly6C-236-R, m-CD121b-352-R or GAPDH-128R (Marker)), and adding sterile distilled water to a total volume of 10 μL. The qPCR reaction program includes: (1) amplification: 94°C pre-denaturation for 30 s; 94°C denaturation for 5 s, 60°C annealing for 15 s, 72°C extension for 10 s, a total of 40 cycles, to obtain a qPCR amplification curve (Fig. Figure 5 ); (2) melting: 95°C for 15 s, 60°C for 60 s, 95°C for 1 s, to obtain a qPCR melting curve (Fig. Figure 6 ). According to the RT-qPCR amplification results, taking the mRNA expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the standard, the mRNA relative expression levels of CX3CR1, Ly6C, F4 / 80, and CD121b are calculated (Fig. Figure 7 , Figure 8 , Figure 9 and Figure 10 ).

[0087] Among them, the primer sequences involved in RT-PCR and qPCR can be seen in Table 3. In Table 3, the upstream primer m-CX3CR1-162-F and the downstream primer m-CX3CR1-162-R are used to confirm the mRNA expression of CX3CR1, the upstream primer m-F4 / 80-181-F and the downstream primer m-F4 / 80-181-R are used to confirm the mRNA expression of F4 / 80, the upstream primer m-Ly6C-236-F and the downstream primer m-Ly6C-236-R are used to confirm the mRNA expression of Ly6C, the upstream primer m-CD121b-352-F and the downstream primer m-CD121b-352-R are used to confirm the mRNA expression of CD121b, and the upstream primer GAPDH-128F (Marker) and the downstream primer GAPDH-128R (Marker) are used to confirm the mRNA expression of the internal reference gene GAPDH.

[0088] Table 3

[0089]

[0090] Figure 7mRNA relative expression graph of CX3CR1, Figure 8 mRNA relative expression graph of F4 / 80, Figure 9 mRNA relative expression graph of Ly6C, Figure 10 mRNA relative expression graph of CD121b. It can be found that Ly6C is expressed in all Raw264.7, indicating that Raw264.7 cells are Ly6C positive cells, which have the prerequisite basis for transforming into intestinal macrophages; when the mRNA expression of CX3CR1, F4 / 80 and CD121b is up-regulated, it indicates that the number of Raw264.7 differentiation into intestinal macrophages increases; when the mRNA expression of CX3CR1, F4 / 80 and CD121b is up-regulated and the mRNA expression of Ly6C is down-regulated, it indicates that Raw264.7 is transdifferentiated into intestinal macrophages and the inflammatory state is down-regulated.

[0091] Figures 7-9 It is shown that compared with the control group, OPO alone treatment has no significant effect on the mRNA expression levels of CX3CR1, F4 / 80 and CD121b, but can significantly up-regulate the mRNA expression level of Ly6C. Compared with the control group, 2’-FL alone treatment significantly down-regulates the mRNA expression level of CX3CR1, and has no significant effect on the mRNA expression levels of F4 / 80, Ly6C and CD121b. Compared with the control group, LNnT alone treatment has no significant effect on the mRNA expression levels of CX3CR1 and CD121b, but significantly down-regulates the mRNA expression level of F4 / 80 and significantly up-regulates the mRNA expression level of Ly6C. At the overall level, compared with OPO, 2’-FL and LNnT alone treatment, the binary composition (OPO combined with 2’-FL and 2’-FL combined with LNnT) treatment has a promoting effect on the mRNA expression levels of CX3CR1, F4 / 80 and CD121b, and an inhibitory effect on the expression level of Ly6C. At the overall level, compared with the binary composition treatment, the ternary composition treatment of OPO, 2’-FL and LNnT has a promoting effect on the mRNA expression levels of CX3CR1, F4 / 80 and CD121b, and the A+ (B+C)=25:1 group has a significant inhibitory effect on the expression of Ly6C gene.

[0092] The above results show that OPO, 2'-FL, LNnT alone has no obvious promoting effect on the transdifferentiation of monocytes into intestinal macrophages. The binary composition of OPO combined with 2'-FL has obvious promoting effect on the transdifferentiation of monocytes into intestinal macrophages, and the promoting effect is obviously better than that of single component. The binary composition of 2'-FL combined with LNnT has no obvious promoting effect on the transdifferentiation of monocytes into intestinal macrophages. The ternary composition of OPO, 2'-FL and LNnT has obvious promoting effect on the transdifferentiation of monocytes into intestinal macrophages, and is better than single component and binary composition.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A composition characterized in that, The composition comprises 1,3-dioleic acid-2-palmitic acid triglyceride and human milk oligosaccharide.

2. The composition of claim 1, wherein, The mass ratio of the 1,3-dioleic acid-2-palmitic acid triglyceride and the human milk oligosaccharide is (2-25):

1.

3. The composition according to claim 1 or 2, characterized in that, The human milk oligosaccharide comprises 2'-fucosyllactose.

4. The composition of claim 3, wherein, The mass ratio of the 1,3-dioleic acid-2-palmitic acid triglyceride and the 2'-fucosyllactose is (2-25):

1.

5. The composition of claim 3, wherein, The human milk oligosaccharide further comprises lacto-N-neotetraose.

6. A nutritional composition, characterized in that, The composition of any one of claims 1-5 and at least one of raw milk, skim milk, lactose, whey powder, edible vegetable blend oil, docosahexaenoic acid oil powder, eicosapentaenoic acid oil powder.

7. Use of the composition of any one of claims 1-5 and / or the nutritional composition of claim 6 in promoting differentiation of intestinal immune cells.

8. Use of the composition of any one of claims 1-5 and / or the nutritional composition of claim 6 in the preparation of an intestinal immune cell differentiation agonist.

9. Use of the composition of any one of claims 1-5 and / or the nutritional composition of claim 6 in the preparation of a medicament for preventing, treating or adjuvant treatment of diseases related to intestinal immune function.

10. Use of the composition of any one of claims 1-5 and / or the nutritional composition of claim 6 in the preparation of a food, health food, health-care food, functional food or special medical purpose food for improving intestinal immune function.

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