Extraction method and application of dermal fat cell lipid component

Abstract

The invention discloses an extraction method and application of a dermal fat cell lipid component. The extraction method comprises the following steps: (1) extracting primary dermal fibroblasts from a 0th-day mouse; (2) carrying out planking culture on the primary dermal fibroblasts by using a DMEM + + + culture medium until the cell adherence density reaches 100%, then carrying out in-vitro adipogenic differentiation induction culture by using an adipogenic differentiation culture medium, then carrying out maintenance culture by using a maintenance culture medium until the cells are in an early fat state, and separating to obtain supernate of early differentiation culture cells; and (3) extracting and separating the supernate obtained in the step (2) by using a Matyash method to obtain the dermal fat cell lipid component. According to the method, lipid and protein components in the early differentiated fat cell culture supernatant are extracted and separated, and the obtained lipid component has a remarkable immune regulation function and can remarkably inhibit the inflammatory state of macrophages and proliferation of keratinocytes.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for extracting lipid components of dermal adipocytes and an application thereof. Background Art

[0002] Psoriasis is a common chronic inflammatory skin disease caused by the combined effects of genetic and environmental factors, leading to autoimmune-mediated pathological changes. Its typical morphological features are red thickened plaques with clear borders and covered with silvery scales. Psoriasis patients often have multiple comorbidities, such as psoriatic arthritis, hypertension, obesity, type 2 diabetes, dyslipidemia and cardiovascular disease, which bring great burdens to patients' lives and psychology. It is estimated that about 125 million people worldwide (about 1.7% of the world's population) are affected by psoriasis.

[0003] The histological characteristics of psoriasis are mainly reflected in three aspects: first, the epidermis is thickened, and excessive proliferation of keratinocytes leads to accumulation of scales; second, incomplete keratinization leads to the presence of cell nuclei in the keratinized layer; finally, the dermal blood vessels are dilated and prominent, and the dilated and twisted blood vessels extend to the tips of the dermal papillae. In addition, a large number of infiltrations of T lymphocytes, macrophages, mast cells and neutrophils appear in the dermis and epidermis. These cells communicate through cytokines such as TNFα, IFNγ and IL-1, mediate the interaction between the innate immune system and the acquired immune system, activate keratinocytes, drive excessive proliferation of the epidermis and produce more inflammatory factors, thereby forming an inflammatory cycle that maintains the continued development of psoriasis.

[0004] At present, there is no specific drug for psoriasis. Common treatment methods include topical therapy, phototherapy, traditional therapy and biologic therapy. Topical therapy often uses drugs such as glucocorticoids and vitamin D derivatives, but there are side effects such as long treatment cycles and skin irritation. For patients with moderate to severe psoriasis, phototherapy and photochemotherapy are effective but time-consuming and are usually only used for short-term control of the disease. In recent years, the development of biologics has provided new methods for the treatment of psoriasis. Currently, the approved biologics are mainly recombinant monoclonal antibodies or receptor fusion proteins. There are four main types of biologics used to treat moderate to severe psoriasis: anti-TNFα, anti-IL17, anti-IL12 / 23p40 and anti-IL23p19. However, the use of biologics is risky, and it is easy to develop drug resistance. The disease is prone to relapse after discontinuation of medication, and its highly specific targeting of inflammatory mediators may lead to changes in clinical and immunological characteristics. Summary of the invention

[0005] The present invention aims to overcome the defects of the prior art and provide a method for extracting lipid components of dermal adipocytes.

[0006] Another object of the present invention is to provide the use of the dermal adipocyte lipid component obtained by the above extraction method.

[0007] The technical solution of the present invention is as follows:

[0008] A method for extracting lipid components from dermal adipocytes comprises the following steps:

[0009] (1) Primary dermal fibroblasts were extracted from mice on day 0 after birth;

[0010] (2) The primary dermal fibroblasts are plated and cultured with DMEM+++ medium until the cell attachment density reaches 100%, and then induction culture is performed in vitro with adipogenic differentiation medium, and then maintenance culture is performed with maintenance medium until the cells are in an early fat state, and the supernatant of the early differentiated cultured cells is separated;

[0011] (3) Extracting and separating the supernatant obtained in step (2) by the Matyash method to obtain the dermal adipocyte lipid component.

[0012] In a preferred embodiment of the present invention, the formula of the DMEM+++ medium is shown in the following table:

[0013] .

[0015] Further preferably, the formula of the adipogenic differentiation medium is shown in the following table:

[0016] Reagents Dosage Dexamethasone 2μM 3-Isobutyl-1-methylxanthine 250μM Indomethacin 200μM insulin 10 μg / mL DMEM+++ .

[0017] Further preferably, the formula of the maintenance medium is shown in the following table:

[0018] Reagents Dosage insulin 10 μg / mL DMEM+++ .

[0019] The use of the dermal adipocyte lipid component obtained by the above extraction method in preparing a psoriasis treatment composition.

[0020] In a preferred embodiment of the present invention, the psoriasis treatment composition further comprises bee sponge spicules.

[0021] A psoriasis treatment composition, whose effective components include the dermal adipocyte lipid components obtained by the above extraction method.

[0022] In a preferred embodiment of the present invention, bee sponge spicules are also included.

[0023] Further preferably, the effective components are the dermal adipocyte lipid component and bee sponge spicules obtained by the extraction method.

[0024] The beneficial effects of the present invention are:

[0025] 1. The present invention extracts and separates lipid and protein components in the culture supernatant of early differentiated adipocytes, and the obtained lipid components have significant immunoregulatory function and can significantly inhibit the inflammatory state of macrophages and the proliferation of keratinocytes.

[0026] 2. The serum-free culture medium used in the present invention is used for maintaining the culture of adipocytes, which does not affect the production and secretion of anti-inflammatory substances, thereby ensuring the maintenance of anti-inflammatory activity during the culture process.

[0027] 3. The present invention mixes the lipid components in the culture supernatant of early differentiated adipocytes with bee sponge spicules to prepare a composition that can effectively relieve inflammation of the back skin of psoriasis mice, effectively achieving the effect of treating psoriasis and having good market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The adipogenic differentiation timeline of dermal fibroblasts and the time points for collecting the cell culture supernatant in Example 1 of the present invention are shown.

[0029] Figure 2 The RNA expression level of the characteristic genes of adipocytes during the adipogenic differentiation of dermal fibroblasts in Example 1 of the present invention is shown

[0030] Figure 3 The expression levels of inflammatory factor RNA after co-culture of macrophages and adipocyte culture supernatant in Example 2 of the present invention are shown.

[0031] Figure 4 The phenotype of psoriasis mice after eAd_CM treatment in Example 2 of the present invention is shown.

[0032] Figure 5 The in vitro anti-inflammatory effects of the culture supernatants of adipocytes collected from cultures at different fetal bovine serum concentrations in Example 3 of the present invention are compared.

[0033] Figure 6 The expression levels of inflammatory factor RNA after co-culture of macrophages with each component in eAd_CM in Example 5 of the present invention are shown.

[0034] Figure 7 The phenotype of psoriasis mice after combined treatment with eAd_lipids and bee sponge spicules in Example 6 of the present invention is shown.

[0035] Figure 8The lipidomics analysis of the culture supernatant of adipocytes at different differentiation stages in Example 7 of the present invention is shown. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0037] Example 1 Extraction, adipogenic differentiation and collection of culture supernatant of primary dermal fibroblasts

[0038] The mice on day 0 after birth were taken out, decapitated, and the limbs were cut off. The skin was peeled off, and after removing the excess fat and mucosal tissue, the skin was immersed in 2 mL of DMEM containing 2 mg / mL Dispase II and incubated overnight at 4°C with rotation. The next day, HBSS buffer (HBSS++, the formula is shown in Table 1) containing 20 mg / mL bovine serum albumin and antibiotics was prepared, and the epidermis was carefully removed, washed with 1×PBS, HBSS++ was added, and then minced into small pieces (1-2 mm wide strips). The minced tissue was transferred to a centrifuge tube. The dermis of each newborn mouse required 10 mL of digestion solution (HBSS+1 mg / mL Dnase I+2 mg / mL collagenase IV). After adding the digestion solution, it was heated in a 37°C water bath for 5 minutes, and then digested on a shaker at 37°C for 2 hours. After the digestion was completed, the centrifuge tube was placed in ice for 5 minutes to terminate the digestion. The digested tissue suspension was filtered through 100 μm and 40 μm filters to remove hair, tissue and other incompletely digested tissues, and the filtrate was collected and centrifuged at 4°C and 1400 rpm for 5 min. The supernatant was removed and the red blood cells were lysed. After centrifugation, the cells were resuspended in preheated DMEM+++ medium (the formula of DMEM+++ medium is shown in Table 2) and cultured at 37°C and 5% CO2.

[0039] Table 1 HBSS++ formulation

[0040]

[0041] After the cells have attached to the wall for 8-12 hours, fresh DMEM+++ medium (formula as shown in Table 2) is replaced and culture is continued at 37°C, 5% CO2. The same fresh DMEM+++ medium is then replaced every 2 days, and culture is carried out at 37°C, 5% CO2 until the cell density reaches 100%. 2 days later, the medium is replaced with adipogenic differentiation medium (as shown in Table 3, 2 μM dexamethasone, 250 μM 3-isobutyl-1-methylxanthine, 200 μM indomethacin, 10 μg / mL insulin are added to DMEM+++), and differentiation culture is carried out at 37°C, 5% CO2. Figure 1As shown, fresh adipogenic differentiation medium was replaced on the 2nd day of differentiation culture, and fresh maintenance medium was replaced on the 3rd day of differentiation culture (the formula is shown in Table 4, 10 μg / mL insulin was added to DMEM+++), and maintenance culture was carried out at 37°C and 5% CO2, and fresh maintenance medium was subsequently replaced every 3 days.

[0042] Table 2 Formulation of DMEM+++

[0043]

[0044] Table 3 Formulation of adipogenic differentiation medium

[0045] Reagents Dosage brand model Dexamethasone 2μM Sigma D4902 3-Isobutyl-1-methylxanthine 250μM Sigma I5879 Indomethacin 200μM Sigma I8280 insulin 10 μg / mL Sigma 91077C DMEM+++

[0046] Table 4 Formulation of maintenance medium

[0047] Reagents Dosage brand model insulin 10 μg / mL Sigma 91077C DMEM+++

[0048] At the time before adipogenic differentiation, at the beginning of adipogenic differentiation, and at 6h, 1d, 2d, 3d, 6d, and 9d after induction of adipogenic differentiation, the corresponding culture medium was removed, trizol was added and the cells were slowly pipetted to lyse the cells, and the cells were collected in 1.5mL centrifuge tubes to extract RNA. qRT-PCR experiments were performed to detect the expression levels of adipocyte characteristic genes, so as to distinguish adipocytes at different differentiation stages.

[0049] The results are as follows Figure 2As shown, compared with undifferentiated cells and other differentiation time points, adipocytes on the 3rd day of differentiation culture were in an early adipocyte (eAd) state, characterized by the peak expression of the antimicrobial peptide Camp; and as the number of days of adipocyte differentiation increased, cells on the 9th day of differentiation were in a mature adipocyte (mAd) state, characterized by a significant loss of Camp expression (less than 10% of eAd) and maintained Fabp4 expression (maintained at 50% to 100% of eAd). Therefore, in this embodiment, the high expression of Camp is used as the definition index of eAd, and the relevant research on defining the period of adipocyte differentiation can be found in the research paper: Zhang LJ, Guerrero-Juarez CF, Chen SX, Zhang X, Yin M, Li F, Wu S, Chen J, Li M, Liu Y, Jiang SIB, Hata T, Plikus MV, Gallo RL. Diet-induced obesity promotes infection by impairment of the innate antimicrobial defense function of dermal adipocyte progenitors. Sci Transl Med. 2021 Jan 20; 13 (577): eabb5280. Among them, the early fat state (eAd) is equivalent to the Early adipocytes mentioned in the above research paper, and the mature fat state (mAd) is equivalent to the Adipocytes (AD) mentioned in the above research paper.

[0050] The cell culture supernatants obtained from differentiation day -2 to 0, differentiation day 3 to 6, and differentiation day 9 to 12 were collected and recorded as undif_CM, eAd_CM, and mAd_CM, respectively. Figure 1 At the same time, the cell-free blank maintenance medium placed under the same culture conditions for 3 days was collected as a control, recorded as BM. The collected cell culture supernatant was centrifuged at 4°C and 3000 rpm for 5 minutes to remove dead cells or cell debris, and then frozen in a -80°C refrigerator for subsequent analysis.

[0051] Example 2 Anti-inflammatory property test of dermal adipocyte culture supernatant

[0052] In vitro anti-inflammatory performance test: Peritoneal macrophages were extracted from C57BL / 6 mice aged 2 to 3 months, and cultured in DMEM++ medium (as shown in Table 5) at 37°C and 5% CO2. Fresh DMEM++ medium was replaced every other day to remove non-adherent cells. After continuing to culture for 24 hours, the medium was changed, and BM, undif_CM, eAd_CM and mAd_CM prepared in Example 1 were added respectively. The co-culture system was DMEM++: CM = 1:1 (volume ratio). After 2 hours of co-culture, LPS was added to activate macrophage inflammation. After 12 hours of stimulation, the cells were collected to extract RNA, and qRT-PCR experiments were performed to detect the expression levels of inflammatory factors in macrophages. The results are as follows: Figure 3 As shown, eAd_CM can significantly downregulate the RNA levels of Il1b, Nos2 and Cxcl2, indicating that only early adipocytes can secrete anti-inflammatory substances in large quantities to exert unique anti-inflammatory effects.

[0053] Table 5 DMEM++ formulation

[0054]

[0055] In vivo anti-inflammatory performance test: 7-8 week old C57BL / 6 female mice were randomly divided into two groups. The backs were depilated, and then 40 mg of imiquimod cream was applied to a 2 cm × 2 cm area to induce psoriasis-like lesions for six consecutive days. Starting from the second day of application, 100 μL of BM or eAd_CM was intradermally injected into the back skin of the two groups of mice every morning and evening. On the seventh day, the non-lesional and lesional areas of the back of the mice were collected, and the PASI score and qRT-PCR experiment were used to analyze the therapeutic effect of eAd_CM on psoriasis-like inflammation in mice. The results are as follows: Figure 4 As shown in the figure, after eAd_CM injection, the levels of inflammatory factor Il1b and epidermal cell proliferation (Krt6a) decreased, indicating that the degree of psoriasis-like lesions in mice was alleviated, reflecting the good effect of eAd_CM in the treatment of psoriasis.

[0056] Example 3 Test of the culture effect of serum-free medium

[0057] According to the method in Example 1, adipogenic differentiation of dermal fibroblasts was induced in vitro. On the third day of differentiation, maintenance medium containing 10% fetal bovine serum, 5% fetal bovine serum, 2% fetal bovine serum, 1% fetal bovine serum and serum-free medium were used to culture cells. The concentration of fetal bovine serum did not affect the differentiation efficiency and growth of adipocytes. On the sixth day of differentiation, the adipocyte culture supernatant was collected, and the difference in in vitro anti-inflammatory effect of the adipocyte culture supernatant containing different fetal bovine serum concentrations was compared according to the method in Example 2. The results are shown in FIG. Figure 5As shown, the concentration of fetal bovine serum does not change the ability of adipocytes to secrete anti-inflammatory substances. Therefore, this example uses serum-free medium to culture early adipocytes on days 3 to 6 of differentiation, and collects adipocyte culture supernatants for subsequent experiments in Examples 4 to 7.

[0058] Example 4 Lipid extraction from early adipocyte culture supernatant

[0059] The lipids, polar metabolites and proteins in serum-free BM and eAd_CM were extracted and separated using the Matyash method. The specific implementation scheme is as follows:

[0060] Take out serum-free BM and eAd_CM from the -80℃ refrigerator and thaw, add methyl tert-butyl ether and methanol, the volume ratio of methyl tert-butyl ether, methanol and sample is 10:3:2.5, vortex for 1min, and sonicate in an ice water bath for 10min. Centrifuge at 4℃ and 4000rpm for 15min. After centrifugation, it can be observed that the solution is divided into two layers and a white precipitate appears. The upper solution is the lipid layer, and the white precipitate is the protein. They are collected in new centrifuge tubes respectively. The liquid in each component is blown clean by nitrogen blowing at room temperature, and freeze-dried to completely remove the liquid. Finally, serum-free maintenance medium is used to resuspend each component to the original volume before extraction, and after fully dissolving, it is frozen in a -80℃ refrigerator for subsequent analysis.

[0061] Example 5 In vitro efficacy verification of lipids

[0062] According to the method in the "In vitro anti-inflammatory performance test" in Example 2, it is verified whether the components extracted and separated from eAd_CM can exert anti-inflammatory effects. The specific implementation plan is as follows:

[0063] Peritoneal macrophages were extracted from 2-3 month old C57BL / 6 mice. Fresh DMEM++ medium was replaced every other day after plating to remove non-adherent cells. After 24 hours of culture, the medium was changed and BM, eAd_lipids and eAd_proteins were added respectively. The co-culture system was DMEM++: eAd_lipids (or eAd_proteins) = 1:1. After 2 hours of co-culture, LPS was added to activate macrophage inflammation. After 12 hours of stimulation, cells were collected to extract RNA, and qRT-PCR experiments were performed to detect the expression level of inflammatory factors in macrophages. The results are shown in the figure. Figure 6 As shown, eAd_lipids can significantly downregulate the RNA levels of Il1b and Cxcl2, indicating that only lipids secreted by early adipocytes can stably exert significant anti-inflammatory effects.

[0064] Example 6 Preparation of lipid and bee sponge spicule composition and in vivo efficacy verification

[0065] In order to further verify the in vivo efficacy of eAd_lipids, this example uses bee sponge spicules and lipids to prepare a composition and apply it to a psoriasis-like mouse model. Bee sponge spicules can help lipids better penetrate into the skin to play a therapeutic role. The specific preparation method is as follows: Take 1 mg of bee sponge spicule powder and resuspend it with 100 μL BM_lipids solution or eAd_lipids solution.

[0066] In vivo anti-inflammatory property test: 7-8 week old C57BL / 6 female mice were randomly divided into two groups, their backs were depilated, and 25 mg of imiquimod cream was applied to a circular area of ​​1.77 cm2 on the back to induce psoriasis-like lesions, and the application was continued for six days. Starting from the second day of application, 100 μL of BM_lipids or a mixture of eAd_lipids and bee sponge spicules were dripped on the modeling area on the backs of the two groups of mice every day, and manual massage was performed for 2 minutes to assist absorption. On the seventh day, the non-lesional and lesional areas on the backs of the mice were collected, and the PASI score and qRT-PCR experiment were used to analyze the therapeutic effect of eAd_CM on psoriasis-like inflammation in mice. The results are as follows: Figure 7 As shown, eAd_lipids can reduce the expression of inflammatory factor Il1b and the proliferation of epidermal cells (Krt6a), indicating that the degree of psoriasis-like lesions in mice was alleviated, reflecting the good effect of eAd_lipids in the treatment of psoriasis.

[0067] Example 7 Lipidomic analysis of culture supernatants of adipocytes at different differentiation stages

[0068] In order to further clarify the specific lipid components in early adipocytes that exert anti-inflammatory effects, this example performed lipidomics analysis on the culture supernatant of adipocytes at different differentiation stages. Figure 8 As shown in the figure, eAd_CM is mainly enriched with a large amount of glycerophospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), as well as lysophospholipids, including lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). In contrast, the culture supernatant of mature adipocytes (mAd-CM) is mainly enriched with triglycerides (TG) and free fatty acids (FFAs). The above results indicate that early adipocytes may exert anti-inflammatory effects by secreting glycerophospholipids and lysophospholipids.

[0069] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for extracting lipid components from dermal adipocytes, characterized in that: The steps include: (1) Primary dermal fibroblasts were extracted from mice on day 0 after birth; (2) The primary dermal fibroblasts are plated and cultured with DMEM+++ medium until the cell attachment density reaches 100%, and then induction culture is performed in vitro with adipogenic differentiation medium, and then maintenance culture is performed with maintenance medium until the cells are in an early fat state, and the supernatant of the early differentiated cultured cells is separated; (3) Extracting and separating the supernatant obtained in step (2) by the Matyash method to obtain the dermal adipocyte lipid component.

2. The extraction method according to claim 1, wherein: The formula of the DMEM+++ medium is shown in the following table:

3. The extraction method according to claim 2, wherein: The formula of the adipogenic differentiation medium is shown in the following table: 。 4. The extraction method according to claim 2, characterized in that: The formula of the maintenance medium is shown in the following table: 。 5. Use of the dermal adipocyte lipid fraction obtained by the extraction method according to any one of claims 1 to 4 in preparing a composition for treating psoriasis.

6. The use according to claim 5, characterized in that: The psoriasis treatment composition also includes bee sponge spicules.

7. A psoriasis treatment composition, wherein the effective component comprises the dermal adipocyte lipid component obtained by the extraction method according to any one of claims 1 to 4.

8. A psoriasis treatment composition according to claim 7, characterized in that: Also includes bee sponge spicules.

9. A psoriasis treatment composition according to claim 8, characterized in that: The effective components are the dermal fat cell lipid component and the bee sponge spicules obtained by the extraction method.

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