Adipose-derived stem cell exosome and application thereof in treating colitis and mesentery

Abstract

The invention provides an adipose-derived stem cell exosome and application of the adipose-derived stem cell exosome in treatment of colitis and mesentery. It is verified for the first time that the adipose-derived stem cell exosome CrF-exo from crawling adipose tissue regulates the lymphatic endothelial cell function and promotes lymphatic drainage in colon and mesentery and finally relieves inflammation of the colon and mesentery, and it is found through verification that CrF-exo-miR-132-3p promotes the lymphatic endothelial cell function and CCL21 secretion through an RASA1 / ERK1 / 2 axis; the miR-132-3p is highly expressed in CrF and inflammatory intestines of a CD patient along with increase of lymphatic vessel density; the expression of miR-132-3p in serum of a patient is also increased and is negatively correlated with an inflammation biomarker. The research result provides a new insight for the effect of the mesenteric lymphatic function in CD progress and a potential treatment method for targeting lymphatic vessels to treat CD mesenteric and intestinal lesions.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to adipose stem cell exosomes and their application in treating colitis and mesenteric inflammation. The adipose stem cells are separated from crawling adipose tissue, and the adipose stem cell exosomes can specifically be miR-132-3p. Background Art

[0002] Crohn's disease (CD) is a chronic, recurrent gastrointestinal inflammatory disease that can affect the entire intestinal wall and its surrounding structures. Creeping fat (CrF), the hypertrophic mesenteric adipose tissue (MAT) that surrounds the inflamed intestinal segment, is considered an incompletely understood hallmark of CD, and although recent evidence suggests that CrF may play a protective role in CD disease development, deciphering the detailed mechanisms and pathophysiological relevance of the interaction between creeping fat and intestinal inflammation remains urgent and challenging.

[0003] Lymphatic vessels (LVs) are responsible for transporting lymph fluid, proteins, immune cells, and digested lipids to regional lymph nodes. It has been reported that morphological and functional abnormalities of the mesenteric component in CD patients are associated with intestinal inflammation and disease progression, which is related to the response of lymphatic drainage, adipokines, and functional cytokines. Impaired lymphatic drainage leads to edema, lymphangiogenesis, impaired immune cell trafficking, and lymphatic leakage. Lymphatic leakage in MAT further stimulates adipose tissue proliferation, produces cytokines, affects immune cell function, and aggravates inflammation. This pathological process is considered to be one of the important mechanisms for the formation of CrF.

[0004] Cellular components in CrF exhibit abnormal functions under the influence of inflammation and lipid-rich microenvironment. Adipocytes in CrF are characterized by small size, large number, and release of pro-inflammatory and anti-inflammatory cytokines and adipokines. . Adipose derived stem cells (ADSCs), as an important stromal cell, have strong immune defense regulatory properties and pro-inflammatory effects, which regulate inflammatory diseases through the secretion and delivery of exosomes. Importantly, ADSCs can communicate with surrounding cells by delivering extracellular vesicles (EVs), including microvesicles, exosomes (Exo), and apoptotic bodies; among them, exosomes are produced and secreted by host cells and carry a large number of essential components, including miRNAs, proteins, and metabolites. ADSCs mainly interact and regulate other cell types through exosomes, among which miiRNA plays an important role.

[0005] CD has long been considered a lymphatic system disease. The lymphatic vessels of the intestine and mesentery of CD patients are discontinuous and highly permeable, accompanied by lymphatic leakage and drainage dysfunction. These pathological changes lead to tissue lymphatic congestion and lymphedema, further aggravating chronic inflammation, fibrosis and fat hyperplasia of the mesentery. Methods focusing on stimulating lymphatic vessel density (LVD) and improving lymphatic function have achieved satisfactory results in the treatment of CD. ADSCs pretreated with VEGF-C can effectively enhance lymphatic drainage and reduce intestinal inflammation. Blocking the VEGF-C / VEGFR-3 signaling pathway is a molecular mechanism of lymphangiogenesis, which can lead to lymphangiectasia and aggravation of experimental colitis.

[0006] An increasing number of studies have focused on ADSCs from patients with inflammatory bowel disease (IBD), and abnormal characteristics of IBD-derived ADSCs stem cells have been confirmed, but the effects of CrF-derived ADSCs on CD intestinal and mesenteric lesions are still unclear. It has been reported that ADSCs from patients with UC and CD show abnormal abilities in proliferation and adipogenic differentiation, and subcutaneous ADSCs from CD have been shown to have unique DNA methylation patterns. However, these studies have mainly focused on the external phenotype of their stem cells in vitro. Recent studies have shown that; under the influence of abnormal microenvironments such as hypoxia or inflammation, the anti-inflammatory ability of ADSCs is generally enhanced; CrF-ADSCs show a strong potential in brown differentiation, which has been shown to be an anti-inflammatory mechanism of CD MAT; CrF-derived ADSCs promote fibroblast activation and collagen deposition in intestinal fibrosis; ADSCs from CrF (ADSCs-CrF) show a potential conversion of the browning phenotype, which has been shown to be a compensatory change in CD-regulated intestinal inflammation; ADSCs-CrF activates fibroblasts through exosome delivery.

[0007] However, due to the lack of animal models, the effects of CrF-ADSCs on CD colitis and mesenteritis remain unclear. Summary of the invention

[0008] In order to overcome at least one problem existing in the prior art, and to reveal whether CrF-ADSCs-derived exosomes (CrF-exo) regulate peripheral cell function and affect CD inflammation through exosome delivery, the inventors found that exosomes secreted by CrF ADSCs (CrF-exo) can regulate mesenteric lymphatic function and ultimately affect mesenteric inflammation and colitis. The specific mechanisms include: CrF-exo delivers miR-132-3p to lymphatic endothelial cells (LECs) by targeting the RASA1 / ERK1 / 2 axis, thereby promoting lymphangiogenesis and improving lymphatic function, thereby alleviating colitis and mesenteric inflammation.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The first aspect of the present invention is to provide adipose stem cell exosomes, the separation steps of which include:

[0011] S1. Isolation and culture of adipose stem cells: The adipose tissue harvested from the crawling fat was washed with phosphate-buffered saline to remove red blood cells and digested with type I collagenase; the supernatant was removed by centrifugation, and the spheroid cells were resuspended and stored in an incubator;

[0012] S2. Isolation of exosomes: The isolated adipose-derived stem cells were cultured in a culture medium without exosomes, the culture supernatant was collected, and centrifuged once to remove cells and cell debris; exosomes were separated by a second centrifugation, and the exosome microspheres were centrifuged three times and collected and stored.

[0013] Furthermore, in step S2, the operation steps of one centrifugation are: centrifugation at 300g for 10 min at 4°C, and then centrifugation at 3000g for 30 min.

[0014] Furthermore, in step S2, the secondary centrifugation steps are: centrifugation at 10000g for 30 min and centrifugation at 100000g for 70 min.

[0015] Furthermore, in step S2, the three centrifugation steps are centrifugation at 100000g for 70 minutes.

[0016] Furthermore, the adipose stem cell exosomes include miR-132-3p, which is denoted as CrF-exo-miR-132-3p.

[0017] A second aspect of the present invention provides an application of adipose stem cell exosomes, which is selected from at least one of the following applications: application in the preparation of a drug for treating Crohn's disease, application in the preparation of a drug for treating colitis and mesenteric inflammation, and application in the preparation of a drug for regulating lymphatic function; wherein the adipose stem cells are isolated from crawling adipose tissue.

[0018] Furthermore, the adipose stem cell exosomes are prepared by any of the separation steps described in the first aspect.

[0019] Furthermore, the adipose stem cell exosomes are used to prepare a drug having at least one of the following functions: colonization in the colon and mesentery; alleviation of weight loss and shortening of colon length; alleviation of colon thickening and changes in vascular pattern; reduction of endoscopic and histological scores; reduction of weight-to-body weight ratio; reduction of adipocyte size; increase in Ucp-1 protein expression; increase in browning phenotype of mesenteric adipocytes (MAT); reduction in CD4 + T cells and F4 / 80+ The results showed that the cytokine levels of TNF-α, IL-1β and IL-6 in the colon and mesentery were significantly increased, while the number of macrophages was decreased; the levels of proinflammatory factors Tnf-α, IL-1β and IL-6 were decreased; the lymphatic vessel density (LVD) in the colon and mesentery was increased; the mesenteric lymphatic drainage was increased; the proliferation, migration and tube formation of lymphatic endothelial cells (HLECs) were enhanced; the CCL21 production of HLECs was increased; and the regulatory function of HLECs in the migration of immune cells was enhanced.

[0020] Furthermore, the exosomes include miR-132-3p, which is denoted as CrF-exo-miR-132-3p.

[0021] The third aspect of the present invention is to provide an application of exosome CrF-exo-miR-132-3p, which is selected from at least one of the following applications: application in the preparation of RASA1 inhibitors and / or ERK1 / 2 pathway activators, application as a marker in screening RASA1 regulators and / or ERK1 / 2 pathway regulators, application in the preparation of active CD detection reagents, application in the preparation of drugs for treating Crohn's disease, application in the preparation of drugs for treating colitis and mesenteric inflammation, and application in the preparation of drugs for regulating lymphatic function.

[0022] Furthermore, the active CD detection reagent is a reagent for detecting the expression level of CrF-exo-miR-132-3p in the patient's CrF, inflammatory bowel or serum.

[0023] Furthermore, the adipose stem cell exosomes regulate lymphatic function by delivering CrF-exo-miR-132-3p, including: CrF-exo-miR-132-3p promotes lymphatic endothelial function by targeting RASA1 and activating the ERK1 / 2 signaling pathway; CrF-exo-miR-132-3p targets RASA1 expression in LECs to promote lymphangiogenesis and lymphatic drainage, thereby alleviating spontaneous mesenteritis and colitis.

[0024] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0025] Mesenteric fat wrapping and thickening (i.e. creeping fat CrF) are typical features of Crohn's disease (CD), which is characterized by lymphangiogenesis and lymphatic dysfunction. However, it is still unclear whether ADSC-exo in CrF (CrF-exo) can regulate lymphatic function and further affect the occurrence of mesenteric inflammation and colitis in CD patients. The present invention uses ADSCs and exosomes isolated from CrF of CD patients and healthy mesenteric adipose tissue as research objects for experiments, and confirms that: compared with Ctrl-exo, CrF-exo significantly reduces IL-10 by promoting lymphatic drainage function. -- / -Spontaneous enteritis and colitis in mice; the mechanism by which CrF-exo promotes the function of lymphatic endothelial cells (LECs) was revealed through the miR-132-3p / RASA1 / ERK1 / 2 axis in vitro; miR-132-3p expression was significantly upregulated in CrF and corresponding intestines of active CD patients, lymphatic vessel density increased, adipocyte size decreased, and elevated serum miR-132-3p levels were also positively correlated with inflammatory biomarkers.

[0026] The present invention verifies for the first time that CrF-exo regulates LECs function and promotes lymphatic drainage in the colon and MAT, and ultimately reduces inflammation in the colon and MAT. Specifically, CrF-exo-miR-132-3p promotes LECs function and CCL21 secretion through the RASA1 / ERK1 / 2 axis, and miR-132-3p is highly expressed in CrF and inflammatory bowel of CD patients with increased LVD; serum miR-132-3p is also increased in patients and negatively correlated with inflammatory biomarkers. The above research results provide new insights into the role of mesenteric lymphatic function in CD progression and potential therapeutic approaches targeting lymphatic vessels (LVs) to treat CD mesenteric and intestinal lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0028] Figure 1 Schematic diagram of the analysis of the identification results of adipose-derived stem cells (ADSCs) and their exosomes in one embodiment of the present invention; wherein, Part A: Flow cytometry characterization of ADSCs in MAT; Part B: Oil red O staining showing adipose differentiation of ADSCs from CrF; Part C: Transmission electron microscopy showing the typical structure of exosomes; Part D: Western blot detection of the expression of TSG101, CD63, CD9 and ALIX exosome proteins; Part E: Nanoparticle tracking analysis reveals the size of exosomes;

[0029] Figure 2 Figure 1 is a schematic diagram of the results of CrF-ADSCs exosomes circulating into the mesentery and colon of mice in one embodiment of the present invention; Part A: Exosomes labeled with DiR dye were injected into the IL-10 - / - In vivo in mice; Part B: DiR-labeled exosomes detected in the colon and mesentery by imaging system; Part C: IF staining confirmed IL-10 - / - DiR-labeled exosomes in mouse mesentery;

[0030] Figure 3In one embodiment of the present invention, CrF-exo reduces IL-10 - / - Schematic diagram of the results of spontaneous colitis and mesenteric inflammation in mice I; among them, the clinical manifestations of the four groups of mice were compared, Part A: the weight curve of mice injected with exosomes for four weeks; Part B: endoscopic characteristics of the colon; Part C: colon length analysis; Part D: endoscopic examination results; Part E: endoscopic score of colitis; Part F: histological score of colitis; Part G: HE staining of colon and mesentery; Part H: mesenteric changes; Part I: ratio of mesenteric weight to body weight;

[0031] Figure 4 In one embodiment of the present invention, CrF-exo reduces IL-10 - / - Schematic diagram of spontaneous colitis and mesenteric inflammation in mice II; among them, the clinical manifestations of four groups of mice are compared. Part A: Colon and mesenteric F4 / 80 + Macrophages and CD4 + Immunohistochemical staining of T cells; BD section: relative expression of Tnf-α (B), IL-1β (C), IL-6 (D) mRNA in colon tissue; EG section: relative expression of Tnf-α (E), IL-1β (F), IL-6 (G) mRNA in mesentery; Figure 3 and Figure 4 The data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 5 per group;

[0032] Figure 5 The figure is a schematic diagram of the results of CrF-exo enhancing lymphatic endothelial function in vivo in one embodiment of the present invention; wherein, Part A: Immunohistochemical staining of Lyve-1 positive lymphatic endothelium in mouse colon and mesentery; Parts B and C: Lymphatic vessel density (LVD) analysis of colon (B) and mesentery (C); Part D: Detection of functional lymphatic vessels after oral administration of BODITY dye; Part E: Quantification of functional lymphatic vessels in mesentery; Part F: Evans blue staining to detect colon lymphatic drainage;

[0033] Figure 6 It is a schematic diagram of the results of CrF-exo enhancing lymphatic endothelial function in vitro in one embodiment of the present invention; wherein, Part A: exosomes taken up by lymphatic endothelial cells (HLECs) in vitro; Part B: HLECs migration and tube formation under CrF-exo culture; Parts C and D: quantification of HLECs migration (C) and tube formation (D) after incubation with CrF-exo; Part E: CCK-8 detection of HLECs proliferation cultured with CrF-exo; Part F: ELISA analysis of CCL21 secretion by HLECs cultured with CrF-exo; Figure 5 and Figure 6 The data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, n = 5 per group;

[0034] Figure 7 Schematic diagram of the results of improving lymphatic endothelial function by CrF-exo through the delivery of miR-132-3p in one embodiment of the present invention I; wherein, Part A: heat map of high-throughput sequencing of miRNAs of CrF-exo and Ctrl-exo; Part B: qPCR detection of the relative expression of miR-132-3p in CrF-exo and Ctrl-exo; Part C: expression of miR-132-3p in HLECs cultured with CrF-exo; Part D: inhibition of CrF-exo -miR-132-p; Part E: quantification of HLECs migration and tube formation after inhibition of CrF-exo-miR-132-p; Part F: quantification of HLECs tube formation after inhibition of CrF-exo-miR-132-p; Part G: CCK-8 detection of HLECs proliferation after CrF-exo-miR-132-p inhibition; Part H: amount of CCL21 secreted by HLECs after CrF-exo-miR-132-p inhibition;

[0035] Figure 8 Schematic diagram II of the results of CrF-exo improving lymphatic endothelial function through the delivery of miR-132-3p in one embodiment of the present invention; wherein, Part A: HLECs migration and tube formation after transfection with miR-132-3p mimics or inhibitors; Parts B and C: Analysis of the effects of different miR-132-3p expressions on HLECs migration (B) and tube formation (C); Part D: HLECs proliferation at different miR-132-3p expression levels; Part E: CCL21 secretion in HLECs conditioned medium containing miR-132-3p mimics or inhibitors; Figure 7 and Figure 8 The data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001;

[0036] Fig. 9Schematic diagram of the results of CrF-exo-miR-132-3p promoting lymphatic endothelial function by targeting RASA1 and activating ERK1 / 2 signaling pathway in one embodiment of the present invention I; wherein, Part A: Prediction of miR-132-3p downstream genes using four bioinformatics databases, TargetScan, miRDB, miRTarBase and miRanda; Part B: GO enrichment analysis of 12 common targets predicted by the four databases; Part C: Construction of a luciferase reporter vector containing WT and 3′-UTR intramutation (MUT) binding sites; Part D: Luciferase activity detected in HLECs treated with miR-132-3p mimics and normal controls (NC); Part E: Western blot of RASA1 and p-ERK1 / 2 expression in HLECs after inhibition of miR-132-3p in CrF-exo blot analysis; Part F, G: quantitative analysis of RASA1 (F) and p-ERk1 / 2 expression (G) in HLECs after inhibition of miR-132-3p in CrF-exo; Part H: western blot detection of RASA1 and p-ERK1 / 2 protein expression in HLECs with different miR-132-3p expression; Part I, J: analysis of RASA1 expression (I) and p-ERk1 / 2 activation (J) in HLECs with different miR-132-3p expression;

[0037] Fig.10 Schematic diagram II of the results of CrF-exo-miR-132-3p promoting lymphatic endothelial function by targeting RASA1 and activating the ERK1 / 2 signaling pathway in one embodiment of the present invention; wherein, parts AC: migration and tube formation of HLECs overexpressing RASA1; part D: CCK-8 method for detecting the proliferation of HLECs overexpressing RASA1; part E: the relationship between HLECs CCL21 secretion and RASA1 overexpression; part FH: migration and tube formation of HLECs after pre-incubation with U0126; part I: CCK-8 method for detecting the proliferation of HLECs after pre-incubation with U0126; part J: CCL21 secretion of HLECs after pre-incubation with U0126; Fig. 9 and Fig.10 The data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001;

[0038] Fig.11 This is a schematic diagram of the results of CrF-exo-miR-132-3p targeting Rasa1 expression in HLECs and promoting lymphangiogenesis and lymphatic drainage in vivo in one embodiment of the present invention; wherein, parts A and B: IL-10 - / -IF double staining of Lyve-1 and Rasa1 in mouse colon (A) and mesentery (B); C and D: IL-10 - / - Immunohistochemical staining of Lyve-1 in mouse colon (C) and mesentery (D); Parts E and F: IL-10 - / - LVD analysis of mouse colon (E) and mesentery (F); Parts G and H: Fluorescence microscopic appearance (G) and quantitative analysis (H) of functional lymphatic vessels after oral administration of BODITY dye; Part I: Evans blue staining of colonic lymphatic drainage; Data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0039] Fig.12 In one embodiment of the present invention, CrF-exo-miR-132-3p reduces IL-10 - / Schematic diagram of the results of mesenteric inflammation and colonic inflammation in mice I; Part A: Weight curve of mice injected with exosomes for four weeks; Part B: Colon length; Part C: Quantitative analysis; Part D: Endoscopic images; Part E: Endoscopic scores of colitis in four groups; Part F: Histological scores of colitis; Part G: HE staining of colon and mesentery; Part H: Gross characterization of mesentery; Part I: Ratio of mesentery weight to body weight;

[0040] Fig.13 In one embodiment of the present invention, CrF-exo-miR-132-3p reduces IL-10 - / Schematic diagram of the results of mesenteric inflammation and colonic inflammation in mice II; Part A: F4 / 80-positive macrophages and CD4-positive T cells in the colon and mesentery; Part BD: Relative expression of Tnf-α (B), IL-1β (C), and IL-6 (D) mRNA in colon tissue; Part EG: Relative expression of Tnf-α (E), IL-1β (F), and IL-6 (G) mRNA in the mesentery; Fig.12 and Fig.13 The data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 5 per group;

[0041] Fig.14The figure is a schematic diagram of the results that the increased expression of miR-132-3p in CD patients is closely related to LVD and inflammation in one embodiment of the present invention; wherein, Part A: expression of miR-132-3p in CrF, MAT resection margin (RMAT) and non-CD MAT (Ctrl-MAT); Part B: miR-132-3p levels in inflammatory bowel (D), intestinal resection margin (R) and non-CD bowel (Ctrl); Parts C and D: IHC (D) and LVD analysis (C) of LYVE-1 in CrF, RMAT and Ctrl-MAT; Parts E and F: LYVE-1 staining of small intestine samples using IHC method (F) and LVD method (E); Parts G and H: Spearman correlation analysis of miR-132-3p and LVD in mesentery (G) and intestine (H); Part I : qPCR detection of the expression of miR-132-3p in the serum of healthy control group (Ctrl), active CD patients (A-CD) and remitted CD patients (R-CD); Part J, K: Spearman correlation analysis of serum miR-132-3p with CRP (J) and IL-6 (K) in CD patients; Part L: Mechanism diagram of exosomal miR-132-3p-mediated lymphatic function regulation; Data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0043] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0044] In the following examples, female C57BL / 6WT mice (15 weeks old), weighing 25-30 g, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Female IL-10 - / - Mice were purchased from Cyagen Biosciences, Santa Clara, CA, USA.

[0045] In the following examples, human lymphatic endothelial cells (HLECs) and endothelial cell culture medium (ECM) were purchased from Wuhan Saios Company, and ECM contained 1% endothelial cell growth factor (ECGF), penicillin-streptomycin (P / S) and 5% fetal bovine serum (FBS). CCK-8 kit was purchased from Shanghai Yeasun Biotechnology Company, Ltd. (Shanghai, China). Matrigel was purchased from BD Biosciences (Bedford, MA, USA). ERK1 / 2 pathway inhibitor (U0126) was purchased from Selleckchem (Shanghai, China). miRNA mimics, miRNA inhibitors and transfection reagents were from Guangzhou Ribobio Co., Ltd. (Guangzhou Ribobio Co., Ltd.). RASA1 overexpression plasmid was designed and synthesized by Shanghai Genome Technology Co., Ltd. (Genomeditech). Antibodies used for western blot: anti-RASA1 (#ab40677), anti-TSG101 (#ab125011), anti-CD9 (#ab236630), anti-CD63 (#ab134054), anti-ALIX (#ab275377) and anti-Tubulin (#ab68193) were from Abcam (Cambridge, MA, USA), and anti-Phospho-p44 / 42 mitosis-activated protein kinase (MAPK) (ERK1 / 2) (#9101) and anti-p44 / 42 MAPK (ERK1 / 2) (#9102) were from Celll Signaling Technology (CST, Danvers, MA, USA).

[0046] In the following examples, patients and samples: Mesenteric adipose tissue (MAT), intestinal and serum samples were collected from January 2020 to December 2022 at the Tenth People's Hospital Affiliated to Tongji University, Shanghai and Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine; Creeping fat (CrF) and corresponding inflammatory bowel samples were collected from 25 patients undergoing intestinal resection and non-CD MAT; Normal MAT and intestinal tissue were obtained from 10 patients with colon cancer as controls (Ctrll). For serum analysis, samples were collected from another 35 CD patients and 10 healthy controls (HC). The CD Activity Index (CDAI) was used to evaluate the disease activity of CD patients, which was divided into active (>150) and remission (0-150).

[0047] In the following examples, human lymphatic endothelial cells (HLECs) were cultured in ECM supplemented with 5% FBS, 1% P / S (100 U / mL) and 1% ECG. After the HLECs concentration reached 70%, exosomes under different conditions were added at a final concentration of 10 μg / mL for 24 hours. The conditioned medium and cells were then collected for further experiments.

[0048] In the following examples, cell transfection and transduction: Negative control (miR-NC) and miR-132-3p inhibitor (miR-132-3pin) were purchased from RiboBio and transfected into cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions when cell confluence reached 50%. The final concentration of miR-132-3p inhibitor was 100 nM.

[0049] In the following embodiments, the experimental methods involved include:

[0050] Cell proliferation assay: CCK-8 kit was used to determine cell viability, and the optical density value (OD value) proportional to the number of living cells was used to determine cell viability. The absorbance at 450nm was measured using an enzyme marker. All experiments were repeated three times.

[0051] Cell migration assay: Lymphatic endothelial cells (LECs) migration assay was performed using a transwell chamber with an 8 μm pore size insert (Corning Costar). 4 LECs were suspended in serum-free medium, treated with designated reagents, placed in the upper chamber for migration assay, and medium supplemented with 5% FBS was placed in the lower chamber. After 12 hours, cells that had migrated through the membrane to the lower surface were fixed, stained, and counted.

[0052] Tube formation test: Matrigel tube formation test was performed by placing the matrix solution in a 96-well plate (100 μL / well) and gelling at 37°C for 30 minutes. 4 LECs were inoculated into the coated wells, 100 μL of ECM (containing indicator reagent) was added, and cultured at 37°C, 5% CO2 for 2 h. The LEC tube formation was observed under a microscope and photographed. The total tube length was calculated using Image J software (National Institutes of Health, Bethesda, MD, USA).

[0053] Immunofluorescence (IF) staining: MAT and intestinal samples were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned at a thickness of 5 μm. Rabbit anti-Lyve-1 (1:500, ab218535, Abcam) and rabbit anti-Rasa1 (1:100, ab40677, Abcam) were used to detect the expression of specific proteins. Secondary antibodies labeled with Cy3 (red) or Alexa 488 (green) (Servicebio) and DAPI (Servicebio, Wuhan, China) were used. IF was observed with a fluorescence microscope (Nikon). Colocalization analysis was calculated using the Manders colocalization coefficient based on pixel intensity correlation measurement.

[0054] Real-time quantitative polymerase chain reaction (qRT-PCR): Total RNA from MAT samples and HLECs was isolated using TRIzol reagent (Ambion, Thermo Fisher, Germany). Concentration and purity were determined using a NanoVue spectrophotometer (GE Healthcare, Germany). TM cDNA was synthesized using a RT kit (Takara, Ostu, Shiga, Japan). qRT-PCR was performed using a 7900HT fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) and TB Green Premix Ex Taq TM The relative mRNA expression levels were evaluated by the 2-ΔΔCT method and normalized to GAPDH expression.

[0055] Immunohistochemical staining (IHC): MAT and intestinal specimens were collected, fixed with 10% paraformaldehyde for 24 h, embedded in paraffin, and sliced ​​into 5 μm thick sections. After rehydration with 80% methanol, PBS, and PBS containing 12% BSA, they were incubated with primary antibodies at 4°C overnight. The next day, the slides were washed with TBS-Tween and incubated with secondary antibodies. Standard immunohistochemical staining was performed. The primary antibody LYVE-1 was purchased from Abcam (Cambridge, UK) at a ratio of 1:200. LYVE-1 was detected in the intestine and MAT under a 200x magnification microscope. + The lymphatic vessel density (LVD, per mm 2 ).

[0056] Western Blotting: Using Micro BCA TM After protein samples were quantified using the protein Assay Kit (Beyotime, Shanghai, China), 10 μg of protein was electrophoresed on 7.5% or 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking, the membranes were incubated with the following primary antibodies: TSG101 (1:1000), CD9 (1:1000), CD63 (1:1000), ALIX (1:1000), Tubulin (1:5000), RASA1 (1:1000), ERK1 / 2 (1:1000), p-ERK1 / 2 (1:1000) at 4°C overnight, and then incubated with the above anti-mouse or anti-rabbit secondary antibodies (1:5000) at 37°C for 1 hour. Finally, the blots were scanned using the Odyssey dual-color infrared laser imaging system (LI-COR Biosciences, Lincoln, NB, USA). ImageJ software was used to quantify the intensity of the selected bands.

[0057] miRNA target prediction: The bioinformatics databases TargetScan, miRDB, miRanDa, and mirTarBase were used to predict the target genes of miRNAs. The 12 common differentially expressed genes in the four databases were further analyzed. Gene ontology (GO) enrichment analysis was used to identify important pathways. P < 0..05 was used as the cutoff standard for significant enrichment.

[0058] Endoscopic Surgery: The experimental endoscopic device, called the “Coloview system,” consisted of a microendoscope (scope outer diameter 1.9 mm), a xenon light source, a three-chip camera, and an air pump (Karl Storz, Tuttlingen, Germany) to achieve regulated inflation of the mouse colon. The endoscopic procedure was observed on a color monitor and digitally recorded on magnetic tape (DSR-20MDP; Sony, Cologne, Germany).

[0059] Example 1 - Isolation and identification of exosomes from adipose-derived stem cells

[0060] This embodiment is a preferred method for isolating and identifying exosomes from adipose-derived stem cells (see Yin Y, Yang J, Pan Y, et al.. Chylomicrons-Simulating Sustained Drug Release in Mesenteric Lymphatiics for the Treatment of Crohn's-Like Colitis. J Crohns Colitis 2021; 15(4): 631-646.), which specifically includes the steps:

[0061] (1) Isolation and culture of adipose-derived stem cells (ADSCs);

[0062] Mesenteric adipose tissue (MAT) (from creeping fat (CrF)) was washed with phosphate buffered saline (PBS) to remove red blood cells and digested with 0.1% collagenase type I (Sigma-Aldrich) at 37°C with constant shaking. After centrifugation, the supernatant was removed and the spheroid cells were resuspended in dulbecco's modified eagle medium (DMEM) / high glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin and stored in a 5% CO2 humidified incubator at 37°C. Most ADSCs were used for passage 3 to passage 6.

[0063] (2) Isolation of exosomes;

[0064] The isolated and cultured adipose stem cells were cultured in exosome-free culture medium for 48 hours. The culture supernatant was collected and centrifuged at 300g for 10 minutes at 4°C, and then at 3000g for 30 minutes to remove cells and cell debris. The samples were then centrifuged at 10,000g for 30 minutes and 100,000g for 70 minutes to separate exosomes. Finally, the exosome microspheres were centrifuged at 100,000g for 70 minutes and stored at -80°C. The exosomes of ADSCs isolated from CrF cells of CD patients were the experimental group (CrF-exo), and the exosomes of ADSCs isolated from healthy mesenteric adipose tissue were used as the control group (Ctrl-exo).

[0065] (3) Identification of exosomes;

[0066] The identification of exosomes mainly included: the morphological observation of the isolated CrF-exo and Ctrl-exo was performed by transmission electron microscopy (TEM; FEI Tecnai Spirit 120kV). The diameter of the exosomes was detected using an LM10NTA device (Malvern). The exosomes were then dissolved in radioimmunoprecipitation assay (RIPA) buffer, and the protein concentration was determined using a biciinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). Western blotting was used to detect the expression of exosome-specific surface markers (TSG101, CD9, CD63, and ALIX).

[0067] (4) Analysis of identification results of adipose-derived stem cells (ADSCs) and their exosomes;

[0068] like Figure 1 As shown in Figure 2, ADSCs were isolated from CrF and normal MAT (Ctrl-MAT) of 25 CD patients, and flow cytometry confirmed the expression of classic mesenchymal positive markers CD90, CD44, CD29 and negative marker CD45 ( Figure 1 To further confirm the differentiation potential of ADSCs, lipid droplets and Oil Red O staining were detected in differentiated CrF ADSCs and Ctrl-MAT ADSCs ( Figure 1 The particles secreted by CrF-exo and Ctrl-exo were observed to have typical exosome morphology by transmission electron microscopy (TEM). Figure 1 WB analysis confirmed the expression of CD9, CD63, TSG101 and Alix, typical markers of exosomes ( Figure 1Nanoparticle tracking analysis (NTA) showed that the particle size of CrF-exo and Ctrl-exo was between 100-200 nm ( Figure 1 (as shown in Part E of FIG.

[0069] (4) ADSC-CrF exosomes enter IL-10 - / - Mouse mesenteric and colonic circulation;

[0070] IL-10 - / - The mouse is a widely accepted animal model for spontaneous colitis and mesenteric inflammation that mimics human Crohn's disease (CD), showing marked lymphatic dysfunction and mesenteric hypertrophy.

[0071] To investigate the effect of CrF-exo on CD progression, the distribution of exosomal CrF-exo in the colon and mesentery was verified, as Figure 2 As shown in part A, DiR fluorescently labeled exosomes were injected into the IL-10 - / - In mice, specifically: DiR dye was used to label exosomes according to the manufacturer's instructions, 2 mg / mL of dye was added to the exosome solution, incubated for 10 minutes, and then centrifuged at 100,000 g for 70 minutes to remove excess dye; 200 μg of DiR dye (Invitrogen) labeled exosomes were injected into the tail vein of mice; 12 hours later, mice were killed and fluorescent signals were detected using the IVIS Lumina II in vivo imaging system (PerkinElmer, Thermo Fisher). After 12 hours, IL-10 was detected under a fluorescence microscope. - / - Strong DiR fluorescence signals in mouse mesentery and colon ( Figure 2 In addition, IF staining confirmed the presence of abundant DiR-labeled exosomes in mesenteric tissues.

[0072] Therefore, the above results indicate that both CrF-exo and Ctrl-exo effectively colonize IL-10 - / - The colon and mesentery of mice ( Figure 2 (as shown in Part C of FIG.

[0073] Example 2 - CrF-exo mitigates IL-10 - / - Spontaneous colitis and mesenteric inflammation in mice

[0074] This example verifies the effect of CrF-exo on spontaneous colitis and mesenteric inflammation using IL-10 - / - Mice are used as an animal model, specifically including the following steps:

[0075] (1) IL-10 - / -Mouse exosome treatment: IL-10 - / - Mice developed spontaneous colitis and hypertrophic mesenteric mesenteritis at week 15. To evaluate the role of CrF-exo in the disease process, exosomes were injected into the 15-week IL-10 - / - The mice were divided into four groups: WT mice group, IL-10 - / - Mouse group, CrF-exo+IL-10 - / - Mouse group, Ctrl-exo+IL-10 - / - Mouse group (Con-exo+IL-10 - / - The exosomes were administered on the 2nd and 5th day of each week, 200 μg / mouse each time. The body weight was recorded weekly during the treatment. The mice were killed at the end of the 19th week, and the colon and MAT samples were collected for further study.

[0076] (2) Experimental results;

[0077] IL-10 - / - Mice began to develop obvious colitis and mesenteric inflammation from 15 weeks of age, and IL-10 - / - The mesentery of the mice also showed obvious hypertrophy. At week 19, compared with WT mice, IL-10 - / - Mice showed spontaneous weight loss and shortened colon length ( Figure 3 In addition, under high-resolution endoscopy, IL-10 - / - Mice showed colon thickening and altered vascular patterning ( Figure 3 Compared with WT mice, IL-10 - / - These phenotypes in mice showed higher endoscopic and histological scores ( Figure 3 However, the above changes could be alleviated by 4 weeks of CrF-exo treatment, but not by Ctrl-exo treatment. - / - Mice had a significantly higher ratio of MAT (mesenteric adipose tissue) weight to body weight, which could be reversed by CrF-exo intervention ( Figure 3 In addition, it was found that after injection of CrF-exo, IL-10 - / - The size of adipocytes in MAT of mice was smaller than that of WT mice ( Figure 3 of the G section).

[0078] Based on the recent studies showing that fat browning in MAT of CD can be a feature of anti-inflammatory mechanism, we further investigated the expression of uncoupling protein-1 (Ucp-1), a typical marker of fat browning in mice.- / - A significant increase in Ucp-1 protein expression was detected in the MAT of mice, suggesting that the tendency of fat browning after CrF-exo treatment may play a potential protective role in inflammation.

[0079] Macrophages and CD4 + T cells are the two most common cell components infiltrating CrF. This study showed that compared with WT mice, IL-10 - / - CD4 + T cells and F4 / 80 + The number of macrophages increased ( Figure 4 CrF-exo, but not Ctrl-exo, effectively suppressed the inflammation of these immune cells. - / - The expression levels of pro-inflammatory factors Tnf-α, IL-1β, and IL-6 mRNA in the mesentery and colon of mice were also increased. Similarly, these pathological changes could be restored after 4 weeks of CrF-exo treatment ( Figure 4 of the BG part).

[0080] Therefore, the above experimental data indicate that CrF-exo treatment can effectively alleviate the expression of IL-10 - / - Progression of spontaneous inflammation in the murine mesentery and colon.

[0081] Example 3 - CrF-exo enhances lymphatic drainage and LECs (lymphatic endothelial cells) function

[0082] This example verifies the role of CrF-exo in regulating lymphatic function, which specifically includes the steps of:

[0083] IL-10 - / - Mice show obvious lymphatic dysfunction, and previous studies have confirmed that in the chronic colitis model induced by sodium trinitrobenzene sulfonate (TNBS), the enhancement of lymphatic drainage function can alleviate colonic inflammation. Therefore, in this example, it is presumed that improving mesenteric lymphatic function can effectively reduce not only intestinal inflammation, but also mesenteric inflammation.

[0084] (1) Based on the fact that ADSCs have been shown to improve LECs function through in vitro exosome delivery, this example further investigated whether CrF-exo affects IL-10 by regulating lymphatic function. - / - In mice with colitis and mesenteric inflammation, Lyve-1, a LECs-specific marker, was used to assess lymphatic vessel density. - / - Lymphangiogenesis and lymphatic vessel density (LVD) were significantly increased in the colon and mesentery of mice, affecting lymphangiogenesis ( Figure 5AC part).

[0085] (2) Fluorescent lymphangiography experiment (see Su X, Brassard A, Bartolomucci A, et al. Tumour extracellular vesicles induce neutrophil extracellular traps to promote lymphnode metastasis. J Extracell Vesicles 2023Aug; 12(8): e12341.): Wild type (WT) and IL-10 -- / - Mice were orally administered with long-chain fluorescein isothiocyanate (FITC)-labeled fatty acids (BODIPY FLC16, Thermo) to study lymphatic drainage. A total of 1 mg of the product was dissolved in 5 mL of vegetable oil, and 100 μL of the solution was orally administered to each mouse. Fluorescent images of mesenteric lymphatic drainage were obtained under a dissecting microscope 6 hours after administration (Leica). Lymphatic drainage function was assessed by counting the number of functional lymphatic vessels per field in the mesentery.

[0086] In vivo, IL-10 was expressed 7 h after intragastric administration of Bodipy FL-C16 as a fluorescent lipid tracer. - / - Compared with WT mice, mice had impaired mesenteric lymphatic drainage and weaker fluorescence signals in mesenteric lymph vessels ( Figure 5 (D). Compared with Ctrl-exo treatment, IL-10 - / - Mice showed a significant increase in mesenteric lymphatic drainage ( Figure 5 of the E section).

[0087] (3) Evans blue dye in vivo validation of lymphatic drainage test: 10 micrograms (1%) of Evans blue dye (Sigma-Aldrich, St. Louis, MO, USA) in 10 μL PBS was injected into the rectal mucosa of anesthetized mice using a Hamilton syringe. The mice were killed 16 hours later. The distal colon tissue was incubated in 1 mL of formamide (Sangon Biotech, Shanghai, China) at 55°C overnight, and Evans blue dye was extracted from it (i.e., Evans blue dye that can be specifically taken up by intestinal lymphatic vessels can be extracted 16 hours after submucosal injection of Evans blue dye in the distal rectum). The background-subtracted absorbance at 630 nm was measured using a microplate reader (Biotek, Vermont, USA). The concentration of the extracted dye was calculated using a standard curve and expressed as the absolute amount of dye remaining in the colon.

[0088] Compared with WT mice, IL-10- / - The extraction of Evans blue dye was significantly impaired in mice. However, the above-mentioned impaired extraction of Evans blue dye was reversible by systemic delivery of CrF-exo, as less dye remained in the distal rectum ( Figure 5 of the Regulations).

[0089] (4) To further verify the regulatory effect of CrF-exo on lymphatic endothelial function, exosomes were labeled with the fluorescent dye PKH26 and incubated with HLECs in vitro for 24 hours. Specifically, exosomes were labeled with PKH26 dye (Sigma Aldrich) according to the manufacturer's instructions. 2 mg / mL PKH26 dye was added to the exosome solution, incubated for 10 minutes, and then centrifuged at 100,000 g for 70 minutes to remove excess dye. To observe the uptake of exosomes by LECs, 10 μg / mL PKH26-labeled exosomes were incubated with human lymphatic endothelial cells (HLECs) for 24 hours. The cells were then washed with PBS to remove exosomes that were not internalized by HLECs, and 4,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) was used to observe the internalization of exosomes in HLECs using a confocal imaging system (Zeiss LSM880).

[0090] Under confocal fluorescence microscopy, it was observed that both CrF-exo and Ctrl-exo were taken up by HLECs ( Figure 6 In addition, HLECs co-incubated with CrF-exo had significantly enhanced proliferation, migration, and tube formation functions ( Figure 6 In addition, it was found that the CCL21 production of HLECs was significantly increased after injection of CrF-exo ( Figure 6 F part), indicating that CrF-exo can affect the regulatory function of LECs in immune cell migration.

[0091] Therefore, the above experimental results show that CrF-exo can enhance lymphatic physiology, growth and immune regulation functions both in vivo and in vitro.

[0092] Example 4-CrF-exo improves lymphatic endothelial function through miR-132-3p

[0093] This example further studies the composition of CrF-exo and finds that it improves lymphatic endothelial function through miR-132-3p, specifically including the following steps:

[0094] (1) miRNA is the most common mediator for executing exosome functions. In order to reveal the molecular mechanism of CrF-exo regulating the lymphatic system, this example performed high-throughput sequencing of miRNA in CrF-exo exosomes. The heat map showed that compared with Ctrl-exo, 47 miRNAs were upregulated and 17 miRNAs were downregulated in CrF-exo (fold-change ≥ 1.5, p < 0.05) ( Figure 7 Part A of the .

[0095] (2) Previous studies have shown that miR-132-3p may be a regulator of LEC function in ADSCs-derived exosomes. Compared with Ctrl-exo, miR-132-3p is highly expressed in CrF-exo. This example further confirmed by qPCR that the expression of miR-132-3p was increased in both CrF-derived ADSCs and ADSCs-derived exosomes ( Figure 7 HLECs incubated with CrF-exo also showed up-regulation of miR-132-3p levels ( Figure 7 Therefore, the above experimental results show that CrF-exo regulates lymphatic function by delivering miR-132-3p.

[0096] (3) To further verify the role of miR-132-3p, the miR-132-3p inhibitor was transfected into CrF-exo and co-incubated with HLECs. The results showed that the promotion of HLECs proliferation, migration and tube formation induced by CrF-exo was completely eliminated by inhibiting miR-132-3p ( Figure 7 The elevated CCL21 secretion induced by CrF-exo in HLECs was also reversed after inhibition of exosomal miR-132-3p ( Figure 7 H section).

[0097] (4) We further investigated whether miR-132-3p directly affects cell function in HLECs. miR-132-3p mimics and miR-132-3p inhibitors were transfected into HLECs, respectively. In HLECs transfected with miR-132-3p mimics, miR-132-3p levels were significantly upregulated, and the proliferation, migration, and tube formation functions of HLECs were also significantly enhanced. In contrast, after miR-132-3p inhibition, these cell functions and the secretion of CCL21 were inhibited ( Figure 8 AE part).

[0098] Therefore, the above experimental results indicate that CrF-exo improves lymphatic function through the delivery of miR-132-3p.

[0099] Example 5-CrF-exo-miR-132-3p promotes lymphatic endothelial function by targeting RASA1 and activating the ERK1 / 2 signaling pathway

[0100] This example further studies the molecular mechanism of CrF-exo-miR-132-3p in regulating lymphatic endothelial function, specifically including the following steps:

[0101] (1) Four bioinformatics databases (Targetscan, miRDB, miRTarBase, and miRanda) were used to predict the downstream genes of miR-132-3p in LEC. A total of 12 genes were predicted in these four databases ( Fig. 9 Part A of the study), through the analysis of GO enrichment, it was found that MAPK signal ranked first ( Fig. 9 Part B). Among the potential predicted targets, RASA1 is an important regulator of the MAPK pathway and can effectively inhibit ERK1 / 2 and signal transduction downstream of the MAPK pathway. In addition, RASA1 also plays an important role in regulating endothelial function, and the activation of ERK1 / 2 has also been shown to increase the production of chemokine CCL21 in endothelial cells.

[0102] (2) In this example, WT and mutant MUT 3'-UTR sequences containing potential binding sites were constructed, and the luciferase reporter gene method was used to verify whether miR-132-3p could directly bind to the 3'-UTR of RASA1 in HLECs. The results showed that the luciferase activity of the WT group and the MUT group was equivalent, while after overexpression of miRNA-132-3p in the WT group, the luciferase activity was significantly inhibited ( Fig. 9 C and D of the .

[0103] In addition, the study found that the decreased expression of RASA1 after CrF-exo incubation was due to a decrease in protein levels rather than mRNA, which may be related to the post-transcriptional regulation of miR-132-3p. After inhibition of miR-132-3p, CrF-exo lost its downregulatory effect on RASA1 in HLECs ( Fig. 9 Transfection of HLECs with miR-132-3p mimics significantly inhibited the expression of RASA1, whereas transfection with miR-132-3p inhibitors had the opposite effect ( Fig. 9 In addition, the phosphorylation of ERK1 / 2 in HLECs showed the opposite changes to that of RASA1 ( Fig. 9 This is consistent with a role for RASA1 as a regulator of ERK1 / 2 activation.

[0104] (3) To verify the regulatory role of RASA1 in lymphatic endothelial function, this example used plasmid transfection to achieve overexpression of RASA1 in HLECs. By plasmid transfection, it was detected that the expression of RASA1 was significantly increased, while the activation of ERK1 / 2 was further inhibited. Compared with the control group, the function of HLECs with high RASA1 levels was significantly impaired, but there was no difference in the function of HLECs with overexpression of RASA1 incubated with CrF-exo ( Fig.10 AD).

[0105] ERK1 / 2 activation can regulate endothelial function, and U0126 is considered a classic inhibitor of ERK1 / 2. The experimental results showed that pre-incubation with U0126 inhibited the activation of ERK1 / 2 and abolished the enhanced endothelial function of HLECs stimulated by CrF-exo treatment ( Fig.10 In HLECs, RASA1 overexpression reduced CCL21 secretion, whereas CrF-exo abolished this effect ( Fig.10 E), however, incubation with U0126 reversed this effect as expected ( Fig.10 of the J section).

[0106] Therefore, the above experimental results indicate that CrF-exo-miR-132-3p promotes lymphatic endothelial function by targeting RASA1 and activating the ERK1 / 2 signaling pathway.

[0107] Example 6 - In vivo, CrF-exo-miR-132-3p targets RASA1 expression in LECs to promote lymphangiogenesis and lymphatic drainage

[0108] This example further verifies the effect of CrF-exo-miR-132-3p on lymphatic function, and specifically includes the steps of:

[0109] In IL-10 - / - After mice were treated with exosomes, the co-localization of RASA1 and Lyve-1 in LECs was further observed and analyzed by IF double staining. - / - After mice were treated with CrF-exo, the expression of RASA1 in mesenteric and colonic LECs was significantly inhibited, and Lyve-1 + The RASA1 fluorescence signal in LECs was weak ( Fig.11 Meanwhile, antagonism or inhibition of miR-132-3p rescued CrF-exo-induced suppression of RASA1 in mesenteric and colonic lymphatic vessels ( Fig.11 Part A and B of the

[0110] IL-10 in CrF-exo transfected with miR-132-3p inhibitor- / - Mice treated with IL-10 - / - Mice showed similar LVD in the mesentery and colon ( Fig.11 Moreover, the CrF-exo-mediated promotion of mesenteric and colonic lymphatic drainage was reversed by miR-132-3p antagonism ( Fig.11 of the GI portion).

[0111] Therefore, the above experimental data show that IL-10 - / - LVD and lymphatic drainage in the mouse mesentery and colon were both regulated by miR-132-3p contained in CrF-exo.

[0112] Example 7-CrF-exo-miR-132-3p reduces IL-10 - / - Mesenteric and colonic inflammation in mice

[0113] This example studies the effect of CrF-exo-miR-132-3p on IL-10 - / - Effects of IL-10 on enteritis and colitis in mice - / - Mice were used as an animal model. After exosomal miR-132-3p was antagonized, the effect of CrF-exo on IL-10 was eliminated. - / - The results showed that IL-10 - / - Mice with miR-132-3p antagonism and untreated IL-10 - / - There were no significant differences in body weight change, colon length, colitis score, and mesenteric weight / body weight ratio between the mice ( Fig.12 After inhibition of CrF-exo-miR-132-3p, immune cell infiltration and inflammatory production in the mesentery and colon remained unchanged ( Fig.13 Consistent with the changes in mesenteric weight and adipocyte size, inhibition of exosomal miR-132-3p significantly reversed the IL-10 - / Browning phenotype of mouse mesenteric adipocytes.

[0114] Therefore, the above experimental data indicate that CrF-exo-miR-132-3p promotes lymphangiogenesis in colon and MAT, enhances lymphatic drainage function, and further alleviates IL-10 - / Spontaneous mesenteritis and colitis in mice.

[0115] Example 8 - Elevated expression of miR-132-3p is closely associated with LVD and inflammation in CD patients

[0116] In order to further verify the relationship between miR-132-3p and disease progression in CD patients, intestinal and mesenteric samples were collected from 25 CD patients and 10 non-CD patients in this example to detect the expression of miR-132-3p. Fig.14 As shown in A and B, the miR-132-3p levels in inflammatory bowel (D) and corresponding mesentery (CrF) were significantly higher than those in resection margin tissue (R) and control samples (Ctrl and Ctrl-MAT). Fig.14 The CF part of the study showed that the LVD of the inflammatory bowel (D) and the corresponding mesentery (CrF) was significantly higher than that of the marginal tissue (R and RMAT) and the control samples. The level of miR-132-3p was significantly higher in the mesentery (r = 0.4555, p = 0.0221, Fig.14 G part) and intestinal tract (r = 0.6119, p = 0.0009, Fig.14 The H part) is positively correlated.

[0117] Further investigation of the role of miR-132-3p upregulation in CD progression, such as Fig.14 As shown in Part I of Figure 2, serum miR-132-3p levels were higher in patients with active CD (n=25) compared with patients with inactive CD (n=10) or healthy controls (n=10). In addition, serum miR-132-3p expression was negatively correlated with CRP and IL-6 levels (r=-0.5668, p=0.0092; r=-0.6674, p=0.0013, Fig.14 J and K parts), respectively, showing their anti-inflammatory effects in the disease process.

[0118] From the above examples, it can be seen that CrF-exo can significantly improve lymphatic function and alleviate colitis and mesenteric inflammation through the miR-132-3p / RASA1 / ERK1 / 2 axis, which provides new insights into the treatment and mechanism of CD ( Fig.14 of the L part).

[0119] The present invention uses IL-10 - / - Mice showed obvious intestinal lesions and mesenteric hypertrophy at 15 weeks of age, and 4 weeks of CrF-ADSC intervention effectively alleviated the IL10 - / -Disease progression in the colon and mesentery of mice. In order to explore the specific mechanism, miRNA high-throughput sequencing was used, and it was found that miR-132-3p was highly expressed in CrF-ADSCs and their exosomes, and could be delivered to LECs. It was also verified that CrF-exo promoted lymphogenesis and improved lymphatic endothelial function, but these effects were eliminated by the antagonism of exosomal miR-132-3p. In addition, the present invention also used bioinformatics databases to predict 12 potential targets that were highly enriched in MAPK signaling. The study found that CrF-exo delivered miR-132-3p to LECs through the RASA1 / ERK1 / 2 axis, improved lymphatic function, and inhibited IL-10 - / - Expression of RASA1 in the colon and mesenteric lymphatic vessels of mice. Similarly, antagonism of exosomal miR-132-3p abolished the effects of CrF-exo. Thus, injection of CrF-exo regulates IL-10 through miR-132-3p / RASA1 / ERK1 / 2-dependent improvement of lymphatic endothelial function. - / - Colitis and mesenteric inflammation in mice. The present invention also detected upregulated miR-132-3p levels in CrF and its surrounding intestine in CD patients, which was positively correlated with increased LVD, verifying that CrF-exo-miR-132-3p can induce MAT browning by regulating lymphatic function. In addition, serum miR-132-3p levels were also increased in CD patients and negatively correlated with inflammatory biomarkers such as CRP and IL-6, further verifying its anti-inflammatory effect.

[0120] In summary, this paper first discovered that CrF-exo improves lymphatic function and alleviates colitis and mesenteritis through the miR-132-3p / RASA1 / ERK1 / 2 axis, revealing a new role of lymph in CrF involved in the CD process and providing new insights into the treatment and mechanism of CD.

[0121] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Application of adipose stem cell exosomes, characterized in that: The application is selected from at least one of the following applications: application in the preparation of a drug for treating Crohn's disease, application in the preparation of a drug for treating colitis and mesenteric inflammation, and application in the preparation of a drug for regulating lymphatic function; wherein the adipose stem cells are derived from crawling adipose tissue.

2. The use according to claim 1, characterized in that: The exosome separation step comprises: S1. Isolation and culture of adipose stem cells: The adipose tissue harvested from the crawling fat was washed with phosphate-buffered saline to remove red blood cells and digested with type I collagenase; the supernatant was removed by centrifugation, and the spheroid cells were resuspended and stored in an incubator; S2. Isolation of exosomes: The adipose-derived stem cells obtained by separation and culture were cultured in a culture medium without exosomes, and the culture supernatant was collected and centrifuged once to remove cells and cell debris; exosomes were separated by a second centrifugation, and the exosome microspheres were centrifuged three times and collected and stored.

3. The use according to claim 2, characterized in that: In step S2, the operation steps of the first centrifugation are: centrifugation at 300g for 10 min at 4°C, and then centrifugation at 3000g for 30 min; and / or, the operation steps of the second centrifugation are: centrifugation at 10000g for 30 min and centrifugation at 100000g for 70 min; and / or, the operation steps of the third centrifugation are centrifugation at 100000g for 70 min.

4. The use according to claim 1, characterized in that: The exosomes of adipose stem cells are used to prepare a drug having at least one of the following functions: colonization in the colon and mesentery; alleviation of weight loss and shortening of colon length; alleviation of colon thickening and changes in vascular pattern; reduction of endoscopic and histological scores; reduction of weight-to-body weight ratio; reduction of adipocyte size; increase of Ucp-1 protein expression; increase of browning phenotype of mesenteric adipocytes; reduction of CD4 + T cells and F4 / 80 + The number of macrophages was reduced; the expression levels of pro-inflammatory factors Tnf-α, IL-1β and IL-6 mRNA were reduced; the LVD of colon and mesentery was increased; the mesenteric lymphatic drainage was increased; the proliferation, migration and tube formation of lymphatic endothelial cells were enhanced; and the CCL21 production of lymphatic endothelial cells was increased; Enhance the regulatory function of lymphatic endothelial cells in immune cell migration.

5. The use according to any one of claims 1 to 4, characterized in that: The exosomes include miR-132-3p, which is recorded as CrF-exo-miR-132-3p.

6. The use according to claim 5, characterized in that: The application is the application of CrF-exo-miR-132-3p, and the application of CrF-exo-miR-132-3p is selected from at least one of the following applications: application in the preparation of RASA1 inhibitors and / or ERK1 / 2 pathway activators, application as a marker in screening RASA1 regulators and / or ERK1 / 2 pathway regulators, application in the preparation of active CD detection reagents, application in the preparation of drugs for treating Crohn's disease, application in the preparation of drugs for treating colitis and mesenteric inflammation, and application in the preparation of drugs for regulating lymphatic function.

7. The use according to claim 5, characterized in that: The adipose stem cell exosomes regulate lymphatic function by delivering CrF-exo-miR-132-3p, including: CrF-exo-miR-132-3p promotes lymphatic endothelial function by targeting RASA1 and activating the ERK1 / 2 signaling pathway; CrF-exo-miR-132-3p targets RASA1 expression in lymphatic endothelial cells to promote lymphangiogenesis and lymphatic drainage, thereby alleviating spontaneous mesenteritis and colitis.

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