Icam-1 markers and uses thereof

Abstract

The present application provides ICAM-1 markers and applications thereof. Specifically, the present application provides an application of ICAM-1 and a modulator thereof in promoting or inhibiting differentiation of adipose-derived stem cells into adipocytes, and an application of ICAM-1 or a detection reagent thereof in (a) detecting adipose-derived stem cells, and / or (b) judging a risk of obesity of a test subject, and a corresponding diagnostic kit and method. The present application also provides a non-therapeutic method for preparing adipocytes in vitro.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, more particularly to ICAM-1 and its application in identifying adipose stem cells and regulating adipocyte differentiation. BACKGROUND

[0002] Obesity is manifested by an increase in adipose tissue, which includes both hypertrophy of adipocytes, excess lipid intake and accumulation, and hyperplasia of adipocytes. Mature adipocytes are not mitotically competent, so that hyperplasia of adipocytes is caused by differentiation of adipocyte precursors into new adipocytes. In adults, adipose tissue is renewed at a rate of 10% per year, and in obese individuals, the rate of elimination of adipocytes is not different from that in normal individuals, but the rate of new adipocyte recruitment is significantly higher than that in normal individuals, resulting in hyperplasia of adipocytes. In rodents, it is generally believed that when obesity is induced by high-fat diet, the size of adipocytes is initially increased, and as the time of high-fat feeding is prolonged, the number of adipocytes is also gradually increased. By using genetically modified mice that can mark newly formed adipocytes, it is found that in the early stage of obesity, adipogenic differentiation is not obvious, but in the later stage, a large number of adipose stem cells differentiate into newly formed adipocytes, especially in visceral adipose tissue. Therefore, obesity is accompanied by adipogenic differentiation of adipose stem cells, which is an important cause of obesity in both humans and rodents. However, the definition of these adipose stem cells and the cellular and molecular level regulation mechanisms of their adipogenic differentiation in vivo, especially in the stage of obesity, are not clear.

[0003] Although the in vitro differentiation process of adipocytes and its molecular mechanism have been established, the regulation of adipocyte differentiation in vivo is still to be studied. Many scholars have previously established that Sca-1, CD34, CD29, CD24, PDGFR-β and PDGFR-α can mark adipocyte precursors, but these markers cannot well define specific adipocyte differentiation types.

[0004] Therefore, there is an urgent need in the art to develop new molecules that can identify adipose stem cells and mark adipocyte differentiation. SUMMARY

[0005] The present application relates to the field of biotechnology, more particularly to ICAM-1 and its application in identifying adipose stem cells and regulating adipocyte differentiation.

[0006] In a first aspect of the present application, there is provided the use of an ICAM-1 inhibitor for the manufacture of a preparation or composition for promoting differentiation of adipose stem cells into adipocytes.

[0007] In another preferred embodiment, the adipose stem cells are ICAM-1 positive adipose stromal cells.

[0008] In another preferred embodiment, the adipose stem cells are CD45 - CD31 - Sca-1 + PDGFR-α + ICAM-1 + cells.

[0009] In another preferred embodiment, the adipose stem cells express adipogenic differentiation regulatory genes.

[0010] In another preferred embodiment, the adipogenic differentiation regulatory genes are selected from the group consisting of Pparg, Cebpa, Cebpb, Cebpg, Gata2, Gata3, Irs1, Pparg, Cebpa, and Fabp4, or a combination thereof.

[0011] In another preferred embodiment, the adipose stem cells express signature molecules selected from the group consisting of Sca-1, CD34, CD29, CD24, Pdgfr-β, Zfp423, or a combination thereof.

[0012] In another preferred embodiment, the preparation or composition is further used for adipose tissue remodeling.

[0013] In another preferred embodiment, the ICAM-1 inhibitor specifically inhibits the expression or activity of ICAM-1.

[0014] In another preferred embodiment, the ICAM-1 inhibitor comprises a MicroRNA, an siRNA, an shRNA, or a combination thereof.

[0015] In another preferred embodiment, the ICAM-1 inhibitor comprises an antibody.

[0016] In another preferred embodiment, the ICAM-1 is derived from a human or a non-human mammal.

[0017] In another preferred embodiment, the composition is a pharmaceutical composition.

[0018] In another preferred embodiment, the pharmaceutical composition comprises (a) an ICAM-1 inhibitor; and (b) a pharmaceutically acceptable carrier.

[0019] In another preferred embodiment, the pharmaceutical composition is in a dosage form selected from the group consisting of an oral dosage form, an injectable dosage form, and a topical pharmaceutical dosage form.

[0020] In a second aspect of the present application, there is provided use of ICAM-1 or an enhancer thereof for the manufacture of a preparation or a composition for inhibiting differentiation of adipose tissue-derived stem cells into adipocytes.

[0021] In another preferred embodiment, the preparation or the composition is used for maintaining undifferentiated state of the adipose tissue-derived stem cells.

[0022] In another preferred embodiment, the ICAM-1 enhancer specifically enhances expression or activity of ICAM-1.

[0023] In a third aspect of the present application, there is provided an in vitro non-therapeutic method for preparing adipocytes, the method comprising the steps of:

[0024] (a) providing ICAM-1 positive adipose stromal cells;

[0025] (b) culturing the adipose stromal cells under conditions suitable for differentiation of adipocytes, thereby obtaining a cell population comprising differentiated adipocytes; and

[0026] (c) isolating the adipocytes from the cell population.

[0027] In another preferred embodiment, the adipose stromal cells are CD45 - CD31 - Sca-1 + PDGFR-α + ICAM-1 + cells.

[0028] In another preferred embodiment, the ICAM-1 positive adipose stromal cells are adipose tissue-derived stem cells.

[0029] In another preferred embodiment, in step (b) and step (c), the expression level of ICAM-1 is detected, thereby determining the degree of differentiation of the adipose stromal cells into adipocytes.

[0030] In another preferred embodiment, the expression level of ICAM-1 of the adipose stromal cells decreases as the degree of differentiation of the adipose stromal cells into adipocytes increases.

[0031] In another preferred embodiment, in step (b), the expression of ICAM-1 of the adipose stromal cells is inhibited, thereby promoting differentiation of the adipose stromal cells into adipocytes.

[0032] In another preferred embodiment, in step (b), the expression level of ICAM-1 of the adipose stromal cells gradually decreases as the culturing proceeds.

[0033] In another preferred embodiment, in step (b), the adipocyte cells are isolated from the cell population when the cell population substantially does not express ICAM-1.

[0034] In another preferred embodiment, the substantial lack of expression means that the ratio N1 / N2 of the number of cells expressing ICAM-1 N1 to the total number of cells in the cell population N2 is less than or equal to 5%, preferably less than or equal to 1%.

[0035] In a fourth aspect of the present application, there is provided a method for inhibiting the differentiation of adipose stem cells into adipocytes in vitro, which method comprises maintaining the expression level of ICAM-1 in the adipose stem cells.

[0036] In another preferred embodiment, the maintaining the expression level of ICAM-1 comprises adding ICAM-1 or a promoter thereof to the culture system of the adipose stem cells.

[0037] In a fifth aspect of the present application, there is provided the use of ICAM-1 or a detection reagent thereof for the manufacture of a detection kit for (a) detecting adipose stem cells, and / or (b) determining the risk of obesity in a test subject.

[0038] In another preferred embodiment, the kit further comprises FABP4 or a detection reagent thereof.

[0039] In another preferred embodiment, the adipose stem cells have adipogenic differentiation capacity.

[0040] In another preferred embodiment, the adipose stem cells can differentiate into adipocytes, resulting in an increase in the number of adipocytes.

[0041] In another preferred embodiment, the detecting the adipose stem cells comprises:

[0042] (i) detecting whether the sample contains adipose stem cells, and / or

[0043] (ii) detecting the number of adipose stem cells contained in the sample.

[0044] In another preferred embodiment, the sample is a tissue sample, preferably the tissue sample comprises adipose tissue, more preferably the tissue is perivascular adipose tissue.

[0045] In another preferred embodiment, the kit detects ICAM-1 + in the sample, thereby detecting the adipose stem cells.

[0046] In another preferred embodiment, the determining comprises auxiliary determination and / or pre-treatment determination.

[0047] In another preferred embodiment, the judging is based on a ratio of ICAM-1 + cell proportion A1 to the corresponding ICAM-1 + cell proportion A0 of the normal population. If A1 is significantly higher than A0, it indicates that the test subject has a high risk of developing obesity.

[0048] In another preferred embodiment, the judging further comprises judging the risk of the test subject developing obesity based on a ratio of FABP4 + cell proportion B1 to the corresponding FABP4 + cell proportion B0 of the normal population. If B1 is significantly lower than B0, it indicates that the test subject has a high risk of developing obesity.

[0049] In another preferred embodiment, the "significantly higher than" means A1 / A0≥1.25, preferably A1 / A0≥1.5, more preferably A1 / A0≥2.0.

[0050] In another preferred embodiment, the "significantly lower than" means B0 / B1≥1.25, preferably B0 / B1≥1.5, more preferably B0 / B1≥2.0

[0051] In another preferred embodiment, the number of the normal population is at least 100, preferably at least 300, more preferably at least 500, most preferably at least 1000.

[0052] In another preferred embodiment, the detection reagent comprises a protein chip, a nucleic acid chip, or a combination thereof.

[0053] In another preferred embodiment, the detection reagent comprises an ICAM-1 specific antibody.

[0054] In another preferred embodiment, the ICAM-1 specific antibody is coupled with or carries a detectable label.

[0055] In another preferred embodiment, the detectable label is selected from the group consisting of a chromophore, a chemiluminescent group, a fluorophore, an isotope, and an enzyme.

[0056] In another preferred embodiment, the ICAM-1 specific antibody is a monoclonal antibody or a polyclonal antibody.

[0057] In a sixth aspect of the present application, a diagnostic kit is provided, which comprises a container containing ICAM-1 or a detection reagent thereof; and a label or an instruction indicating that the kit is used for (a) detecting adipose stem cells, and / or (b) judging the risk of a test subject developing obesity.

[0058] In another preferred embodiment, the kit further comprises FABP4 or a detection reagent thereof.

[0059] In another preferred embodiment, the ICAM-1 and FABP are used as standards.

[0060] In another preferred embodiment, the kit further comprises sample pre-treatment reagents and instructions for use.

[0061] In another preferred embodiment, the instructions describe the detection method and the method for judging based on the value of Al.

[0062] In another preferred embodiment, the kit further comprises standards of ICAM-1 gene sequence and protein.

[0063] In a seventh aspect of the present application, a method for judging the risk of obesity of a test subject is provided, comprising the steps of:

[0064] (a) providing a sample of the test subject;

[0065] (b) determining the proportion of ICAM-1 + cells in the sample;

[0066] (c) comparing the proportion of ICAM-1 + cells in step (b) with the proportion of ICAM-1 + cells in a normal population, and if Al is significantly higher than A0, it indicates that the test subject has a high risk of obesity.

[0067] In another preferred embodiment, the method further comprises determining the proportion of FABP4 + cells in the sample, B1, and comparing B1 with the proportion of FABP4 + cells in a normal population, B0, and if B1 is significantly lower than B0, it indicates that the test subject has a high risk of obesity.

[0068] In another preferred embodiment, the test subject is a human or a non-human mammal.

[0069] In another preferred embodiment, the test sample is a tissue sample, preferably an adipose tissue sample.

[0070] In an eighth aspect of the present application, a use of stromal cells is provided, wherein the stromal cells are isolated from adipose tissue and positive for ICAM-1, and wherein the stromal cells are used to prepare a cell preparation for adipose tissue remodeling.

[0071] Preferably, the adipose tissue remodeling comprises remodeling of adipose tissue in facial, buttock, and breast regions.

[0072] In another preferred embodiment, the remodeling comprises adipose tissue remodeling in cosmetic applications, and adipose tissue remodeling in wound repair.

[0073] In another preferred embodiment, the formulation further includes an ICAM-1 inhibitor.

[0074] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0075] Figure 1 Display ICAM-1 + Adipose stromal cells possess the potential for directed differentiation into adipose stem cells. Specifically, this is achieved by using flow cytometry to separate CD31 cells from visceral adipose tissue. - CD45 - Adipose-derived stromal cells are used for single-cell analysis.

[0076] Figure 1 A shows the analysis of CD31 in visceral adipose tissue using flow cytometry. - CD45 - Expression of Sca-1 and PDGFR-α in adipose stromal cells.

[0077] Figure 1 B shows the levels of ICAM-1 in CD31 in visceral adipose tissue (epididymal fat) and subcutaneous adipose tissue (groin fat) using flow cytometry. - CD45 - Sca-1 + PDGFR-α + Expression levels in a cell population.

[0078] Figure 1 C displays sorting CD31 - CD45 - Sca-1 + PDGFR-α + ICAM-1 in cell population + and ICAM-1 - Cells were analyzed using real-time PCR to detect the expression levels of genes related to adipogenic differentiation and adipose-derived stem cells.

[0079] Figure 1 D shows the CD31 from wild-type mouse adipose tissue. - CD45 - Sca-1 + PDGFR-α + ICAM-1 isolated from cell population - CD31 in cells and GFP mouse adipose tissue - CD45 - Sca-1+ PDGFR-a + ICAM-1 isolated in cell population + Cells were co-cultured and observed for spontaneous adipogenic differentiation.

[0080] Figure 2 ICAM-1 shown + Adipogenic differentiation of adipose stem cells in vivo.

[0081] Figure 2 A shows that mTmG mice were crossed with Icaml-CreERT2 knock-in mice to activate the recombinase with tamoxifen to construct ICAM-1 adipose stem cell tracing mice.

[0082] Figure 2 B shows that ICAM-1 in adipose tissue development in neonatal mice was observed by whole-mount fluorescent staining technique of adipose tissue. + Adipogenic differentiation of adipose stem cells in vivo.

[0083] Figure 2 C shows that ICAM-1 in adipose tissue development in neonatal mice was observed by whole-mount fluorescent staining technique of adipose tissue. + Adipogenic differentiation of adipose stem cells in vivo.

[0084] Figure 3 ICAM-1 shown under obesity conditions + Evolution characteristics of adipose stem cells.

[0085] Figure 3 A shows that Fabp4-Cre; mTmG mice were constructed to study adipose stem cells in adipogenic differentiation.

[0086] Figure 3 B shows that the expression level of ICAM-1 in adipose precursor cells in adipogenic differentiation state in adipose tissue was analyzed by flow cytometry.

[0087] Figure 3 C shows that the expression level of ICAM-1 in adipogenic differentiation state in adipose precursor cells in adipose tissue and subcutaneous adipose tissue under normal diet and high-fat induced obesity was analyzed by flow cytometry. + FABP4 (EGFP marker) expression level of adipose stem cells.

[0088] Figure 3 D and Figure 3 E is a statistical analysis of the repetition of the experiment shown. Figure 3

[0089] Figure 3 F shows that adipocytes (Adi) were obtained by flow cytometry sorting, ICAM-1 + EGFP​+ (I + G + ), ICAM-1 + EGFP - (I + G - ) and ICAM-1 - (I - ) cells and transcriptome analysis and correlation analysis.

[0090] Figure 3 G shows the differentially expressed genes of mature adipocytes, I + G + cells, I + G - cells and I - cells, mainly involved in PPAR signaling, adipogenesis and lipolysis, fatty acid biosynthesis and fatty acid elongation.

[0091] Figure 4 It is shown that ICAM-1 negatively regulates the directional differentiation of adipose stem cells.

[0092] Figure 4 A shows the body weight changes of wild-type (WT) mice and ICAM-1 - / - mice under normal diet and high-fat diet.

[0093] Figure 4 B shows the changes in fat tissue weight of wild-type (WT) mice and ICAM-1 - / - mice under normal diet and high-fat diet.

[0094] Figure 4 C shows the results of fluorescence staining analysis of the size of adipocytes in adipose tissue.

[0095] Figure 4 D shows the statistical results of fluorescence staining analysis of the size of adipocytes in adipose tissue.

[0096] Figure 4 E-4H respectively show that the WT mice bone marrow is transplanted into the irradiated WT mice and ICAM-1 - / - mice for bone marrow reconstruction, and given high-fat diet, the body weight changes of mice at different time points Figure 4 E) and the changes in fat tissue weight Figure 4 F), as well as the size of adipocytes in adipose tissue analyzed by fluorescence staining Figure 4 G) and statistical analysis Figure 4 H).

[0097] Figure 4I shows ICAM-1 - / - mice and ICAM-1 + / + Mice were crossed with Fabp4-Cre; mTmG mice, and flow cytometry was used to analyze the adipogenic potential of adipocyte stem cells in the absence of ICAM-1. Statistical analysis was performed.

[0098] Figure 4 J shows statistical analysis of flow cytometry analysis as shown in 4I for multiple mice

[0099] Figure 4 K shows western blot analysis of GFP content in adipocyte stem cells to determine adipocyte neogenesis in the presence of ICAM-1.

[0100] Figure 5 ICAM-1 negatively regulates adipogenic differentiation of adipocyte stem cells.

[0101] Figure 5 A-5D shows adipocyte stem cells isolated from WT mice and ICAM-1 - / - mice were subjected to adipogenic differentiation, and the expression of adipogenic differentiation-related genes, including Pparg Figure 5 A), Cebpa Figure 5 B), Fabp4 Figure 5 C), and Plin1 Figure 5 D), was detected at different time points.

[0102] Figure 6 ICAM-1 negatively regulates adipogenic differentiation of adipocyte stem cells.

[0103] Figure 6 A shows the expression levels of Rho-GTP, Rho-GDP, and total Rho in adipocyte stem cells derived from WT mice and ICAM-1 - / - mice using active Rho GTPases pull-down experiments.

[0104] Figure 6 B shows F-actin cytoskeleton staining results.

[0105] Figure 6 C shows the addition of DMSO or 10 μM Y-27632 (a ROCK inhibitor) during in vitro differentiation of adipocyte stem cells derived from WT mice and ICAM-1 - / - mice, and oil red staining was used to observe the adipogenic differentiation of adipocyte stem cells.

[0106] Figure 6D shows the detection of Perilipin A protein expression in WT and ICAM-1 - / - Perilipin A protein expression in mouse-derived adipose stem cells after adipogenic differentiation.

[0107] Figure 6 E, Figure 6 F shows the detection of Pparg - / - mRNA levels in WT and ICAM-1 Figure 6 E) and Fabp4 Figure 6 F) mRNA levels in mouse-derived adipose stem cells after adipogenic differentiation.

[0108] Figure 6 G shows the detection of Pparg - / - mRNA levels in WT and ICAM-1

[0109] Figure 6 H-6K shows the detection of Pparg Figure 6 H), Cebpa Figure 6 I), Fabp4 Figure 6 J) and Plin1 Figure 6 K) mRNA levels in mouse-derived adipose stem cells after adipogenic differentiation.

[0110] Figure 6 L-6N shows the detection of Pparg - / - mRNA levels in WT and ICAM-1 Figure 6 L) mice after 2 months of high fat diet. Figure 6 M) and the statistical analysis of subcutaneous fat weight changes (6N).

[0111] Figure 7 shows the role of ICAM-1 in the recognition and regulation of human adipose stem cells.

[0112] Figure 7 A shows the detection of ICAM-1 expression in human adipose tissue adipose precursor cells by flow cytometry analysis.

[0113] Figure 7 B shows the detection of ICAM-1 + tissue localization of adipose stem cells in human adipose tissue by immunofluorescence.

[0114] Figure 7C shows the expression changes of ICAM-1 and FABP4 in the adipogenic differentiation process of adipose-derived stem cells detected by Real time PCR.

[0115] Figure 7 D shows the expression of ICAM-1 in human adipose-derived stem cells knocked down by ICAM-1 siRNA.

[0116] Figure 7 E shows the adipogenic differentiation ability of cells observed by oil red staining after the expression of ICAM-1 in human adipose-derived stem cells is knocked down by ICAM-1 siRNA.

[0117] Figure 7 F-7G respectively shows the expression of adipogenic related genes and Rho GTP activity in adipogenic differentiation process of adipose-derived stem cells interfering with the expression of ICAM-1.

[0118] Figure 7 H-7J respectively shows the expression of adipogenic differentiation related genes (PPARG, CEBPA, FABP4) in adipose-derived stem cells after activating Rho with RA2 after interfering with the expression of ICAM-1.

[0119] Figure 7 K-7L respectively shows the correlation analysis of the expression level of ICAM-1 and the body fat ratio BMI index, ICAM-1 expression intensity and CD31 - CD45 - Fabp4 in adipose stromal cells + Expression level of adipose precursor cells. DETAILED DESCRIPTION

[0120] The present inventors have made extensive and in-depth research and for the first time accidentally found a new molecule for identifying adipose-derived stem cells. Specifically, the present application provides the use of ICAM-1 and its modulators in promoting or inhibiting the differentiation of adipose-derived stem cells into adipocytes, and the use of ICAM-1 or its detection reagent in (a) detecting adipose-derived stem cells, and / or (b) judging the risk of obesity in a test subject, and the corresponding diagnostic reagent kit and method. The present application also provides a non-therapeutic method for preparing adipocytes in vitro. Experiments show that ICAM-1 + Adipose-derived stem cells are located around blood vessels in adipose tissue, have the ability of spontaneous adipogenic differentiation, can be differentiated into adipocytes in vitro and in vivo, and participate in the development and remodeling of adipose tissue. In addition, ICAM-1 + The number of adipose-derived stem cells is proportional to the increase and increase of obese adipose tissue, and can be used to guide the diagnosis of obesity. On this basis, the present application is completed.

[0121] TERMS

[0122] As used herein, the term "committed fat precursor cell" or "fat precursor cell" refers to a mesenchymal stem cell that has begun to lose pluripotency in fat tissue and become a precursor cell that can differentiate into a fat cell.

[0123] As used herein, the term "stromal reserve cell" refers to a class of cells in the fat stroma that have ambiguous differentiation properties, which can have some adipogenic differentiation potential but less than the fat precursor cell.

[0124] As used herein, the term "fat stromal cell" refers to a class of cells in the fat tissue that are not blood cells and not endothelial cells, which have many mesenchymal stem cell properties

[0125] As used herein, the term "fat stem cell" refers to a stem cell that can differentiate into a fat cell.

[0126] ICAM-1

[0127] ICAM-1 (Intercellular adhesion molecule-1, ICAM-1, CD54) is a widely studied cell surface adhesion molecule that is a type I transmembrane protein with a molecular weight ranging from 80 to 114 kDa depending on its degree of glycosylation, and the unglycosylated ICAM-1 has a molecular weight of 60 kDa (38). The extracellular portion of ICAM-1 contains 453 amino acids, mostly hydrophobic amino acids, which form five immunoglobulin (Ig) -like domains. The extracellular portion is connected to a very short (28 amino acid) cytoplasmic tail by a hydrophobic, 24 amino acid transmembrane region. Its cytoplasmic tail lacks a classical signaling motif, but has a tyrosine residue that can play an important role in its signaling. The gene sequence of ICAM-1 contains 7 exons, with exon 1 encoding the signal peptide, exons 2-6 encoding one of the five Ig domains, and exon 7 encoding the transmembrane region and cytoplasmic tail.

[0128] The ligands of ICAM-1 include the β2 integrins LFA-1 (CD11a / CD18) and Mac-1 (CD11b / CD18) on leukocytes, fibrinogen, and rhinoviruses.

[0129] ICAM-1 plays an important role in both innate and adaptive immune responses. It mediates leukocyte transmigration across the vascular wall into inflammatory sites, and also regulates the interaction of antigen presenting cells (APCs) and T cells, participating in immunological synapse formation. ICAM-1 can transmit signals from outside to inside. The cytoplasmic tail of ICAM-1 is only 28 amino acids long, and lacks known kinase activity and protein interaction domains that can recruit downstream signaling molecules. However, it has many positively charged amino acids and a tyrosine residue (Y512). At present, many signaling molecules and adaptor proteins have been found to be associated with ICAM-1 pathway in different cells, especially the molecules related to actin cytoskeleton, including α-actinin, ERM proteins, cortactin, and β-tubulin. In B cells, ICAM-1 cross-linking can activate Src family kinases, such as p53 / p56Lyn. A very important molecule in the signaling pathway of ICAM-1 is small GTPase Rho, a member of the Ras superfamily of G proteins. Rho and downstream Rho associated kinase (ROCK) play an important role in regulating cytoskeletal rearrangement and maintaining cell morphology. The clustering of ICAM-1 induced by antibody cross-linking or co-culture with monocytes is accompanied by the co-localization of ERM proteins and the assembly of stress fibers. This process requires the activation of RhoA, and the cytoplasmic tail of ICAM-1 plays an important role in this process: the clustering of ICAM-1 lacking the cytoplasmic tail cannot activate Rho protein. The activation and inactivation of Rho are strictly regulated by many factors, including guanine exchange factors (GEFs), GTPase activating proteins (GAPs) and guanine nucleotide dissociation inhibitor (GDI). The specific mechanism of ICAM-1 activating Rho is not clear, but ERM proteins and Rho-GDI may play an important role in it. On endothelial cells, ICAM-1 binds to LFA-1 or Mac-1 on leukocytes, activates downstream Rho and ROCK, causes cytoskeletal rearrangement and morphological changes, thereby mediating leukocyte transmigration across the blood vessel and into the inflammatory tissue.

[0130] The obtained ICAM-1 positive adipose stromal cells can be used for medical cosmetology, such as remodeling of adipose tissue.

[0131] ICAM-1 inhibitors and promoters

[0132] The present application provides the use of ICAM-1 inhibitors in promoting the differentiation of adipose stem cells into adipocytes, and the use of ICAM-1 or its promoters in inhibiting the differentiation of adipose stem cells into adipocytes. Wherein, the ICAM-1 inhibitor specifically inhibits the expression or activity of ICAM-1, and the ICAM-1 promoter specifically promotes the expression or activity of ICAM-1.

[0133] Based on the above-mentioned use, the present application further provides a non-therapeutic method for preparing adipocytes in vitro, which comprises the steps of:

[0134] (a) providing an ICAM-1 positive adipose stromal cell;

[0135] (b) culturing the adipose stromal cell under conditions suitable for the differentiation of adipocytes, thereby obtaining a cell population comprising differentiated adipocytes; and

[0136] (c) isolating the adipocytes from the cell population.

[0137] RNA interference (RNAi)

[0138] In the present application, an effective ICAM-1 inhibitor is an interfering RNA.

[0139] As used herein, the term "RNA interference (RNAi)" refers to the fact that some small double-stranded RNAs can efficiently and specifically block the expression of specific genes in vivo, promote the degradation of mRNA, and induce cells to exhibit the phenotype of specific gene deletion, which is also called RNA intervention or RNA interference. RNA interference is a highly specific gene silencing mechanism at the mRNA level.

[0140] As used herein, the term "small interfering RNA (siRNA)" refers to a short double-stranded RNA molecule that can target specific mRNA for degradation with homologous complementary sequence mRNA, which is the RNA interference pathway.

[0141] In the present application, the interfering RNA includes siRNA, shRNA and corresponding constructs.

[0142] A typical construct is double-stranded, and its positive or negative strand contains the structure shown in Formula I:

[0143] Seq 正向 -X-Seq 反向 Formula I

[0144] wherein,

[0145] Seq 正向 is a nucleotide sequence of an ICAM-1 gene or fragment;

[0146] Seq 反向 is a nucleotide sequence substantially complementary to Seq 正向 .

[0147] X is a spacer sequence between Seq 正向 and Seq 反向 , and the spacer sequence is not complementary to Seq 正向 and Seq 反向 .

[0148] In a preferred embodiment of the present application, Seq 正向 , Seq 反向 has a length of 19-30 bp, preferably 20-25 bp.

[0149] In the present application, a typical shRNA is shown in Formula II,

[0150]

[0151] wherein,

[0152] Seq’ 正向 is a RNA sequence or sequence fragment corresponding to Seq 正向 .

[0153] Seq’ 反向 is a sequence substantially complementary to Seq’ 正向 .

[0154] X’ is nothing; or is a spacer sequence between Seq’ 正向 and Seq’ 反向 , and the spacer sequence is not complementary to Seq’ 正向 and Seq’ 反向 .

[0155] || represents a hydrogen bond formed between Seq 正向 and Seq 反向 .

[0156] In another preferred embodiment, the spacer sequence X has a length of 3-30 bp, preferably 4-20 bp.

[0157] wherein, Seq 正向 the target gene to which the sequence is directed includes (but is not limited to) Beclin-1, LC3B, ATG5, ATG12, or a combination thereof.

[0158] Compositions and methods of administration

[0159] The present application also provides a composition for promoting or inhibiting the differentiation of adipose stem cells into adipocytes, which contains an ICAM-1 inhibitor or promoter as an active ingredient. The composition includes, but is not limited to, a pharmaceutical composition, a food composition, a dietary supplement, a beverage composition, etc.

[0160] In the present application, the ICAM-1 inhibitor can be directly used for medical cosmetology, for example, for the remodeling of adipocytes. In using the ICAM-1 inhibitor of the present application, other components can also be used simultaneously, for example, in combination with adipose stem cells.

[0161] The present application also provides a pharmaceutical composition, which contains a safe and effective amount of the ICAM-1 inhibitor or promoter of the present application and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, powder, and combinations thereof. The pharmaceutical preparation should be matched with the mode of administration. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition, such as tablets and capsules, can be prepared by a conventional method. The pharmaceutical composition, such as needles, solutions, tablets, and capsules, should be manufactured under sterile conditions. The pharmaceutical composition of the present application can also be prepared in the form of a powder for aerosol inhalation. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the ICAM-1 inhibitor of the present application can be used in combination with other therapeutic agents.

[0162] The pharmaceutical composition of the present application can be administered to a desired subject (such as a human and a non-human mammal) by a conventional method. Representative modes of administration include, but are not limited to, oral administration, injection, aerosol inhalation, etc.

[0163] In using the pharmaceutical composition, a safe and effective amount of the ICAM-1 inhibitor is administered to a mammal, wherein the safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases, not more than about 8 mg / kg body weight, and preferably, the dose is about 10 μg / kg body weight to about 1 mg / kg body weight. Of course, the specific dose should also take into account the route of administration, the patient's health condition, etc., which are within the skill of a skilled physician.

[0164] Detection reagent

[0165] The detection reagent of the present application includes a protein chip, a nucleic acid chip, or a combination thereof.

[0166] In another preferred embodiment, the detection reagent of the present application further comprises an ICAM-1 specific antibody.

[0167] Protein chips are high throughput monitoring systems that monitor the interaction between protein molecules through the interaction of target molecules and capture molecules. Capture molecules are generally pre-immobilized on the chip surface and antibodies are widely used as capture molecules due to their high specificity and strong binding characteristics with antigens. The effective immobilization of antibodies on the chip surface is very critical for the study of protein chips, especially in terms of the consistency of the immobilized antibodies, which is important for enhancing the sensitivity of the protein chip. Protein G is an antibody binding protein that specifically binds to the FC fragment of antibodies and has been widely used to immobilize different types of antibodies. The protein chip for detecting ICAM-1 of the present application can be prepared by various techniques known to those skilled in the art.

[0168] Nucleic acid chips, also known as DNA chips, gene chips or gene microarrays, refer to the in-situ synthesis of oligonucleotides on a solid support or the direct immobilization of a large number of DNA probes on the surface of the support in a micro-printing manner, followed by hybridization with labeled samples, and the genetic information of the sample can be obtained by detecting and analyzing the hybridization signal. In other words, gene chips are constructed by microfabrication technology, in which tens of thousands or even millions of DNA fragments (gene probes) with specific sequences are regularly arranged and fixed on a 2 cm 2 square silicon wafer, glass slide or other support to form a two-dimensional DNA probe array, which is very similar to an electronic chip on a computer, hence the name gene chip.

[0169] The present application relates to polyclonal and monoclonal antibodies specific for human ICAM-1, and in particular to monoclonal antibodies. Herein, "specific" means that the antibody binds to the human ICAM-1 gene product or fragment. Preferably, it refers to those antibodies that bind to the human ICAM-1 gene product or fragment but do not recognize and bind to other non-related antigen molecules. The antibodies of the present application include those that can bind to and inhibit the function of the human ICAM-1 protein, and also include those that do not affect the function of the human ICAM-1 protein. The present application also includes antibodies that can bind to the human ICAM-1 gene product in modified or unmodified form.

[0170] The present application includes not only intact monoclonal or polyclonal antibodies, but also antibody fragments having immunological activity, such as Fab' or (Fab)2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules (Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies having the binding specificity of a murine antibody but retaining a portion of the antibody from a human.

[0171] The antibodies of this invention can be prepared using various techniques known to those skilled in the art. For example, purified human ICAM-1 gene product or its antigenic fragment can be administered to animals to induce the production of polyclonal antibodies. Similarly, cells expressing human ICAM-1 protein or its antigenic fragment can be used to immunize animals to produce antibodies. The antibodies of this invention can also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al.). Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol .6:292,1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas (Elsevier, NY, 1981). The antibodies of this invention include antibodies that block the function of human ICAM-1 protein and antibodies that do not affect the function of human ICAM-1 protein. The various antibodies of this invention can be obtained using fragments or functional regions of the human ICAM-1 gene product through conventional immunoassay techniques. These fragments or functional regions can be prepared using recombinant methods or synthesized using a peptide synthesizer. Antibodies binding to the unmodified form of the human ICAM-1 gene product can be produced by immunizing animals with the gene product generated in prokaryotic cells (e.g., E. coli); antibodies binding to the post-translational modified form (e.g., glycosylated or phosphorylated proteins or peptides) can be obtained by immunizing animals with the gene product generated in eukaryotic cells (e.g., yeast or insect cells).

[0172] Detection methods and test kits

[0173] This invention provides a detection method and a detection kit utilizing ICAM-1 and its detection reagents.

[0174] Specifically, the present invention provides a kit comprising a container containing ICAM-1 or its detection reagent; and a label or instruction manual indicating that the kit is used for (a) detecting adipose stem cells and / or (b) assessing the risk of obesity in test subjects.

[0175] This invention also provides a method for determining the risk of obesity in test subjects, comprising the steps of:

[0176] (a) Provide a sample of the test subjects;

[0177] (b) Determination of ICAM-1 in the sample + The cell ratio is A1;

[0178] (c) comparing the proportion of ICAM-1 + If A1 is significantly higher than A0, it indicates that the test subject has a high risk of obesity.

[0179] In another preferred embodiment, the method further comprises determining the proportion of FABP4 + cells B1 in the sample, and comparing B1 with the proportion of FABP4 + cells B0 in the normal population, if B1 is significantly lower than B0, it indicates that the test subject has a high risk of obesity.

[0180] The main advantages of the present application include:

[0181] (a) The present application finds that ICAM-1 + Adipose stem cells have the ability of spontaneous adipogenic differentiation, which can be differentiated into adipocytes in vitro and in vivo, and participate in the development and remodeling of adipose tissue.

[0182] (b) The present application finds that ICAM-1 + The number of adipose stem cells is positively correlated with the increase and increase of obese adipose tissue, and can be used to guide the diagnosis of obesity.

[0183] (c) The present application finds that ICAM-1 has a negative regulatory effect on the in vivo adipogenic differentiation of human adipose precursor cells, and the expression level of ICAM1 in human adipose precursor cells gradually decreases with adipogenic differentiation.

[0184] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, which are usually carried out according to the conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0185] General materials and methods

[0186] ICAM-1- / - mice (B6.129S4-Icam1tm1Jcgr / J) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Fabp4-Cre (B6.Cg-Tg(Fabp4-cre)1Rev / JNju) mice, mTmG (B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / JNju) mice were purchased from Nanjing Model Animal Center. Icam1-CreERT2 knockin mice were constructed by Southern Model Organism Center, and CreERT2 expression sequence was directly inserted into the start codon ATG of Icam1 gene by Cas9 technology.

[0187] Tamoxifen induction of cell lineage tracing in vivo

[0188] Postnatal Icam-1-CreRET2; mTmG mice were injected i.p. with 200 μg / mice Tamoxifen for 3 consecutive days from P1 to P3. Tamoxifen was prepared in corn oil as a stock solution of 20 mg / ml. When mice were 4-6 weeks old, they were euthanized and adipose tissues were analyzed for EGFP+ adipocytes.

[0189] Measurement of body fat ratio in mice

[0190] High fat induced obese mice were analyzed for adipose and other "lean" tissues using a Body Composition Analyzer. Data were collected for 2-3 times consecutively for each mouse and the mean values were taken.

[0191] Culture of adipose stromal cells

[0192] Sorted CD31 - CD45 - Sca-1 + PDGFR-α + Adipose stromal cells, and CD31 - CD45 - Sca-1 + PDGFR-α + ICAM-1 + and CD31 - CD45 - Sca-1 + PDGFR-α + ICAM-1 - were cultured in DMEM low glucose medium with 10% FBS. In some experiments, adherent adipose mononuclear cells were directly cultured in DMEM low glucose medium with 10% FBS. Immune cells and vascular endothelial cells were removed by medium change and passaging to obtain pure adipose stromal cells.

[0193] Induction of adipogenic differentiation of stromal cells

[0194] Prepare the differentiation medium by adding 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 50 μM indomethacin, 10 μg / ml insulin, and 0.5 μM dexamethasone to 10% FBS DMEM high-glucose medium. Once the adipocytes have reached 100% confluence, change the differentiation medium every two days until differentiation is complete, which takes approximately 5 days.

[0195] Example 1

[0196] ICAM-1-expressing adipose stromal cells are potential adipose-derived stem cells.

[0197] By analyzing non-endothelial cells and non-leukocytes (CD31) in adipose tissue rich in adipose stem cells - CD45 - Cellular characteristics of cells (cells) revealed CD31 - CD45 - The stromal cells are mostly CD34 + and CD29 + It is also PDGFR-α + Sca-1 + ( Figure 1 A), the latter being two characteristic surface molecules of mesenchymal stromal cells (MSCs).

[0198] Further analysis of adipose stromal cells using flow cytometry revealed CD45 - CD31 - The stromal cells were mostly Sca-1 + PDGFR-α + These cell populations can all be classified as ICAM-1 + and ICAM-1 - Two groups, we found this group of CD45 - CD31 - Sca-1 + PDGFR-α + Approximately 50% of cells in the inguinal adipose tissue (subcutaneous adipose tissue) are ICAM-1 positive, and approximately 80% of cells in the epididymal adipose tissue (visceral adipose tissue) are ICAM-1 positive. Figure 1 B).

[0199] CD45 was obtained by flow cytometry sorting. - CD31 - Sca-1 + PDGFR-α+ ICAM-1 + and CD45 - CD31 - Sca-1 + PDGFR-α + ICAM-1 - Two groups of stromal cells were analyzed genetically, revealing that characteristic molecules of preadipocytes, Pdgfrb and Zfp423, as well as adipogenic differentiation-related molecules, Pparg, Cebpa, and Fabb4, were present in ICAM-1. + High expression in cells ( Figure 1 C). This result indicates that preadipocytes are primarily found in ICAM-1-positive stromal cells, and ICAM-1... + adipose stromal cells (CD31) - CD45 - Sca-1 + PDGFR-α + It is rich in adipose stem cells and adipose precursor cells.

[0200] To further examine ICAM-1 + Whether adipose-derived stem cells possess the potential for spontaneous adipogenic differentiation, and sorting ICAM-1. + adipose stromal cells (CD31) - CD45 - Sca-1 + PDGFR-α + ) and ICAM-1 - adipose stromal cells (CD31) - CD45 - Sca-1 + PDGFR-α + The spontaneous adipogenic differentiation capacity of wild-type ICAM-1 cells was analyzed. Considering that spontaneous adipogenic differentiation may be the result of interactions between different cell populations through paracrine mechanisms, wild-type ICAM-1 cells were used in this study. - stromal cells and ICAM-1 in EGFP mice + Mixed culture of stromal cells allows for the tracking of different cell populations without affecting their interactions.

[0201] The results showed that during the initial adherent growth phase, cells from both sources exhibited fibroblast morphology. In the later culture stage (day 8), spontaneous adipogenic differentiation occurred, with lipid droplets accumulating in some cells. Interestingly, the vast majority of spontaneously adipogenic differentiated cells were EGFP. + This indicates that ICAM-1 + Adipose stromal cells may be adipose stem cells ( Figure 1 D).

[0202] Meanwhile, we also performed reverse co-culture, in which wild type ICAM-1 + cells and EGFP expressing ICAM-1 - cells were mixed together, and found that all spontaneously adipogenic differentiated cells were ICAM-1 + cells. In addition, ICAM-1 - adipose stromal cells and ICAM-1 + adipose stromal cells were cultured separately, and it was found that only ICAM-1 + cells could spontaneously adipogenic differentiate. These results indicated that ICAM-1 + adipose stromal cells are adipose stem cells with the ability of directional adipogenic differentiation.

[0203] Example 2

[0204] ICAM-1 + adipose stem cells in vivo

[0205] To fully verify whether ICAM-1 + adipose stromal cells are adipose stem cells, i.e., whether they can generate mature adipocytes in vivo, we made Icam1-CreERT2 knock-in mice: using CRISPR / Cas9 technology, we knocked in a CreERT2 expression cassette at the ATG site of the ICAM-1 gene by homologous recombination. In this Icam1-CreERT2 knock-in mouse, cells expressing ICAM-1 will also express CreERT2, which does not have recombinase activity itself and needs to be combined with Tamoxifen to activate its recombinase activity. The mTmG tracer reporter mouse expresses a cell membrane-localized red fluorescent protein, tdTomato, in all cells and tissues in the absence of Cre recombinase; when Cre recombinase is present, the tdTomato expression sequence is deleted by recombination, and the downstream cell membrane-localized EGFP is expressed, and the progeny cells of the cells will only express cell membrane-localized EGFP. Therefore, we crossed the Icam1-CreERT2 knock-in mouse with the tracer reporter mouse mTmG, and treated the newborn mice with Tamoxifen to activate the recombinase activity. The activated CreERT2 recombinase in ICAM-1 + cells will cut off the tdTomato expression sequence between the two loxP sites on the Rosa26 locus, and initiate the expression of EGFP on the following sequence, so that ICAM-1 + cells and their derived progeny cells will express EGFP Figure 2 A).

[0206] Results show that when neonatal mice are treated with Tamoxifen, EGFP adipocytes can be detected in their subcutaneous and visceral adipose tissues in adulthood (Fig. 2A). Figure 2 B) More importantly, when high-fat diet-induced obese mice are treated with Tamoxifen at an early stage, EGFP adipocytes can be observed at a later stage of obesity (Fig. 2C). Since adipocytes do not express ICAM-1, the above results suggest that ICAM-1 + Adipose stem cells participate in adipogenesis and obesity by differentiating into mature adipocytes.

[0207] Example 3

[0208] ICAM-1 in obesity + Adipogenic differentiation of adipose precursor cells

[0209] Next we investigate this population of ICAM-1 + Relationship between adipose precursor cells and obesity. To this end, we introduce another lineage tracing system. FABP4 is a marker expressed by adipose stem cells when they differentiate into adipocytes. We cross Fabp4-Cre mice with mTmG tracing mice, and in the offspring, adipose precursor cells that have undergone adipogenic differentiation to the early adipocyte stage expressing Fabp4 will express EGFP (Fig. 3A). We feed Fabp4-Cre; mTmG mice with normal and high-fat diets, respectively, and analyze the stromal cells (CD45 - CD31 - Sca-1 + ) in inguinal and epididymal adipose tissues for EGFP + expressing early differentiated adipocytes by flow cytometry.

[0210] We find that in normal chow diet, compared with littermate Fabp4-Cre mice, there are only a small number of EGFP + adipose precursor cells in the adipose tissues of Fabp4-Cre; mTmG adult mice, and they are mainly CD31 - CD45 - Sca-1 + ICAM-1 + ( Figure 3 B), suggesting that they are derived from ICAM-1 + adipose stem cells, which still maintain the surface molecular phenotype of adipose stem cells, ICAM-1 + adipose stem cells participate in the normal turnover of adipocytes. Importantly, when these mice are induced to be obese with high-fat diet, there are a large number of EGFP expressing early adipocytes in both adipose tissues, which still maintain ICAM-1 +Surface molecular profile of adipose stem cells (CD31 - CD45 - Sca-1 + ICAM-1 + ( Figure 3 C-D), indicating that obesity induces adipocyte neogenesis, and these newly differentiated adipocytes are mainly derived from ICAM-1 + adipose stem cells. Meanwhile, we found these EGFP + ICAM-1 + early adipocytes in obese adipose tissue, which are all located around blood vessels, with the same localization as ICAM-1 + adipose stem cells ( Figure 3 E).

[0211] To further identify these ICAM-1 + EGFP + cells, we sorted mature adipocytes, ICAM-1 + EGFP + , ICAM-1 + EGFP - and ICAM-1 - cells from obese mice for RNA-seq analysis. We found that the gene expression profile of ICAM-1 + EGFP + subgroup is very similar to that of ICAM-1 + EGFP - subgroup, with a correlation coefficient of 0.98 ( Figure 3 F). Compared with other subgroups, ICAM-1 + EGFP + cells have a similar gene expression pattern to adipocytes ( Figure 3 F), especially when focusing on genes involved in adipocyte characteristic signaling pathways ( Figure 3 G). Among these adipocyte characteristic genes, adipocytes have the highest correlation with ICAM-1 + EGFP + , followed by ICAM-1 + EGFP - cells, and the lowest correlation with ICAM-1 - . These results indicate that ICAM-1 + EGFP + cells are an intermediate product of adipogenic differentiation, derived from ICAM-1 + EGFP - adipose stem cells.

[0212] Example 4

[0213] ICAM-1 negatively regulates terminal differentiation of adipocyte precursor cells

[0214] Based on the above studies, it has been demonstrated that ICAM-1 expression on adipose stem cells and adipocyte precursor cells, during adipogenesis, however, mature adipocytes do not express ICAM-1, and ICAM-1 expression gradually decreases during adipogenic differentiation. This expression profile is similar to the characteristic genes of adipocyte precursor cells, Pref-1, GATA2 / GATA3, which have the function of resisting adipogenic differentiation and maintaining the undifferentiated state of adipocyte precursor cells, from which we speculate that ICAM-1 can play the same regulatory role. Studies have found that compared with wild-type mice, ICAM-1 - / - Mice significantly increased in body weight and adipose tissue weight under both normal diet and high-fat diet conditions, and the increase in adipose tissue was not dependent on the increase in adipocyte volume (Fig. 4A-D). Since ICAM-1 is expressed on immune cells, in order to rule out the effect of immune cell ICAM-1 deletion on obesity, we performed a bone marrow replacement experiment, and found that even if the immune cells of ICAM-1 - / - Mice whose immune cells were replaced with wild-type mouse immune cells were still more prone to obesity Figure 4 E-F). Since the proliferation of fat includes both cell enlargement and increase, we found by analysis that ICAM-1 - / - Mice did not significantly increase in adipocyte size Figure 4 G-H), indicating that the increase in adipocyte number played an important role in their obesity, which was a result of the over-differentiation of adipose stem cells.

[0215] To determine the contribution of the increase in adipocyte number to the occurrence of obesity, we crossed ICAM-1 - / - Mice and Fabp4-Cre; mTmG mice were crossed, and it was found that compared with ICAM-1 + / + ICAM-1 - / - ICAM-1 + The adipogenic differentiation of intermediate cells in Fabp4-Cre; mTmG mice was significantly increased Figure 4 I-K), indicating that ICAM-1 deletion can promote the adipogenic differentiation process of adipose stem cells in vivo. Compared with wild-type adipose stem cells, ICAM-1 - / - Primary adipocyte precursor cells of ICAM-1 Figure 4 H) differentiated faster Figure 5 A-D). Therefore, ICAM-1 negatively regulates the terminal differentiation of adipose stem cells.

[0216] Example 5

[0217] ICAM-1 maintains the undifferentiated state of adipose stem cells through Rho and ROCK

[0218] To further investigate the molecular mechanism of ICAM-1 in controlling adipogenic differentiation, one of the most important components in the downstream signaling of ICAM-1 is the small GTPase Rho. We found that ICAM-1 - / - The activated form of Rho (Rho-GTP) in the activated adipose precursor cells was significantly less than that in wild-type precursor cells, and the inactive Rho-GDP was higher than that in wild-type precursor cells Figure 6 A). Activated Rho can regulate the formation of intracellular stress fibers through ROCK. By performing fluorescent immunoassay on F-actin, we found that there was a large amount of tightly structured stress fibers in wild-type precursor cells, and the fiber bundles of F-actin and ICAM-1 clusters were co-localized; while in ICAM-1 - / - precursor cells, the density of stress fibers was significantly lower than that in wild-type stromal cells, and the structure was loose, and the fiber bundles were rare Figure 6 B). This indicates that ICAM-1 can activate Rho and ROCK in adipose precursor cells, and play an important role in the assembly of stress fibers and the construction of cytoskeleton.

[0219] Rho and ROCK can negatively regulate adipogenic differentiation through cytoskeleton-dependent or insulin signaling-dependent ways, and our RNA-seq data also supports the role of Rho GTPase in adipogenic differentiation. To test whether Rho and ROCK are involved in the inhibitory effect of ICAM-1 on adipogenic differentiation of adipose stem cells, we treated adipose stem cells with the inhibitor Y-27632 of ROCK. We found that compared with the DMSO treatment group, Y-27623 could significantly accelerate the adipogenic differentiation of wild-type adipose stem cells, while the effect on ICAM-1 - / - adipose stem cells was not obvious Figure 6 C). At the same time, we analyzed the expression level of the mature adipocyte characteristic protein Perilipin A, and found that Y-27632 could significantly increase the expression level of this protein in wild-type adipose stem cells, while the effect on ICAM-1 - / - adipose stem cells was not obvious Figure 6 C). Moreover, inhibiting ROCK could significantly increase the expression of adipogenic differentiation-related proteins and genes in wild-type mouse adipose stem cells, including Perilipin A, Pparg and Fabp4, while the effect on ICAM-1 - / - mouse-derived adipose stem cells was not obvious Figure 6ICAM-1 inhibits adipogenic differentiation of adipose stem cells through Rho-ROCK pathway.

[0220] To verify whether Rho GTPase activity can reverse the excessive adipogenic differentiation caused by ICAM-1 deficiency, we used Rho activator II (RA2), a Rho activator that constitutively activates Rho GTPase. We found that RA2 significantly inhibited ICAM-1 - / - adipogenic differentiation of adipose stem cells, but had no obvious effect on wild-type adipose stem cells Figure 6 G). Consistently, activation of Rho GTPase in ICAM-1 - / - precursor cells led to a broad reduction of adipogenic genes, including Pparg, Cebpa, Fabp4, and Plinl, while only Pparg and Fabp4 were significantly changed by Rho GTPase activation in wild-type cells Figure 6 H-K). Importantly, the expression difference of adipogenic genes between wild-type and ICAM-1 - / - precursor cells was abolished by Rho GTPase activation Figure 6 H-K). These results confirmed that ICAM-1 regulates adipogenic differentiation through Rho GTPase.

[0221] To test whether ICAM-1 regulates adipogenic differentiation through Rho GTPase in vivo, we injected RA2 locally into the right inguinal fat pad of mice and showed the effect of local Rho GTPase activation by comparing with the left fat pad. By treating mice with high-fat diet for 10 weeks with RA, we found that the excessive adipogenic differentiation of ICAM-1 - / - mice was attenuated, and the inguinal fat pads on both sides were asymmetric Figure 6 L). This asymmetry was not observed in RA2-treated WT mice and PBS-treated mice Figure 6 L). We collected these fat tissues for weighing and found that RA2 significantly reduced fat weight in ICAM-1 - / - but not in WT mice Figure 6 M-N).

[0222] Example 6

[0223] ICAM-1 negatively regulates human adipose precursor cell differentiation

[0224] We first analyzed the expression level of ICAM-1 in human adipose tissues. Currently, there is no well-accepted marker for human adipose precursor cells. We found that ICAM-1 was expressed in human CD31 - CD45- ICAM-1 is widely expressed in adipose stromal cells Figure 7 A). These ICAM-1 + cells are also mainly located around blood vessels as in mouse adipose tissue Figure 7 B). To examine the regulatory role of ICAM-1 in human adipose stem cells, we isolated primary human adipose stromal cells for adipogenic differentiation induction. We found that, consistent with mouse, the expression level of ICAM1 in human adipose precursor cells gradually decreased with adipogenic differentiation Figure 7 C). When the expression of ICAM1 was knocked down by siRNA Figure 7 D), the adipogenic differentiation of human adipose precursor cells was significantly enhanced Figure 7 E), the expression of adipogenic genes including PPARG, CEBPA and FABP4 was significantly increased Figure 7 F), indicating that ICAM-1 negatively regulates the adipogenic differentiation of human adipose stem cells. Notably, the knockdown of ICAM-1 led to the decrease of Rho GTPase activity in human adipose stem cells Figure 7 G). When human adipose stem cells were treated with RA2 during differentiation, the enhanced adipogenic differentiation of ICAM-1 knockdown cells was abolished Figure 7 H-J). Therefore, ICAM-1 also has the ability to negatively regulate the terminal differentiation of human adipose stem cells.

[0225] To examine the physiological role of ICAM-1 in human adipose precursor cells, we collected human adipose tissue samples from patients undergoing plastic surgery and analyzed the expression levels of ICAM-1 and FABP4 on CD31 - CD45 - adipose stromal cells by flow cytometry. The expression level of ICAM-1 was significantly correlated with the body mass index (BMI) of the subjects Figure 7 K), a result similar to that observed in mice. We used linear regression analysis to examine the correlation between the expression level of ICAM-1 and the proportion of FABP4 + adipose precursor cells. Given the strong correlation between BMI and the expression of ICAM-1, we introduced an interaction term of BMI and ICAM-1 MFI to obtain a corrected linear model. On this basis, we found that the proportion of FABP4 + adipose precursor cells was significantly negatively correlated with the expression level of ICAM-1 Figure 7 K-L), indicating that ICAM-1 negatively regulates the adipogenic differentiation of human adipose precursor cells in vivo.

[0226] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to readers of the disclosure and the appended claims.

Claims

1. The use of an ICAM-1 inhibitor, characterized in that, This preparation is intended to develop a formulation or composition for promoting spontaneous adipogenic differentiation of adipose-derived stem cells into adipocytes; wherein the adipose-derived stem cells are CD45. - CD31 - Sca-1 + PDGFR-α + ICAM-1 + The cells, and the adipose stem cells are ICAM-1 positive adipose stromal cells, and the adipose stem cells express the regulatory genes Pparg, Cebpa and Fapp4 for adipogenic differentiation.

2. The use as described in claim 1, characterized in that, The adipose-derived stem cells also express regulatory genes for adipogenic differentiation selected from the following group: Cebpb, Cebpg, Gata2, Gata3, Irs1, or combinations thereof.

3. The use as described in claim 1, characterized in that, The adipose-derived stem cells further express characteristic molecules selected from the group consisting of CD34, CD29, CD24, Pdgfr-β, Zfp423, or combinations thereof.

4. The use as described in claim 1, characterized in that, The formulations or compositions described herein are also used for the remodeling of adipose tissue.

5. A method for preparing adipocytes in vitro without therapeutic effect, characterized in that, The method includes the following steps: (a) Provide an ICAM-1 positive adipose stromal cell, wherein the adipose stromal cell is a CD45 cell. - CD31 - Sca-1 + PDGFR-α + ICAM-1 + The cells, specifically the adipose stromal cells, express the regulatory genes Pparg, Cebpa, and Fapp4 for adipogenesis differentiation; (b) Culture the aforementioned adipocytes to induce spontaneous adipogenic differentiation, thereby obtaining a cell population containing differentiated adipocytes; and (c) Isolate adipocytes from the cell population.

6. The method as described in claim 5, characterized in that, In steps (b) and (c), the expression level of ICAM-1 was detected to determine the degree of differentiation of adipocytes into adipocytes in the cell population.

7. The method as described in claim 5, characterized in that, As the degree of differentiation of adipose stromal cells into adipocytes increases, the expression level of ICAM-1 in the adipose stromal cells decreases.

8. The method as described in claim 5, characterized in that, In step (b), ICAM-1 expression in the adipocytes is inhibited, thereby promoting spontaneous adipogenic differentiation of the adipocytes into adipocytes.

9. The method as described in claim 5, characterized in that, In step (b), when the cell population does not substantially express ICAM-1, adipocytes in the cell population are isolated.

10. The use of an ICAM-1 or its detection reagent, characterized in that, This kit is used to prepare a detection kit for (a) detecting adipose-derived stem cells and (b) determining the risk of obesity in test subjects; wherein the adipose-derived stem cells are CD45. - CD31 - Sca-1 + PDGFR-α + ICAM-1 + The cells, and the adipose stem cells are ICAM-1 positive adipose stromal cells, and the adipose stem cells express the regulatory genes Pparg, Cebpa and Fapp4 for adipogenic differentiation.

11. The use as described in claim 10, characterized in that, The kit also contains FABP4 or its detection reagent.

12. The use as described in claim 10, characterized in that, The determination is based on the ICAM-1 of the samples from the test object. + Cell proportion A1 and corresponding ICAM-1 in normal population + If the cell proportion A1 is significantly higher than A0 compared to A0, it indicates that the test subject has a high risk of developing obesity.

13. The use as described in claim 10, characterized in that, The determination also includes using FABP4 from samples from the test subject. + Cell ratio B1 and FABP4 in normal population + If the cell proportion B1 is significantly lower than B0 compared to B0, it indicates that the test subject has a high risk of developing obesity.

14. The use of a stromal cell, characterized in that, The stromal cells are ICAM-1 positive stromal cells isolated from adipose tissue, and the stromal cells are CD45. - CD31 - Sca-1 + PDGFR-α + ICAM-1 + The stromal cells express the regulatory genes Pparg, Cebpa, and Fapp4 for adipogenic differentiation, wherein the stromal cells are used to prepare a cell preparation for the remodeling of adipose tissue.

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