CN113151506B

CN113151506B - Atherosclerosis related cell marker molecule and application thereof

Info

Publication number: CN113151506B
Application number: CN202110548355.4A
Authority: CN
Inventors: 石小峰; 吉训明; 刘桂友; 张永彪; 朱尚明
Original assignee: Brain Disorders Research Center Of Capital Medical University (beijing Institute For Brain Disorders)
Current assignee: Brain Disorders Research Center Of Capital Medical University (beijing Institute For Brain Disorders)
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2022-01-14
Anticipated expiration: 2041-05-19
Also published as: CN113151506A

Abstract

The invention discloses atherosclerosis-related cell marker molecules and application thereof, and utilizes a method of single cell sequencing combined with bioinformatics analysis to discover the marker molecules which can be used for identifying, characterizing and optionally enriching or separating smooth muscle cell subsets; the invention also discloses that the smooth muscle cell subsets have significant difference in atherosclerosis.

Description

Atherosclerosis related cell marker molecule and application thereof

Technical Field

The invention belongs to the field of biological medicines, and relates to an atherosclerosis related cell marker molecule and application thereof.

Background

Atherosclerosis (AS) is a chronic inflammatory disease typically characterized by the formation of fibrofatty lesions under the intima of arteries. Worldwide, AS is the most important cause of cardiovascular and cerebrovascular and peripheral vascular diseases (e.g., myocardial infarction, ischemic stroke, gangrene of the lower extremities, etc.). The susceptibility of AS in different parts of the human arterial system is significantly different, wherein the abdominal aorta, iliac artery, carotid artery and coronary artery are the four most susceptible parts of AS, and the subclavian artery, aortic arch and thoracic aorta are not easy to generate AS. Due to the lack of an ideal large animal model capable of simulating human AS, the understanding of the characteristics and the occurrence mechanism of AS susceptible parts is very limited at present. An ideal large animal model similar to the human AS diseased part is constructed, and the characteristic difference, the cell composition difference, the cell proportion difference and the gene expression difference of the AS susceptible part and the AS less susceptible part are compared, so that the effective way for explaining the generation mechanism is provided.

ApoE of AS large animal model constructed in earlier stage of project group^-/-Beagle, Apo^E-/-Beagle dogs have advantages beyond the reach of other AS animal models, can spontaneously develop AS without high fat diet induction, and have a disease site highly similar to that of human AS, and also exhibit clinical endpoint events highly similar to that of human AS. ApoE^-/-Beagle dogs are the most ideal big animal model for researching the characteristics and the occurrence mechanism of AS susceptible parts at present.

The study of multiomics can reveal the change of gene expression profile in AS lesion process in a panoramic way, but most of the previous study of omics focuses on the tissue level, the cells are analyzed AS a homogeneous whole, and the obtained omics data are often the mixed average result of a plurality of cell groups, so that the dynamic change process of omics of each cell group is difficult to reflect directly. The traditional sequencing method ignores the heterogeneity of tissue structure and cells (Bondarev O, et al (2019) Identification of pathological stress sensitivity indexes in endogenous cells. Cardiovasular research 115(10): 1487) 1499), and the obtained omics data are often the mixed average result of various cell groups, so that the dynamic change process of each cell group in the disease occurrence and development process is difficult to directly analyze. In real plaque tissue, the involved cell types are extremely complex, and the heterogeneity and plasticity of the various cells during disease progression is further complicated. Therefore, solving the problems of disordered progression of cellular timing and cellular heterogeneity in the plaque is the central importance of deeply revealing the pathogenesis of AS.

The problem was solved by the advent of Single cell transcriptome sequencing technology (scRNA-seq) (Saliba AE, Westernmann AJ, Gorski SA, & Vogel J (2014) Single-cell RNA-seq: advances and future variations. nucleic acids research 42(14):8845 + 8860). The scRNA-seq can reveal the overall appearance of cellular heterogeneity and define key characteristics of cell subsets at unprecedented resolution, can draw a panoramic cell map of AS lesion blood vessels, characterize the functional state of each cell and identify key cell subsets and potential regulatory factors from the panoramic cell map through single cell sequencing, and provides a new means for personalized diagnosis and treatment of AS.

Disclosure of Invention

In the research, a systemic AS large animal model ApoE-/-beagle dog is used AS a research object, and the susceptibility mechanism of AS is analyzed at the single cell level. The inventor of the invention utilizes a method of single cell transcriptome sequencing combined with bioinformatics analysis, separates and characterizes cell subsets capable of reflecting organism AS by analyzing single cell gene expression profiles of a disease group and a normal group, and further researches and determines new characteristic genes expressed by the cell subsets and the correlation between the characteristic genes and AS, thereby completing the invention. The research has important significance for revealing the occurrence mechanism of AS-related diseases, expanding the early intervention time window of the diseases and continuously reducing the incidence of cardiovascular and cerebrovascular diseases.

In a first aspect the present invention provides a biomarker for identifying or detecting a smooth muscle cell subpopulation, said biomarker comprising the gene TNC, or a protein or protein fragment expressed by said gene.

Further, the biomarker TNC is highly expressed in smooth muscle cell subpopulations.

A second aspect of the invention provides an agent or kit for identifying or detecting a smooth muscle cell subpopulation/diagnosing atherosclerosis, the agent or kit comprising a binding agent capable of binding to a TNC gene or a protein or protein fragment expressed therefrom in smooth muscle cells.

Further, the reagent or kit further comprises a binding agent capable of binding to a biomarker of smooth muscle cells.

Further, the biomarkers include one or more of ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM2, CALD1, GPR38, HEXIM1, CDH5, SM22 a.

Further, the biomarker is selected from one or more of ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM 2.

Further, TNC is highly expressed in smooth muscle cell subsets.

Further, the kit comprises a container or containers holding one or more of the binding agents.

Further, the kit further comprises instructions.

Further, the binding agent is selected from a nucleic acid, a ligand, an enzyme, a substrate and/or an antibody.

Further, a reporter molecule is attached to the binding agent.

Further, the reporter molecule is selected from a fluorescent substance, a radioactive substance and/or an enzyme.

Further, the nucleic acid includes a probe that hybridizes to the gene.

Further, the nucleic acid includes a primer for amplifying the gene.

Further, the antibody is an antibody that binds to a protein or a fragment of a protein encoded by the gene.

A third aspect of the invention provides a smooth muscle cell subpopulation expressing TNC.

Further, the smooth muscle cell subpopulation is highly expressing TNC.

A fourth aspect of the invention provides a method of enriching a smooth muscle cell subpopulation according to the third aspect of the invention, comprising:

contacting smooth muscle cells with a binding agent that binds to a TNC gene or a protein or protein fragment expressed thereby; sorting smooth muscle bound to the binding agent to provide an enriched smooth muscle cell subpopulation.

Further, the sorting techniques include fluorescence activated cell sorting, magnetic assisted cell sorting, substrate assisted cell sorting, laser mediated cleavage, fluorimetry, flow cytometry or microscopy.

In a fifth aspect the invention provides a method of identifying a subpopulation of smooth muscle cells in a test cell population, comprising the steps of:

and detecting the level of the TNC gene in the cell population to be detected, wherein if the TNC gene is highly expressed in the cell, the cell is a smooth muscle cell subgroup.

Further, the method may further comprise the step of identifying smooth muscle cells.

Further, the step of identifying smooth muscle cells comprises detecting a biomarker of smooth muscle cells.

Further, the biomarkers include one or more of ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM2, CALD1, GPR38, HEXIM1, CDH 5.

A sixth aspect of the invention provides the use of any one of,

1) use of a biomarker according to the first aspect of the invention for identifying, screening or sorting a subpopulation of smooth muscle cells;

2) use of a biomarker according to the first aspect of the invention in the manufacture of a product for the diagnosis/treatment of atherosclerosis;

3) use of a reagent or kit according to the second aspect of the invention in the manufacture of a product for identifying or detecting a subpopulation of smooth muscle cells;

4) use of a reagent or kit according to the second aspect of the invention in the manufacture of a product for the diagnosis of atherosclerosis;

5) use of a subpopulation of smooth muscle cells according to a third aspect of the invention for the manufacture of a product for diagnosing atherosclerosis;

6) use of a subpopulation of smooth muscle cells according to a third aspect of the invention for screening a candidate agent for the treatment of atherosclerosis;

7) use of a subpopulation of smooth muscle cells according to a third aspect of the invention for the manufacture of a medicament for the treatment of atherosclerosis.

Further, smooth muscle cell subsets exhibit high expression in sites susceptible to atherosclerosis.

Further, the susceptible site includes abdominal aorta, iliac artery, carotid artery, and coronary artery.

Further, the smooth muscle cell subpopulation exhibited low expression in non-susceptible sites of atherosclerosis.

Further, non-susceptible sites include the subclavian artery, the aortic arch and the thoracic aorta.

A seventh aspect of the present invention provides a method of screening for a cellular marker molecule comprising the steps of:

1) separating single cells and performing lysis;

2) constructing a sequencing library and performing single cell sequencing;

3) and (4) performing bioinformatics analysis on the sequencing result, and searching for cell marker molecules.

Further, the step of bioinformatics analysis includes:

1) sequencing data quality control and expression quantity quantification;

2) cell clustering analysis;

3) the marker gene is identified.

Further, the data quality control conditions are as follows:

each gene is expressed in at least 3 cells;

the number of genes expressed per cell is >200 and < 4000;

mitochondrial genes accounted for < 20% per cell.

Further, the conditions for screening the marker gene were:

adjusted p-value<0.05；

log(fold change)>0.5。

the invention has the advantages and beneficial effects that:

the invention utilizes the single cell sequencing technology and biological informatics analysis to research the single cell gene expression profile of artery tissue and discover a new gene for representing the smooth muscle cell subgroup.

The invention utilizes the single cell sequencing technology to discover the TNC for the first time^highThe smooth muscle cell subgroup is obviously up-regulated in a sample of a susceptible part of an atherosclerosis group by detecting TNC^highThe proportion of the smooth muscle cell subset can determine whether the subject suffers from atherosclerosisSclerosis or the risk of atherosclerosis, thereby intervening in the treatment early and improving the life and the quality of life of the subject.

Definitions and detailed description of the invention

The term "and/or" as used in this application in phrases such as "a and/or B" is intended to include both a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

The term "single cell transcriptome" is a new technology for amplification and sequencing of whole transcriptomes at the single cell level, which is based on high throughput sequencing after amplification of isolated single cell micro-whole transcriptome RNA. In the present invention, single cell sequencing can be performed using any single cell transcriptome sequencing technique that has been disclosed, including but not limited to Tang RNA-Seq, Smart-Seq/Smart-Seq2, CEL-Seq, Quartz-Seq, STRT-seqUMI techniques. The Tang RNA-Seq technique uses poly-T bases as primers to synthesize cDNA and adds poly-base A at the 3' end as the poly-T base binding site of the second cDNA strand, and the amplification is carried out by PCR reaction. The Smart-Seq technology is a single-cell RNA sequencing method capable of detecting full-length mRNA, and can improve the coverage rate of the 5' end of the mRNA. The Smart-seq2 technology improves the primers in the Smart-seq into Locked Nucleotide (LNA) modification, and improves the amplification efficiency of second strand cDNA. CEL-Seq was amplified by in vitro transcription, and bias was removed by linear amplification. The Quartz-seq adopts an inhibition PCR technology to form a hairpin structure from the second strand cDNA of the small segment, thereby reducing the pollution of the small segment. The STRT-seqUMI technology combines molecular markers with microfluidic technology to quantitatively estimate the expression of the initial mRNA.

For the purposes of this application, the terms "selecting", "sorting", "dividing" or "isolating" a selected cell, cell population or cell subpopulation may be used interchangeably and, unless the context indicates otherwise, refer to removing a selected cell or defined subset of cells from a tissue sample and separating from other cells and contaminants not within the parameters defining such cell or cell population. An isolated smooth muscle cell will generally not be contaminated by other cell types and has the ability to allow self-renewal that leads to the production of differentiated progeny. However, when the process produces a cell population, it is understood that it is impractical to provide a composition having absolute purity. In such cases, the cell population is "enriched" for selected cells, wherein these selected cells are then present in the absence of various contaminants (including other cell types) that do not substantially interfere with the function or characteristics of the selected cell subpopulation.

The term "enriched" or variants thereof is used herein to describe a population of cells in which the proportion or percentage of cells of a particular cell type or the proportion or percentage of a plurality of particular cell types is increased in the population when compared to an untreated population of cells (e.g., cells in their natural environment).

In one example, the term "enriched" refers to a proportion or percentage of smooth muscle cells that is greater than the proportion or percentage of smooth muscle cells in the cell population that originally contained it.

The terms "marker", "marker" or "cellular marker" are synonymous and refer to any trait or characteristic in the form of a chemical or biological entity. The label may be morphological, functional or biochemical in nature. In preferred embodiments, the markers are differentially or preferentially expressed by particular cell types, or by cells under certain conditions (e.g., cytokines or surface antigens or membrane proteins or cytoplasmic proteins expressed at particular points in the cell cycle or under particular extracellular matrices, etc. more particularly, in the present invention, are those markers that indicate cells or cell subsets by virtue of their expression levels.

In the present invention "marker" means any moiety that can be used to identify a desired smooth muscle cell. For example, the marker may be a polypeptide molecule expressed on the cell of interest, such as a "surface marker". A specific marker may be present only in, or encompass, a cell of interest, or the detectable level of the marker in the cell of interest is sufficiently high compared to other cells so that the cell of interest can be identified using any of a variety of methods known in the art. One skilled in the art will appreciate that expression is a relative term and that other cell types have different expression.

By "smooth muscle cell" is meant any cell that exhibits at least one phenotypic characteristic of a smooth muscle cell in the muscle cell lineage. In one example, smooth muscle cells may express one or more markers, including ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM2, CALD1, GPR38, HEXIM1, CDH5, and/or SM22 a.

In a typical assay for detection and/or isolation, a population of cells is contacted with at least one label-specific "reagent" and the presence of the complex formed is detected, either directly or indirectly. Methods for isolating and selecting cells based on the expression of cell surface markers are familiar to the skilled person.

The isolated or selected cells bound to the binding agent can then be isolated. Separation of the cells can be achieved by any method known in the art, including affinity-based interaction, affinity panning, magnetic beads (e.g., Dynabeads), or flow cytometry.

In one example, cells are selected using the binding agents described herein and optionally one or more other cell surface markers, and Fluorescence Activated Cell Sorting (FACS) or Magnetic Activated Cell Sorting (MACS) is used^TM) To isolate the cells. In one example, MACS is used^TMTo select cells, wherein the cells are desired for the composition to be studied in vivo or administered in vivo.

In another example, cells can be isolated by binding to a fixed support and then harvested by removing the cells from the support.

Harvesting of the cells may be accomplished by collecting the isolated cells in a suitable container or collection dish, test tube, or the like.

In various embodiments, a smooth muscle cell-enriched cell population is obtained according to a method using a surface marker disclosed herein. However, it will be appreciated that the cells may include one or more other cell surface markers, typically markers known to be expressed on smooth muscle cells.

Regardless of which marker is ultimately selected to identify or characterize the selected cell subpopulation, the actual monitoring or analysis may be carried out using any of a number of standard techniques well known to those skilled in the art. For example, cell surface marker expression can be analyzed by immunoassays including, but not limited to, western blotting, immunohistochemistry, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement binding assays, immunoradiometric assays, fluorescent immunoassays, immunofluorescence assays, protein a immunoassays, laser capture microdissection, large scale multiparametric mass cytometry, flow cytometry, and FACS analysis.

The terms "binding agent", "binding molecule" and "binding entity" are synonymous and may be used interchangeably. In the context of the present invention, the binding agent binds to, recognizes, interacts with, or otherwise associates with a selectable marker on the subpopulation of cells. Exemplary binding agents may include, but are not limited to, antibodies or fragments thereof, antigens, aptamers, nucleic acids (e.g., DNA and RNA), proteins (e.g., receptors, enzymes, enzyme inhibitors, enzyme substrates, ligands), peptides, lectins, fatty acids or lipids, and polysaccharides. For example, in some embodiments of the invention, the binding agent comprises an antibody or fragment thereof, a nucleic acid (e.g., DNA and RNA). As alternative embodiments, the nucleic acid includes, but is not limited to, primers, probes.

The term "antibody" is used in the broadest sense and specifically covers, for example, monoclonal antibodies, polyclonal antibodies, antibodies with polyepitopic specificity, single chain antibodies, multispecific antibodies and antibody fragments. Such antibodies can be chimeric, humanized, human and synthetic.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, such variants typically being present in minor amounts, except for possible variants that may arise during the course of production of the monoclonal antibody. Such monoclonal antibodies typically include an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from a collection of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the selected target binding sequence may be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

Monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remaining portion of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

An "antibody fragment" comprises a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

"functional fragments" of an antibody of the invention refer to those fragments that retain the binding of the polypeptide with substantially the same affinity as the intact full chain molecule from which they are derived and that are active in at least one assay, such as in a mouse model, or in vitro, the biological activity of an antigen to which the antibody fragment binds.

In various embodiments, the antibody is labeled with a detectable moiety. The detectable moiety may be selected from: a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a radioactive moiety, an enzymatic moiety and a second antibody. "detectable moiety" is meant to include any moiety that can be detected and the relative amount and/or relative position of that moiety determined.

The detectable moiety may be a fluorescent moiety and/or a luminescent moiety and/or a chemiluminescent moiety, which may be detected when exposed to a particular condition. For example, the fluorescent moiety may require exposure to radiation (i.e., light) at a particular wavelength and intensity to cause excitation of the fluorescent moiety, thereby enabling it to emit detectable fluorescence at the particular wavelength that can be detected.

Alternatively, the detectable moiety may be an enzyme capable of converting a (preferably undetectable) substrate into a detectable product that can be visualized and/or detected. Examples of suitable enzymes to be used may be those known for use in assays such as ELISA. Examples include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.

Alternatively, the detectable moiety may be a radioactive atom for imaging. Suitable radioactive atoms include those for scintigraphic studies^99mTc and¹²³I. other readily detectable moieties include, for example, spin labels for Magnetic Resonance Imaging (MRI), e.g.¹²³I、¹³¹I、¹¹¹In、¹⁹F、¹³C、¹⁵N、¹⁷O, gadolinium, manganese or iron. Obviously, to facilitate detection of the detectable moiety, the agent to be detected (e.g., one or more proteins in the test sample and/or control sample described herein and/or the like) isAntibody molecules for detection of the selected protein) must have sufficient of the appropriate atomic isotope.

Radiolabels or other labels may be incorporated into the reagents of the invention (i.e. the proteins present in the sample of the method of the invention and/or the binding agents of the invention) in a known manner. For example, if the binding moiety is a polypeptide, it may be biosynthesized or may be synthesized by chemical amino acid synthesis using an appropriate amino acid precursor (containing, for example, fluorine-19 in place of hydrogen). Such as^99mTc、¹²³I、¹⁸⁶Rh、¹⁸⁸Rh and¹¹¹labels such as In may be attached, for example, via a cysteine residue In the binding moiety. Yttrium-90 may be attached via a lysine residue. Methods for conjugating other detectable moieties (e.g., enzymatic, fluorescent, luminescent, chemiluminescent, or radioactive moieties) to proteins are well known in the art.

It is understood that the binding agents may be used simultaneously or sequentially. For example, cells may be first selected based on cell surfaces bound to ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM2, CALD1, GPR38, HEXIM1, CDH5, and/or SM22 a, then isolated by flow cytometry, then further selected with a binding agent that binds to another cell surface marker, and then further subjected to flow cytometry. Alternatively, cells may be selected using binding agents that bind to all cell surface markers simultaneously, wherein one of the binding agents may be labeled with, for example, FITC (fluorescein isothiocyanate) and the second binding agent labeled with, for example, Phycoerythrin (PE), which facilitates separation by flow cytometry.

According to the invention, cells selected according to the methods disclosed herein comprise a population of cells having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 95%, 96%, 97%, 98%, 99% or 100% smooth muscle cells. In one example, the selected or isolated cells have at least about 70% smooth muscle cells. In another example, the selected or isolated cells have at least about 80% smooth muscle cells. In another example, the selected cells have at least about 90% smooth muscle cells.

The invention also provides smooth muscle cell-enriched cell populations according to the methods described herein. In one example, the cell population has at least about 70% smooth muscle cells. In another example, the cell population has at least about 80% smooth muscle cells. In another example, the cell population has at least about 90% smooth muscle cells.

In various embodiments, the isolated cells comprise a population of cells having at least about 85% smooth muscle cells.

In various embodiments, the invention can also provide a purified population of cells comprising at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% smooth muscle cells. In other examples, the purified population of cells can include at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% smooth muscle cells having ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM2, CALD1, GPR38, HEXIM1, CDH5, and/or SM22 a surface marker.

The terms "expression" and "gene expression" are synonymous and mean that a cell converts genetic information stored in a DNA sequence during its life through transcription and translation into a biologically active protein molecule.

The terms "increased expression" and "high expression" are synonymous and refer to an increased copy number of a gene transcript, and/or increased translation, as compared to normal levels.

In the present invention, the term "primer" means 7 to 50 nucleic acid sequences capable of forming a base pair (base pair) complementary to a template strand and serving as a starting point for copying the template strand. The primers are generally synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer does not necessarily need to be completely identical to the sequence of the template, and may be sufficiently complementary to hybridize with the template. Additional features that do not alter the basic properties of the primer may be incorporated. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of more than one nucleic acid with a homolog, and modification between nucleic acids.

The term "probe" as used herein refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

As the probe, a labeled probe in which a disease-detecting polynucleotide is labeled, such as a fluorescent label, a radioactive label, or a biotin label, can be used. Methods for labeling polynucleotides are known per se. The presence or absence of the test nucleic acid in the sample can be checked by: immobilizing the test nucleic acid or an amplification product thereof, hybridizing with the labeled probe, washing, and then measuring the label bound to the solid phase. Alternatively, the polynucleotide for disease detection may be immobilized, a nucleic acid to be tested may be hybridized therewith, and the nucleic acid to be tested bound to the solid phase may be detected using a labeled probe or the like. In this case, the polynucleotide for detecting a disease bound to the solid phase is also referred to as a probe. Methods for assaying test nucleic acids using polynucleotide probes are also well known in the art. The process can be carried out as follows: the polynucleotide probe is contacted with the test nucleic acid at or near Tm (preferably within ± 4 ℃) in a buffer for hybridization, washed, and the hybridized labeled probe or template nucleic acid bound to the solid phase probe is then measured.

The size of the polynucleotide used as a probe is preferably 18 or more nucleotides, more preferably 20 or more nucleotides, and the entire length of the coding region or less. When used as a primer, the polynucleotide is preferably 18 or more nucleotides in size, and 50 or less nucleotides in size. These probes have a base sequence complementary to a specific base sequence of a target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA or RNA, or they may be polynucleotides in which part or all of the nucleotides are substituted with artificial nucleic acids such as PN, LNA, ENA, GNA, TNA, etc.

The primers or probes of the invention can be chemically synthesized using a solid phase support of phosphoramidite or other well known methods. The nucleic acid sequence may also be modified using a number of means known in the art. Non-limiting examples of such modifications are methylation, capping, substitution with one or more analogs of a natural nucleotide, and modification between nucleotides, for example, modification of an uncharged linker (e.g., methyl phosphate, phosphotriester, phosphoimide, carbamate, etc.), or modification of a charged linker (e.g., phosphorothioate, phosphorodithioate, etc.).

Reagent kit

The invention also provides kits for identifying, characterizing and/or enriching, or isolating smooth muscle cell subsets as described herein. Such kits may be used in a clinical setting for patient diagnostic purposes or in research for characterization and/or enrichment of smooth muscle cell populations. Kits according to the invention will comprise one or more containers comprising the binding agent and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, 96-well plates, and the like. The container may be formed from a variety of materials such as glass or plastic. Such a container contains one or more compositions comprising a binding agent effective for analyzing smooth muscle cells and optionally providing enriched or isolated cells or cell subpopulations as described herein. Such kits will typically contain a preparation of one or more binding agents in a suitable container, in the case of multiple binding agents, the binding agents may be in the same or different containers. The kit may also contain other pharmaceutically acceptable preparations for use in diagnosis or for labeling or modifying blocked binding agents.

More specifically, these kits may have a single container containing one or more binding agents, with or without additional components, or they may have different containers for each component. Where a combination reporter is provided for incorporation, the individual solutions can be combined in molar equivalents or premixed where one component exceeds the remaining components. Alternatively, the binding agent and any optional reporter of the kit may be maintained separately within different containers prior to administration to a subject or use in vitro. The kit may also comprise second/third container means for holding sterile, pharmaceutically acceptable buffers or other diluents, such as bacteriostatic water for injection (BWFI), Phosphate Buffered Saline (PBS), Ringer's solution and dextrose solution.

When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution. However, the components of the kit may be provided as dry powders. When the reagent or component is provided as a dry powder, the powder may be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in another container.

The terms "subject" or "patient" may be used interchangeably. The term includes, but is not limited to, humans, non-human animals, e.g., non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic subjects such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs, and the like. The term does not indicate a specific age or gender. Thus, adult and neonatal subjects, as well as fetuses, whether male or female, are contemplated. The term "subject" also includes living organisms susceptible to a disorder or disease state as generally disclosed throughout this specification (but not limited thereto). Examples of subjects include humans, dogs, cats, cows, goats, and mice, including transgenic species.

The terms "non-human animal" and "non-human mammal" are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, as well as non-mammals such as chickens, amphibians, reptiles, and the like. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal replacement, such as a dog, as a model of disease.

Drawings

FIG. 1 is a cellular landscape map of an arterial vessel; wherein, A: t-SNE cell grouping map after sample combination; b: t-SNE pattern of major cell population; EC, endothiral cells; SMC, smooth muscle cells; FB, fibroblastis; MFB, myofibroblast; m φ, macropositive cells.

FIG. 2 is a graph of the change in the direction of differentiation of subclavian and iliac artery SMC cells; wherein, A: SMC cell differentiation of subclavian arteries; b: SMC cells of the iliac arteries differentiated.

FIG. 3 is a graph of pseudo-time traces of SMC cells and MFB cells; wherein, A: arrangement of SMC cells and MFB cells on a pseudo-time trajectory; b: (ii) arrangement of each subpopulation of cells on a pseudo-time trajectory; c: and simulating a time sequence.

FIG. 4 is ApoE^-/-Change pattern of cell subpopulations of subclavian and iliac arteries of beagle dogs.

FIG. 5 is a graph showing the expression of TNC in canine SMC cells.

FIG. 6 is a t-SNE plot of the major cell population of carotid plaque.

FIG. 7 is a graph of pseudo-time traces of human SMC cells and MFB cells.

FIG. 8 is a graph showing the expression of TNC in human SMC cells.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1 Single cell sequencing of AS model animals

1. Sample collection

Collecting ApoE of large animal model of atherosclerotic disease^-/-Artery tissue of beagle and WT dogs, including carotid, subclavian, aortic arch, coronary, thoracic, abdominal and iliac arteries. The isolated artery was snap frozen with liquid nitrogen and stored at-80 ℃ for RNA extraction and Bulk RNA-seq. In addition, a part of fresh subclavian and iliac arteries was washed clean with pre-cooled physiological saline repeatedly, and then placed in an ice cell culture dish, and RPMI 1640 cell culture medium containing 10% FBS was added for single cell dissociation and scRNA-seq.

2. RNA extraction and Bulk RNA-seq

Total RNA extraction from tissues was performed using Trizol method, RNA purity was analyzed by a NanoDrop Spectrophotometer, and RNA concentration and integrity were analyzed by an Agilent 2100 Bioanalyzer. After the RNA quality was qualified, a sequencing Library was constructed using the TruSeq Stranded mRNA Library Prep Kit (Illumina). The sequencing platform used Novaseq 6000platform (Illumina) for paired-end 150bp sequencing. And removing the joint and the low-quality reads from the raw data obtained by sequencing to obtain clean data. Clean data was then aligned to the canine reference genome using HISAT2 (Canfam 3). Aligned reads were assembled and quantified using StringTie. Differential gene expression analysis was performed using R-package DESeq2, with differential gene screening criteria of adjusted p-value<0.01，|log₂(fold change)|> 1。

3. Preparation of Single cell suspensions

WT beagle dog and ApoE^-/-The subclavian and iliac arteries of beagle dogs were used for single cell dissociation and scRNA-seq. After washing the above tissue sample 3 times with pre-cooled serum-free RPMI 1640 cell culture medium, the perivascular fat and connective tissue were carefully stripped off with sterile ophthalmic surgical scissors and forceps, and the tissue was minced, and the vascular single cell digest (450U/ml Collagenase I, 250U/ml Collagenase XI, 120U/ml Hyaluronidase, 120U/ml DNase I) was added and digested for 1h in a 37 ℃ thermostat water bath. The single cell suspension obtained by digestion is sequentially filtered by 70 μm and 40 μm cell sieves, then RPMI 1640 cell culture solution is added, and centrifugation is carried out for 5min at 4 ℃ and 500 g. Then according to the kitThe instructions lyse the red blood cells, kill the cells, and remove debris. The cell activity is identified by using Countstar, the number of living cells in the single cell suspension is determined to be more than 85%, and the total number of the cells is determined>1×10⁵And (4) respectively.

4. ScRNA-seq and data Pre-processing

Using a commercialization platform 10x

Chromium^TMscRNAseq was performed, and the Single Cell library construction Kit was Single Cell 3' Solution v3 Reagent Kit (10 × Genomics). Firstly, Gel Beads (Gel Beads) containing barcode, high-quality single cells, enzyme mixture and oil are mixed to form GEMs (water-in-oil microsystems). Cells were lysed in GEMs, pooled and paired-end 150bp sequencing was performed using an Illumina NovaSeq 6000 sequencer. Single cell transcriptome raw sequencing data were preprocessed by CellRanger V3.1.0(https:// support.10Xgenomics. com) to obtain a gene-cell matrix file, reference genome canFam 3.

5. Cell grouping and marker Gene identification

The gene-cell matrix file was analyzed downstream using the R package Seurat v3. The cell filtration criteria were: each gene is expressed in at least 3 cells, with a gene factor >200 and <4000 expressed per cell, with a mitochondrial gene fraction of < 20% per cell. Filtered data were transformed by normalized and log and then identified for the hypervariable genes. Dimension reduction was further performed by Principal Component Analysis (PCA), and after calculating principal components using the "JackStraw" function, the first 20 Principal Components (PCs) were selected for cell clustering. The resolution parameter is set to 0.5 when the cells are clustered. Cells were visualized using the T-distributed stored genetic near embedding (T-SNE) method after clustering. The marker gene of each cell cluster was identified using the function "FindMarkers" or "findalmarkers" in the R package, sourtat v3, with the marker gene screening criteria for different cell clusters being adjusted p-value <0.05, log (fold change) > 0.5.

6. RNA Rate (RNA velocity) analysis and cell differentiation potential analysis

RNA velocity analysis was performed by the method of La Manno, and specific and unspliced transcripts were obtained for each cell using Scvelo to estimate the state of each cell. The differentiation rate per cell was calculated by normalization and log transformation. Visualization was then performed, with the differentiation rate of each cell being shown on top of the t-SNE cell population map previously plotted by saurta. In addition, the cell dynamics is deduced through the CellRank function, the fate probability of converting the cell dynamics into other states is calculated, and the differentiation potential of the cell dynamics is judged.

7. Construction of cell trajectories by time-simulated analysis

Single cell trajectories for specific cell types were constructed using R-pack Monocle2(version 2.9). The differential genes for each cluster were calculated by the differential GeneTest function, ranked according to p-value, and the top 1000 genes were selected for time-fitting analysis. All cells were dimensionality reduced using DDRTree algorithm and sorted according to a pseudo-time trajectory.

8. Results

1) WT and ApoE^-/-Digesting and dissociating the part (subclavian artery) which is not easy to be sensed and the part (iliac artery) which is easy to be sensed of the beagle dog into single cell suspension by enzyme, then carrying out scRNA-seq to obtain raw data, and sequencing the saturation>75% of reads aligned to the reference genome>79% average basis factor detected per cell>1200. WT beagle dog and ApoE^-/-The number of cells captured by subclavian and iliac arteries of beagle dogs was 9386, 7412, 9759 and 6902, respectively.

2) Cell filtration was performed according to the following criteria: each gene is expressed in at least 3 cells, and the number of genes expressed per cell>200 and<4000 mitochondrial Gene proportion per cell<20 percent. After filtration, we obtained 29517 high-quality effective cells in total, among which 8893 effective cells in the subclavian artery, 6755 effective cells in the iliac artery, and ApoE in WT beagle dog^-/-8567 effective cells of the subclavian artery and 5302 effective cells of the iliac artery of beagle dog. Cells from the arterial samples were pooled and unbiased cell clustering and grouping was performed. A total of 22 cell clusters were obtainedMajor cell types include: endothelial cells (cluster2,9,11,12,14,18), smooth muscle cells (cluster3,4,5,10), fibroblasts (cluster0, 1,7,8), myofibroblasts (cluster6,16), macrophages (cluster13,19,21), T cells (cluster17) and neuronal cells (cluster15) (fig. 1). The cellular composition is consistent with the anatomy of the intima, media and adventitia of the blood vessel.

Endothelial Cells (ECs) can be divided into 6 subpopulations, typical marker genes are PECAM1, VWF, ICAM2, CDH5, EGFL7 and ESAM. Smooth Muscle Cells (SMCs) can be divided into 4 subgroups, typical marker genes being ACTA2, MYH11, TAGLN, CNN1, MYL9 and TPM 2. Fibroblasts (FB) can be divided into 4 subgroups, typical marker genes being ELN, DCN, COL3A1, COL1A1, FMOD and PI 16. Myofibroblasts (MFB) can be divided into 2 subpopulations. Macrophages (M.phi.) can be divided into 3 subgroups, with typical marker genes being CD68, C1QA, C1QB, C1QC, CD163 and AIF 1. Typical marker genes for T cells are CD3D, CD3E, CD69, IL7R and CCL 5.

3) RNA rate analysis can predict the future state of each cell, by which the direction and course of differentiation of cells in healthy arteries and atherosclerotic lesion vessels can be delineated, and arrows are added to the t-SNE cell grouping map to indicate the direction and directional flow of differentiation of cells.

Compared with the WT canine subclavian artery, the SMC3 subset cells had stronger and clearer differentiation direction in the WT canine iliac artery, indicating that the SMC3 subset cells were in an unstable transcriptional state and the turnover rate of intracellular RNA was faster, which may be related to the iliac artery adapting to stronger blood flow pressure (FIG. 2A, B). In ApoE^-/-In the iliac artery at the site of susceptibility of beagle dogs, the SMC3 subset cells could differentiate into MFB cells, suggesting that the SMC3 subset cells may undergo reprogramming and cell fate transition processes, which are closely related to the phenotypic plasticity of SMC cells (fig. 2B).

The potential differentiation association of cells from the SMC3 subgroup and the MFB1 subgroup was further confirmed by time-simulated analysis (fig. 3).

4) By comparing the proportion of all cell subsets in each sample, it was found that although the total number of smooth muscle cell SMC cells was significantly reduced in both subclavian and iliac arteries of ApoE-/-beagle dogs, the number of cells of the SMC3 subset was significantly increased (fig. 4).

5) The differences between the four sub-populations of SMC cells were compared and TNC gene was found to be specifically expressed in sub-population SMC3 by hypermutagen analysis, according to the screening criteria of adjusted p-value <0.05, log (fold change) >0.5 (FIG. 5).

Example 2 Single cell sequencing of AS patients

To determine TNC^highThe SMC3 subset was also present in human AS, and human carotid plaque scRNA-seq data (n 3 patients) were further analyzed in the same manner AS in example 1.

Human carotid plaque cells were divided into 25 clusters by unbiased clustering, with the major cell types ECs, SMCs, MFBs, M phi cells, T cells, B cells and mast cells (fig. 6). It is pointed out that in human carotid endarterectomy tissue specimens of carotid atherosclerosis, most of the SMCs in the media of the arteries and FBs in the adventitia may be lost. In human carotid plaques, the gene expression patterns of SMC-associated and MFB-associated genes (ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM2, ELN, DCN, COL3a1, COL1a1, FMOD) are very similar to ApoE-/-dogs.

Trace analysis revealed the inter-cluster relationship of SMC3 and MFB cells (fig. 7). TNC was specifically expressed in the SMC3 subset of human carotid plaques (fig. 8). Thus, the SMC3 subgroup in human carotid plaque was associated with ApoE-/-canine TNC^highThe SMC3 subgroup has a broad molecular commonality.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. An agent or kit for identifying, enriching or isolating a smooth muscle cell subpopulation/diagnosing atherosclerosis, characterized in that it comprises a binding agent capable of binding to a TNC gene or a protein or protein fragment expressed thereof in smooth muscle cells, said TNC being highly expressed in the smooth muscle cell subpopulation; the reagent or kit further comprises a binding agent capable of binding to a biomarker of smooth muscle cells.

2. The reagent or kit of claim 1, wherein the biomarker comprises one or more of ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM2, CALD1, GPR38, HEXIM1, CDH 5.

3. The reagent or kit of claim 2, wherein the biomarker is selected from one or more of ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM 2.

4. A reagent or kit as claimed in any of claims 1 to 3, wherein the kit comprises a container or containers holding one or more of the binding agents.

5. The reagent or kit of claim 4, wherein the kit further comprises instructions.

6. The reagent or kit of claim 1, wherein the binding agent is selected from a nucleic acid, a ligand, an enzyme, a substrate and/or an antibody.

7. The reagent or kit of claim 6, wherein the binding agent has a reporter molecule attached thereto.

8. The reagent or kit according to claim 7, wherein the reporter molecule is selected from a fluorescent substance, a radioactive substance and/or an enzyme.

9. The reagent or kit according to claim 6, wherein the nucleic acid comprises a probe that hybridizes to the gene.

10. The reagent or kit of claim 6, wherein the nucleic acid comprises a primer that amplifies the gene.

11. The reagent or kit of claim 6, wherein the antibody is an antibody that binds to a protein or protein fragment encoded by the gene.

12. A smooth muscle cell subpopulation that overexpresses TNC.

13. A method of enriching the smooth muscle cell subpopulation of claim 12, comprising:

14. The method of claim 13, wherein the sorting technique comprises fluorescence activated cell sorting, magnetic assisted cell sorting, substrate assisted cell sorting, laser mediated cleavage, fluorimetry, flow cytometry or microscopy.

15. A method for identifying a subpopulation of smooth muscle cells in a test cell population, comprising the steps of:

16. The method of claim 15, further comprising the step of identifying smooth muscle cells.

17. The method of claim 16, wherein the step of identifying smooth muscle cells comprises detecting a biomarker of smooth muscle cells.

18. The method of claim 17, wherein the biomarkers comprise one or more of ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM2, CALD1, GPR38, HEXIM1, CDH 5.

19. The method of claim 18, wherein the biomarker is selected from one or more of ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM 2.

20. Use according to any one of the following:

1) use of a biomarker comprising the gene TNC, or a protein or protein fragment expressed by said gene, for identifying, screening or sorting a subpopulation of smooth muscle cells;

2) use of a biomarker comprising the gene TNC, or a protein or protein fragment expressed by said gene TNC, in the manufacture of a product for diagnosing atherosclerosis; the gene TNC is highly expressed in atherosclerosis;

3) use of a reagent or kit comprising a binding agent capable of binding to a TNC gene or an expressed protein or protein fragment thereof in smooth muscle cells for the manufacture of a product for identifying, enriching or isolating a subpopulation of smooth muscle cells;

4) use of a reagent or kit comprising a binding agent capable of binding to a TNC gene or a protein or protein fragment thereof expressed in smooth muscle cells in the manufacture of a product for the diagnosis of atherosclerosis; the TNC is highly expressed in a smooth muscle cell subpopulation;

5) use of a smooth muscle cell subpopulation according to claim 12 for the preparation of a product for the diagnosis of atherosclerosis.

21. The use of claim 20, wherein the subpopulation of smooth muscle cells exhibits high expression in a site susceptible to atherosclerosis.

22. The use of claim 21, wherein the susceptible site comprises the abdominal aorta, iliac arteries, carotid arteries, and coronary arteries.

23. The use of claim 22, wherein the subpopulation of smooth muscle cells exhibits low expression in non-susceptible sites of atherosclerosis.

24. The use of claim 23, wherein the non-susceptible sites include the subclavian artery, the aortic arch and the thoracic aorta.

25. The use of claim 20, wherein the reagent or kit of 3) or 4) further comprises a binding agent that binds to a biomarker of smooth muscle cells.

26. The use of claim 25, wherein the biomarkers comprise one or more of ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM2, CALD1, GPR38, HEXIM1, CDH 5.

27. The use of claim 26, wherein the biomarker is selected from one or more of ACTA2, MYH11, TAGLN, CNN1, MYL9, TPM 2.

28. The use according to any one of claims 20, 25 to 27, wherein the kit comprises a container or containers containing one or more of the binding agents.

29. The use of claim 20, wherein the kit further comprises instructions.

30. Use according to claim 20 or 25, wherein the binding agent is selected from a nucleic acid, a ligand, an enzyme, a substrate and/or an antibody.

31. The use of claim 30, wherein the binding agent has a reporter molecule attached thereto.

32. The use according to claim 31, wherein the reporter molecule is selected from a fluorescent substance, a radioactive substance and/or an enzyme.

33. The use of claim 30, wherein said nucleic acid comprises a probe that hybridizes to said gene.

34. The use of claim 33, wherein said nucleic acid comprises a primer that amplifies said gene.

35. The use of claim 30, wherein the antibody is an antibody that binds to a protein or protein fragment encoded by the gene.