阅读说明：本技术 一种检测分析血液中特定细胞外囊泡的方法及其用途 (Method for detecting and analyzing specific extracellular vesicles in blood and application thereof ) 是由 鞠佃文 范佳君 章旭耀 陈伟 栾静韵 朱泽国 梁嫣煦 叶丽 于 2019-04-22 设计创作，主要内容包括：本发明属于生物技术领域,涉及检测分析血液中特定细胞外囊泡的方法及其用途。尤其是检测分析血液中特定细胞外囊泡的数量和比例,有助于预测患者动脉粥样硬化病程及其急性心血管事件的发生几率。本发明还提供了根据检测动脉粥样硬化患者外周血中细胞外囊泡数量,CD47<Sup>+</Sup>细胞外囊泡以及Annexin V<Sup>+</Sup>细胞外囊泡的比例结果,用于制备预测患者动脉粥样硬化病程的水平及其发生急性心血管事件的几率的制剂或工具模型；其中尤其是细胞外囊泡、CD47<Sup>+</Sup>细胞外囊泡以及Annexin V<Sup>+</Sup>细胞外囊泡用于制备检测及治疗动脉粥样硬化制剂中的应用。该发明有望成为临床心血管疾病的预测提供新技术支持和检测分析方法。(The invention belongs to the technical field of biology, and relates to a method for detecting and analyzing specific extracellular vesicles in blood and application thereof. Especially, the quantity and the proportion of specific extracellular vesicles in blood are detected and analyzed, which is helpful for predicting the atherosclerotic course and the occurrence probability of acute cardiovascular events of patients. The invention also provides a method for detecting the number of extracellular vesicles, CD47, in peripheral blood of an atherosclerotic patient + Extracellular vesicles and Annexin V + The proportion result of the extracellular vesicles is used for preparing a preparation or a tool model for predicting the level of the atherosclerotic course of the patient and the probability of the patient suffering from an acute cardiovascular event; among these are especially extracellular vesicles, CD47 + Extracellular vesicles and Annexin V + Application of extracellular vesicles in preparation of preparations for detecting and treating atherosclerosis. The invention is expected to provide a new technical support and a detection analysis method for the prediction of clinical cardiovascular diseases.)

1. A method for detecting and analyzing specific extracellular vesicles in blood, which comprises detecting and analyzing the quantity and proportion of the specific extracellular vesicles in the blood, and predicting the level of atherosclerotic process and the probability of acute cardiovascular events.

2. The method of claim 1, comprising the steps of,

1) collecting the case information of healthy people and atherosclerosis patients:

detecting pathological changes in coronary artery of a patient through coronary angiography and intravascular ultrasound, calculating the area of stenosis in the blood vessel of the patient and the proportion of the stenosis in the blood vessel of the patient, and determining: healthy subjects, early AS patients, patients with coronary heart disease, and patients prone to myocardial infarction;

2) collecting and detecting extracellular vesicles in peripheral blood, and performing flow cytometry sorting and counting on the extracellular vesicles in a test sample by flow cytometry;

3) analyzing and comparing the level of extracellular vesicles in peripheral blood of an atherosclerotic patient with that of a healthy person,

4) analysis and comparison of CD47 in peripheral blood of atherosclerotic patients and healthy people⁺(ii) the level of extracellular vesicles

5) Analysis and comparison of Annexin V in peripheral blood of atherosclerotic patients and healthy people⁺The level of extracellular vesicles of (a);

6) analysis and comparison of extracellular vesicle levels in peripheral blood and CD47 in atherosclerotic and healthy persons⁺The level of extracellular vesicles is correlated with the degree of disturbance of lipid metabolism.

3. The method according to claim 1 or 2, wherein said extracellular vesicles are exosomes having a diameter below 200nm and extracellular microvesicles having a diameter in the range of 200-2000 nm.

4. The method of claim 1 or 2, wherein said CD47⁺Extracellular phaseVesicles and Annexin V⁺The extracellular vesicles are extracellular vesicles carrying CD47 and Annexin V at positions including the outer surface, the inner surface and the interior of the vesicles.

5. The method of claim 1 or 2, wherein said extracellular vesicles, CD47⁺Extracellular vesicles and Annexin V⁺Extracellular vesicles derived from: macrophage-derived, platelet-derived, endothelial cell-derived, monocyte-derived, or smooth muscle cell-derived; macrophage-derived extracellular vesicles are preferred.

6. A method according to claim 1 or 2, wherein the positions of CD47 and Annexin V in the vesicle are the levels of CD47 and Annexin V on the outer surface of the vesicle.

7. The method of claim 1 or 2, wherein said atherosclerotic patient is: patients with chronic atherosclerosis, patients with coronary heart disease, patients with long-term (> 3 years) hypertension or patients with acute coronary syndrome.

8. The method of claim 7, wherein said atherosclerotic patients are chronic atherosclerotic patients and coronary heart disease patients.

Technical Field

The invention belongs to the technical field of biological detection, and relates to a method for detecting and analyzing specific extracellular vesicles in blood and application thereof. In particular, the number and proportion of specific extracellular vesicles in the blood to be analyzed; the method of the invention is helpful for predicting the course of atherosclerosis and the occurrence probability of acute cardiovascular events of patients.

Background

Studies have shown that atherosclerosis, which is characterized pathologically by the accumulation of lipid cells under the endothelium and atheromatous or fatty deposits in the intima, is one of the common cardiovascular diseases associated with serious health problems and is also the leading cause of morbidity and mortality [1 ]. The process of coronary atherosclerosis can obstruct blood flow, cause blockage of distal blood vessels, and cause related acute cardiovascular events such as acute myocardial infarction, unstable angina pectoris and the like, thereby seriously threatening the life of patients.

Research shows that the formation of atherosclerosis is divided into three stages, firstly, coronary artery vascular endothelial cells are activated by various harmful stimuli such as hypertension, hyperglycemia, hyperlipidemia, oxidative stress and the like, adhesion molecules such as ICAM-1 and the like are secreted to promote the adhesion of leucocytes, meanwhile, the endothelial permeability is increased and the extracellular matrix components under the endothelium are changed to promote the accumulation of low-density lipoprotein (ox-LDL) modified by oxidation on the artery wall and the low-density lipoprotein (ox-LDL) is identified by scavenger receptors to enter macrophages to form foam cells; then, the cell factors and growth factors secreted by endothelial cells induce the proliferation and migration of smooth muscle cells to form a fibrous cap consisting of fibrous tissues, smooth muscle cells, macrophages and T lymphocytes; finally, damaged endothelial cells, foam cells, large amounts of lipids, calcium deposits, pathological vascular remodeling constitute the plaque core [2 ]. At present, the specific molecular mechanism of atherosclerosis is not clear, so that a very clear diagnosis method and a very clear therapeutic drug for atherosclerosis are not available in clinical practice.

Acute Cardiovascular events (Acute Cardiovascular events) are Acute ischemic heart diseases such as Acute Coronary Syndromes (ACS) caused by continuous progression of blood vessels due to long-term atherosclerosis. Studies have shown that the main cause of acute cardiovascular events is acute ischemic syndrome of the heart due to fresh thrombosis secondary to rupture or erosion of unstable atherosclerotic plaques in the coronary arteries. Due to the lack of necessary diagnostic and prognostic methods, acute cardiovascular events are currently one of the leading causes of death in atherosclerotic patients. According to statistics, the number of people dying from atherosclerosis and complicated acute cardiovascular diseases reaches 1500 ten thousand, and among them, China accounts for 1/5[3 ]; therefore, it is important to further study the mechanism of atherosclerosis and to discover molecules therefrom that can be used as a predictor and prognosis of the disease.

Extracellular vesicles are small vesicles secreted by cells and having a size of about 20-10000nm, and these vesicles are responsible for cell-to-cell communication, and although release of apoptotic bodies during apoptosis has been reported for a long time, vesicles released from cell membranes by healthy cells have been recently attracting attention, and they mainly include microvesicles (microviscles), apoptotic bodies (apoptotic bodies), exosomes (exosomes), and the like [4 ];

the exosome is an extracellular vesicle surrounded by phospholipid bilayers, has the diameter of 20-200nm, can be released by resting cells or induced release, is released by the fusion of a polyplast and a cell membrane after an endocytosis body is inwards sunken to form a multivesicular body (MVB), and not only can transversely transmit mRNA and miRNA, an oncogenic receptor (oncogenic receptor) and an AIDS virus particle (HIV particle), but also has antigen presentation, immune activation and immune suppression activities, and expresses markers [5] such as D63, CD81, CD9, LAMP1, TSG101 and the like;

microvesicles are extracellular vesicles produced on the surface of cells such as endothelial cells or platelets [6], a factor which has been found to precipitate in plasma which does not contain platelets but has thrombin activity was found in 1946, and dust (platelet dust) which separates platelets from fresh plasma by ultracentrifugation was studied in 1967 and defined as microvesicles; microvesicles are thought to be produced by platelets, erythrocytes or epithelial cells and have a procoagulant function, wherein the microvesicles are membrane structures surrounded by phospholipid bilayers with a diameter of 200-100nm, and are generated by the outward budding of cell membranes, the generation of the microvesicles is regulated, and the activation of cell surface receptors, apoptosis or the increase of intracellular calcium ion concentration can induce the generation of the microvesicles [7 ];

apoptotic bodies were proposed by Kerr et al in 1972 [8], the Robert Horvitz team to study apoptosis in cell lineage development in nematodes is yet another milestone for apoptotic body study; . The diameter of the apoptotic body is over 1000nm, the apoptotic body is generated by bubbling in the process of apoptosis, can horizontally transmit DNA and oncogene, presents T cell antigenic determinant when being taken by phagocyte, and has immunosuppressive effect.

It has been reported that atherosclerotic coronary lesions, from early to late stages, contain extracellular vesicles that increase their release under activation, apoptosis, inflammation and hypoxia, and that may be derived from leukocytes, erythrocytes, smooth muscle cells and endothelial cells, and that may play a role in the initiation and progression of atherosclerotic coronary lesions due to their effects on inflammation, thrombosis, angiogenesis, cell survival and endothelial homeostasis [9 ].

Also studies have disclosed that circulating extracellular vesicle levels are associated with various cardiovascular risk factors, including smoking, dyslipidemia, diabetes and hypertension or with subclinical atherosclerotic lesions, and extracellular vesicles can exert pro-inflammatory and anti-inflammatory effects in various circumstances; most in vitro studies indicate that extracellular vesicles increase proinflammatory cytokine release mediated from endothelial cells and leukocytes, particularly interleukin 6 and interleukin 8, which promotes monocyte adhesion to endothelium and facilitates monocyte migration to plaque [9 ]; extracellular vesicles of endothelial origin may also regulate monocyte activation by NFKB; it has also been shown that extracellular vesicles interact with vascular endothelium and promote dysfunction of cells, that endothelial extracellular vesicles reduce Nitric Oxide (NO) production by endothelial cells, possibly by modification of NO synthase phosphorylation or local oxidative stress, leading to altered vascular tone and damage to endothelial coronary atherosclerosis protection [10 ]; in contrast, extracellular vesicles isolated in vitro from healthy individuals or normal conditions had no effect on endothelial function, indicating that the effects of extracellular vesicles are largely influenced by their composition, including cell origin, local environment and cell stimulation, etc.; in addition, endothelial extracellular vesicles and large platelet extracellular vesicles can also increase endothelial permeability, delivering apoptotic protease-3 and Rho-kinase to target cells to increase local apoptosis; extracellular vesicles isolated from human coronary atherosclerotic plaques can directly transfer ICAM-1 to target cells, and ICAM-1 expression can also be regulated by miR-222 present in extracellular vesicles [11 ]; arachidonic acid and oxidized phospholipids carried by extracellular vesicles also contribute to increased leukocyte adhesion, transferring the original atheromatous CCL5 from the platelet extracellular vesicles to endothelial cells to increase leukocyte adhesion [12 ]; the above results indicate that extracellular vesicles released under pathological conditions can promote the infiltration of leukocytes and low-density lipoproteins (LDL) in the vascular endothelium, thus favoring the development of atherosclerotic lesions.

At present, although some studies suggest that extracellular vesicles may be involved in the regulation of atherosclerotic disease processes, no study has yet clearly shown that extracellular vesicles are significantly linked to clinical atherosclerotic progression and the occurrence of acute cardiovascular events associated therewith; meanwhile, as the effect of the extracellular vesicles is influenced by the source and the components of the extracellular vesicles, reports showing that the extracellular vesicles derived from macrophages are related to the atherosclerosis are not found yet.

References relevant to the present invention are:

[1]Campbell KA,Lipinski MJ,Doran AC,Skaflen MD,Fuster V,McNamara CA.Lymphocytes and the adventitial immune response in atherosclerosis.Circ Res.2012；110:889-900.

[2]Childs BG,Baker DJ,Wijshake T,Conover CA,Campisi J,van Deursen JM.Senescent intimal foam cells are deleterious at all stages ofatherosclerosis. Science.2016；354:472-7.

[3]Mariscalco G,Wozniak MJ,Dawson AG,Serraino GF,Porter R,Nath M,etal. Body Mass Index and Mortality Among Adults Undergoing Cardiac Surgery:ANationwide Study With a Systematic Review and Meta-Analysis.Circulation.2017；135:850-63.

[4]Sasso L,Hosamuddin H,Emanueli C.Extracellular vesicles at thecross-line between basic science and clinical needs.Microcirculation.2017；24.

[5]Fu M,Gu J,Jiang P,Qian H,Xu W,Zhang X.Exosomes in gastric cancer:roles, mechanisms,and applications.Mol Cancer.2019；18:41.

[6]Agrahari V,Agrahari V,Burnouf PA,Chew CH,Burnouf T.ExtracellularMicrovesicles as New Industrial Therapeutic Frontiers.Trends Biotechnol.2019.pii:S0167-7799(18)30335-4.

[7]Melki I,Tessandier N,Zufferey A,Boilard E.Platelet microvesiclesin health and disease.Platelets.2017；28:214-221.

[8]Hauser P,Wang S,Didenko VV.Apoptotic Bodies:Selective Detection inExtracellular Vesicles.Methods Mol Biol.2017；1554:193-200.

[9]Maas SL,Breakefield XO,Weaver AM.Extracellular Vesicles:UniqueIntercellular Delivery Vehicles.Trends Cell Biol.2017；27:172-188.

[10]Loyer X,Zlatanova I,Devue C,Yin M,Howangyin KY,Klaihmon P,et al.Intra-Cardiac Release of Extracellular Vesicles Shapes Inflammation FollowingMyocardial Infarction.Circ Res.2018；123:100-106.

[11]Chistiakov DA,Orekhov AN,Bobryshev YV.Cardiac ExtracellularVesicles in Normal and Infarcted Heart.Int J Mol Sci.2016；17.pii:E63.

[12]Franciszkiewicz K,Boutet M,Gauthier L,Vergnon I,Peeters K,Duc O,et al. Synaptic release of CCL5 storage vesicles triggers CXCR4 surfaceexpression promoting CTL migration in response to CXCL12.J Immunol. 2014；193:4952-61.。

disclosure of Invention

The invention aims to provide a method for detecting and analyzing specific extracellular vesicles in blood and application thereof, in particular to a method for detecting and analyzing the quantity and proportion of the specific extracellular vesicles in the blood, and the method is helpful for predicting the atherosclerotic course and the occurrence probability of acute cardiovascular events of patients.

The invention discloses a method for detecting and analyzing the number and proportion of specific extracellular vesicles in blood, in particular to a method for detecting and analyzing extracellular vesicles and Annexin V in peripheral and coronary blood of a patient⁺And CD47⁺Extracellular vesicles further useful for predicting the course of atherosclerosis and the incidence of acute cardiovascular events in a patient.

The method of the invention comprises the following steps:

1) collecting the case information of healthy people and atherosclerosis patients:

detecting pathological changes in coronary artery of a patient through coronary angiography and intravascular ultrasound, calculating the area of stenosis in the blood vessel of the patient and the proportion of the stenosis in the blood vessel of the patient, and determining: healthy subjects, early AS patients, patients with coronary heart disease, and patients prone to myocardial infarction;

2) collecting and detecting extracellular vesicles in peripheral blood, and performing flow cytometry sorting and counting on the extracellular vesicles in a test sample by flow cytometry;

3) analyzing and comparing the level of extracellular vesicles in peripheral blood of an atherosclerotic patient with that of a healthy person,

4) analysis and comparison of CD47 in peripheral blood of atherosclerotic patients and healthy people⁺(ii) the level of extracellular vesicles

5) Analysis and comparison of Annexin V in peripheral blood of atherosclerotic patients and healthy people⁺The level of extracellular vesicles of (a);

6) analysis and comparison of extracellular vesicle levels in peripheral blood and CD47 in atherosclerotic and healthy persons⁺The level of extracellular vesicles is correlated with the degree of disturbance of lipid metabolism.

In the invention, the total number of extracellular vesicles and CD47 are shown by detection analysis and comparison⁺Number of extracellular vesiclesThe amount of CD47 gradually increases with the progression of the patient's atherosclerotic process (mainly referring to the degree of coronary stenosis in the patient)⁺The extracellular vesicles are less than 8 percent in the peripheral blood of normal people, and the proportion of the extracellular vesicles in the peripheral blood of coronary heart disease patients reaches 15 to 20 percent along with the development of the atheromatous course; furthermore, Annexin V⁺The extracellular vesicles in the blood are less than 10% in the peripheral blood of normal people, the distribution in the blood of the patients with atherosclerosis at each stage is about 25% at most, and the proportion in the blood of the patients with acute coronary syndrome or the patients with acute coronary syndrome is about to reach 50%.

In the present invention, the extracellular vesicles refer to exosomes having a diameter of 200nm or less and extracellular microvesicles having a diameter in the range of 200-2000 nm.

In the present invention, the CD47⁺Extracellular vesicles and Annexin V⁺By extracellular vesicles is meant extracellular vesicles carrying CD47 and Annexin V. The location of which includes the outer surface of the vesicle, the inner surface and the interior of the vesicle.

In the present invention, the extracellular vesicle, CD47⁺Extracellular vesicles and Annexin V⁺Extracellular vesicles may be derived from sources including, but not limited to: macrophage source, platelet source, endothelial cell source, monocyte source, smooth muscle cell source. But most notably refers to extracellular vesicles of macrophage origin.

In the invention, the positions of CD47 and Annexin V in the vesicle mainly refer to the level of CD47 and Annexin V on the outer surface of the vesicle.

In the present invention, reference to atherosclerotic patients includes, but is not limited to: patients with chronic atherosclerosis, patients with coronary heart disease, patients with long-term (> 3 years) hypertension, and patients with acute coronary syndrome.

In the invention, the patients suffering from atherosclerosis, preferably chronic atherosclerosis and coronary heart disease.

The invention further provides a method for detecting the number of extracellular vesicles, CD47, in the peripheral blood of an atherosclerotic patient⁺Extracellular vesicles and Annexin V⁺CellsThe proportion result of the outer vesicles is used for preparing a tool model for predicting the level of the atherosclerotic course of a patient and the probability of the patient suffering from an acute cardiovascular event; among these are especially extracellular vesicles, CD47⁺Extracellular vesicles and Annexin V⁺Application of extracellular vesicles in preparation of preparations for detecting and treating atherosclerosis.

In the invention, the extracellular vesicles can be combined with triglyceride, total cholesterol and LDL-C which are relevant indexes of lipid metabolism to prepare preparations for predicting and diagnosing atherosclerosis.

Drawings

Fig. 1, angiography of coronary vessels and intravascular ultrasound analysis of a patient/healthy person.

Figure 2 flow cytometric measurement of extracellular vesicle levels in peripheral blood of patients/healthy persons.

FIG. 3, CD47 in peripheral blood of patient/healthy person⁺Analysis of extracellular vesicle levels.

FIG. 4, Annexin V in peripheral blood of patient/healthy person⁺Analysis of extracellular vesicle levels.

FIG. 5, correlation analysis of the level of extracellular vesicles in the peripheral blood of a patient with an index of lipid metabolism.

FIG. 6, CD47 in patient's peripheral blood⁺And (3) analyzing the correlation between the extracellular vesicle level and the lipid metabolism index.

Detailed Description