CN1928111B - Correlation analysis of circulation endothelium progenitor cell and coronary artery pathological changes - Google Patents
Correlation analysis of circulation endothelium progenitor cell and coronary artery pathological changes Download PDFInfo
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Abstract
The present invention discloses the correlation between circular endothelial progenitor cell (EPC) and coronary artery pathological change. It is test proved that the number and migration and adhesion power of circular EPC is high correlated with the stenosis degree of coronary artery, so that circular EPC may be used as the reference index for diagnosing the stenosis degree of coronary artery. The present invention also provides method and kit for detecting the stenosis degree of coronary artery based on the said correlation.
Description
Technical field
The present invention relates to biology field.Relate more specifically to the dependency between circulation endothelium progenitor cell and the coronary artery pathological changes (especially degree of stenosis).The invention still further relates to method and test kit based on this correlation detection coronary artery pathological changes.
Background technology
Coronary artery pathological changes is the healthy diseases of a kind of serious harm people.
From people such as Asahara in 1997 first at the external human peripheral CD34 that successfully induces
+Cytodifferentiation has been (Asahara T, Murohara T, Sullivan A.et al.Isolation of putativeprogenitor endothelial cells for angiogenesis.Science1997 since the endotheliocyte; 275:964-7), more and more evidences shows in the marrow of growing up and has endothelial progenitor cells (EPCs), and marrow EPCs is mobilized to peripheral blood when blood vessel injury, and differentiation is also whole and to damage location, participates in the blood vessel endothelium repair process.
Complete tunica intima is to keeping antiotasis and blood vessel running balance plays an important role.Multiple Hazard Factor stimulate by oxidation and cause endothelial injury, and then cause atherosclerosis (Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation2002; 105:1135-43).
In recent years bibliographical information circulation EPCs can repair damage inner membrance (Shi Q, Raffi S, Wu MH, et al.Evidence for circulating bone marrow-derived endothelial cell.Blood1998; 92:362-7).Yet,, before the present invention, but never confirmed the dependency of EPCs quantity and function and coronary artery pathological changes though there is the people to infer that EPCs quantity and inner skin cell function, cardiovascular risk factors have certain dependency.
In sum, in order to prevent and treat coronary artery pathological changes as early as possible, this area presses for the correlative factor of seeking coronary artery pathological changes, and method, the test kit of exploitation detection coronary artery pathological changes, and relevant medicine.
Summary of the invention
Purpose of the present invention just provides the method and the detection kit of a kind of complementary diagnosis (especially early diagnosis) coronary artery pathological changes.
In a first aspect of the present invention, a kind of method of screening the candidate therapeutic agent of treatment coronary artery pathological changes is provided, comprise step:
(a) in test group, in the culture of circulation endothelium progenitor cell, add material standed for to be screened, cultivate and detect the quantity of circulation endothelium progenitor cell; And, in control group, under the situation of not adding material standed for to be screened, cultivate and detect the quantity of circulation endothelium progenitor cell;
(b) quantity with circulation endothelium progenitor cell in the quantity of circulation endothelium progenitor cell in step (a) test group and the control group that does not add material standed for compares, if the quantity of the circulation endothelium progenitor cell of test group is significantly higher than control group statistically, just show that this material standed for is the candidate therapeutic agent of treatment coronary artery pathological changes.
In another preference, described method also comprises step:
(b ') detects the transfer ability of circulation endothelium progenitor cell in the test group, and with control group in the transfer ability of circulation endothelium progenitor cell compare, wherein the transfer ability of the circulation endothelium progenitor cell of test group is significantly higher than control group statistically, just shows that this material standed for is the candidate therapeutic agent of treatment coronary artery pathological changes.
In another preference, described method also comprises step:
(b ") detects the adhesive capacity of circulation endothelium progenitor cell in the test group; and with control group in the adhesive capacity of circulation endothelium progenitor cell compare; wherein the adhesive capacity of the circulation endothelium progenitor cell of test group is significantly higher than control group statistically, just shows that this material standed for is the candidate therapeutic agent of treatment coronary artery pathological changes.
In another preference, described coronary artery pathological changes is a coronary stricture.
The present invention also comprises the candidate therapeutic agent that obtains with the aforesaid method screening.
In a second aspect of the present invention, a kind of purposes of specificity marker thing of circulation endothelium progenitor cell is provided, be used to prepare the reagent or the test kit of the coronary artery pathological changes of detection.Preferably, described specificity marker thing is selected from: CD133, KDR or vWF.
In another preference, described coronary artery pathological changes is a coronary stricture.
In a third aspect of the present invention, a kind of purposes that promotes the promotor of circulation endothelium progenitor cell quantity is provided, be used to prepare the medicine for the treatment of coronary artery pathological changes.
In another preference, described coronary artery pathological changes is a coronary stricture.
In a fourth aspect of the present invention, the method of a kind of detection coronary stricture (or susceptibility) is provided, it comprises step: the quantity that detects circulation endothelium progenitor cell in the individuality to be detected, and compare with the circulation endothelium progenitor cell quantity of normal population (normal control), if the quantity of circulation endothelium progenitor cell is lacked 30% than normal control and (is preferably lacked 50% in the individuality to be detected, more preferably lack 70%), just show that this individuality coronary stricture possibility is higher than normal population.
Description of drawings
Fig. 1 has shown the relation of CD133/KDR positive cell number, change of serum C RP concentration and coronary artery pathological changes.
Fig. 2 has shown the relation of CD133/KDR positive cell number, change of serum C RP concentration and Gensini scoring
Fig. 3 has shown the EPCs phenotypic evaluation.
Fig. 4 has shown the relation of EPCs transfer ability and coronary artery pathological changes.
Fig. 5 has shown the relation of EPCs adhesive capacity and coronary artery pathological changes.
Embodiment
The inventor is by extensive and deep research, and the inventor finds and confirmed the dependency between circulation endothelium progenitor cell (EPC) and the coronary artery pathological changes (especially degree of stenosis) first.There are high dependency in the quantity of circulation endothelium progenitor cell and degree of stenosis, thereby can be used as the complementary reference index of diagnosis degree of stenosis.The invention provides based on this dependency method and the test kit that detects coronary artery pathological changes is provided.
Particularly, the inventor does pathology severity and Hazard Factor analysis to 104 routine coronarography patients (getting rid of acute coronary syndrome, myocardial infarction); As the EPCs marker, detect patient's CD133/KDR double labeling cells quantity with CD133/KDR with flow cytometer; 93 routine patients are made the peripheral blood mononuclear cell differentiation culture, detect EPCs migration and adhesive capacity.
Test-results proves that coronarography positive person EPCs quantity significantly reduces (p＜0.01) than negative patient; EPCs quantity and Gensini scoring be negative correlation (n=49, r=-0.318, p=0.016).Peripheral blood EPCs quantity and age, super sensitive C-reactive protein (hs-CRP) are negative correlation (p=0.001,0.012); Hypertension and familial history of coronary artery disease patient EPCs quantity significantly reduce (p=0.042,0.043).Patients with coronary heart disease EPCs transfer ability also significantly descends than the normal people, and many pathologies are more singly propped up pathology decline more remarkable (p＜0.05).EPCs transfer ability and Gensini scoring be negative correlation (n=55, r=-0.315, p=0.021).Patients with coronary heart disease EPCs adhesive capacity is than normal people significantly descend (p＜0.05).
This shows that the SCHD patient, circulation EPCs quantity, migration and adhesive capacity descend than the normal people, and quantity, transfer ability are relevant with the coronary artery pathological changes degree.EPCs quantity and change of serum C RP concentration negative correlation relevant with cardiovascular risk factors.Because circulation EPCs has the blood vessel repair, therefore patients with coronary heart disease angiogenesis ability increases the weight of with degree of stenosis and weakens, multiple cardiovascular risk factors especially CRP may promote atherosclerotic generation, development by influencing EPCs quantity, function.
As used herein, term " EPC " refers to circulation endothelium progenitor cell (endothelial progenitorcells).
As used herein, term " CRP " refers to the C-reactive protein, and it is a kind of Inflammatory Mediators.
Based on new discovery of the present invention, circulation endothelium progenitor cell has many-sided new purposes.These purposes include, but is not limited to: directly as pharmacological agent coronary artery pathological changes, especially coronary stricture.In addition, also can screen the material that promotes circulation endothelium progenitor cell quantity, the material that these preliminary screening go out can constitute a screening storehouse, so that people finally can therefrom filter out the medicine that can effectively treat coronary artery pathological changes (especially coronary stricture).
On the other hand, the specificity marker thing of circulation endothelium progenitor cell also can be used for the present invention, by detecting the circulation endothelium progenitor cell that has these specificity markers (thing) in the individuality, can measure the quantity of circulation endothelium progenitor cell, thus the complementary index of the early stage auxiliary diagnosis coronary artery pathological changes (especially coronary stricture) of conduct.
Utilize circulation endothelium progenitor cell,, can filter out with circulation endothelium progenitor cell interactional material takes place, as inhibitor, agonist or antagonist etc. by various conventional screening methods.
The agonist of promotion circulation endothelium progenitor cell quantity or promotor etc. when using (administration) in treatment, can promote the quantity of circulation endothelium progenitor cell, and then improve the symptom of coronary artery pathological changes (especially coronary stricture).Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenously or subcutaneous administration.
The present invention also provides a kind of pharmaceutical composition, and it contains promotor and the pharmaceutically acceptable carrier or the vehicle of the promotion circulation endothelium progenitor cell quantity of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.The dosage of activeconstituents is the treatment significant quantity, for example every day about 0.1 microgram/kg body weight-Yue 10 mg/kg body weight.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization circulation endothelium progenitor cell quantity levels.These detected results can be used for auxiliary judgment or diagnosis coronary artery pathological changes (especially coronary stricture).
A kind of method that detects circulation endothelium progenitor cell quantity is to utilize the specific marker thing of circulation endothelium progenitor cell to detect, and it comprises: the specific antibody of sample with anti-circulation endothelium progenitor cell specificity marker thing contacted; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample circulation endothelium progenitor cell.Can measure the quantity of the cell (being circulation endothelium progenitor cell) that has this specificity marker thing in conjunction with equipment such as flow cytometers.
Major advantage of the present invention is:
Confirmed the dependency between circulation endothelium progenitor cell (EPC) and the coronary artery pathological changes (especially degree of stenosis) first, thereby be that coronary artery pathological changes (especially coronary stricture) diagnosis (especially early stage auxiliary diagnosis) and treatment provide new way.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Method
One. research object
In March, 2004, the inpatient of 104 routine underwent coronary radiographies (CAG) was research object (a CAG positive or negative) to May.Get rid of malignant tumour, peripheral blood vessel pathology, proliferative retinopathy patient; Get rid of nearly 2 months troubles acute coronary syndromes, acute myocardial infarction, inflammation, acute hemorrhages or the history and take statins or the erythropoietin person of transfusing blood; Get rid of the preceding women of menopause.
Two. general clinical data
Collect all patient's basic documents: sex, age, height, body weight, blood pressure.Detail knowledge medical history: essential hypertension, diabetes, coronary heart disease, cerebrovascular disease, nephropathy, smoking history, history of drinking history, mode of life (whether moving), familial history of coronary artery disease.And do the Routine Test Lab inspection: on an empty stomach and postprandial blood sugar, total cholesterol, LDL-C (LDL-C), highdensity lipoprotein-cholesterol (HDL-C), triglyceride level, creatinine, uric acid.
Three. echocardiography
Use the U.S. Hewlett Packard M2410A-M2408A of company type color Doppler ultrasonography instrument and the patient is carried out two dimension, M type and TTDE inspection, frequency probe 2.5MHz.With reference to U.S. ultrasonic cardiogram association proposed standard.
Press the Devereux formula and calculate left ventricular mass (LVM), LVM (g)=1.04[(LVDd+IVST+PWT)
3-LVDd
3]-13.6.
Body surface area (BSA)=0.0061 * height (cm)+0.0128 * body weight (kg)-0.1529.
Left ventricular mass index (LVMI) adopts formula: LVMI (g/m
2)=LVM/BSA (body surface area).
Four. coronarography
With electing property of Judkins method coronarography.Blood vessel and stenosis with quantitative coronary angiography (QCA) appraisal coronary artery pathological changes.With angiostenosis 〉=50% as positive criteria.Pathology is involved left anterior descending branch (LAD), one of (LCX), arteria coronaria dextra (RCA) are circled round for single pathology in a left side, is many pathologies more than or equal to two persons.The left coronary artery main stem pathology is two pathologies.Estimate the severity of coronary artery pathology in two kinds of modes: 1. the coronary artery number (being divided into single pathology, two pathologies and three pathologies) that involves of pathology; 2.Gensini scoring (is undertaken by the described mode of following document: Gensini GG.A moremeaningful scoring system for determining the severity of coronary heartdisease.Am J Cardiol 1983; 51:606).
Five. Serum hs-CRP, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) are measured
Serum hs-CRP concentration adopts super quick CRP test kit, and (Dade Behring Inc.Marburg German) measures with immune scattering turbidimetry method.Sensitivity is 0.175mg/L.ICAM-1 and VCAM-1 serum-concentration are used sICAM-1 and sVCAM-1 ELISA test kit, and ((DIACLONE, Besancon France) measure.Sensitivity is 8ng/ml and 0.6ng/ml.
Six. flow cytometry is surveyed circulation EPCs quantity
The Judkins conduit inserts femoral artery and gets blood 150ul before the coronary angiography, add the PE mark CD133 mouse anti human mono-clonal IgG1 antibody (MACS, Bergisch Gladbach, Germany), the mouse anti human KDR mono-clonal IgG1 antibody (R﹠D of APC mark, Minneapolis, USA).Homotype contrast for the mouse IgG1 of PE mark and APC mark (Becton Dickinson, Franklin Lakes, USA).Adopt the BD FACSCalibur of company flow cytometer to carry out flow cytometry with CellQuest software.On forward angle light scatter light and the two-parameter point diagram of lateral angle scattered light lymphocyte populations is established window, totally 60000 of collecting cell numbers are with the percentage of the statistics KDR/CD133 of this colony cell.
The separation of seven .EPC, cultivation
Get peripheral blood 10ml on an empty stomach, obtain mononuclearcell with density gradient centrifugation in the 4h.With mononuclearcell be seeded in be coated with fibronectin (FN) culture plate (BD Biosciences, CA, USA).(Cambrex, Walkersville MD) cultivated 4 days EGM substratum (containing 10% foetal calf serum, penicillin 100U/ml, Streptomycin sulphate 100U/ml), washed non-adherent cell off with PBS, changed nutrient solution and continued to be cultured to 7d, and attached cell is carried out CYTOCHEMICAL ANALYSIS.
Eight .EPCs identify
With 0.125% trypsin treatment attached cell, human vessel endothelium growth factor resisting (KDR) monoclonal antibody (R﹠D with the PE mark, Minneapolis, USA) and the Von Willebrand factor (vWF) antibody (DAKO, Glostrup, Denmark) labeled cell carries out flow cytometry.
Nine .EPCs transfer abilities detect
As above-mentioned collection attached cell and counting.With 600 μ l nutrient solution and vascular endothelial growth factor
(VEGF, (New York USA) descends the chamber, with 2 * 10 for Corning Costar Inc., Corning 50ng/ml) to place transwell (8-μ m aperture)
4EPCs is suspended in 200 μ l nutrient solutions and injects upward chamber, cultivates 24h, scrapes off the not migratory cell above the filter membrane, fixes with 75% ice ethanol, and HE dyeing selects 5 fields of microscope (* 200) counting to move to the cell of low layer at random.
Ten .EPCs adhesive capacities detect
Collect attached cell with 0.25% tryptic digestion, be suspended in the EGM nutrient solution, counting, 1 * 10
5EPCs is layered on and is coated with the FN culture plate, cultivates 30min at 37 ℃, the counting attached cell.
11. statistical study:
All data are represented with mean number ± standard deviation.Be for data processing with the SPSS statistical software.CRP concentration is converted to the form of natural logarithm to reach normal distribution.Analyze CD133/KDR with multifactor stepwise regression method
+The influence factor of cell.Judge CD133/KDR with partial regression analysis and covariance analysis
+The relation of cell quantity, EPCs migration, adhesive capacity and severity degree of coronary.
The result
One. selected patients clinical data
80 examples are suspected or clinical confirmation patients with coronary heart disease is selected object.Be divided into three groups by the coronary angiography result, i.e. coronarography feminine gender, single pathology and Duo Zhi pathology group.Three groups of patient age, weight index, hypertension, smoking, drink, familial history of coronary artery disease, fasting plasma glucose and blood lipid level all do not have significant difference, but the CHD group male sex (p＜0.01), diabetic history (p＜0.05) and alcohol user (p＜0.05) significantly increase; The serum creatinine clearance rate obviously reduces (p＜0.01) (seeing Table 1).
Table 1. is selected in the patients clinical data
| Amount to | CAG(-) | Single vascular disease | Many vascular disease | The P value | |
| N | 104 | 44 | 35 | 25 | |
| Age (year) | 63.9±10.6 | 61.5±9.98 | 63.03±10.01 | 64.60±10.48 | 0.23 |
| BMI(Kg/m 2) | 24.69±3.67 | 23.80±3.24 | 25.07±3.96 | 25.80±3.80 | 0.11 |
| Sex (male sex, %) | 77(74.0％) | 25(57％) | 29(82％) | 23(92％) | ＜0.01 |
| Medical history, n (%) | |||||
| Hypertension | 55(52.9％) | 18(45％) | 20(57.1％) | 17(60％) | 0.21 |
| Diabetes | 12(11.5％) | 1(2.3％) | 6(17.1％) | 5(20.0％) | 0.049 |
| Present smoker | 37(35.6％) | 16(36.3％) | 13(37.1％) | 8(40％) | 0.87 |
| The alcohol user | 10(9.6％) | 2(4.5％) | 6(17.1％) | 2(24％) | 0.02 |
| The CAD family history | 27(26.0％) | 9(20.5％) | 13(37.1％) | 5(20％) | 0.37 |
| LVMI(g/cm 2) | 119.33±28.78 | 114.3±25.46 | 120.43±20.81 | 126.64±26.62 | 0.51 |
| The blood test |
| Fasting plasma glucose (mmol/L) | 5.75±1.11 | 5.49±0.68 | 5.84±1.27 | 6.07±1.40 | 0.15 |
| Serum cholesterol (mmol/L) | 4.81±1.05 | 4.81±0.90 | 4.80±1.22 | 4.82±0.85 | 0.89 |
| Serum triglyceride (mmol/L) | 1.71±0.90 | 1.66±0.90 | 1.73±1.11 | 1.76±0.53 | 0.88 |
| Serum hdl-cholesterol (mmol/L) | 1.26±0.41 | 1.36±0.49 | 1.18±0.31 | 1.18±0.34 | 0.39 |
| Serum LDL-cholesterol (mmol/L) | 2.79±0.78 | 2.77±0.70 | 2.81±0.94 | 2.82±0.70 | 0.92 |
| Urea (umol/L) | 346.27±85.65 | 334.35±75.14 | 348.11±84.01 | 364.69±103.61 | 0.65 |
| Ccr(ml/min/1.73m 2) | 76.55±21.32 | 83.53±22.27 | 75.74±17.72 | 65.40±19.96 | 0.03 |
CAD: coronary artery pathological changes;
BMI: weight index;
LVMI: left ventricular mass index (left ventricular mass index);
CAG (-): normal coronary angiography (normal coronary angiography);
Ccr: CCr (creatinine clearance).
Two. peripheral blood CD133/KDR
+The relation of cell and Hazard Factor
Analyze the predicting function of multiple Hazard Factor with multiple regression procedure to EPCs.Find age, hs-CRP, hypertension, familial history of coronary artery disease relevant with EPCs quantity (seeing Table 2).
The relation of table 2 cardiovascular risk factors and circulation EPCs quantity
| Standardized regression coefficient | (SE) | The P value | |
| Age | -0.390 | (0.001) | 0.001 |
| Ln?hs-CRP | -0.2291 | (0.003) | 0.012 |
| Hypertension | -0.166 | (0.005) | 0.042 |
| The CAD family history | -0.170 | (0.005) | 0.043 |
Hs-CRP: highly sensitive C activated protein (C reactive protein);
Ln: natural logarithm;
CAD: coronary artery pathological changes;
SE: standard deviation
Three .CD133/KDR
+Cell quantity, hs-CRP are with the relation of coronary artery pathological changes degree
The coronary artery quantity of involving by pathology is divided into three groups with the patient, proofreaies and correct with age, sex and body surface area, does covariance analysis.The negative group of coronary angiography EPCs quantity is (0.064 ± 0.004%) % (n=44); Single pathology group is (0.043 ± 0.004%) % (n=35); Many pathology groups be (0.030 ± 0.005%) (n=25).Compare with the negative group of coronary angiography, single pathology and Duo Zhi pathology group EPCs quantity significantly reduce (p＜0.05; P＜0.01), many pathology persons more singly prop up the also significantly reduction (p＜0.05) of pathology person EPCs quantity.Three groups of patient CRP serum-concentrations also have marked difference, and the negative group of coronary angiography ln CRP is 0.077 ± 0.015, (n=44); Single pathology group is 0.725 ± 0.125 (n=35); Many pathology groups be 1.343 ± 0.206 (n=25) (Fig. 1).
Proofread and correct with age, sex and body surface area, to coronarography positive patient CD133/KDR
+The partial regression analysis is done in cell quantity, hs-CRP and Gensini scoring, finds CD133/KDR
+Cell quantity and Gensini scoring be negative correlativing relation (n=60, r=-0.355, P=0.006), hs-CRP and Gensini the mark relation of being proportionate (n=49, r=0.476, P=0.001) (Fig. 2).
Four .EPCs phenotypic evaluation
To peripheral blood mononuclear cell adherent culture seven days, can find that cell is fusiformis, identify with the flow cytometer pair cell, find cell expressing endothelium mark vWF (100%), KDR (96%) (Fig. 3).
The relation of five .EPCs transfer abilities and coronary artery pathological changes
EPCs plays an important role in angiogenic process to the transfer ability of VEGF.By detection to 38 routine coronarography negative patients and 55 routine patients with coronary heart disease circulation EPCs transfer abilities, find that (EPCs moves 38.73 ± 1.37/*200 to CAG (-) person, n=38) the EPCs transfer ability is significantly higher than single pathology (EPCs moves 34.33 ± 1.58/*200, n=27) and many pathology persons (EPCs moves 29.46 ± 1.66/*200, n=27) and EPCs transfer ability and Gensini scoring be inverse ratio (n=55, r=-0.315, p=0.021) (Fig. 4).
The relation of six .EPCs adhesive capacities and coronary artery pathological changes
Coronarography positive person EPCs adhesive capacity significantly descends (p＜0.05), but adhesive capacity and the no correlationship of Gensini scoring (n=55, r=-0.196, p=0.159) (Fig. 5)
Discuss
The present invention measures circulate EPCs quantity, functionally active of SCHD patient, finds that SCHD patient EPCs quantity, transfer ability, adhesive capacity reduce than the normal people; Find that first EPCs quantity is relevant with degree of stenosis with shift function; And find in clinical study that for the first time circulation EPCs quantity and level of serum hs-CRP are negative correlativing relation.
Being the EPCs sign with CD133/KDR detects the patient EPCs that circulates, and finds: the coronarography positive person EPCs quantity that circulates significantly reduces than the coronarography negative patient; And many pathology persons more singly prop up pathology person reduction trend; EPCs quantity and coronary angiography Gensini scoring are negative correlation.The above results can be inferred: the patient that coronary artery pathological changes is serious, EPCs produce, mobilize and reduce, and the transformation period shortens.
Circulation EPCs quantity is few, and during blood vessel injury, the local cytokine that discharges as VEGF, SDF-1 etc., produces chemotaxis to EPCs, makes it be able to play a role at ishemic part.Therefore except that EPCs quantity, the shift function of EPCs plays a crucial role in angiogenic process, and VEGF is considered to most important inducible factor.The present invention is inducible factor with VEGF, and circulation EPCs shift function is detected, and finds the negative group of CAG, single pathology group, there were significant differences for many pathology group EPCs transfer abilities, and transfer ability is marked with Gensini and is negative correlation.Prompting circulation EPCs transfer ability increases the weight of with the coronary artery pathological changes degree and weakens.VEGF participates in angiogenesis by the migration of VEGFR-2 (KDR) inducing endothelial cell, therefore points out EPCs to participate in this process by KDR.But it is relevant with coronary artery pathological changes that the present invention does not find that KDR expresses, so may have the downstream signal transduction pathway and make the decline of EPCs transfer ability.
Another important mechanisms that EPCs participates in angiogenesis is the cell adhesion effect, and EPCs adheres to by cell-cell adhesion and cell-extracellular matrix and is integrated into the endothelial injury position, repairs blood vessel.The present invention finds that patients with coronary heart disease EPCs significantly descends to the FN adhesion function, but does not find that its adhesion function is relevant with severity degree of coronary.
Coronary heart disease EPCs quantity and biological function change, and may be mediated by following mechanism:
At first, Angiotensin II and active oxygen (ROS) are the materials that plays an important role in the pathogenesis of coronary artery pathological changes, the bibliographical information Angiotensin II promotes that by inducing oxidation to stimulate EPCs is aging, apoptosis (Imanishi T, Hano T, NishioI.Angiotensin II accelerates endothelialprogenitor cell senescence through induction of oxidative stress.JHypertens 2005; 23:97-104.).
Secondly, eNOs can quicken EPCs and mobilize, and patients with coronary heart disease blood vessel endothelium generation nitric oxide synthetase (eNOs) function is impaired, thereby EPCs quantity is reduced.To patients with coronary heart disease, hypoxia inducible VEGF discharges minimizing, may also be to reduce the important factor that EPCs mobilizes, breaks up, moves.In addition, the ox-LDL that plays an important role in Atherosclerosis can suppress the EPCs differentiation, influences EPCs migration, vasculogenesis ability.On the other hand, the chronic blood vessel endothelium injury that atherosclerosis is followed can continue to consume circulation EPCs and participate in the endothelium repairing, thereby reduces more powerful EPCs in the circulation.Cardiovascular risk factors comprises that inflammatory factor CRP may also play an important role in this process.Complete tunica intima is kept the blood vessel normal function, and endothelial injury can cause atherosclerosis.And circulation EPCs is " cell bank " of repairing the damage endothelium.Under normal circumstances there are running balance in endothelial injury and EPCs between repairing, and keep interior film integrality, and EPCs quantity/function minimizing may influence the endothelium reparation, and film integrality is impaired in causing, and have urged atherosclerosis and have taken place, develop.The present invention finds that EPCs quantity and coronary artery pathology severity are remarkable negative correlativing relation, and it is to bring out one of cause of coronary heart disease mechanism that the endothelium that is not enough to repair damage to the EPCs of peripheral blood is mobilized in prompting in the marrow.
The present invention finds that CD133/KDR positive cell quantity and change of serum C RP level are negative correlation.(George J such as George, Goldstein E, Abashidze S, et al.Circulating endothelialprogenitor cells in patients with unstable angina:association withsystemic inflammation.Eur Heart J 2004; 25:1003-8.) discovery EPCs quantity and the positive correlation of CRP concentration in the research of coronary heart disease (comprising SCHD and acute coronary artery syndrome) EPCs.Cardiac muscular tissue's ischemic that acute coronary artery syndrome produces may induce VEGF to discharge, thereby increases EPCs differentiation, mobilization, increases circulation EPCs quantity; And CRP is the inflammatory index, significantly increases in the acute inflammation process.In recent years document prompting hs-CRP not only is important cardiovascular risk factors, still is atherosclerotic important participation material simultaneously.In vitro tests finds that CRP significantly reduces EPCs quantity, delays the endothelium specificity marker and occurs, and promotes the EPCs apoptosis, makes EPCs angiogenesis ability impaired.Other has test prompting: CRP to suppress its angiogenesis ability by reducing the EPCs secretion various kinds of cell factor.The inventor finds the negative correlativing relation of EPCs and CRP first from clinical study, confirm that further CRP can reduce EPCs quantity.Because CRP is the important predictor of cardiovascular event, and EPCs is relevant with CRP, so circulation EPCs quantity can become the biology sign of predicting cardiovascular disease.The present invention shows that also circulation EPCs quantity is relevant with cardiovascular risk factors such as age, hypertension, familial history of coronary artery disease.Therefore, this prompting conventional risk factors and CRP can reduce circulation EPCs quantity, hinder the endothelium reparation, promote atherosclerotic lesion.
Because circulation EPCs quantity is relevant with the coronary stricture severity with function, be that the angiogenesis ability that degree of stenosis and EPCs participate in is negative correlation, therefore this prompting, in a part of coronary artery pathological changes, the multiple factor that comprises CRP, may hinder the endothelium reparation by reducing EPCs quantity and function, quicken atherosclerosis generation, development.
Screening promotes the promotor of EPC quantity
Get peripheral blood 10ml on an empty stomach, obtain mononuclearcell with density gradient centrifugation in the 4h.With equal amts (about 1 * 10
5) mononuclearcell be inoculated in respectively 6 be coated with fibronectin (FN) culture plate (BD Biosciences, CA, USA).
In 3 culture plates of control group, do not adding the EGM substratum of any compound to be tested (containing 10% foetal calf serum, penicillin 100U/ml, Streptomycin sulphate 100U/ml) (Cambrex, Walkersville, MD) the middle cultivation 4 days.
In 3 culture plates of control group, be EGM substratum (containing 10% foetal calf serum, penicillin 100U/ml, Streptomycin sulphate 100U/ml) (Cambrex, Walkersville, MD) the middle cultivation 4 days of 0.1M compound to be tested having added concentration.
Cultivate after 4 days, wash non-adherent cell in test group and the control group off, change nutrient solution and continue to be cultured to 7d, respectively attached cell is carried out CYTOCHEMICAL ANALYSIS with PBS.
With 0.125% trypsin treatment attached cell, with human vessel endothelium growth factor resisting (KDR) monoclonal antibody (R﹠amp of PE mark; D, Minneapolis, USA) (Denmark) labeled cell carries out flow cytometry for DAKO, Glostrup with the Von Willebrand factor (vWF) antibody.
Compare with the circulation endothelium progenitor cell quantity (mean values of 3 culture plates) of control group, if the circulation endothelium progenitor cell quantity of test group (mean values of 3 culture plates) exceeds 30%, think that then this test compounds is the promotor that more effectively promotes EPC quantity; If the circulation endothelium progenitor cell quantity of test group (mean values of 3 culture plates) exceeds 50%, think that then this test compounds is the promotor of effectively promotion EPC quantity.
Screening promotes the compound of circulation endothelium progenitor cell migration and adhesive capacity
For the promotor of the promotion EPC quantity that filters out among the embodiment 2, further measure it and whether have migration of the circulation endothelium progenitor cell of promotion and adhesive capacity:
(a) the EPCs transfer ability detects
Collect attached cell and counting as embodiment 2.With 600 μ l nutrient solutions and vascular endothelial growth factor (VEGF, 50ng/ml) place transwell (8-μ m aperture) (Corning Costar Inc., Corning, New York, USA) chamber down is with 2 * 10
4EPCs is suspended in 200 μ l nutrient solutions and injects upward chamber, cultivates 24h, scrapes off the not migratory cell above the filter membrane, fixes with 75% ice ethanol, and HE dyeing selects 5 fields of microscope (* 200) counting to move to the cell of low layer at random.
(b) the EPCs adhesive capacity detects
Collect attached cell with 0.25% tryptic digestion, be suspended in the EGM nutrient solution, counting, 1 * 10
5EPCs is layered on and is coated with the FN culture plate, cultivates 30min at 37 ℃, the counting attached cell.
The result shows that 2 promotor that promote EPC quantity for preliminary screening obtained all do not promote circulation endothelium progenitor cell migration and adhesive capacity.This prompting promotes the propagation of EPC to relate to different approach with migration and adhesion.
Utilize circulation endothelium progenitor cell specificity marker thing to detect the test kit of coronary stricture susceptibility
Prepare a test kit, it contains: (a) the CD133 mouse anti human mono-clonal IgG1 antibody of PE mark (MACS, Bergisch Gladbach, Germany) and (b) the mouse anti human KDR mono-clonal IgG1 antibody (R﹠amp of APC mark; D, Minneapolis, USA).
To 10 coronary stricture patients and 10 test group that the normal people volunteer forms, insert femoral artery with the Judkins conduit and get blood 150ul.Under the double-blind method condition, add the PE mark CD133 mouse anti human mono-clonal IgG1 antibody (MACS, Bergisch Gladbach, Germany), the mouse anti human KDR mono-clonal IgG1 antibody of APC mark (R﹠D, Minneapolis, USA).Homotype contrast for the mouse IgG1 of PE mark and APC mark (Becton Dickinson, Franklin Lakes, USA).Adopt the BD FACSCalibur of company flow cytometer to carry out flow cytometry with CellQuest software.On forward angle light scatter light and the two-parameter point diagram of lateral angle scattered light lymphocyte populations is established window, totally 50000 of collecting cell numbers are with the percentage of the two positive cells of the statistics KDR/CD133 of this colony.Simultaneously all testers are carried out coronarography.
Judging criterion:
(a) percentage＞0.05% of the two positive cells of KDR/CD133 is represented no coronary stricture;
(b) percentage＜0.045% of the two positive cells of KDR/CD133, expression coronary stricture or coronary stricture susceptibility are higher than normal population;
(c) percentage of the two positive cells of KDR/CD133 does not have the value of clarifying a diagnosis between 0.045% and 0.05%.
The result:
Percentage＜0.045% of 8 bit test persons' the two positive cells of KDR/CD133 is arranged, wherein have 7 coronary stricture (single pathology or many pathologies) is arranged.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (2)
1. a method of screening the candidate therapeutic agent of treatment coronary artery pathological changes is characterized in that, comprises step:
(a) in test group, in the culture of circulation endothelium progenitor cell, add material standed for to be screened, cultivate and detect the quantity of circulation endothelium progenitor cell; And, in control group, under the situation of not adding material standed for to be screened, cultivate and detect the quantity of circulation endothelium progenitor cell;
(b) quantity with circulation endothelium progenitor cell in the quantity of circulation endothelium progenitor cell in step (a) test group and the control group that does not add material standed for compares, if the quantity of the circulation endothelium progenitor cell of test group is significantly higher than control group statistically, just show that this material standed for is the candidate therapeutic agent of treatment coronary artery pathological changes;
And described method also comprises: measure the influence of this material standed for to the concentration of super sensitive C-reactive protein in the quantity of circulation endothelium progenitor cell in the peripheral blood and the serum, and select to cause the quantity of circulation endothelium progenitor cell in the peripheral blood to become the material standed for of negative correlation as the candidate therapeutic agent for the treatment of coronary artery pathological changes with the concentration of super sensitive C-reactive protein in the serum.
2. the method for claim 1 is characterized in that, described coronary artery pathological changes is a coronary stricture.
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| 汪海娅 等.稳定性冠心病患者循环内皮祖细胞与冠状动脉病变严重程度的分析.中华心血管病杂志33 5.2005,33(5),425-427. |
| 汪海娅等.稳定性冠心病患者循环内皮祖细胞与冠状动脉病变严重程度的分析.中华心血管病杂志33 5.2005,33(5),425-427. * |
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