CN110333359A - A kind of analysis different size HDL promotees the method and application of Cholesterol Efflux ability - Google Patents
A kind of analysis different size HDL promotees the method and application of Cholesterol Efflux ability Download PDFInfo
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Abstract
The present invention relates to high-density lipoprotein functional examination technical field, method and application that a kind of analysis different size HDL promotees Cholesterol Efflux ability are specifically disclosed, the lipoprotein of this approach includes the following steps (1) separation sample to be tested obtains multiple groups HDL；(2) cell strain is inoculated in cultivation plate hole, the culture solution that the cholesterol containing labelled with radioisotope is added marks cell, makes cell lotus rouge and label；(3) it is incubated for related film cholesterol transporter gene expression in guidance cultivation plate hole inner cell strain；(4) obtained HDL is added separately to through being incubated in step (3) treated cultivation plate hole；(5) draw the supernatant culture solution in cultivation plate hole, remove in supernatant culture solution and be added into scintillation solution after residual cells, calculate every group of HDL promotes the discharge rate of cholesterol in cell.Method provided by the invention can deeper into assessment sample to be tested in different size HDL promote Cholesterol Efflux ability and major cholesterol transport body function between relationship.
Description
Technical field
The present invention relates to high-density lipoprotein functional examination technical field more particularly to a kind of analysis different size HDL promotees gallbladder
The method and application of sterol outflow ability.
Background technique
Cardiovascular disease is to endanger the number one killer of human health, dead due to caused by cardiovascular disease in the world
Number is died higher than other any diseases.Atherosclerosis and its caused coronary heart disease are a kind of shapes common in cardiovascular disease
Formula, the arterial elasticity that shows as pathologically lower, artery wall thickening, coronal lipid and immunocyte and necrosis occur on vascular wall
The accumulation of tissue causes lumen of vessels stenosis narrow in turn, causes Oligemia even blood vessel blockage.Research shows that atherosclerosis
It is a chronic inflammation processes, macrophage plays key effect in its Forming Mechanism.It is thin that macrophage is divided into monocyte
After born of the same parents, macrophage can swallow the OxLDL ELISA rich in cholesterol, fat drips and apoptotic cell fragment etc. finally at
It is deposited in vascular wall to participate in forming coronary artery for foam cells.
High-density lipoprotein (High Density Lipoprotein, HDL) is by cholesterol, phosphatide, apolipoprotein group
At the lipoprotein complexes containing 200 multiple proteins, be a kind of to have the important lipoprotein of protective effect, research card to angiocarpy
The real lipoprotein can transport the reverse cholesterol being especially in the macrophage of coronary vasodilator in human body perimeter systems back liver
And transport to bile fluid, it is excreted finally by excrement.The first step of this inverse transport process, i.e. blood plasma middle-high density rouge egg
The white turn-over capacity to cholesterol in arterial wall macrophage is to measure its function important indicator.
Existing research shows in the foam cells formed by macrophage, ATP binding cassette transporter ABCA1
It plays an important role during cholesterol efflux with ABCG1, about 70% cholesterol efflux is situated between by ABCA1 and ABCG1
Lead progress.Experiment in vivo proves that ABCA1 and ABCG1 can promote the activity of reverse cholesterol transport, and ABCA1 and ABCG1 exist
The defect that missing in myeloide hematopoietic cell can cause HDL to outflow, to make the bulk deposition of cholesterol ester and then accelerate
The formation of atherosclerosis.
Different synthesis and metabolic stage, high-density lipoprotein are presented different albumen and form, aquation density, and particle is big
It is small, charge power etc..According to its aquation density, HDL2, HDL3 and VHDL can be classified as；If it is strong according to its charge, and can incite somebody to action
HDL points are HDL2b, 2B, 3a, 3b, 3c.There are certain differences for different types of HDL its functional mechanism, thus to different type
The functional examination of HDL have important clinical meaning.
In the prior art, usually only HDL concentration total in blood plasma is measured or only will be total obtained in blood plasma
HDL carries out the functional test of reverse cholesterol transport, to analyze the risk etc. of prediction cardiovascular disease, there is no HDL is based on it
Granular size carries out functional test after distinguishing again, therefore, a kind of new analysis method is studied, for vast cardiovascular patient
Person, enterprise and society have huge potential clinical value and economic benefit.
Summary of the invention
In view of this, in order to make up for the deficiencies of the prior art, it is different big that the main object of the present invention is to provide a kind of analysis
The method that small HDL promotees Cholesterol Efflux ability, this method can deeper into assessment sample to be tested in HDL promote Cholesterol Efflux ability
And the relationship between major cholesterol transhipment body function.The present invention can be used for studying lipid metabolism in scientific research especially different
Influence of the HDL of size to disease can be used in clinic to vascular diseases especially atherosclerosis and its related disease
Disease includes the discovery of the new biomarkers of diabetes and obesity etc., early diagnosis and prognostic analysis.
To achieve the above object, the technical solution of the present invention is as follows:
A method of analysis different size HDL promotees Cholesterol Efflux ability, comprising the following steps:
(1) lipoprotein for separating sample to be tested, obtains multiple groups HDL, and the granular size of every group of HDL is different；
(2) cell strain is inoculated in cultivation plate hole, the culture solution that the cholesterol containing isotope labelling is added marks cell, makes
Cell lotus rouge and label, the quantity of cultivation plate hole are no less than the group number of HDL in step (1)；
(3) it is incubated for related film cholesterol transporter gene expression in guidance cultivation plate hole inner cell strain；
(4) every group of HDL obtained in step (1) is added separately to through incubating in step (3) treated cultivation plate hole
It educates；
(5) the supernatant culture solution in cultivation plate hole is drawn, removes in supernatant culture solution and is added after residual cells to scintillation solution
In, with isotope intensity in liquid scintillation counter detection culture solution, calculate every group of HDL promotes the outflow of cholesterol in cell
Rate.
Preferably, the isolated method in step (1) is fast protein liquid chromatography (Fast Protein Liquid
Chromatography, FPLC) chromatography.
Preferably, cell strain described in step (2) includes mouse macrophage strain and human body cell strain.
Preferably, related film cholesterol transporter gene described in step (3) includes ABCAl and non-ABCAl, step
(3) specifically:
Be added cAMP be incubated for guidance cell strain in ABCAl expression, or do not add cAMP be incubated for guidance cell strain in ABCAl and
Non- ABCAl, the i.e. expression of total cholesterol transport protein are used to that HDL is mediated to promote Cholesterol Efflux, and the temperature of incubation is 37 DEG C,
The time of incubation is 16h；Wherein, non-ABCAl is mainly ABCG1.
Preferably, the temperature being incubated in step (4) is 37 DEG C, and the time of incubation is 4h.
Preferably, the sample to be tested includes human plasma, human serum, the serum composition of separation, hdl particle, natural production
One of object, biomolecule and synthesis compound are a variety of.
The second object of the present invention be to provide it is a kind of the above method is used to analyze promote Cholesterol Efflux ability to disease
Application in influence.
Preferably, the disease includes atherosclerosis, diabetes and obesity and related disease.
The beneficial effects of the present invention are:
HDL is based on after its granular size separated, then measures its function to it, this method can deeper into assessment it is to be measured
HDL in sample promotees Cholesterol Efflux ability and the relationship between major cholesterol transhipment body function；The present invention is in scientific research
Can be used for studying lipid metabolism and be especially influence of the different size of HDL to disease, will deeper into exploration disease mechanisms and
It was found that new therapy target；Can be used in clinic include to vascular diseases especially atherosclerosis and its related disease
The discovery of the new biomarkers of diabetes and obesity, early detection and prognostic analysis etc..
Detailed description of the invention
Fig. 1 is the flow diagram for the method that present invention analysis different size HDL promotees Cholesterol Efflux ability；
Fig. 2 is the present invention by the health of FPLC chromatography and the plasma lipoprotein constitutional diagram of obesity crowd；
Fig. 3 is the total cholesterol outflow ratio bar graphs (packet of health and obese people HDL component 32,34 and 37 of the invention
It includes to be mediated by ABCA1 and be mediated with non-ABCA1, non-ABCA1 is mainly ABCG1).
Fig. 4 is that the present invention is based on the cholesterol efflux ratio bar graphs of the ABCA1 HDL component 32,34 and 37 mediated；
Fig. 5 is cholesterol efflux ratio bar graphs of the present invention by the non-ABCA1 HDL component 32,34 and 37 mediated.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is general
Logical technical staff every other embodiment obtained without creative efforts belongs to what the present invention protected
Range.
It in addition, the technical solution between each embodiment of the present invention can be combined with each other, but must be general with this field
Based on logical technical staff can be realized, it will be understood that when the combination of technical solution appearance is conflicting or cannot achieve this
The combination of technical solution is not present, also not the present invention claims protection scope within.
The present invention provides a kind of method of analysis different size HDL rush Cholesterol Efflux ability, comprising the following steps:
(1) lipoprotein for separating sample to be tested, obtains multiple groups HDL, and the granular size of every group of HDL is different；
(2) cell strain is inoculated in cultivation plate hole, the culture solution that the cholesterol containing isotope labelling is added marks cell, makes
Cell lotus rouge and label, the quantity of cultivation plate hole are no less than the group number of HDL in step (1)；
(3) it is incubated for related film cholesterol transporter gene expression in guidance cultivation plate hole inner cell strain；
(4) every group of HDL obtained in step (1) is added separately to through incubating in step (3) treated cultivation plate hole
It educates；
(5) the supernatant culture solution in cultivation plate hole is drawn, removes in supernatant culture solution and is added after residual cells to scintillation solution
In, with isotope intensity in liquid scintillation counter detection culture solution, calculate every group of HDL promotes the outflow of cholesterol in cell
Rate.
HDL is based on after its granular size separates by the present invention, then its function is measured to it, this method can deeper into comment
Estimate the HDL in sample to be tested and promotees Cholesterol Efflux ability and the relationship between major cholesterol transhipment body function；The present invention exists
Can be used for studying lipid metabolism in scientific research and be especially influence of the different size of HDL to disease, will deeper into exploration disease
The mechanism therapy target new with discovery；It can be used in clinic to vascular diseases especially atherosclerosis and its related disease
Disease includes detection and prognostic analysis of diabetes and obesity etc..
Further, the method that the lipoprotein of sample to be tested is separated described in step (1) is fast protein liquid chromatography
(Fast Protein Liquid Chromatography, FPLC) chromatography, compared with traditional ultracentrifugation method, this point
The influence to HDL albumen physical property that may cause by ultracentrifugation can be reduced from method.Pass through many experiments, this separation
Method can with the blood plasma of gradient separations mouse and people, to the HDL analysis sample of variable grain size after separation can deeper into
It solves it and promotees Cholesterol Efflux ability.
Further, cell strain described in step (2) includes mouse macrophage strain and human body cell strain.
Further, related film cholesterol transporter gene described in step (3) includes that atp binding cassette turns
Transport body A1 (ABCA1) and non-Liver X Receptors (non-ABCA1)；Wherein, non-ABCA1 is mainly ABCG1；
Step (3) specifically:
Addition cAMP is incubated for ABCA1 expression in guidance cell strain, or does not add cAMP incubation and guide total gallbladder in cell strain solid
Alcohol transport protein (including by ABCA 1 mediate and non-ABCA1 mediate-be mainly ABCG1) expression, be used to mediate HDL promote gallbladder consolidate
Alcohol outflow, the temperature of incubation are 37 DEG C, and the time of incubation is 16h.The transhipment access of HDL is mediated by specific addition cAMP,
The transhipment cholesterol function by Liver X Receptors (ABCA1) can be measured, while by being not added with
The measurement of the sample of cAMP can calculate the HDL gallbladder of non-Liver X Receptors (predominantly ABCG1) mediation
The function of sterol efflux.It compares in existing most technology and detects single transhipment body function with single experiment, the method can be same
The function that different size HDL flows out cholesterol by two kinds of important transporters is measured in one experiment simultaneously, thus the method is more
It is efficient and convenient.
Further, the temperature being incubated in step (4) is 37 DEG C, and the time of incubation is 4h；The sample to be tested includes people
Blood plasma, human serum, the serum composition of separation, hdl particle, natural products, biomolecule and synthesis one of compound or
It is a variety of.
Further, the present invention propose it is a kind of by the above method be used to analyze promote Cholesterol Efflux ability in sickness influence
Application.
Further, the disease includes atherosclerosis, diabetes and obesity and related disease.
Promote the method for Cholesterol Efflux ability to analysis different size HDL proposed by the present invention below by way of specific embodiment
And application is specifically described: now promoting Cholesterol Efflux function as embodiment using clinical detection crowd's blood plasma, illustrates this detection method
Working principle and operating procedure.Research purpose is to measure the plasma cholesterol outflow ability and and healthy control group of adiposis patient
It compares.Obesity is to cause one of the risk factor of cardiovascular disease especially coronary heart disease, the HDL concentration as caused by obesity
And its variation of rush Cholesterol Efflux function has important connection with atherosclerosis, however, different size of HDL in obesity
The variation of function relative healths crowd is also unknown.This experiment purpose is that different size of HDL promotees gallbladder in measurement adiposis patient blood plasma
Sterol flows out the difference of ability and health group.
Embodiment 1 measures difference HDL in obesity crowd blood plasma and promotees total cholesterol discharge rate
It is incubated for as shown in Figure 1, mouse macrophage J774 cell strain is inoculated in cultivation plate hole, then with containing3H is same
Cell in the cholesterol culture solution label cultivation plate hole of position element label, then in the case where not adding cAMP, at 37 DEG C
The expression for being incubated for 16 hours to induce total cholesterol transport protein；The blood plasma in obesity crowd is taken, Obese is labeled as, by it
Blood plasma goes out the HDL molecule of gradient magnitude using FPLC chromatography, as shown in Fig. 2, 32 are chosen respectively, the component at 34,37, generation
The different HDL component of three kinds of granular sizes of table, it should be noted that 32,34,37 be the label on abscissa in Fig. 2, numerical value
Show that the particle of this group of HDL molecule is smaller more greatly；Three kinds of groups are added separately in cell culture plate well, and are incubated at 37 DEG C
4 hours, the supernatant culture solution in cultivation plate hole is then drawn, remaining cell in supernatant culture solution is centrifugated out, by supernatant
Liquid is detected in culture solution after being added in scintillation solution with liquid scintillation counter3H isotope intensity is finally used and is not added with the thin of cAMP
Born of the same parents flow out the discharge rate that cholesterol isotope intensity calculates total cholesterol divided by cell total cholesterol isotope intensitometer, and with gallbladder
The component of sterol draws histogram marked as abscissa, by ordinate of the discharge rate of cholesterol, as a result such as the black column in Fig. 3
Shown in shape figure.
Embodiment 2 measures difference HDL in healthy population blood plasma and promotees total cholesterol discharge rate
Mouse macrophage J774 cell strain is inoculated in cultivation plate hole and is incubated for, then with containing3H isotope labelling
Cell in cholesterol culture solution label cultivation plate hole is incubated for 16 hours at 37 DEG C then in the case where not adding cAMP
To induce the expression of total cholesterol transport protein；The blood plasma in healthy population is taken, Healthy control is labeled as, by its blood
Slurry goes out the HDL molecule of gradient magnitude using FPLC chromatography, as shown in Fig. 2, choosing 32 respectively, the component at 34,37 is obtained
The different HDL component of three kinds of granular sizes；Three kinds of groups are added separately in cell culture plate well, and small in 37 DEG C of incubations 4
When, then draw the supernatant culture solution in cultivation plate hole, be centrifugated out remaining cell in supernatant culture solution, by supernatant plus
With in liquid scintillation counter detection culture solution after entering in scintillation solution3H isotope intensity, finally with the cell stream for being not added with cAMP
Cholesterol isotope intensity calculates the discharge rate of total cholesterol divided by cell total cholesterol isotope intensitometer out, and with cholesterol
Component marked as abscissa, histogram is drawn by ordinate of the discharge rate of cholesterol, as a result such as the white histogram in Fig. 3
It is shown.
Embodiment 3 measures difference HDL in obesity crowd blood plasma and is based on ABCA1 rush Cholesterol Efflux rate
Mouse macrophage J774 cell strain is inoculated in cultivation plate hole and is incubated for, then with containing3H isotope labelling
Cholesterol culture solution marks cell, and cAMP is then added in cell, is incubated for 16 hours at 37 DEG C to induce ABCA1 to express；
The blood plasma in obesity crowd is taken, Obese is labeled as, its blood plasma is gone out to the HDL particle of gradient magnitude using FPLC chromatography,
As shown in Fig. 2, choosing 32 respectively, the component at 34,37 obtains the different HDL component of three kinds of granular sizes；By three kinds of components
Jia Ru not be into cell culture plate well, and be incubated at 37 DEG C 4 hours, then draw the supernatant culture solution in cultivation plate hole, centrifugation
Remaining cell in supernatant culture solution is isolated, detects culture solution with liquid scintillation counter after supernatant is added in scintillation solution
In3H isotope intensity, finally with the cell outflow cholesterol isotope intensity of addition cAMP divided by cell total cholesterol isotope
Intensitometer calculates the discharge rate by the ABCA1 cholesterol mediated, and with the component of cholesterol marked as abscissa, with cholesterol
Discharge rate is that ordinate draws histogram, as a result as shown in the black histogram in Fig. 4.
Embodiment 4 measures difference HDL in healthy population blood plasma and is based on ABCA1 rush Cholesterol Efflux rate
Mouse macrophage J774 cell strain is inoculated in cultivation plate hole and is incubated for, then with containing3H isotope labelling
Cholesterol culture solution marks cell, and cAMP is then added in cell, is incubated for 16 hours at 37 DEG C to induce ABCA1 to express；
The blood plasma in healthy population is taken, Healthy control is labeled as, its blood plasma is gone out into gradient magnitude using FPLC chromatography
HDL particle, as shown in Fig. 2, choosing 32 respectively, the component at 34,37 obtains the different HDL component of three kinds of granular sizes；It should
Three kinds of groups are added separately in cell culture plate well, and are incubated at 37 DEG C 4 hours, and the supernatant culture in cultivation plate hole is then drawn
Liquid is centrifugated out remaining cell in supernatant culture solution, is examined after supernatant is added in scintillation solution with liquid scintillation counter
It surveys in culture solution3H isotope intensity, it is finally solid divided by the total gallbladder of cell with the cell outflow cholesterol isotope intensity of addition cAMP
Alcohol isotope intensitometer calculates the discharge rate by the ABCA1 cholesterol mediated, and with the component of cholesterol marked as abscissa, with
The discharge rate of cholesterol is that ordinate draws histogram, as a result as shown in the white histogram in Fig. 4.
Embodiment 5 calculates difference HDL in obesity crowd blood plasma and is based on non-ABCA1 rush Cholesterol Efflux rate
It can be obtained with the corresponding measurement result for subtracting 3 each component of embodiment of the measurement result of 1 each component of embodiment by non-
The Cholesterol Efflux rate that ABCA1 (mainly by ABCG1) is mediated；Equally with the component of cholesterol marked as abscissa, with cholesterol
Discharge rate be ordinate draw histogram, as a result as shown in the black histogram in Fig. 5.
Embodiment 6 calculates difference HDL in healthy population blood plasma and is based on non-ABCA1 rush Cholesterol Efflux rate
It can be obtained with the corresponding measurement result for subtracting 4 each component of embodiment of the measurement result of 2 each component of embodiment by non-
The Cholesterol Efflux rate that ABCA1 (mainly by ABCG1) is mediated；Equally with the component of cholesterol marked as abscissa, with cholesterol
Discharge rate be ordinate draw histogram, as a result as shown in the white histogram in Fig. 5.
To sum up, the experimental results showed that, it is representative big in component 32,34 and 37, neutralize in short grained HDL, it is fat
Patient's total cholesterol discharge rate and the Cholesterol Efflux rate mediated by ABCA1 and health group are relatively poorer without apparent statistics
It is different；However in the HDL Cholesterol Efflux mediated by non-ABCA1, the i.e. contained short grained HDL of the FPLC component 37 of adiposis patient promotees
Cholesterol Efflux ability is markedly less than healthy population, and both statistical analysis, which compare, significant difference, (P < 0.05)；So
And biggish HDL promotees Cholesterol Efflux ability contained by component 32 and 34 and human normal plasma does not show statistical difference.It is real
Test and show compared with healthy group, the total cholesterol discharge rate of adiposis patient does not show statistical difference, however after FPLC separation and
With cAMP mediate or non-mediation HDL Cholesterol Efflux rate the results show that the lesser HDL of particle was mediated in non-ABCA 1
Apparent decrease is shown in outflow, foundation is provided for further research, also hence it is demonstrated that examining to different size HDL function
The necessity and importance of survey.
In the description of this specification, reference term " embodiment ", " another embodiment ", " other embodiments " or "
The description of one embodiment~X embodiment " etc. mean specific features described in conjunction with this embodiment or example, structure, material or
Person's feature is included at least one embodiment or example of the invention.In the present specification, to the schematic table of above-mentioned term
Stating may not refer to the same embodiment or example.Moreover, specific features, structure, material, method and step or the spy of description
Point can be combined in any suitable manner in any one or more of the embodiments or examples.
It should be noted that, in this document, the terms "include", "comprise" or its any other variant are intended to non-row
His property includes, so that the process, method, article or the device that include a series of elements not only include those elements, and
And further include other elements that are not explicitly listed, or further include for this process, method, article or device institute it is intrinsic
Element.In the absence of more restrictions, the element limited by sentence " including one ... ", it is not excluded that including
There is also other identical elements in the process, method of the element, article or device.
The serial number of the above embodiments of the invention is only for description, does not represent the advantages or disadvantages of the embodiments.
The above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to preferred embodiment to this hair
It is bright to be described in detail, those skilled in the art should understand that, it can modify to technical solution of the present invention
Or equivalent replacement should all cover without departing from the objective and range of technical solution of the present invention in claim of the invention
In range.
Claims (8)
1. a kind of method that analysis different size HDL promotees Cholesterol Efflux ability, which comprises the following steps:
(1) lipoprotein for separating sample to be tested, obtains multiple groups HDL, and the granular size of every group of HDL is different；
(2) cell strain being inoculated in cultivation plate hole, the culture solution that the cholesterol containing labelled with radioisotope is added marks cell,
Make cell lotus rouge and label, the quantity of cultivation plate hole is no less than the group number of HDL in step (1)；
(3) it is incubated for related film cholesterol transporter gene expression in guidance cultivation plate hole inner cell strain；
(4) every group of HDL obtained in step (1) is added separately to through being incubated in step (3) treated cultivation plate hole；
(5) the supernatant culture solution in cultivation plate hole is drawn, removes in supernatant culture solution and is added after residual cells into scintillation solution, is used
Liquid scintillation counter detects isotope intensity in culture solution, calculate every group of HDL promotes the discharge rate of cholesterol in cell.
2. the method that analysis different size HDL according to claim 1 promotees Cholesterol Efflux ability, which is characterized in that step
Suddenly the isolated method in (1) be fast protein liquid chromatography (Fast Protein Liquid Chromatography,
FPLC) chromatography.
3. the method that analysis different size HDL according to claim 1 promotees Cholesterol Efflux ability, which is characterized in that step
Suddenly cell strain described in (2) includes mouse macrophage strain and human body cell strain.
4. the method that analysis different size HDL according to claim 1 promotees Cholesterol Efflux ability, which is characterized in that step
Suddenly related film cholesterol transporter described in (3) includes ABCA1 and non-ABCA1,
Step (3) specifically:
CAMP is added and is incubated for ABCA1 expression in guidance cell strain, or does not add cAMP and is incubated for ABCA1 and non-in guidance cell strain
The expression of ABCA1 is used to that HDL is mediated to promote Cholesterol Efflux, and the temperature of incubation is 37 DEG C, and the time of incubation is 16h.
5. the method that analysis different size HDL according to claim 1 promotees Cholesterol Efflux ability, which is characterized in that step
Suddenly the temperature being incubated in (4) is 37 DEG C, and the time of incubation is 4h.
6. the method that analysis different size HDL according to claim 1 promotees Cholesterol Efflux ability, which is characterized in that institute
Stating sample to be tested includes human plasma, human serum, the serum composition of separation, hdl particle, natural products, biomolecule and synthesis
One of compound is a variety of.
7. method described in any one of claims 1 to 6 is promoting Cholesterol Efflux ability in sickness influence for analyzing
Using.
8. according to claim 7 promote Cholesterol Efflux ability to the application in sickness influence for analyzing, feature exists
In the disease includes atherosclerosis, diabetes and obesity.
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