CN107807245A - A kind of intracellular cholesteryl susceptibility and the measuring method and its diagnostic reagent of positioning - Google Patents

Abstract

The invention discloses a kind of intracellular cholesteryl susceptibility and the measuring method and its diagnostic reagent of positioning, PBMC cells including fresh separated and smear are fixed, free cholesterol, endoplasmic reticulum and the double probes of the special fluorescence of mitochondria are incubated, and double probes marks, ICF be quantitative and four big steps of correction.Evidence shows：Blood cholesterol level does not have clear and definite positive correlation with the cholesterol concentration in sustainer and coronary tissue and the degree of atherosclerosis.There are multiple cholesterol ponds in human body, in the presence of certain virulence factor, the redistribution that can occur to organelle of cholesterol, detection and localization intracellular cholesteryl level (rather than simple measurement serum cholesterol concentration) have prior meaning.

Description

A kind of intracellular cholesteryl susceptibility and the measuring method and its diagnostic reagent of positioning

Technical field

The present invention relates to a kind of intracellular cholesteryl susceptibility and the measuring method and its diagnostic reagent of positioning.

Background technology

Cardiovascular and cerebrovascular disease is the first killer of human health, and metabolism disorder of blood lipid, particularly blood plasma cholesterol level Increase, be cause artery sclerosis generally acknowledged at present, the important risk factor of cardiovascular and cerebrovascular disease, therefore detect blood plasma cholesterol level Have become clinically one of widely used risk assessment mode at present.But we can not ignore such a fact： Clinically many patients with lipid are not high, also not fat, but suffer from artery sclerosis (heart infarction) in the same old way；In addition, suffer from Other diseases Crowd, such as chronic renal disease, diabetes, autoimmune disease (systemic loupus erythematosus etc.) etc., these patient features It is all to be associated with chronic inflammation, blood cholesterol levels are not high, but the Hazard ratio normal population for suffering from angiocardiopathy wants high 30-50 Times.Because the incidences of disease such as China's diabetes, chronic renal disease, autoimmunity disease increase year by year, the morbidity of diabetes B patient Rate has reached 11.8%, and the incidence of disease of CKD causes these cholesterolemias also up to 10%, also autoimmune disease etc. It is horizontal not high, but the total number of persons of angiocardiopathy may occur close to 300,000,000 (crying ' hiding morbidity crowd ').Fallen ill for concealment Crowd, because cholesterolemia is not high, according to traditional risk assessment means, many people do not arrange for the treatment of blood fat disorder Apply, and in fact our research is found：Such patient causes to be metabolized due to cellular cholesterol sensor (SCAP) dysfunction The exception of stable state, intracellular cholesteryl increases (cholesterol spatial abnormal feature) extremely, and may occur in which the leptin suppression of fat-reducing medicament.Cause This this kind of patient not only needs lipid-lowering therapy, and also needs to Intensive Lipid-lowing treatment.Regrettably do not have at present to this phenomenon Enough attention, neither one are gone back for the early prediction of this kind of ' concealment morbidity crowd ' and the index of risk assessment, so as to indulge in The prevention and treatment of the cardiovascular and cerebrovascular disease of this kind of crowd are missed.Therefore the clear and definite individual blood cholesterol levels why having are not Height, but truly have serious artery sclerosis to occurThe personal blood cholesterol levels that have are high, but the risk factor of artery sclerosis is also not necessarily Directly proportional to blood cholesterol level, still not individual never have is solved important scientific problems for this, while is also that puzzlement is faced Bed doctor and the important clinical problem of patient.

The great metabolic disorder disease in China, including cerebrovascular disease incidence of disease rises year by year caused by artery sclerosis, Because the disease is lethal, disability rate is high, diagnosis and treatment cost is high, causes huge financial burden to family and society.In fact very More patients are not or fat because blood fat is not high, do not draw attention before ill, do not prevented and treated effectively Measure, this kind of patient will account for total cardiovascular event more than 50%, therefore be a significant clinical problems.

Due to the difference of cholesterol sensor function, different individuals is variant to the sensitiveness of cholesterol toxicity；In addition Inflammation stress can disturb expression and the function of cholesterol sensor, cause cholesterol to be redistributed from blood circulation to tissue, lead Cholesterol in blood circulation has been caused to reduce and intracellular (organelle) cholesterol increase.So on the one hand explain why some are suffered from Person's blood plasma cholesterol level is not high, but the reason for cardiovascular mortality increase；It is proposed new theory simultaneously：Neither one safety Cholesterolemia concentration；Cell is to determine lipid mediation to the susceptibilitys of intracellular cholesterol levels, rather than simple serum lipid concentrations Organ injury determinant；Detection and localization intracellular cholesteryl level (rather than simple measurement serum cholesterol concentration) With prior meaning.

Brown and Goldstein are reported first:Cell is adjusted by a negative-feedback dependent on intracellular cholesterol levels The control of section system, the activity of ldl receptor (LDLr) is in a normal condition all the time, and thus win 1985 annual promise shellfishes That Medicine.At present, the molecular mechanism about the looped system has illustrated clearer, that is, passes through Insig- The lipid within endothelial cells homeostasis regulation that SCAP-SREBPs is carried out.Cholesterol modulation element conjugated protein (Sterol Regulatory element binding proteins, SREBPs) be LDLr genes transcription factor, can be with LDLr genes The albumen that cholesterol modulation element 1 (SRE-1) in promoter is specifically bound, so as to strengthen LDLr transcription. SREBPs activation is adjusted by intracellular cholesterol levels, and SREBP degrading activity Protein S CAP (SREBP cleavage- Activating protein, SCAP) and insulin-induced gene (Insulin Induced Gene, Insig) in SREBPs Active Regulation in again play an important role.SCAP is a kind of memebrane protein of endoplasmic reticulum, and matter net is combined in the cell with SREBPs for it Into compound, stabilization is played to SREBPs, and its activity is adjusted by controlling SREBPs to be positioned on subcellular fraction.Insig- 1 (Insulin Induced Gene, Insig) is incorporated in a memebrane protein in endoplasmic reticulum, in the cell cholesterol concentration Gao Shi, combined with SCAP sterol sensitizing range, SCAP is locked in endoplasmic reticulum.When cell needs cholesterol, SCAP will be with Insig-1 is separated, and SCAP-SREBPs compounds are transferred to golgiosome from endoplasmic reticulum, in the work of two protease (S1P, S2P) Under, SREBPs is cleaved, and the N- terminal fragments with transcriptional activity enter in nucleus, promote LDLr and HMGCoAR genes Transcription.When intracellular cholesterol levels raise, Insig-1 is combined with SCAP sterol sensitizing range, makes SCAP-SREBP compound Thing is trapped in endoplasmic reticulum, causes the expression by inhibitation system of LDLr and HMGCoAR genes, so as to prevent the intake of hypercellularity, close Generating cholesterol.SCAP directly controls the conjunction of intake and endogenous cholesterol of the cell to Exogenous Cholesterol to the sensitiveness of cholesterol Into, maintain intracellular cholesteryl homeostasis play extremely important effect.

Our understanding to cholesterol susceptibility are former first to be ignored but highly important clinical picture in some by people:Face Much suffer from the patient of arteriosclerosis disease on bed, its serum lipid concentrations is not high.Although there are a lot of other factors such as：Hypertension, High cysteine mass formed by blood stasis etc. can explain part case, but cannot avoid such a fact：No matter whether cholesterolemia increases, There is the deposition of a large amount of foam wanshings and cholesterol in atherosclerotic plaque.

Secondly, although metabolism disorder of blood lipid, particularly blood plasma cholesterol level increases extremely, is generally recognised as an important cause at present The hazards of atherosclerosis, and the obvious increased one of the main reasons of cardiovascular disease incidence rate, some clinical Study and also once showed there is certain correlation between blood LDL cholesterol or T-CHOL and the generation of artery sclerosis, but This correlation is very faint, and the individual for mainly concentrating on extreme hypercholesterolemia is found, and is confined to White man, male, coronary artery；And there is no discovery to have significant correlation (1-4) in Black people, women or other arterial walls.Enter one Step analysis is found：This faint correlation be due to clinical study selection limitation caused by, show and much face Bed research only selects the patient of hypercholesterolemia, if cholesterolemia concentration to be more than to 350mg/ml (9mmol/l) individual Remove from clinical trial, or random selection crowd makes a search, cholesterolemia and AS correlations also disappear (11) therewith.These Evidence shows：Blood cholesterol level and the cholesterol concentration and atherosclerosis in sustainer and coronary tissue Degree there is no clear and definite positive correlation.

Furthermore with other chronic inflammation disease crowds, such as chronic renal disease, diabetes, LADA disease Disease etc., these patient's blood cholesterol levels are not also high, but the Hazard ratio normal population of angiocardiopathy wants high 30-50 times, these Result of study demonstrates under chronic inflammatory state (CRP increases are characterized), and the risk for suffering from angiocardiopathy substantially increases, Reflect demonstrate,proved Ross etc. (12) in 1999 propose atherosclerosis be a kind of theory of diseases associated with inflammation；Prompt simultaneously：Very In big a part of patient, some factors (such as inflammatory reaction) may change the metabolism and distribution of cholesterol, so as to result in The risk of blood cholesterol levels and angiocardiopathy is not into positive correlation.Cholesterolemia is normal, not equal to not suffering from artery sclerosis Risk.Therefore simple blood cholesterol levels (especially under chronic inflammatory state), it is not to assess angiocardiopathy/artery sclerosis Risk and instruct the efficiency index of clinical lipid-lowering therapy！

So whether inflammation index can be used for weighing cardiovascular riskCRP is an important inflammation index, in recent years Substantial amounts of research prompting CRP is probably the important risk factor of angiocardiopathy, but further investigations have shown that：CRP and the heart The special correlation of vascular diseases is very low, illustrates that increasing for CRP can predict angiocardiopathy currently without any evidence Occurrence and development (13).PEPYS etc. is proved by the ultrapure CRP of Human C-reactiveprotein trangenic mice and injection：The CRP of people is a urgency Property reactive protein, does not aggravate inflammatory reaction, does not aggravate the formation (14) of artery sclerosis plate yet；Also someone is clear and definite recently simultaneously It is not the special risk factor (15) of artery sclerosis to propose CRP.Therefore, it is cardiovascular to find and establish new special assessment The risk indicator of disease/artery sclerosis is the new problem that we face.

Since LDLr has found, LDLr is in regulation blood fat and the role in the generation, development of atherosclerosis Receive much attention always.LDLr also turns into the target spot of clinical reducing blood lipid treatment.Statins is most commonly used both at home and abroad at present Fat-reducing medicament, it is a HMGCoAR inhibitor, and its mechanism of action is to reduce cell by suppressing HMGCoAR activity The synthesis of inner cholesterol, by negative-feedback regu- lation, increase hepatocyte cell LDLr expression, such liver cell can increase to follow to blood The intake of LDL-C in ring, blood LDL-C concentration is reduced, so as to effectively reduce the incidence of disease of angiocardiopathy.At present Substantial amounts of clinical research shows that in population, statins can reduce cholesterol in blood plasma and reach 30-50%, and can have The incidence of disease of the angiocardiopathy of the reduction by 27% of effect, but with chronic inflammation disease such as diabetes, chronic kidney disease In the patient of disease, the cardiovascular protective effect of statins is but under suspicion.A nearest entitled 4D extensive, multicenter Prospective Clinical research shows that atorvastatin (20mg/day) can not effectively reduce diabetes, hemodialysis patient The risk (16) of angiocardiopathy.This clinical trial let us is thought deeply：Why statins works in part crowd, But do not worked in majority of populationsThus, We conducted in-depth study, as a result prompt：With chronic inflammation disease Crowd may have resistance to a certain degree to statins.

Our research further shows that：Under chronic inflammatory state, peripheral cells LDLr this sensitive feedback Regulation imbalance, so as to which peripheral cells LDLr is converted into " lowering resistance type " from " lowering susceptible type ", make peripheral cells surface LDLr becomes insensitive (similar liver cell) to the inhibitory action of cholesterol mediation, peripheral cells is absorbed excessive courage Sterol, foam cells is formed, and cause the resistance of statins.This Species sensitivity change can with 50% inhibiting rate (IC50) quantify (7).This may be also statins idle one of the main reasons in this kind of people at highest risk.We This discovery, cause the concern of blood fat research field, global Cardio Kidney incorporated by NKF The guide of Diabetes lipid-lowering therapies.Importantly, this research also proposed the concept of cholesterol susceptibility and possible Detection method.

At present, sugar tolerance experiment is extensively with the early diagnosis with diabetes/metabolic syndrome, but few resistance on fat The report of the experiment of amount, it is primarily due to the diversity of lipid components and the complexity of fat metabolism.Fatty tolerance this Name is that Fahmay proposes in nineteen fifty-two earliest.Then they determine postprandial 3,5,7 by giving patient one High cholesterol diet The content of hour cholesterolemia, has dynamically observed the change of blood fat, also has later using fat tolerance test analysis people rich in sweet The lipoprotein (TRL) of oily three esters removes delay incidence and its influence to blood plasma lipide.Research finds that fat loading is postprandial, blood The change of fat be mainly breast waste particle rich in triglycerides (TG) lipid change it is obvious, removing substantially delay, fat Fat high TG responses after the meal and the horizontal negatively correlated (P of reduction of plasma hdl cholesterol (HDL-C)<0.01).Also grind Study carefully and show：Free fatty (FFA) level has notable rise in artery sclerosis illness group after fat meal, prompts the FFA generations of confusion Thank to the risk for being possibly used for assessing the normal CAD of blood fat, and possibly as an early stage index (17) of metabolic syndrome.These Method, reflect tolerance of the body to fat metabolism by dynamically monitoring the change of plasma lipid profile, mainly to TG's and FFA The influence of metabolism, but cholesterol is to cause artery sclerosis " the first killer ", this simple fat-tolerance test is not to courage The metabolism of sterol and its risk relations of artery sclerosis judge, and simple dynamic measures the change of blood fat, can not be effective Ground illustrates sensitiveness of the body tissue cell to cholesterol.

There are some researches show LDLr level and function point analysis are similar with other peripheral tissues cell LDLr in normal pbmc (21；22).Because the PBMC in blood circulation is easily obtained, there is good operability, in the prediction, diagnosis in artery sclerosis Having wide should have prospect.In order to inquire into its feasibility, the PBMC (n=5) of our separating health normal persons, and and various concentrations LDL co-incubations in vitro, measure the level of cell LDLr mRNA and albumen, it is suppressed then to calculate the 50%LDLr that sends as an envoy to LDL concentration (IC50).Preliminary result shows：Healthy People PBMC is as vascular smooth muscle cells, in normal physiological shape Under state, reflections of the LDLr to LDL is very sensitive, shows as " lowering susceptible type ", and IC50 is less than 25 μ g/ml.Meanwhile we also can The SCAP to be played an important role in LDLr regulations expression is detected on PBMC.In order to inquire into, whether this method is than simple blood fat It is more sensitive, more accurately reflect the danger of artery sclerosis, we are normal using cholesterolemia, but with arteriosclerotic PBMC, IC50 is measured with same method.The result of preliminary experiment shows：It was found that this kind of patient IC50 is significantly raised, IC50 is prompted There is preferably correlation than cholesterolemia and artery sclerosis.

Because under high smectic state, the horizontal suppressed susceptibilitys of cell LDLr/HMGcoAR directly reflect cell and courage are consolidated The sensitiveness of alcohol, it also reflects SCAP function.

Traditional simple index presence ' blind area ' with cholesterolemia as cardiovascular and cerebrovascular risk assessment.Therefore how to eliminate The blind area of cholesterol risk assessment, the new risk assessment index for concealment morbidity crowd is found, so that 300,000,000 concealment hairs Patient group obtains effective early stage risk assessment, early intervention and treatment, has become international and national important clinical demand.

The content of the invention

The technical problems to be solved by the invention are to provide a kind of plasma cholesterol concentration using safety, and cell is to intracellular The susceptibility of cholesterol levels, rather than simple serum lipid concentrations, it is the key factor for determining lipid mediation organ injury, we Wish to measure LDL-C to LDLr/HMGcoAR inhibiting rate to measure special susceptibility of the cell to cholesterol, can be thorough Bottom changes traditional mode on blood fat measurement and risk assessment, has very important clinical value.

The present invention is achieved through the following technical solutions：A kind of measuring method of cholesterol susceptibility, including LDL marks The preparation of quasi- product, four big step of cell culture processes, RNA extraction method and one-step method fluorescence quantitative RT-RCR；

Wherein, the preparation of LDL standard items comprises the following steps that：

First, healthy premenopausal volunteers blood plasma is taken, adds antioxidant；

2nd, below 10000g centrifuges 30min, removes chylomicron；

Three then add sodium bromide blood plasma is adjusted to 1.006kg/L, 40000r/min ultracentrifugation 18h with density, suction out Upper strata milky white liquid and sublevel weak yellow liquid, collect lower floor's liquid；

4th, it is 1.063gkg/L to add sodium bromide (NaBr) and adjust its whole density, again 40000r/min ultracentrifugations 24h, suction out the orange-yellow liquid in upper strata and obtain LDL；

Wherein, cell culture processes comprise the following steps that：

First, by separating health people and the PBMC of sick people, it is quick that fraction cell is directly used in detection PBMC inner cholesterols Sensor SCAP expression；

2nd, RPMI1640 culture medium of the most cells containing 10% no fat serum, is planted in 12 orifice plates, and culture 12 is small When；

Three and then the LDL-C of the various concentrations with preparing in aforementioned manners, concentration is respectively 0,25,50,100, 200 μ l/ml, it is incubated 24 hours altogether, then extracts mRNA in following manner；

Wherein, RNA extraction method is that PBMC RNA is extracted using Trizol, then separates RNA；

Wherein, one-step method fluorescence quantitative RT-RCR, two processes of reverse transcription and PCR are completed using one-step method, it is not necessary to take again Go out cDNA；

Reagent includes buffer solution, the RT-PCR MIX by optimization, dNTP, Sybr green I dyestuffs and MgCl2 solution Mixture, and the expression of the SCAP with quantitative fluorescence PCR detection PBMC is intended that, and various concentrations LDL stimulates lower LDLr With HMGCoAR mRNA level in-site, and calculate 50%LDLr and HMGCoAR be suppressed when LDL-C concentration, reflection cell/ Body, according under various concentrations LDL states, LDLr and HMGCoAR mRNA level, calculates to the susceptibility of cholesterol IC50。

As preferable technical scheme, the antioxidant is 100umol/L EDTA, 20umol/L Butylated Hydroxytoluenes.

A kind of detection kit of the measurement of cholesterol susceptibility, the reagent of several parts, quantitative fluorescence including more than RT-PCR reagents can be adapted to any quantitative real time PCR Instrument.

A kind of method for the accuracy for verifying cholesterol susceptibility IC50, (one) U937 cells are used containing 10% no fat serum RPMI1640 culture mediums, plant in 12 orifice plates, cultivate 12 hours, the LDL courages of the various concentrations with preparing in aforementioned manners are consolidated Alcohol is incubated 24 hours altogether, then extracts cell total rna, one-step method fluorescence quantitative RT-RCR, according to various concentrations LDL with Trizol Under state, LDLr and HMGCoAR mRNA level, and amount effect relation curve is made with Excel softwares, fit linear regression side Journey calculates mRNA IC50.

(2) method transfected with gene, the cell line of different expression SCAP overexpressions is established on Chinese hamster ovary celI, is turned The SCAP of various concentrations Chinese hamster ovary celI has been contaminated, with the RPMI1640 culture mediums containing 10% no fat serum, has been planted in 12 orifice plates, Culture 12 hours, cell total rna then is extracted with Trizol, SCAP expression is detected using quantitative fluorescence PCR, with PBMC Inner cholesterol sensor SCAP expression compares, and checks kit to SCAP in PBMC by the use of this cell as positive control The accuracy and sensitivity of the above method quantitatively detected, and various experiment conditions and production technology are carried out according to testing result Adjustment, makes the accuracy of kit and sensitivity reach the standard of technical requirements.

The beneficial effects of the invention are as follows：According to previous work basis, it is proposed that：The plasma cholesterol of neither one safety Concentration, cell are to determine lipid mediation organ injury to the susceptibilitys of intracellular cholesterol levels, rather than simple serum lipid concentrations Key factor.It is desirable that measurement LDL-C measures spy of the cell to cholesterol to LDLr/HMGcoAR inhibiting rate Different susceptibility.If succeeded in developing, this will thoroughly change traditional mode on blood fat measurement and risk assessment, have very Important clinical value.

Embodiment

The preparation of LDL standard items

Healthy premenopausal volunteers blood plasma is taken, adds antioxidant (100umol/L EDTA, 20umol/L Butylated Hydroxytoluenes), 10000g 30min is centrifuged below, removes chylomicron, and then plus blood plasma is adjusted to 1.006kg/L, 40000r/min by sodium bromide with density Ultracentrifugation 18h, upper strata milky white liquid (VLDL) and sublevel weak yellow liquid (IDL) are suctioned out, collect lower floor's liquid, add It is 1.063gkg/L that sodium bromide (NaBr), which adjusts its whole density, again 40000r/min ultracentrifugations 24h, and it is orange-yellow to suction out upper strata Liquid obtains LDL.

Cell culture processes

By separating health people and the PBMC of sick people, fraction cell is directly used in detection PBMC inner cholesterol sensors SCAP expression；RPMI1640 culture medium of the most cells containing 10% no fat serum, is planted in 12 orifice plates, is cultivated 12 hours (synchronization), then with LDL-C (0,25,50,100, the 200 μ l/ of the various concentrations prepared in aforementioned manners Ml) it is incubated 24 hours altogether, then extracts mRNA in following manner.

RNA extraction method

The RNA of high quality is the key for detecting SCAP gene expressions.Extract enough RNA's from limited cell at present Technical comparing is ripe, and Trizol is a kind of new total serum IgE extraction agent, includes the materials such as guanidinium isothiocyanate, can crush rapidly thin Born of the same parents, suppress the nuclease that cell discharges.We intend extracting PBMC RNA using Trizol, then separate RNA.

One-step method fluorescence quantitative RT-RCR

Two processes of reverse transcription and PCR are completed using one-step method, it is not necessary to further take out cDNA.Reagent is included by the slow of optimization Fliud flushing, RT-PCR MIX (mixed enzyme being made up of reverse transcriptase and Taq archaeal dna polymerases, not only can cDNA synthesis again can PCR expansion Increase), dNTP, Sybr green I dyestuffs and MgCl2 solution mixtures.We are intended that with quantitative fluorescence PCR detection PBMC's SCAP expression, and various concentrations LDL stimulate lower LDLr and HMGCoAR mRNA level in-site, and calculate 50%LDLr and The concentration (IC50) of LDL-C when HMGCoAR is suppressed, susceptibility of the reflection cell/body to cholesterol.According to different dense Spend under LDL states, LDLr and HMGCoAR mRNA level, calculate IC50.

According to above technology platform, plan develops the detection kit of the measurement of cholesterol susceptibility, including the above Several parts reagent.Quantitative fluorescence RT-PCR reagents can be adapted to any quantitative real time PCR Instrument, such as：lightcycler、 ABI7000, roto-gene, FTC2000, icycler etc..

We carry out the monocytic series (U937) of the in vitro culture of employment and our laboratories have built up markization IC50 measuring method, to verify cholesterol susceptibility IC50 accuracy.U937 cells are used containing 10% no fat serum RPMI1640 culture mediums, plant in 12 orifice plates, cultivate 12 hours (synchronization), with the various concentrations that prepare in aforementioned manners LDL-C (0,25,50,100,200 μ l/ml) is incubated 24 hours altogether, then extracts cell total rna, one-step method with Trizol Fluorescence quantitative RT-RCR, according under various concentrations LDL states, LDLr and HMGCoAR mRNA level, and made with Excel softwares Go out amount effect relation curve, fit equation of linear regression and calculate mRNA IC50 (50% inhibition concentration).

Meanwhile our methods for will being transfected with gene, the thin of different expression SCAP overexpressions is established on Chinese hamster ovary celI Born of the same parents system (note：With the SCAP cDNA of varying number, the SCAP cDNA of our laboratory possessors expression plasmid), transfect not With the SCAP of concentration Chinese hamster ovary celI, with the RPMI1640 culture mediums containing 10% no fat serum, plant in 12 orifice plates, culture 12 Hour (synchronization), cell total rna then is extracted with Trizol, SCAP expression is detected using quantitative fluorescence PCR, with PBMC inner cholesterol sensors SCAP expression compares, and checks kit in PBMC by the use of this cell as positive control The accuracy and sensitivity for the above method that SCAP is quantitatively detected, and according to testing result to various experiment conditions and production technology It is adjusted, the accuracy of kit and sensitivity is reached the standard of technical requirements.

We have demonstrated the hair of cholesterol sensor (degree) and artery sclerosis in cell biology and zoopery Raw, development has substantial connection.In the present embodiment, volunteer (note of the age 18-60 year will be selected：Agree to participate in this experiment simultaneously Signature Written informed consent person) and it is divided into 4 groups：Normal control population's (medical center), normal lipid but is diagnosed as AS groups, high Cholesterolemia but AS groups simultaneously are diagnosed as without AS groups, hypercholesterolemia, every group of 60 people.

The diagnostic criteria of hypercholesterolemia using domestic China generally used in the diagnostic criteria formulated in 1997, i.e., T-CHOL (TC) in every liter of blood is more than 5.7 mMs (i.e.>5.7mmol/L) raised for cholesterol levels.Atherosclerosis Change using medical history, physical examination and auxiliary examination as diagnosis basis, at present using thickness in Doppler ultrasound technology detection carotid artery intima Degree (IMT) and patch are formed, it has also become judge the generally acknowledged index of Peripheral atherosclerosis lesion, carotid intimal medial thickness (IMT)>1.0mm is intimal thickening, IMT>1.2mm is considered as patch and formed.

Plan uses quantitative fluorescence PCR technology, measures normal population and the water of sick people's IC50 and SCAP gene expression It is flat, establish the baseline of normal health crowd's cholesterol susceptibility；And inquire into cholesterol susceptibility and the water of SCAP gene expressions Equal itself and blood cholesterol levels and the correlation of artery sclerosis.

The foregoing is only a specific embodiment of the invention, but protection scope of the present invention is not limited thereto, any The change or replacement expected without creative work, it should all be included within the scope of the present invention.Therefore, it is of the invention Protection domain should be determined by the scope of protection defined in the claims.

Claims (7)

1. A kind of 1. measuring method of cholesterol susceptibility, it is characterised in that：Preparation, cell culture processes including LDL standard items, Four big step of RNA extraction method and one-step method fluorescence quantitative RT-RCR；

   Wherein, the preparation of LDL standard items comprises the following steps that：

   (1) healthy premenopausal volunteers blood plasma, is taken, adds antioxidant；

   (2), below 10000g centrifuges 30min, removes chylomicron；

   (3) and then add sodium bromide that blood plasma is adjusted into 1.006kg/L with density, 40000r/min ultracentrifugation 18h, in sucking-off Layer milky white liquid and sublevel weak yellow liquid, collect lower floor's liquid；

   (4) it is 1.063gkg/L, to add sodium bromide (NaBr) and adjust its whole density, again 40000r/min ultracentrifugations 24h, suction out the orange-yellow liquid in upper strata and obtain LDL；

   Wherein, cell culture processes comprise the following steps that：

   (1), by separating health people and the PBMC of sick people, it is sensitive that fraction cell is directly used in detection PBMC inner cholesterols Device SCAP expression；

   (2), RPMI1640 culture medium of the most cells containing 10% no fat serum, is planted in 12 orifice plates, and culture 12 is small When；

   (3) then the various concentrations with preparing in aforementioned manners LDL-C, concentration is respectively 0,25,50,100,200 μ L/ml, it is incubated 24 hours altogether, then extracts mRNA in following manner；

   Wherein, RNA extraction method is that PBMC RNA is extracted using Trizol, then separates RNA；

   Wherein, one-step method fluorescence quantitative RT-RCR, two processes of reverse transcription and PCR are completed using one-step method, it is not necessary to further take out cDNA；

   Reagent includes buffer solution, the RT-PCR MIX by optimization, dNTP, Sybr green I dyestuffs and the mixing of MgCl2 solution Thing, and be intended that the expression of the SCAP with quantitative fluorescence PCR detection PBMC, and various concentrations LDL stimulate lower LDLr and HMGCoAR mRNA level in-site, and the concentration of LDL-C when 50%LDLr and HMGCoAR is suppressed is calculated, reflect cell/machine Body, according under various concentrations LDL states, LDLr and HMGCoAR mRNA level, calculates IC50 to the susceptibility of cholesterol.
2. 2. the measuring method of cholesterol susceptibility as claimed in claim 1, it is characterised in that：The antioxidant is 100umol/L EDTA, 20umol/L Butylated Hydroxytoluenes.
3. A kind of 3. detection kit of the measurement of cholesterol susceptibility, it is characterised in that：Including several parts of claim 1 Reagent, quantitative fluorescence RT-PCR reagents can be adapted to any quantitative real time PCR Instrument.
4. A kind of 4. method for the accuracy for verifying cholesterol susceptibility IC50, it is characterised in that：

   (1), RPMI1640 culture medium of the U937 cells containing 10% no fat serum, is planted in 12 orifice plates, is cultivated 12 hours, The LDL-C of various concentrations with preparing in aforementioned manners is incubated 24 hours altogether, then extracts cell total rna with Trizol, One-step method fluorescence quantitative RT-RCR, according under various concentrations LDL states, LDLr and HMGCoAR mRNA level, and use Excel Software makes amount effect relation curve, fits equation of linear regression and calculates mRNA IC50；

   (2), the method transfected with gene, the cell line of different expression SCAP overexpressions, transfection are established on Chinese hamster ovary celI The SCAP of various concentrations Chinese hamster ovary celI, with the RPMI1640 culture mediums containing 10% no fat serum, is planted in 12 orifice plates, is trained Support 12 hours, then extract cell total rna with Trizol, SCAP expression is detected using quantitative fluorescence PCR, in PBMC Cholesterol sensor SCAP expression compares, and checks that kit is determined SCAP in PBMC by the use of this cell as positive control The accuracy and sensitivity of the above method of detection are measured, and various experiment conditions and production technology are adjusted according to testing result It is whole, the accuracy of kit and sensitivity is reached the standard of technical requirements.
5. A kind of 5. position finding and detection method of intracellular cholesteryl, it is characterised in that：The PBMC cells and smear of fresh separated are simultaneously solid Determine, the double probes incubations of fluorescence that free cholesterol and endoplasmic reticulum are special, mark, ICF are quantitative and correct four big steps；

   Wherein, the PBMC cells of fresh separated and smear, are comprised the following steps that：

   (1), by separating health people and the PBMC of sick people, and with centrifugation smearing machine smear；

   (2), cell is fixed with 3% paraformaldehyde；

   Wherein, free cholesterol and the double probes of the special fluorescence of endoplasmic reticulum are incubated, and mark, are comprised the following steps that：

   It is incubated with the double probes of the special fluorescence of cholesterol dyestuff or cholesterol sensor (albumen or special peptide) and endoplasmic reticulum, mark Note；

   Wherein, ICF is quantitative and corrects four big steps, comprises the following steps that：

   With the content of free cholesterol in fluorescence microscopy organelle and with Sigma Scan Pro software to fluorescence Quantified, and result is corrected with the cell gross area.
6. A kind of 6. detection and localization kit of intracellular cholesteryl, it is characterised in that：Including several parts of claim 1 Reagent.
7. A kind of 7. method of the accuracy of the detection and localization of intracellular cholesteryl, it is characterised in that：

   (1) in order to assess the correctness of the method, it will also select density gradient ultracentrifugation method separation endoplasmic reticulum and directly survey Amount endoplasmic reticulum and the method for mitochondria cholesterol prove the accuracy of double fluorescence probe methods；

   (2) clinical assessment：Crowd is selected with reference to Part I, and we will be determined with double fluorescence probe labelling method intracellular cholesteryls The content of position fast diagnosis reagent measurement normal population and sick people's PBMC inner cholesterols, is established in normal health crowd PBMC The baseline of cholesterol level；And inquire into its correlation between blood cholesterol levels, artery sclerosis.