CN104667261A - Application of recombinant human fibroblast growth factor 21 in prevention and treatment of atherosclerosis and related diseases - Google Patents
Application of recombinant human fibroblast growth factor 21 in prevention and treatment of atherosclerosis and related diseases Download PDFInfo
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Abstract
The invention discloses a genetic engineering protein-recombinant human fibroblast growth factor 21 capable of preventing and treating atherosclerosis and related diseases. The recombinant human FGF21 protein obtains patent protection by an applicant (the potential number: ZL200810223501.0). The recombinant human FGF21 protein is capable of effectively relieving and controlling pathogenesis of atherosclerosis by promoting external flow of fat in vascular endothelial cells and reducing accumulation of fat in blood vessel endothelium. Therefore, compared with other listed medicines, the recombinant human FGF21 protein has the advantage that prevention and treatment can be carried out with respect to the sources of atherosclerosis and a coronary heart disease. In addition, through analysis of a lot of clinical data, the inventor discovers that the FGF21 can be applied to auxiliary diagnosis of indexes of the atherosclerosis and the coronary heart disease, and has remarkable correlation in content increase in serum and morbidity of the coronary heart disease. In view of wide action of the recombinant human FGF21 protein in prevention, treatment and diagnosis of the atherosclerosis, the inventor believes that the clinical application value of the recombinant human fibroblast growth factor 21 is great.
Description
Technical field
The present invention relates to the albumen with pharmacological action, be specifically related to the application of a kind of albumen in diagnosis or treatment and coronary heart disease relevant disease.
Background technology
Atherosclerosis (Atherosclerosis, AS) is the important pathologic basis of cardiovascular and cerebrovascular disease, be also an important pivot of metabolic syndrome, ductus arteriosus wall thicken, follow the string and tube chamber to reduce be the common feature of various AS.Epidemiology and clinical research show: AS mainly occurs in the mid-aged population of more than 40 years old, within 49 years old, make fast progress later, but in some between twenty and fifty people even postmortems of child, also once find that their tremulous pulse had early stage atherosclerotic lesion.Coronary atherosclerotic heart disease refers to that coronary atherosclerosis makes lumen of vessels stenosis or occlusion, or cause myocardial ischemia-anoxemia or downright bad and heart disease that is that cause because of coronary artery changing function, be commonly referred to as coronary heart disease (CHD), be called for short coronary heart disease.CHD is the result of AS development, is the commonly encountered diseases of serious harm human health.Diabetes, the meeting of the disease such as hyperlipidemia and obesity is adjoint or bring out atherosclerosis even coronary heart disease.
Epidemiological study result shows, CHD has become the one of the main reasons of human death.In American-European countries, because the number suffering from CHD death accounts for the 1/3-1/2 of human mortality's number.And in China, CHD morbidity in recent years shows a rising trend, add China's large population base, how this controls and treats the development of CHD conditions of patients, is a problem demanding prompt solution.Meanwhile, diabetes, hyperlipidemia, obesity Dou Shi China sickness rate is higher while morbidity presents the metabolic disease of rejuvenation trend.
With regard to current clinical treatment, although statins can significantly alleviate developing of AS, it still fails to suppress completely and reverse the deterioration of the AS state of an illness.Therefore, explore AS morbidity molecular regulation mechanism and confirm new drug target, thus illustrate further AS pathogeny, develop new medicine, all there is important basic research meaning and using value.
FGF2 1 (hereinafter referred to as FGF21) is a polypeptide with 210 amino acid residues, is mainly expressed in liver and fatty tissue.Recent research finds: under pathology and physiological condition, and FGF21 participates in the metabolic process of conditioner body lipid and sugar directly, and a series of metabolic syndromes caused with fat, carbohydrate metabolism disturbance are closely related.In addition, animal and clinical research also find: the morbidity of the metabolism syndrome that FGF21 not only causes with multiple obesity is closely related, what is more important, and the external FGF21 of giving treatment significantly can reduce the level of cardiovascular disease risk factor in diabetic animal body.But, with regard to the research of FGF21 and coronary heart disease, no matter be animal or clinicing aspect, all know little about it.Lipid metabolic disorder is one of main mechanism of AS morbidity, and hyperglycemia is then one of high risk factor of AS morbidity, but FGF21 whether fall ill to AS relevant it by the metabolic process in which approach control agent, these all await further further investigation.
In clinical diagnosis, the early diagnosis of atherosclerosis and these two kinds of diseases of coronary heart disease is all more difficult, and the organ lesion stage only occurring to essence just can make a definite diagnosis.Wherein, for atherosclerosis, lipid examination, the method such as x-ray, ultrasonic and angiography is topmost detection means.And in the inspection of coronary heart disease, most importantly rely on Electrocardiography, and as electrocardiogram stress test, ambulatory electrocardiogram etc.In addition, the inspection method of coronary heart disease also has Nuclear Cardiac Imaging, coronarography, cardiac ultrasonic and intravascular ultrasound, Myocardial Enzymologic inspection etc., and wherein coronarography detects is diagnosis of coronary heart disease " goldstandard ".How to find the index characterizing atherosclerosis and incidence of coronary heart disease probability and just seem particularly important.As lipid examination as the index must looked into inside metabolic disease, whether FGF21 can as the conventional inspection parameter of diagnosing atherosclerotic and coronary heart disease, and the prevention for these diseases has important clinical value.
Summary of the invention
An object of the present invention is to provide a kind of for preventing the method for the incidence of atherosclerosis caused by high fat diet.
In order to achieve the above object, the present invention has investigated the atherosclerotic preventive effect that recombined human FGF21 causes high fat diet.
Cause atherosclerotic preventive effect in order to simulate recombined human FGF21 to high fat diet, the present invention adopts subcutaneous injection 100-400 μ g/kg recombined human FGF21 albumen, then gives the method for high fat diet.
In order to show that injecting recombined human FGF21 in body causes atherosclerotic preventive effect to high fat diet further, this patent is from body weight, blood lipid level, liver metabolism, cholesterol transport, vascular lesion etc. five aspect comparative analysis injection recombined human FGF21 albumen and the difference of not inject between recombined human FGF21 albumen sample.
An object of the present invention is to provide a kind of recombined human FGF21 that may be used for the atherosis and coronary heart disease of prevention of arterial.
Above-mentioned recombined human FGF21 is for according to the applicant, method described in patent ZL200810223501.0 of having obtained the authorization prepares.
Described in above-mentioned patent, the preparation method of recombined human FGF21 is: the method adopting the external restructuring of genetic engineering, build the engineering strain of SUMO molecular chaperones and FGF-21 amalgamation and expression, at 37 DEG C, 1mM IPTG induces 4h, expressing quantity >20%; Western blotting experimental result shows, the FGF21 expressed by vitro recombination can react to each other with FGF21 monoclonal antibody, and this illustrates that recombiant protein is FGF21.In addition, structural analysis, Mass Spectrometric Identification and N-hold analysis result all to show that this seminar successfully obtains highly purified target protein; In addition, the test of adipose cell glucose reabsorption shows, restructuring FGF21 significantly can promote that adipose cell is to the absorption of glucose.Establish reliable and stable, to be easy to amplification pilot process system simultaneously, product purity reaches more than 95%, the response rate is more than 60%, for pharmacy and animal experiment study provide sufficient sample, establish a complete set of quality control index comprising stock solution and finished product simultaneously, people FGF21 is cloned into target expression vector, and successful expression goes out corresponding target protein and has corresponding biological activity.
An object of the present invention is to provide atherosclerosis and there is the evidence of Close relation between coronary heart disease and FGF21.
In order to reach above object, the concentration of FGF21 in inventor's examination normal person and Patients With Coronary Heart Disease.Angina pectoris or acute myocardial infarction coronary heart disease patient are classified as object of study by the existing CHD clinical criteria of " name of ischemic heart desease and the diagnostic criteria " of issuing according to World Health Organization (WHO) (WHO) 1999 and China.135 routine CHD patients (male 67 examples/female 68 example) are incorporated into the research work of this project.Meanwhile, the normal population that 61 examples (male 31 examples/female 30 example) age, sex match is included the matched group studied for this project.Meanwhile, inventor utilizes apo E-deficient mice to simulate atherosclerosis, have detected the concentration of FGF21 in atheromatosis model mouse serum and normal health mouse serum, liver, aorta, kidney and spleen.
An object of the present invention is to provide a kind of clinical assistant diagnosis index that may be used for atherosclerosis and secondary disease coronary heart disease thereof.
In order to achieve the above object, in conjunction with result of study of the present invention, suggestion:
1. when FGF21 is as incidence of coronary heart disease auxiliary diagnostic index, adopt following standard: in human serum, the normal value of FGF21 content is 167.9 ± 15.6ng/L; If in serum, the content of FGF21 reaches or more than 513.5 ± 47.5ng/L, shows that it suffers from the possibility of coronary heart disease, but not as the unique foundation made a definite diagnosis.
2. be adopt following standard as incidence of atherosclerosis auxiliary diagnostic index at FGF21: FGF21 content is abnormal in human serum when raising, and judges that it may suffer from atherosclerosis, and advise that it does and further make a definite diagnosis.
In order to verify that FGF21 can as the feasibility of early stage auxiliary diagnostic index further, inventor has investigated FGF21 content and Abnormal Lipid Metabolism and insulin resistant, triglyceride, the dependency of the physical signs such as ApoA.Result shows that FGF21 and These parameters have significant correlation.
benefit of the present invention:
1. the invention provides probability and the method for the application of recombined human FGF21 albumen at prevention of arterial in atherosis and secondary disease coronary heart disease.On the one hand for the drug development of recombined human FGF21 albumen provides foundation and feasibility; On the other hand, also for provide method from pathogenesis undertaking therapy atherosclerosis.
2. at present for obesity, hyperlipemia, the metabolic disease increased popularity such as diabetes, under atherosclerosis and coronary heart disease lack the overall background of method of early diagnosis, the present invention, using the auxiliary diagnostic index of FGF21 as these two kinds of diseases, is conducive to early diagnosis and course of disease intervention.
Accompanying drawing explanation
Fig. 1 recombined human FGF21 albumen is on the impact of apoE KO mouse growth
Fig. 2 recombined human FGF21 albumen is on the impact of apoE KO lipid of mice
Fig. 3 recombined human FGF21 albumen is on the impact of apoE KO mouse liver athero.
The impact that Fig. 4 recombined human FGF21 albumen damages apoE KO cardiovascular pathological changes mouse liver.
Fig. 5 recombined human FGF21 albumen is on the impact of apoE KO mouse liver lipogenesis, oxidation, the main related gene expression of metabolism.
Fig. 6 recombined human FGF21 albumen is on the impact of the related gene expressions such as apoE KO hepatic tissue cholesterol, bile acid biosynthesis adjustment
Fig. 7 recombined human FGF21 albumen is piled up and the impact of liver metabolism gene signal path apoE mouse liver glycogen
Fig. 8 recombined human FGF21 albumen organizes the impact of ABCA1, ABCG1 to apoE KO mouse small intestine
Fig. 9 recombined human FGF21 albumen is on the impact of apoE KO mouse aorta bow blood vessel wall
Figure 10 recombined human FGF21 albumen is on the impact of apoE KO rat aorta atherosclerotic lesion
The serum FGF21 concentration of Figure 11 patients with coronary heart disease and matched group.
The dependency of Figure 12 serum FGF21 level and Abnormal Lipid Metabolism and insulin resistant
The changing condition of Figure 13 all ages and classes apoE KO atherosclerosis mice disease and serum FGF21 level thereof
The differential expression of Figure 14 FGF21mRNA level in 24 weeks apoE KO mices and WT mice linked groups organ
Figure 15 FGF21 is apoE KO mice and the differential expression of WT rat aorta blood vessel in 24 week age
Detailed description of the invention
Embodiment 1 recombinant human fibroblast cytokine 21 prevention of arterial is atherosis
1. animal grouping and drug treating arrangement
Get ApoE KO mice 30 in 8 week age, be divided into A/B/C tri-groups, often organize 10, in addition, get the consistent C57BL/6j mice of 10 genetic background again as normal control experimental group (D group), A/B/C tri-groups all gives high fat diet, A/B two groups injects restructuring FGF21 albumen by 100 and 400 μ g/kg dosage in hypodermic mode respectively simultaneously, C/D two groups notes subcutaneous injection normal saline in contrast, after two weeks, the unified chow diet that gives of A/B/C/D tetra-groups of mices is fed, in administration process, within every 2 weeks, detect once to Mouse Weight, observe body weight change.
2. sacrifice of animal and tissue sample are collected
Administration put to death mouse after 6 weeks, and by mice overnight fasting, with etherization, eye socket is got blood and collected blood sample, separation of serum, observes the changing condition of blood lipids; After sacrifice, open abdomen thoracic cavity successively, the PBS of sterilizing carries out cardiac perfusion, remained blood in removing blood vessel, then be separated aorta, on aorta is initial, 0.2-0.4cm and abdominal aortic bifurcation place intercept aorta, peel off clean by Supraaortic fat, by frozen for aorta in liquid nitrogen, for separating of extraction RNA and protein sample.Win heart simultaneously, after removing myocardium the latter half cardiac muscular tissue, be embedded in OCT and embed in liquid, prepare for heart tissue frozen section; Other internal organs: spleen, kidney, liver etc., a part is frozen in liquid nitrogen, is used for cooking frozen section, extracts RNA and albumen; A part is fixed on 4% paraformaldehyde, is used for dyeing or doing paraffin section.
3. the specific experiment method of main research
(1) serum FGF21ELISA detects
Get above-mentioned collected mice serum, adopt the mouse FGF21ELISA reagent that Biovendor provides, detect FGF21 serum-concentration.
(2) aortic arch oil red dyeing
The aortic arch heart tissue embedded by above-mentioned OCT, adopts freezing microtome to be cut into the tissue heart section of 10 μm of thickness, and room temperature condition is issued to till dry condition appears in tissue slice, is then positioned over-80 DEG C of refrigerators and keeps for subsequent use; After section room temperature places 30min, be positioned over ddH
22min in O, then places 30min in oil red stain working solution; 60% isopropyl alcohol rinses to clean (about 2min), is then positioned over running water 3min; With water solublity hematoxylin 30s; Coloration result is controlled under mirror; After running water 2min, with oil blackeite 2min in saturated sodium bicarbonate, running water 2min, Fast green rinses 30s, running water 2min; With water-soluble glycerol natural gum mountant mounting.
(3) hepatic glycogen PAS detects
1. test solution configuration
Camoy fixative: dehydrated alcohol 60mL+ glacial acetic acid 10mL+ chloroform 30mL.
Periodic acid alcoholic solution: periodic acid (HIO2H
2o) 0.4g, adds 95% ethanol 35mL, sodium acetate solution (2.72g+ distilled water 100ml) 5mL, distilled water 10mL, to put in brown bottle in 4 DEG C of refrigerators.
SchiffShi liquid: 0.5g basic fuchsin adds in 100mL distilled water, rocks 5min and makes it abundant dissolving.Filter after being cooled to 50 DEG C and add 10mL1N hydrochloric acid.Be cooled to 25 DEG C, add 0.5-1g and lay particular stress on sodium sulfate, at least leave standstill 24h at room temperature, then seal Refrigerator store.
SchiffShi alcohol liquid configures: SchiffShi liquid 11.5mL, 1N HCI0.5mL, dehydrated alcohol 23mL.
Sulfurous sour water: 1% sodium metabisulfite 10mL, 1N HCl10mL, the gum dissolution of distilled water 180mL.
Hematoxylin: haematoxylin 0.9g, 95% ethanol 10mL, ammonium (potassium) Alumen 20g, H
2o200mL.Haematoxylin is dissolved in the ethanol of 95%, potassium alum water-soluble (can heat), then haematoxylin liquid is added in alum steep and mix, add mercury oxide 0.5g after boiling with strong fire, stir rapidly, become darkviolet, in rapid immigration cold water, next day filters, and faces the used time and gets this liquid 95mL and add glacial acetic acid 5mL, cell color can be made clear.
2. staining procedure
After paraffin section de-waxing, distillation washing, periodic acid wine stillness of night 10min, tap water 10min, SchiffShi liquid 10min, running water 5min, hematoxylin 3min (nuclear targeting crosses dark available hydrochloride alcohol differentiation), running water 5min, conventional dehydration, transparent, sealing.
(4) aorta, liver etc. organize RNA to extract and concentration determination
1. taken out by each group of mouse liver tissue being stored in liquid nitrogen, with autoclaving surgical scissors clip 20-30mg hepatic tissue in clean 1.5mL EP pipe, add 1mL Trizol, use electric homogenizer high-speed homogenization tissue, the complete tissue of homogenate is put in and leaves standstill 5min on ice.To aortic tissue, first use the little scissor cut aortic tissue of autoclaving ophthalmologic operation, then carry out with reference to said extracted method.
2. RNA extracts: add 0.2mL chloroform in each pipe, covers tightly EP pipe, and turn upside down mixing 15s, and room temperature leaves standstill 2-3min; 4 DEG C of centrifugal 15min of 12000g on low temperature supercentrifuge will be organized in; Take out EP pipe, now EP pipe divides three layers, gets upper phase in another standby EP pipe.
3. RNA precipitation: add 0.5mL isopropyl alcohol, room temperature leaves standstill 10min (isopropyl alcohol is 0.6-1 times of upper liquid volume), 4 DEG C of centrifugal 10min of 12000g; Abandon supernatant, add 1mL75% ethanol purge precipitation.
4. RNA dissolves: 4 DEG C of centrifugal 5min of 7500g; Abandon supernatant, dry RNA; Amount according to RNA suitably adds DEPC water dissolution, for subsequent use.
5. RNA concentration determination: the RNA of extraction is measured in ultraviolet spectrophotometer 260OD value, 260/280 ratio, RNA concentration (μ g/ μ L)=OD260 × extension rate × 40/1000.
(5) RNA reverse transcription synthesis cDNA
1. according to the RNA concentration detected, with the reaction system of 20 μ L, each reverse transcription 20ng-5 μ g total serum IgE amount.
2. RNA degeneration: by 1 μ L oligo (dT) 12-18 (500 μ g/mL), 1 μ L10mM dNTP mixture (dATP, dTTP, dCTP, dGTP are 10mM, and pH is neutral), RNA joins in the 0.2mL centrifuge tube of nuclease free, add DEPC water to 13 μ L, mixture heats 5min at 65 DEG C, is placed in cooled on ice rapidly.
3. RNA reverse transcription: add 4 μ L5 × the first chain synthesis buffer in microcentrifugal tube, 2 μ L0.1M DTT, by centrifugal for each composition mixing, and hatch 2min at 37 DEG C; 1 μ L M-MLV (reverse transcriptase) is added in system.
4. system is placed in 37 DEG C of 50min in PCR instrument, 70 DEG C of 15min complete reverse, are stored in-20 DEG C of refrigerators for subsequent use.
(6) QPCR detects the expression situation of FGF21mRNA
Experimental designing requirement, by the inquiry of US National library gene bank, the gene order of design mice FGF21 and mRNA expression of gene associated amplimer, sequence is as follows:
mFGF21 F：5’-CTGGGGGTCTACCAAGCATA-3’
R：5’-CACCCAGGATTTGAATGACC-3’
18s RNA F：5’-GGCTGTATTCCCCTCCATCG-3’，
R：5’-CCAGTTGGTAACAATGCCATGT-3’
miNOS F：5’-ACGTTGGATTTGGAGCAGAA-3’
R：5’-TGGTAGGATTTGACTTTGAAGAG-3’
mTGF-β F：5’-GAGACGGAATACAGGGCTTT-3’
R：5’-ACGTGGAGTTTGTTATCTTTGC-3’
mTNF-α F：5’-TGGAACTGGCAGAAGAGG-3’
R：5’-AGACAGAAGAGCGTGGTG-3’
mACC1 F：5’-CCTCCGTCAGCTCAGATAC-3’
R：5’-CATGTGAAAGGCCAAACCAT-3’
mGPAT F：5’-TGGGTTCAAACCTACCTTC-3’
R：5’-GGCTCTCCTTCCATTTCAG-3’
mFAS F：5’-TTCTGGGCCAACCTCATT-3’
R：5’-AGCCTTCCATCTCCTGTCATC-3’
mSCDl F：5’-CCCTGCGGATCTTCCTTAT-3’
R：5’-CCATTCGTACACGTCATTC-3’
mHMGR F：5’-GTATTGCTGGCCTCTTCAC-3’
R：5’-AGCCTGTCAGTTCTTTGTCG-3’
mINSIG-1 F：5’-CCTTGGGCTTGGGATTAC-3’
R：5’-CACCATTATACACAAGAAACTGA-3’
Get the cDNA that above-mentioned obtained reverse is good, adopt SYBR-Green quantitative PCR method, according to reaction system below with carry out QPCR amplification:
SYBR-Green QPCR reaction system:
SYBR-Green QPCR amplification condition:
95℃1min
Do solubility curve analysis simultaneously.
4. case result
(1) recombined human FGF21 albumen delays the body weight growth of apoE KO mice
In this project, applicant observes the impact of recombined human FGF21 albumen on apoE KO mice, found that: recombined human FGF21 albumen significantly suppresses apoE KO mice (tremulous pulse medicated porridge sample animal pattern) body weight to increase.As shown in Figure 1A, compared with matched group apoE KO mice (C group), the body weight increase giving 100 μ g/kg and 400 μ g/kg recombined human FGF21 albumen process mice (A/B two groups) significantly slows down; Analyze shown (Figure 1B) by comparing body weight evolution, in administration 2 during thoughtful three weeks, the body weight increase of 100 μ g/kg and 400 μ g/kg recombined human FGF21 albumen processed group apoE KO mices almost stops, but goes out again the phenomenon slowly increased subsequently.
In addition, the process of recombined human FGF21 albumen also significantly can reduce apoE KO mouse liver tissue weight, as shown in Figure 1 C, the liver weight of 400 μ g/kg dosage groups is significantly lower than matched group (C group) mice (0.99 ± 0.05vs.1.4 ± 0.05g; And also there is significant difference (0.99 ± 0.05vs.1.23 ± 0.08g between 400 μ g/kg groups and 100 μ g/kg, two dosage groups p=0.0004); P=0.0330).In addition, as shown in figure ip, the recombined human FGF21 albumen process of 400 μ g/kg dosage also significantly reduces the ratio (p=0.0309) of apoE KO mouse liver/body weight, but between 100 μ g/kg and 400 μ g/kg, two groups of dosage, liver/body weight ratio not there are differences.
(2) recombined human FGF21 albumen reduces apoE KO mice dyslipidemia level
In this experiment, applicant by systemic applications in apoE KO mice, found that: recombined human FGF21 albumen significantly can reduce abnormal apoE KO lipid of mice situation, the lipid metabolism pressure (table 1) that alleviation high fat diet and apoE gene delection are brought.As shown in Figure 2 A, the process of recombined human FGF21 albumen significantly can reduce serum triglycerides (hereinafter referred to as the TG) level of apoE KO mice, compared with matched group, the recombined human FGF21 albumen of 100 and 400 μ g/kg dosage reduces apoE KO mice serum triglyceride levels 18.8% (p=0.014) and 28.3% (p=0.015) respectively; Simultaneously, as shown in Figure 2 B, the process of recombined human FGF21 albumen also significantly can reduce apoE KO mice serum free fatty (hereinafter referred to as NEFA) level, the recombined human FGF21 albumen of 100 and 400 μ g/kg dosage reduces serum free fatty acid levels 17.6% (p=0.0162) and 33.7% (p=0.0004) respectively, and between two dosage groups, also there is significant difference (p=0.0319) in serum free fatty acid levels; In low density lipoprotein, LDL (hereinafter referred to as LDL), as shown in Figure 2 C, the recombined human FGF21 albumen of 100 and 400 μ g/kg dosage lowers LDL (p=0.0275) with also presenting dose-dependant, and LDL serum levels declines 23.3% (p=0.0427) and 28.8% (p=0.0275) respectively; In T-CHOL (hereinafter referred to as TC), as shown in Figure 2 D, the FGF21 of 100 and 400 μ g/kg dosage lowers TC level with also presenting dose-dependant, and TC serum levels declines 13.7% (p=0.0015) and 16.7% (p=0.0008) respectively; In addition, in high density lipoprotein (hereinafter referred to as HDL) (Fig. 2 E), compared with matched group, 400 μ g/kg dosage group apoE KO mice HDL levels raise 25.2% (p=0.0119); The HDL of 100 μ g/kg dosage groups also has the trend of rising, but does not have significant difference (p=0.096).
Table 1FGF21 process is on the impact of apoE KO mice lipid metabolism
*P<0.05VS control，#P<0.01VS control，
P<0.0001VS control
(3) recombined human FGF21 albumen is on the impact of apoE KO mouse liver metabolism
1. recombined human FGF21 albumen and liver fat are piled up
In this project, applicant finds that recombined human FGF21 albumen significantly can alleviate the accumulation of triglyceride in apoE KO mouse liver tissue.As shown in table 1 and Fig. 3 A, compared with matched group, the recombined human FGF21 albumen of 100 and 400 μ g/kg dosage reduces the content of triglyceride of 46.2% (p=0.0012) and 65.6% (p<0.0001) in liver organization respectively; In addition, between 100 and 400 μ g/kg, two dosage groups, there is the relation of dose-dependent effect in the dosage of the content of triglyceride in liver organization and recombined human FGF21 albumen, also there is significant difference (p=0.0425) between the two.In addition, as shown in Figure 3 B, the content of apoE KO mouse liver free fatty is also significantly lowered in the recombined human FGF21 albumen process of 400 μ g/kg dosage.But oil red O stain experimental result shows: liver organization total lipid content significantly reduces (Fig. 3 C/D) in the apoE KO mice of recombined human FGF21 albumen process.
2. the expression situation of recombined human FGF21 albumen and hepar damnification label
In this project, applicant explores Metabolic stress that recombined human FGF21 albumen and high fat diet and apoE gene delection causes further to the loss situation of liver, result shows, recombined human FGF21 albumen can alleviate high fat diet and the chaotic detrimental effect to liver organization of lipid metabolism.As shown in Fig. 4 A/B, although HE coloration result points out the apoE KO murine liver tissue of this experiment not occur significant damage status in morphology, then start to have occurred significant change in molecular level.In apoE KO matched group, the mrna expression level of hepar damnification label TNF-α (Fig. 4 C), TIMP-1 (Fig. 4 D), IL-1 (Fig. 4 E), TGF-β (Fig. 4 F) and iNOS (Fig. 4 G) significantly rises, and this shows that high fat diet and apoE gene delection have caused damage to a certain degree to the liver of mice; But in the apoE KO mouse liver tissue of recombined human FGF21 albumen process, the mrna expression level of these hepar damnification labels significantly declines; In addition, between 100 and 400 μ g/kg, two dosage groups, these hepar damnification labels also present dose-dependent downward.This illustrates that recombined human FGF21 albumen significantly can alleviate high fat diet and the chaotic damage caused apoE KO mouse liver tissue of lipid metabolism.
3. recombined human FGF21 albumen regulates apoE KO mouse liver cholesterol, lipid metabolism
In this project, applicant adopts recombined human FGF21 albumen process apoE KO mice.Result shows, in matched group apoE KO mice, the mrna expression level comprising the main regulator gene such as liver fat synthesis and metabolism such as Yi Xian Drink-heavily enzyme A carboxylase (ACC1), fatty acid synthetase (FAS), glycerol 3-phosphate acyltransferase (GPAT), 18 Wan Xian Drink-heavily enzyme A desaturase 1 (SCD1) significantly rises, this illustrates in apoE KO mice, and the cholesterol of liver, lipid metabolism are in disorderly situation; Then, after the process of recombined human FGF21 albumen, apoE KO mouse liver comprises ACC1 (Fig. 5 A), FAS (Fig. 5 B), GPAT (Fig. 5 C), SCD1 (Fig. 5 D) significantly reduce in the mRNA level in-site of interior several participation lipogenesis, oxidation regulation and control related gene, but does not make significant difference to the expression of FoxA2 gene.
In lipoprotein metabolism adjustment, matched group apoE KO mouse liver organizes the mrna expression of CD36 significantly to rise, and PON1 then significantly lowers.After the process of recombined human FGF21 albumen, the expression of CD36 gene significantly declines (Fig. 5 E), and the expression of PON1 then occurs raising (Fig. 5 F).This shows that the process of recombined human FGF21 albumen will directly affect the regulation process of apoE KO mice lipoprotein metabolism.
In cholesterol, bile acid biosynthesis adjustment, matched group apoE KO mouse liver organizes HMGR (Fig. 6 A), the mrna expression of INSIG-1 (Fig. 6 B) significantly rises, and the gene expression of INSIG-2 (Fig. 6 C) is then significantly lowered.After the process of recombined human FGF21 albumen, the expression of HMGR and INSIG-1 gene mRNA is then significantly lowered, and the situation significantly risen then appears in INSIG-2 gene expression, but then affects not quite cholesterol 12a hydroxylase B1 (CYP8B1).
In HNF, the process of recombined human FGF21 albumen will significantly raise the expression (Fig. 6 D) of HNF4 α gene, then show significant resistancing action (Fig. 6 E) to PPAR-γ gene expression.In addition, compared with matched group, after the process of recombined human FGF21 albumen, the mrna expression level of apoE KO hepatic tissue Genetyping poD (Fig. 6 F), adiponectin APN (Fig. 6 G) gene significantly rises; What is interesting is, compared with matched group, after the process of recombined human FGF21 albumen, apoE KO mice serum adiponectin level also significantly rises (Fig. 6 H).
4. recombined human FGF21 albumen is on the impact of liver glycogen
In this project, experimental result display (Fig. 7 A/B) of applicant, in the matched group apoE KO mice that high fat is fed, liver glycogen content is in higher level, and this illustrates that the hepatic glycogen of the apoE KO mice of high fat diet is in height accumulation situation; After giving recombined human FGF21 albumen, compared with matched group, the content of apoE KO mouse liver glycogen significantly reduces.This illustrates that the process of recombined human FGF21 albumen can alleviate the accumulation of apoE KO Mouse Liver glycogen in liver organization cell.
5. recombined human FGF21 albumen is to the regulating and controlling effect of liver metabolism associated signal paths
In this project, applicant also observes gene signal path approach relevant in apoE KO mouse liver.WB experimental result display (Fig. 7 C): in the apoE KO mouse liver tissue of High-fat diet, the phosphorylation level of JNK gene is significantly higher than WT mice, and after the process of recombined human FGF21 albumen, JNK phosphorylation level then significantly reduces.This illustrates, high fat diet and apoE genetic flaw excite JNK gene signal, and the process of recombined human FGF21 albumen can suppress high fat diet and apoE genetic flaw to cause JNK gene expression.
In addition, in the apoE KO mouse liver tissue of High-fat diet, the phosphorylation level of p-P38 signal gene is also significantly higher than WT mice, and in recombined human FGF21 albumen process mice, p-P38 phosphorylation level then significantly reduces (Fig. 7 C).This also illustrates, will excite p-P38 gene signal, and the process of recombined human FGF21 albumen can suppress high fat diet and apoE genetic flaw to cause p-P38 gene phosphorylation signal path in high fat diet and apoE genetic flaw.
(4) recombined human FGF21 albumen promotes reverse cholesterol transport
In this project, in order to explore the impact of recombined human FGF21 albumen on apoE KO mice cholesterol metabolism, applicant observes the expression situation of ABCA1, ABCG1 gene relevant to reverse cholesterol transport in each group of apoE KO mouse small intestine tissue.Experimental result shows, compared with matched group poE KO mice, recombined human FGF21 albumen processed group significantly can raise the mrna expression (Fig. 8) of small intestinal ABCA1, ABCG1, shows that recombined human FGF21 albumen can promote the counter transport of single cholesterol.
(5) recombined human FGF21 albumen is on the impact of apoE KO mice vascular lesion
1. recombined human FGF21 albumen reduces apoE KO mouse aorta tunica media thickness
In this project, applicant observes the impact of recombined human FGF21 albumen on apoE KO vascular lesion, as shown in Figure 9, HE Coloration experiment result shows: in apoE KO control mice, the tunica media thickness at ascending aorta bow place obviously thickens, and has a large amount of macrophages infiltration, inside have lipidosis in inner membrance, form foam cell, tube chamber narrows; And in the process of recombined human FGF21 albumen after 6 weeks, apoE KO mice blood vessel media thickness significantly reduces, in inner membrance, the situation of cellular infiltration and lipid accumulation reduces.
2. recombined human FGF21 albumen alleviates apoE KO mice blood vessel plaque area
In order to explore the relation between recombined human FGF21 albumen and atherosclerotic lesion further, applicant observes the changing condition of the formation of apoE KO mice vascular plaque after the process of recombined human FGF21 albumen.In High fat diet apoE KO matched group, opening thoracic cavity aorta vessel has obvious atherosclerosis plaque forming, and perusal is canescence graininess, polypoid, irregular or oval, or is fused into mutually streak with tremulous pulse longitudinal axis.As shown in Figure 10 A, tunica intima oil red coloration result shows, the Lipid Plaque of the aorta vessel inner membrance redness of matched group apoE KO mice significantly increases, and the red Lipid Plaque that the apoE KO mouse aorta blood vessel of recombined human FGF21 albumen processed group occurs then significantly reduces.In addition, also obtain similar result (Figure 10 B) in aortic arch root tissue section oil red O stain result, in apoE KO matched group, the area of the pathological changes speckle in mouse aorta nut portion is 0.16 ± 0.015mm
2, and be 0.13 ± 0.012mm at the area of the pathological changes speckle in recombined human FGF21 albumen processed group apoE KO mouse aorta nut portion
2(p=0.042).
3. recombined human FGF21 albumen reduces the expression of ICAM-1 in vascular tissue
The proinflammatory factor that ICAM-1 (hereinafter referred to as ICAM-1) vascular endothelial cell is impaired.Applicant observes the expression situation of ICAM-1 (hereinafter referred to as ICAM-1) in apoE KO mice lesion vessels tissue after the process of recombined human FGF21 albumen.As shown in the ImmunohistochemistryResults Results of Figure 10 C, in apoE KO matched group, the expression of ICAM-1 is significantly risen, and after FGF21 process, its expression is then significantly lowered.After this illustrates the process of recombined human FGF21 albumen, the ICAM-1 in vascular tissue expresses and significantly lowers, and namely the vascular endothelial cell extent of damage reduces.
The clinical manifestation of embodiment 2FGF21 in patients with coronary heart disease
1.CHD clinical case is collected and clinical analysis method
(1) clinical criteria
Angina pectoris or acute myocardial infarction coronary heart disease patient are classified as object of study by the existing CHD clinical criteria of " name of ischemic heart desease and the diagnostic criteria " of issuing according to World Health Organization (WHO) (WHO) 1999 and China.Concrete diagnostic criteria is as follows: 1. the clinical diagnosis of myocardial infarction occurs that sero-enzyme raises and clinical manifestation is that the symptoms such as chest discomfort are passed judgment on according to patient ECG's testing result, Serologic detection.2. angina pectoris be then according to below clinical manifestation and Testing index as diagnostic criteria, namely main manifestations is the chest discomfort (have a rest or with nitrate preparations then then occur transference cure) reaching 15min after often there is the excited or motion of emotion, and with the detection evidence of following myocardial ischemia, namely bring out the decline of ST wave band or the T ripple >=inverted phenomenon in 2mm place of appearances >=1mm in ischemia experiment electrocardiogram at pharmacological stress test.
(2) case is collected
According to above-mentioned clinical criteria, 135 routine CHD patients (male 67 examples/female 68 example) are incorporated into the research work of this project.Meanwhile, the normal population that 61 examples (male 31 examples/female 30 example) age, sex match is included the matched group studied for this project.All enter the crowd of anthology project research selected before carry out questionnaire survey and clinical biochemical inspection by same Vasculocardiology Deparment doctor, to the crowd having the relevant disease medical histories such as cardiovascular disease, hypertension and diabetes, and used blood fat reducing, resisting hypertension or the CHD patient for the treatment of diabetes medicament all can not enter to elect as the object of study of this project in the past.In addition, all selected objects comprise CHD patient and normal control population all endorsed Written informed consent, and all programs are ratified by Ethics Committee of Wenzhou Medical College.
(3) clinical data is collected and biochemistry detection
After carrying out the assessment of relevant clinical questionnaire survey to all experimenters, overnight fast 10h, collects blood sample, separation of serum specimen ,-80 DEG C of freezing maintenances.The mode that body index detection (height, body weight, body-mass index, waistline and blood pressure) and biochemical indicator are reported with reference to front research is carried out.Insulin resistant carries out insulin resistant coefficient (HOMA-IR) assessment with reference to the organism balance pattern of report in early stage.FGF21 (ELISAkit in serum, Biovendor, Modrice, Czech Republic) and C reactive protein level (CRP ELISAkit, R & D, USA) description that provides according to commercial reagents company detects; Dibit chemiluminescent enzyme-linked immunosorbent immunoabsorption is adopted to detect serum insulin (Diagnostic Products, Los Angeles, CA); At the Clinical Test Lab through certification, fasting glucose, T-CHOL, HDL-C, Apolipoprotein A1 (ApoA1), apolipoprotein B100 (ApoB100), low-density lipoprotein cholesterol and triglyceride are detected.
(4) statistical analysis
The all analyses of this project all adopt statistics software 13-0 version (SPSS, Chicago, IL) to carry out statistical analysis according to the clinical research mode reported in the past.In simple terms, normal distribution data use mean ± SD to describe usually.The data Kolmogorox-Smimov of nonnormal distribution checks, and carries out Logarithm conversion, represent median with interquartile-range IQR before analysis.Adopt between Student's independent samples t-test group and compare.Correct group difference with Pearson's dependency, and correct multiple assay with Bonferroni.By FGF21 in Multiple Regression Analysis Method serum analysis and the relatedness between other parameter such as clinical indices, biochemical indicator.Variable and serum FGF21 are that the parameter of significance relevant (correcting multiple inspection with Bonferroni) enters multiple regression analysis.In all the statistical testing results, P<0.05 is considered to have statistical significance.
2. case result
(1) the FGF21 level that circulates in CHD patients serum significantly raises
Biochemical indicator and the Clinical symptoms of CHD patient and normal healthy controls group are as shown in table 2.The serum FGF21 horizontal extent of 196 routine participants is 11.63-2614.9ng/L, the horizontal asexuality difference of serum FGF21.(table 1 and Figure 11 A) statistic analysis result display after correcting with body-mass index (BMI), compared with matched group FGF21 level (167.9 ± 15.6ng/L), CHD patients serum FGF21 level (513.5 ± 47.5ng/L) significantly exceeds 3 times (P<0.0001); On the other hand, with the CHD patients serum FGF21 level (530.0ng/L of diabetes, [75.6-1941.3], n=61, P=0.034) apparently higher than CHD patient (317.2ng/L, [59.1-680.5], the n=74 not with diabetes, Figure 11 B), this is consistent with the report of the people such as Chen.Although the serum FGF21 level with hypertension CHD patient presents rising trend (Figure 11 C), but with the serum FGF21 level (462.2ng/L of hypertension CHD patient person, [59.1-2614.9], n=97) with without hypertension CHD patient serum FGF21 level (428.2ng/L, [77.8-1069.2] n=38) between there is no significant difference (P=0.0718).In addition, simultaneously with the serum FGF21 level (502.3ng/L of diabetes, hypertensive CHD patient, [157.6-2614.9], n=45) apparently higher than not with the serum FGF21 level (410.5ng/L of the CHD patient of these complication, [77.8-1069.2], n=21, p=0.026, Figure 11 D).Logistic regression analysis shows, serum FGF21 level relevant to the popular independence of CHD (p=0.004).
Clinical and the biochemical indicator of table 2 patients with coronary heart disease and matched group crowd
Data are means±SEM or median(interquartile range).NS，not significant.
(2) relation between serum FGF21 level and Abnormal Lipid Metabolism, insulin resistant
As shown in table 3 and Figure 12, be after correction parameter corrects with BMI, serum FGF21 level significantly and triglyceride, apolipoprotein B100 (r=0.365, P<0.001; R=0.227, P=0.002) being proportionate property, with HDL-C and ApoA (r=-0.313, P<0.001; R=-0.338, P<0.001) in negative correlation.But, between serum FGF21 level and blood total cholesterol (TC), LDL-C in book problem there are no significant dependency.In addition, be after correction parameter corrects with BMI, serum FGF21 level and fasting glucose, fasting insulin and insulin resistance index height correlation (p<0.05), but with insulin secretion index (HOMA-IS) without obvious dependency.
The dependency of table 3 serum FGF-21 level and somatometry parameter and biochemical indicator
*Log transformed before analysis.NS，not significant.#adjusted by BMI，hypertension and diabetes.
(3) serum FGF21 level is independent relevant to Apolipoprotein A1, triglyceride and fasting glucose
In order to whether whether clear and definite serum FGF21 exist independently dependency with human body organism parameter and cardiovascular risk factors, all parameters (Figure 12) of applicant couple and serum FGF21 significant correlation have carried out multiple regression analysis.Analyze display, with diabetes, hypertension and BMI for after correction parameter adjustment, Apolipoprotein A1 (beta coefficient-0.304,95%CI-0.500to-0.173, P<0.001), fasting glucose (beta coefficient0.219,95%C10.013to0.087, P=0.008) and triglyceride (beta coefficient0.213,95%C10.016-0.124, P=0.011) independent relevant to serum FGF21.But other parameters all comprise T-CHOL, HDL-C, low-density lipoprotein cholesterol, apolipoprotein B100, and insulin and insulin resistance index are all excluded in regression analysis process.
The performance of embodiment 3FGF21 in atherosclerosis mouse model
1. animal grouping
By 20 8 week age apoE
-/-male mice is divided into A/B two groups at random, often organizes 10, and wherein A group adopts high lipid food to feed, and B group adopts chow diet diets.Get 10 C57/BL6j normal mouses as experiment contrast group (C group) simultaneously, and with chow diet diets.
2. mice blood specimen collecting
Before zoopery starts, by above-mentioned A/B two groups of overnight fastings, from eyeground vein blood sampling, separation of serum ,-80 DEG C of Refrigerator stores, detect specimen as mice serum FGF21 in 8 week age; Then when respectively 12,24 week age, collect blood sample, observe the changing condition of serum FGF21 cyclical level.
3. sacrifice of animal and tissue sample are collected
When after each treated animal completely 24 week age, by mice overnight fasting, with etherization, eye socket is got blood and is collected blood sample; After sacrifice, open abdomen thoracic cavity successively, the PBS of sterilizing carries out cardiac perfusion, remained blood in removing blood vessel, then be separated aorta, on aorta is initial, 0.2-0.4cm and abdominal aortic bifurcation place intercept aorta, peel off clean by Supraaortic fat, by frozen for aorta in liquid nitrogen, for separating of extraction RNA and protein sample.Win heart simultaneously, after removing myocardium the latter half cardiac muscular tissue, be embedded in OCT and embed in liquid, prepare for heart tissue frozen section; Other internal organs: spleen, kidney, liver etc., a part is frozen in liquid nitrogen, is used for cooking frozen section, extracts RNA and albumen; A part is fixed on 4% paraformaldehyde, is used for dyeing or doing paraffin section.
4. the specific experiment method of main research
(1) serum FGF21ELISA detects
Get above-mentioned collected mice serum, adopt the mouse FGF21ELISA reagent that Biovendor provides, detect FGF21 serum-concentration.
(2) aortic arch oil red dyeing
1. the aortic arch heart tissue embedded by above-mentioned OCT, adopts freezing microtome to be cut into the tissue heart section of 10 μm of thickness, is placed to till dry condition appears in tissue slice, is then positioned over-80 DEG C of refrigerators for subsequent use under room temperature condition;
2., after room temperature of cutting into slices places 30min, be positioned over 2min in ddH2O, then in oil red stain working solution, place 30min; 60% isopropyl alcohol rinses to clean (about 2min), is then positioned over running water 3min with water solublity brazilwood extract dyeing 30s; Coloration result is controlled under mirror;
3. after running water 2min, with oil blackeite 2min in saturated sodium bicarbonate, running water 2min, Fast green rinses 30s, running water 2min; With water-soluble glycerol natural gum mountant mounting.
(3) aorta, liver etc. organize RNA to extract and concentration determination
1. taken out by each group of mouse liver tissue being stored in liquid nitrogen, with autoclaving surgical scissors clip 20-30mg hepatic tissue in clean 1.5mL EP pipe, add 1mL Trizol, use electric homogenizer high-speed homogenization tissue, the complete tissue of homogenate is put in and leaves standstill 5min on ice.To aortic tissue, first use the little scissor cut aortic tissue of autoclaving ophthalmologic operation, then carry out with reference to said extracted method.
2. RNA extracts: add 0.2mL chloroform in each pipe, covers tightly EP pipe, and turn upside down mixing 15s, and room temperature leaves standstill 2-3min; 4 DEG C of centrifugal 15min of 12000g on low temperature supercentrifuge will be organized in; Take out EP pipe, now EP pipe divides three layers, gets upper phase in another standby EP pipe.
3. RNA precipitation: add 0.5mL isopropyl alcohol, room temperature leaves standstill 10min (isopropyl alcohol is 0.6-1 times of upper liquid volume), 4 DEG C of centrifugal 10min of 12000g; Abandon supernatant, add 1mL75% ethanol purge precipitation;
4. RNA dissolves: 4 DEG C of centrifugal 5min of 7500g; Abandon supernatant, dry RNA; Amount according to RNA suitably adds DEPC water dissolution, for subsequent use;
5. RNA concentration determination: the RNA of extraction is measured in ultraviolet spectrophotometer 260OD value, 260/280 ratio, RNA concentration (μ g/ μ L)=OD260 × extension rate × 40/1000.
(4) RNA reverse transcription synthesis cDNA
1. according to the RNA concentration detected, with the reaction system of 20 μ L, each reverse transcription 20ng-5 μ g total serum IgE amount.
2. RNA degeneration: by 1 μ Loligo (dT) 12-18 (500 μ g/mL), 1 μ L10mM dNTP mixture (Datp, dTTP, dCTP, dGTP are 10mM, and pH is neutral), RNA joins in the 0.2mL centrifuge tube of nuclease free, add DEPC water to 13 μ L, mixture, at 65 DEG C of heating 5min, is placed in cooled on ice rapidly;
3. RNA reverse transcription: add 4 μ L5 × the first chain synthesis buffer in microcentrifugal tube, 2 μ L0.1M DTT, by centrifugal for each composition mixing, and hatch 2min at 37 DEG C; 1 μ L M-MLV (reverse transcriptase) is added in system;
4. system is placed in 37 DEG C of 50min in PCR instrument, 70 DEG C of 15min complete reverse, are then stored in-20 DEG C of refrigerators for subsequent use.
(5) QPCR detects the expression situation of FGF21mRNA
Experimental designing requirement, by the inquiry of US National library gene bank, the gene order of design mice FGF21, sequence is as follows:
F：5’-CTGGGGGTCTACCAAGCATA-3’；
R：5’-CACCCAGGATTTGAATGACC-3’。
Simultaneously according to genome GAPDH gene order, design the mrna expression amplimer of mice Actin reference gene, sequence is shown as follows:
F：5’-GGCTGTATTCCCCTCCATCG-3’；
R：5'-CCAGTTGGTAACAATGCCATGT-3’。
Get the cDNA that above-mentioned obtained reverse is good, adopt SYBR-Green quantitative PCR method, according to reaction system below with carry out QPCR amplification:
SYBR-Green QPCR reaction system:
SYBR-Green QPCR amplification condition:
95℃1min
Do solubility curve analysis simultaneously.
(6) histone such as aorta, liver extracts and concentration determination
1. by PMSF and RIPA lysate according to 1:100 proportions Tissue lysates, add 0.5M inhibitors of phosphatases NaF, final concentration is 15mM;
2. each group of mouse liver tissue being stored in liquid nitrogen is taken out, with operating scissors clip 30-50mg hepatic tissue in clean 1.5mLEP pipe, add the lysate that 500 μ L prepare, first tissue is shredded with clean surgical scissors and re-use electric homogenizer high-speed homogenization tissue, the complete sample of homogenate is statically placed in 20min in trash ice, makes the abundant stripping of albumen.
3. the sample abundant suspendible on whirlpool suspendible device will left standstill, in low temperature supercentrifuge 4 DEG C, 12000r/min, centrifugal 15min, draw supernatant, for subsequent use.
4. above extracted albumen is adopted BCA kit measurement determination of protein concentration, specific as followsly to show: add 1 volume BCA reagent B (50:1) by 50 volume BCA reagent A and prepare appropriate BCA working solution, fully mix, for subsequent use; By 0.5mg/mL standard sample according to 0,2.5,5,10,15,20,25 μ L are added in the standard sample wells of 96 orifice plates, use ultra-pure water polishing less than 25 μ L; Protein sample proper proportion diluted adds in 96 orifice plates, and every hole adds 25 μ L; Each hole adds 200 μ LBCA working solutions, places 30min for 37 DEG C; Measure A562, calculate protein concentration according to standard curve.
(7) immunoblotting analysis (Western Blotting) detects
1. SDS-PAGE electrophoresis
Protein sample is diluted for same concentrations, get protein sample 10 μ L (40 μ g total protein), with isopyknic 2 × sample-loading buffer (or 5 × sample-loading buffer, opsin's concentration and determining, loading protein concentration is 30-40 μ g) be mixed after, 100 DEG C are boiled 5min, the centrifugal 5min of 10000g, get supernatant and carry out SDS-PAGE electrophoresis.Voltage is that 80V/110V carries out electrophoresis, can stop electrophoresis when dyestuff arrives gel bottom margin.
2. transferring film
By filter paper, foam-rubber cushion, be positioned over electricity and turn in liquid, soak 30-60min, prepare with size is identical pvdf membrane simultaneously.First pvdf membrane methanol is steeped 30s, then use ddH
2o washes 2min, is then positioned in transfer liquid and soaks 30min.After SDS-PAGE completes, take off glue, concentrated glue is removed, then by foam-rubber cushion, filter paper, glue, film, filter paper, foam-rubber cushion is put on black and white plate in this order, notices that pvdf membrane must be placed on blank, drains air wherein simultaneously, then black and white board clamping is put on shelf, notices that both positive and negative polarity is aimed at.Use the standard wet type membrane-transferring device (use ice chest) of Bio-Rad, can set transferring film electric current is 300mA, the transferring film time is that 60min is (according to the size of destination protein molecular weight, determine the transferring film time, the molecule transferring film 1h of below 60KD, 60-100KD protein molecular transferring film 90min).
3. close
After transferring film, immediately albuminous coat is placed in preprepared PBS, rinsing 1-2min, to wash away the transferring film liquid (step all after transferring film on film, the moisturizing of film must be noted, avoid the drying of film, otherwise very easily produce higher background).Add Western confining liquid, shaking table slowly shakes, room temperature closes 120min.For the antibody that some backgrounds are higher, can close at 4 DEG C and spend the night, wash 3 times with cleaning mixture, each 5min.
4. immune detection
Primary antibodie is hatched: after washing, adds the primary antibodie of having diluted, and on shaking table, slowly 1h (it is not good that primary antibodie hatches 1h, slowly can shake overnight incubation at 4 DEG C) is hatched in shake.Reclaim primary antibodie.Washing: add Westem cleaning mixture, on side-sway shaking table, slowly 5-10min is washed in shake.Wash 3 times altogether.
Two anti-hatch: filter paper exhausts cleaning mixture, and add two resisting of dilute immediately, room temperature, on side-sway shaking table, slow shake hatches 2h.After exhausting cleaning mixture, then add cleaning mixture, washing 5-10min.Wash 3 times altogether, first time washs 10min, washs 5min respectively for the second time, for the third time.
Detect: film filter paper is blotted, hatches 5min with the super quick luminescent solution of ECL.Development, fixing, use exposure, or directly use gel imaging system to take pictures.
5. statistical analysis
All experimental datas all input GraphPad Prism5.0 statistical package and carry out statistical analysis, and P<0.05 is that difference has significance.Major experimental data repeat experiment from three times, and represent with mean ± SD, P<0.05 is that difference has significance.
6. case result
(1) changing condition of apoE KO mice serum FGF21
For exploring the performance of FGF21 in atherosclerosis mice, the serum FGF21 of applicant's desk study apoE KO atherosclerosis mice changes and the age, situation between diet.Experimental result shows, when apoE KO Mouse Age arrives 12 week age, ascending aorta nut portion there is a small amount of plaque (Figure 13 A) in oil red O stain display mice, and during to 24 week age, the area of sclerosis of blood vessels speckle expands (Figure 13 B) further, this illustrates the development along with the age, and apoE KO mouse core vascular lesion also worsens further.In order to explore the relation between FGF21 and atherosclerosis, applicant observes the changing condition of apoE KO mice serum cyclical level, research experiment result shows: apoE KO mice serum FGF21 circulates with advancing age, the deterioration of the state of an illness and significantly increasing; And it is closely related with high fat diet.As shown in fig. 13 c: 8 week age, the serum FGF21 cyclical level of apoE KO mice remained at low levels, and FGF21 serum levels is 175.9 ± 46.78ng/mL; Raise when apoE KO mice gives high fat diet or give normal diet raising 4 weeks, namely when Mouse Age arrives 12 weeks, the apoE KO mice serum FGF21 level that high fat and normal diet are raised all significantly rises, and feed between two groups at normal diet and high fat and there is significant difference (2.03 ± 0.38vs.1.02 ± 0.20ng/mL, p=0.0238); In addition, when the age rises to 24 weeks, the level of the apoE KO mice serum FGF21 that high fat and normal diet are raised rises further, there is significant difference (4.40 ± 1.06vs.1.57 ± 0.28ng/mL in the FGF21 serum circulates level between two groups, p=0.0091), the difference simultaneously between two groups increases further.
(2) FGF21 is correlated with apoE KO mice the differential expression of organ
In order to explore the performance of FGF21 in apoE KO atherosclerosis small mouse model, applicant adopts QPCR method, explores the expression situation of FGF21mRNA each organ in apoE KO mice and the C57BL/6J mice identical with the age with genetic background.FGF21 is the somatomedin being mainly derived from liver organization, and for this reason, first applicant observes the expression situation of FGF21 in liver organization.SABC testing result shows: the C57BL/6J mice identical with the age with genetic background is compared (Figure 14 A), and in the atherosclerosis mouse model in 24 week age, FGF21 albumen significantly raises (Figure 14 B) in liver organization; In addition, QPCR testing result display (Figure 14 C), in WT and apoE KO mouse liver tissue, apoEKO mouse liver FGF21mRNA expression is significantly higher than WT mice (p=0.047).In arterial vascular tissue, applicant finds that in apoE KO rat aorta tissue, FGF21mRNA expression is also significantly also significantly higher than matched group WT mice (p=0.0002), and has higher expression; In addition, in renal tissue, FGF21mRNA expresses and is also significantly higher than matched group WT mice (p=0.048), but its expression is significantly lower than liver and aortic blood tubing; And in spleen tissue, the FGF21mRNA of apoE KO mice expresses the trend also having rising, but compared with matched group WT mice, there is not significant difference in both, and its expression is also remarkable in liver and arterial tissue (Figure 14 C).
(3) expression of FGF21 in atherosclerosis plate in apoE KO mice
For explore FGF21 and atherosclerosis develop between relation, this research observes the expression situation of FGF21 in apoEKO mice atheromatous plaque further.First, applicant observes the disease of apoE KO mice in 24 week age, oil red O stain display (Figure 15 A), compared with same age group matched group, 24 week age, normal diet apoE KO mouse aorta bow was in serious Atheromatosis reason situation, and the thickness of tunica media significantly increases, and in tunica intima tissue, a large amount of red Lipid Plaques occurs, and the area that speckle occupies arteries inside casing significantly increases; And there are no above-mentioned disease in WT mice.Meanwhile, applicant also observes the expression situation of FGF21 in artery plaque, immunohistochemical assay result display (Figure 15 B): in the vascular tissue of apoE KO mice in 24 week age, FGF21 protein expression be significantly higher than genetic background and the age identical WT mice, these FGF21 mainly come across the relevant cell of valnllae vasorum film edge, further, in middle internal film tissue, corresponding FGF21 protein expression is also had.In addition, in arterial vascular tissue, the expression of apoE KO mice FGF21mRNA is also significantly higher than WT matched group (Figure 14 C); In addition, immunoblotting analysis (WB) testing result also shows: the expression of FGF21 in the aortic tissue of apoE KO atherosclerosis mice is significantly higher than the genetic background wild-type mice identical with the age (Figure 15 C/D).
Claims (10)
1. the application of recombinant human fibroblast growth factor 21 (hereinafter referred to as recombined human FGF21) at prevention of arterial in atherosis and relevant disease.
2.FGF21 is as simple coronary heart disease and the application of the morbidity auxiliary diagnostic index of concurrency coronary heart disease that caused by other diseases.
3.FGF21 as the application of atherosclerosis diagnosis auxiliary diagnostic index.
4. claim 1 addresses FGF21, it is characterized by by the independent research and development of applicant and the vitro recombination FGF21 (patent No. ZL200810223501.0) of patented mandate.
5. application according to claim 1, is characterized in that: after recombined human FGF21 vivo medicine-feeding, and the progress of atherosclerotic lesion is eased, and body weight increases and significantly slows down; Meanwhile, the weight of liver organization and liver/body weight ratio reduce.
6. claim 1,5 address application, it is characterized in that:
1) FGF21 albumen of recombinating raises the expression of PON1 (hereinafter referred to as PON1) in Human umbilical vein endothelial cells (being called for short HUVEC cell) by extracellular regulated protein kinase 1/2 (hereinafter referred to as ERK1/2) signal pathway, inhibited apoptosis.
2) recombinate FGF21 albumen by raising the expression of ABCA1 gene, thus impel lipid to flow out from cell, reduce the accumulation of lipid in cell.
7. right 2,3 address FGF21, it is characterized by natural people FGF21, and its information can check in (serial number: AAQ89444.1, GI:37183289) from US National Biotechnology Information center.
8. right 2, the FGF21 described in 7, as incidence of coronary heart disease auxiliary diagnostic index, is characterized by: in human serum, the normal value of FGF21 content is 167.9 ± 15.6ng/L; If in serum, the content of FGF21 reaches or more than 513.5 ± 47.5ng/L, shows that it suffers from the possibility of coronary heart disease, but not as the unique foundation made a definite diagnosis.
9. claim 3, the FGF21 described in 7, as atherosclerosis diagnosis auxiliary diagnostic index, is characterized by: in human serum, FGF21 content is abnormal when raising, and judges that it may suffer from atherosclerosis, and advises that it does and further make a definite diagnosis.
10. claim 2 addresses other diseases is diabetes, hyperlipidemia and obesity.
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| CN108379555A (en) * | 2018-03-28 | 2018-08-10 | 暨南大学 | Applications of the FGF21 in the drug for preparing hypertension and/or angiosis damage that treatment is caused by angiotensinⅡ |
| CN108404119A (en) * | 2018-05-07 | 2018-08-17 | 哈尔滨博翱生物医药技术开发有限公司 | The preparation of FGF-21 analogs and the application in thrombus treatment |
| CN108404119B (en) * | 2018-05-07 | 2020-09-25 | 江苏康缘瑞翱生物医药科技有限公司 | Preparation of FGF-21 analogue and application thereof in thrombus treatment |
| CN109568586A (en) * | 2019-01-22 | 2019-04-05 | 张淑敏 | A kind of function and application of FGF21 gene in treatment hyperlipidemia |
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