CN1699417A - Human blood high density lipoprotein and its preparation method and use - Google Patents
Human blood high density lipoprotein and its preparation method and use Download PDFInfo
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Abstract
The invention relates to a human high density lipoprotein and product, preparation process and use thereof, wherein the apo AI content in the human high density lipoprotein is 77-89%. The preparation process comprises using blood plasma as raw material, extracting HDL through segregation and purification steps. The prepared human high density lipoprotein and products produced thereby can be applied to the preparation for medicaments for treating multiple organ dysfunction syndrome (MODS), cardiovascular system diseases such as atherosclerosis (AS).
Description
Technical field
The present invention relates to a kind of human blood high density lipoprotein, its manufacture method and the application on pharmaceutics thereof, belong to biochemical field.
Background technology
AS is because of the arterial wall lipidosis, fiber and connective tissue proliferation, and cause ductus arteriosus wall to thicken hardening, and elasticity attenuation also forms the common multiple disease of rotten congee sample focus.The arterial lumen obturation that AS causes can make vitals blood supply insufficiencies such as the heart, brain, kidney, thus serious threat human health and life.Treatment AS medicine commonly used clinically at present generally has vasodilation medicine, blood lipid-lowering medicine, antiplatelet drug and thrombolytic drug etc.Though these medicines can resist AS by removing effects such as vasomotor disturbance and regulating blood fat, all can not safe and effective prevention and elimination AS patch.And because AS is a kind of chronic non-inflammation, degeneration and proliferative pathology, the treatment of life-time service some drugs can cause side effects such as cholelithiasis, liver dysfunction.
Epidemiological studies shows that blood fat rising, obesity, hypertension, diabetes and smoking etc. are AS primary hazard factors.But the control to AS at present there is no ideal method and medicine.Present stage control AS is except that dietary control, and general multiselect is with blood fat reducing, vasodilation or at the medicine of the easy trouble factor in pharmacological agent.Therefore find and preparation can prevent, particularly can impel AS to reverse or the new drug that disappears just very necessary.
The massive epidemiology investigation has been found that plasma high density lipoprotein level (HDL) level and atherosclerosis (AS) sickness rate are negative correlation since the seventies.Framingham investigation shows, high family or regional coronary heart disease (CHD) incidence of plasma high density lipoprotein level-cholesterol (HDL-C can represent the index of blood plasma HDL content) obviously reduces.Rifkind etc. find in the crowd of investigation, the every increase of blood plasma HDL-C content 1mg/dL, and the danger of CHD just reduces 2-3%.Helsinki Heart Study (Helsinki cardiac studies) also confirms, as with medicine rising blood plasma HDL-C level, the male CHD incidence can descend 34%.Experimentation on animals also proves, animal such as the family that blood plasma HDL level is lower exempts from, monkey, pig etc. are all easily brought out AS, and the animal that opposite blood plasma HDL content is higher such as ermine, Beijing duck and tree shrew etc. then are difficult for even not producing AS.In addition, cell cultures proves that HDL can promote shifting out of cell (comprising arterial smooth muscle cell) inner cholesterol, and experimentation on animals proves that also HDL has the effect that the AS patch forms and the promotion patch disappears that suppresses.These illustrate that all HDL has the effect of antagonism AS.And, in the long process of prevention and treatment AS, has the incomparable advantage of other synthetic medicine because HDL is the natural drug that derives from human plasma.
HDL mainly is made up of lipophorin and lipid, is that density is the highest but form an extremely inhomogenous class lipoprotein in the blood plasma.The lipoprotein that HDL is made up of protein and lipid, it contains protein 50%～55%, mainly constitute by lipophorin (apo) AI and AII, other contains a spot of apoAIV, CI, CII, CIII, D, E and J, it is about 45%～50% that HDL contains lipid, mainly by phosphatide (PL, 40～60%), cholesterol and cholesteryl ester (total cholesterol TC, 30%～40%) and a small amount of triglyceride level (TG, about 10%) constitute.The content of apo AI is at most and again the main functional protein of HDL in HDL, therefore can reflect the content of HDL with the content of apo AI.
Still the blood plasma of having no way of at present directly prepares the method for HDL.
Summary of the invention
The object of the present invention is to provide a kind of human blood high density lipoprotein and products thereof.
Another object of the present invention provides the manufacture method of human blood high density lipoprotein and products thereof, and this method directly is raw material with blood plasma, preparation HDL.
A further object of the present invention provides above-mentioned human blood high density lipoprotein and products thereof in preparation treatment multiple organ dysfunction syndrome (MODS), cardiovascular system diseases, as AS and in and the application of acute symptom medicine aspect such as bacteriotoxin.
In the human blood high density lipoprotein of the present invention, apo AI content is 77-89%.
The apoAI molecular weight is 27.7KD in the above-mentioned human blood high density lipoprotein.
The human blood high density lipoprotein that can contain 0.1-99% in the human blood high density lipoprotein product of the present invention.
Above-mentioned human blood high density lipoprotein product can add 9～11% sucrose and/or 0.5% albumin and/or 150mg ± 1mg/L vitamins C, makes the human blood HDL preparation for venoclysis.
Above-mentioned HDL is not limited to HDL of the present invention, and HDL of the prior art is suitable for aforesaid combination interworking equally.
The method of the invention may further comprise the steps:
A. get raw blood plasma, dropping buffered soln is 5.0-5.5 to the pH of mixed solution, and 95% ethanol of adding mixed solution 1/4～1/3 volume stirred 1-3 hour down at-5～-2 ℃, left standstill 6-8 hour, the centrifugal supernatant that gets; Add the water for injection of 45-55% supernatant volume to supernatant, drip buffered soln, making pH of mixed is 4.5-6.0, stirs 1-3 hour down at-6～-3 ℃, leaves standstill 1-3 hour, and centrifugal must the precipitation abandoned supernatant;
B. precipitate 0.02～0.1M NaCl solution dissolving, behind accent pH to 5.2 ± 0.5, stirred 2-6 hour centrifugal then must the precipitation down at 0～2 ℃ with 2-3 times of weight; Precipitation is dissolved with 0.1～0.2M NaCl solution of 50-150 times of weight, and regulator solution pH is 7.2 ± 0.5, stirs 6-20 hour down at 2-8 ℃, filters to get filtrate;
C. above-mentioned filtrate adds damping fluid, and making pH is 5.2 ± 0.5, stirs 1-3 hour, under 2～4 ℃, leaves standstill centrifugal collecting precipitation 6-20 hour; Precipitate the 0.1～0.2M NaCl solution dissolving with 50～150 times of amounts, transfer pH to 7.2 ± 0.5, stirring is dissolved precipitation fully, filters, and gets filtrate;
D. filtrate is used the ultra-filtration membrane ultrafiltration, and desalination also concentrates, and must be rich in the solution of HDL, through aftertreatment, gets HDL and products thereof.
Regulate pH in the aforesaid method and use 1mol/LNaHCO
3Solution.
Damping fluid described in the aforesaid method is the HAC/NaAC buffer system of pH=4.Step C can come again after finishing as required, so that reach better purity.
Ultra-filtration membrane among the step D is the 8-70KD ultra-filtration membrane.Preferred 30～50KD ultra-filtration membrane that uses, it has the effect that is further purified.
The described aftertreatment of step D can be: ultrafiltration and concentration liquid adds 9～11% injection sucrose or 0.5% albumin, adds 150mg ± 1mg injection vitamins C by every liter, transfers pH to 7.2 ± 0.5, gets HDL solution; Above-mentioned HDL solution is 60 ± 1 ℃, 10 hours inactivation of viruses in the pasteurizing apparatus of microcomputer automatic control; HDL solution behind the inactivation of viruses, the degerming packing, and in 2～8 ℃ of preservations.
The inventive method prepares the F that human serum albumin and gamma-globulin discard with cold ethanol method
IV-1Be precipitated as raw material, under the condition of pH5.1 ± 0.5,0～2 ℃ and 0.03M NaCl, by washing other plasma proteins of fully removing beyond the HDL.Behind the HDL dissolving crude product again under pH5.1 ± 0.5 (iso-electric point), 2～4 ℃, the condition of 0.15M NaCl reprecipitation refining.The HDL solution of final purification through ultrafiltration, dialysis, concentrate, preparation, 60 ± 1 ℃, pH 6.4～7.4, apolipoprotein AI content 〉=0.5%, purity 〉=95% are made in 10 hours inactivation of viruses and degerming, contain the human blood HDL goods of 10% ± 1% sucrose.In the HDL preparation process, separate each step regulate suitable ethanol concn 6～30%, protein concentration 1～6%, the pH value 4.5～7.5, ionic strength in 0.02～2.0k Ω cm, temperature at-6～10 ℃.
The lipoprotein that HDL is made up of protein and lipid, it contains protein 50%～55%, mainly is made of lipophorin (apo) AI (65%～70%) and AII (20%～25%), and other contains a spot of apoAIV, CI, CII, CIII, D, E and J.It is about 45%～50% that HDL contains lipid, mainly is made of phosphatide (PL, 40～60%), cholesterol and cholesteryl ester (total cholesterol TC, 30%～40%) and a small amount of triglyceride level (TG, about 10%).The content of apoAI is at most and again the main functional protein of HDL in HDL.The content that therefore can reflect HDL with the content of apoAI.The contriver adopts the apolipoprotein AI detection kit (simple immunodiffusion method) of Huaxi Medical Univ lipophorin research department development, and detect apoAI content and represent HDL content with apoAI content in the goods, be qualified with apo AI content 〉=0.5%.
Discover, but have the acceptor of specific combination HDL on the cytolemma of liver cell, arterial smooth muscle cell, endotheliocyte etc.The contriver adopts the liver plasma membrane HDL acceptor and the HDL that are coated in advance on the enzyme plate that specific binding reaction takes place, with the anti-apoAIIgG of horseradish peroxidase-antibody linked thing HRP-the immunology association reaction taking place, detects the biological activity of HDL in the goods by being adsorbed on enzymatic color reaction in receptor-ligand (the HDL)-anti-ligand antibody-enzyme complex on the enzyme plate solid phase more at last.During biological activity determination, with 1 μ g apoAI as 1 HDL activity unit (U).Because HDL goods apoAI content 〉=0.5% promptly is equivalent to contain 5 * 10
5U HDL (theoretical value), thus the contriver with this value 80% as judging the qualified standard of goods, promptly the HDL product biological is active should 〉=4 * 10
5U.
The animal acute toxicity test of HDL product of the present invention: test-results shows that HDL is with intravenous injection and two kinds of administrations of abdominal injection, with maximum concentration (1%) in extracting as maximum administration concentration, with intravenous injection and intraperitoneal injection maximum volume 0.8ml/20g (body weight) administration, successive administration is 3 times in the 12h, administration was observed 7 days continuously, animal is all no abnormal, none death of animal after 7 days is so HDL intravenous injection and intraperitoneal injection mouse maximum tolerated dose are 0.024g/20g (body weight).
The stability test of HDL product of the present invention: according to " new drug (Western medicine) the preclinical study governing principle compilation (pharmacy pharmacology toxicology) " of establishment bureau of drug administration of Ministry of Health of the People's Republic of China in July, 1993 to human blood HDL goods carried out the influence factor test, room temperature (25 ℃) kept sample and investigates and 2～8 ℃ of investigations that keep sample in 14 months in 5 months.The result shows, HDL investigates sample can tolerate the influence that condition changes in the field of circulation and the use, the storage requirement of goods should be strict controlled in preservation under 2～8 ℃ and the lucifuge, can guarantee like this that in 1 year every quality standard that the HDL liquid preparation keeps " Chinese biological goods rules " to require is stablized constant in circulation and use.
The HDL product of the present invention Pharmacodynamic test of active extract relevant: in order to verify the anti-AS effect of inventor's blood plasma HDL goods with therapeutic action, the contriver raises with high fat and causes rabbit As model, when high fat is raised (experimental As prophylactic tria) and high fat raise 10 weeks caused the AS model after (experimental AS therapeutic test), divide low (L), in (M), high (H) three dosage groups (10,50,100mg is in apoAI content) intravenous injection human plasma HDL preparation is (once in a week, amounted to for 10 weeks), lipid and lipid peroxide level from animal subject blood, arterial wall, the liver lipid content, the anti-AS effect of examination of aspects such as gall bladder bile lipid content and arterial wall AS plaque area and checking HDL preparation.
Owing to do not have the anti-AS medicine identical or similar, so the contriver selects lipid regulating agent Fino shellfish specially for positive control drug for use to the HDL mechanism of action.In the selection of negative control, because research has confirmed immunologic injury, the animal pattern that particularly high fat is raised infusion foreign protein repeatedly can quicken generation and the development of AS because of immune response takes place, and the animal experiment of human plasma HDL pharmacodynamics will cause anaphylaxis and the immune response of animal because of injection people HDL foreign protein inevitably, increase the weight of animal pattern AS generation and development that high fat is raised on the contrary, in order to eliminate the influence of foreign protein to the anti-AS effect in rabbit AS animal pattern of observer's blood plasma HDL preparation, the contriver is except that high fat contrast (C) group that injecting normal saline is set, also be provided with HDL in the suitable Plasbumin-25's negative control (A) of dosage group dosage group, the result shows, prevents and treats human AS disease with human plasma HDL preparation the foreign protein reaction can not take place.
It is as follows to test main result:
(1) result of blood fat analysis shows: human plasma HDL preparation does not have AS animal pattern (experimental AS prevention and the therapeutic test) blood fat (effect that cholesterol TC, triglyceride level (TG) and phosphatide (PL) level reduce that high fat is raised.Therefore, the anti-AS effect of HDL does not realize by blood fat reducing (TC, TG) content.
(2) lipid peroxide concentration is measured found that of (experimental AS prophylactic tria), injection HDL preparation, basic, normal, high (L, M, H) three dosage group lipid peroxide concentration level (3.9,2.5,3.2mmol/ml) all significantly reduces (P＜0.05 than control group level (5.4 and 5.0mmol/ml), P＜0.01 and P＜0.001), shows that human plasma HDL preparation has the effect of the rabbit anteserum lipid generation oxidation of the high fat raising of antagonism.
(3) animal pattern thoracic aorta lipid and AS plaque area are measured and are found, rabbit arterial wall lipid content and AS plaque area that high fat is raised significantly increase, and the most serious with negative control (A) treated animal.Each the treated animal arterial wall lipid of injection HDL and AS plaque area reduce or significantly reduce, and be wherein obvious with the middle and high dosage of HDL (M, H) group.For example: prophylactic tria M, H treated animal arterial wall TC, TG content and AS plaque area are respectively 33.8,35.0,28.1,23.7mg/g stem organization and 38.4% (M group), all significantly reduce (P＜0.05) than A treated animal 54.7,38.5mg/g stem organization and 57.6%; The arterial wall lipid assay result of therapeutic test is similar to prophylactic tria, and its L, M, H group AS plaque area are respectively 60.2%, 58.4% and 53.7% than the 79.6% significantly minimizing (P＜0.01 and P＜0.001) of A group AS plaque area.
(4) liver lipid assay result is similar substantially to arterial wall lipid assay result.Prophylactic tria animal livers TC, TG content, H group are 135.8 and 44.0mg/g stem organization, with the apparent in view reductions of 192.1 and 72.8 (mg/g stem organizations) (P＜0.05) of A group; The result of therapeutic test is more outstanding, and L, M, H treated animal liver TC, TG and PL content all obviously reduce (P＜0.05 and P＜0.01) than the A group.
(5) bile lipid content measurement result is opposite with arterial wall and liver lipid content, no matter is AS prevention or therapeutic test, and each treated animal and its bile lipid content of control group comparison of injection HDL are increase trend, and the most obvious with the result of prophylactic tria.AS prophylactic tria H group TC content (337.5mg%, promptly every 100ml contains 337.5mg, down with) be significantly higher than C group (217.2mg%), M and H group PL content (978.7 and 1008.8mg%) are significantly higher than A group (688.3mg%, P＜0.05).
The above results shows that injection human plasma HDL has the effect that As animal pattern arterial wall, liver lipidosis reduce, the discharge of bile lipid increases and arterial wall AS plaque area reduces of adopting high fat to raise that makes.
HDL of the present invention exempts to inject the HDL preparation by giving the hyperlipidemia model man, the anti-AS effect of direct viewing HDL, the experimental result that obtains shows, no matter in high fat moulding, or high fat causes after the rabbit AS model, injection rabbit blood HDL all can make the sedimentary lipid of moulding animal arterial wall and liver, arterial wall AS plaque area significantly reduce, and confirms fully that HDL has to suppress AS and take place and impel AS to reverse or the dual function of disappear (promptly prevent and treat).Therefore HDL of the present invention has good application prospects aspect the new drug of preparation prevention and treatment AS.
Bacteriotoxin mainly is divided into extracellular toxin (exotoxin) and intracellular toxin (endotoxin); The important pathogenic bacteria of some harm humans health such as the extracellular toxin of bacteriums such as diphtheria and tetanus along with its toxinicide and anatoxic succeeding in developing in succession and effectively application, no longer make the people turn pale at the mention of the tiger; Nowadays bacteriotoxic fight has been focused on intracellular toxin.
Medical circle is being perplexed in antiendotoxin treatment at endotoxemia always.Although adopted the organ support and the Intensive Care Therapy technology that become better and approaching perfection day by day, and new drugs such as antiendotoxin antibody, anti-inflammatory cytokines (inflammatory mediator) antibody or inflammatory cytokine receptor antagonist, but systemic inflammatory response syndrome due to the endotoxemia that the severe gram positive bacterial infection causes (SIRS) and resultant multiple organs dysfunction syndromes (MODS), its case fatality rate are but high always.Therefore, intracellular toxin still is a big difficult medical problem.
Intracellular toxin is engulfed, is degraded and remove in that machine is intravital, depends on immune playing an active part in; In addition, vital role has also been brought into play in the detoxification of the perfect internal toxin of detoxifcation mechanism in the body.The immunity of body and detoxification system were both overlapped in many instances or connect difference mutually again each other.After intracellular toxin enters human body, lipoprotein in the blood, particularly the protein ingredient of participation lipid exchange such as high-density lipoprotein (HDL) and transhipment gives the neutralization buffer effect to intracellular toxin with standing in the breach, the neutrophil leucocyte of blood discharges some cationic protein (as bactericidal power/permeability increasing protein) intracellular toxin is given the detoxification unzipping, the alkaline phosphatase that extensively distributes in the tissue also can give dephosphorylation to intracellular toxin, reticuloendothelial system is then given intracellular toxin and is engulfed Degradation, forms body thus to endotoxic detoxification processes.So lipoprotein, particularly high-density lipoprotein (HDL) have the effect of protection body function of detoxification, it has good application prospects in preparation prevention and treatment and aspect bacteriotoxin and the multiple organ dysfunction syndrome MODS new drug.
The present invention directly is raw material with the human plasma, on the basis of a large amount of experiments, pass through purification procedures, therefrom extract the HDL goods, the cold ethanol new process of mass preparation human plasma HDL is provided, and successfully prepared safety, stable, nontoxic, pyrogen-free, aseptic and virus-free pollution, pH 6.4～7.4, apolipoprotein AI content 〉=0.5%, HDL purity 〉=95%, the human blood HDL preparation that contains 10% ± 1% sucrose for venoclysis, lipid and lipophorin are formed and content analysis shows, the lipid of HDL goods and lipophorin composition and content and natural HDL value basically identical, and do not contain low-density lipoprotein (LDL) and the vldl (VLDL) that can impel atherosclerosis to take place.
Description of drawings
Fig. 1 is the precipitation preparation process synoptic diagram that is rich in HDL
E: alcohol concn
T: temperature
Fig. 2 is a HDL process for refining flow process
Fig. 3 is HDL immunoelectrophoresis result, wherein:
1. human normal plasma
2. goat-anti people apoAI antiserum(antisera)
3.HDL goods
Fig. 4 is HDL lipoprotein agarose gel electrophoresis result, wherein:
1. human normal plasma
2.HDL goods
Fig. 5 is HDL preparation SDS-PAGE result, wherein:
1,2,4,5,6,7 is the HDL goods
3 is molecular weight standard
Fig. 6 is HDL bioactive enzyme connection immunodetection program
Fig. 7 is a human blood HDL biological activity typical curve, wherein
■—■：HDL
●—●：LDL
▲—▲：VLDL
Embodiment
Embodiment 1
Get raw blood plasma 1000L, drip the HAC/NaAC buffered soln 25L of pH=4.0, adjust the pH=5.2 of mixed solution, add 95% ethanol 324L, stirred 2 hours down at-5 ℃, left standstill 6 hours, use 142 type continuous centrifuges, centrifugal under 14000r/min, get supernatant 1340L, precipitation 50Kg;
Add water for injection 630L to supernatant, drip pH and be 4.0 HAC/NaAC and palm solution 13.7L off as, making pH of mixed is 4.5, and this moment, protein concentration was 1%, and ionic strength is 0.05, stirred 2 hours down at-3 ℃, left standstill 2 hours, and used 142 type continuous centrifuges, centrifugal under 14000r/min, must precipitate 9Kg, abandon supernatant;
Add 30mmol/L sodium chloride solution 19.8L in precipitation, drip pH and be 4.0 HAC/NaAC and palm solution 14L off as, making pH is 5.5, stirs 4 hours down at 0 ℃, uses 142 type continuous centrifuges, centrifugal under 14000r/min, abandons supernatant, must precipitate 4.65Kg;
In precipitation, add 160mmol/L sodium-chlor 465L, drip 1mol/L NaHCO
3Solution 378ml makes pH of mixed=6.98, stirs 6 hours down at 5 ℃, filters, and gets filtrate 577L;
Drip the HAC/NaAC buffered soln 109mL of pH=4.0 in filtrate, making pH of mixed is 5.16, stirs 1 hour down at 3 ℃, leaves standstill 6 hours, centrifugal under 14000r/min with 142 type continuous centrifuges, abandons supernatant, must precipitate 3.86Kg;
Add 160mmol/L sodium chloride solution 386L in precipitation, this moment, lysate pH was 5.15, dripped 1mol/LNaHCO
3Solution 347ml, making lysate pH is 7.20, stirs 6 hours down at 5 ℃, filters, and gets filtrate 495L;
Use the ultrafiltration of 8-10KD ultra-filtration membrane, concentrate, get concentrated solution 350L, add 35Kg sucrose in the concentrated solution, protein concentration is 18.5g/L, and ethanol content is 0.02%, and specific conductivity is 0.4Ms; 60 ± 1 ℃, 10 hours inactivation of viruses in the pasteurizing apparatus of microcomputer automatic control; HDL behind the inactivation of viruses, apo AI content is 89%, degerming packing finished product 50ml * 6890 bottles, and in 2 ℃ of preservations.
Get raw blood plasma 1000L, drip the HAC/NaAC buffered soln 24.5L of pH=4.0, adjust the pH=5.15 of mixed solution, add 95% ethanol 330L, stirred 2 hours down at-4 ℃, left standstill 8 hours, use 142 type continuous centrifuges, centrifugal under 14000r/min, get supernatant 1270L, precipitation 47Kg;
Add water for injection 646L to supernatant, drip pH and be 4.0 HAC/NaAC and palm solution 14.3L off as, making pH of mixed is 5.2, and this moment, protein concentration was 1.68%, and ionic strength is 0.07, stirred 2 hours down at-6 ℃, left standstill 2 hours, and used 142 type continuous centrifuges, centrifugal under 14000r/min, must precipitate 9.5Kg, abandon supernatant;
Add 30mmol/L sodium chloride solution 19.0L in precipitation, drip pH and be 4.0 HAC/NaAC and palm solution 13.7L off as, making pH is 5.7, stirs 4 hours down at 1 ℃, uses 142 type continuous centrifuges, centrifugal under 14000r/min, abandons supernatant, must precipitate 4.33Kg;
In precipitation, add 160mmol/L sodium-chlor 433L, drip 1mol/LNaHCO
3Solution 387ml makes pH of mixed=7.08, stirs 6 hours down at 6 ℃, filters, and gets filtrate 530L;
Drip the HAC/NaAC buffered soln 115mL of pH=4.0 in filtrate, making pH of mixed is 5.2, stirs 1 hour down at 2 ℃, leaves standstill 6 hours, centrifugal under 14000r/min with 142 type continuous centrifuges, abandons supernatant, must precipitate 3.83Kg;
Add 160mmol/L sodium chloride solution 383L in precipitation, this moment, lysate pH was 5.19, dripped 1mol/LNaHCO
3Solution 368ml, making lysate pH is 7.00, stirs 5 hours down at 5.5 ℃, filters, and gets filtrate 480L;
Use the ultrafiltration of 8-10KD ultra-filtration membrane, concentrate, get concentrated solution 367L, add 36.7Kg sucrose in the concentrated solution, protein concentration is 19g/L, and ethanol content is 0.01%, and specific conductivity is 0.2Ms; 60 ± 1 ℃, 10 hours inactivation of viruses in the pasteurizing apparatus of microcomputer automatic control; HDL behind the inactivation of viruses, apo AI content is 85%, degerming packing finished product 50ml * 7250 bottles, and in 2 ℃ of preservations.
Embodiment 3
Get raw blood plasma 1000L, drip the HAC/NaAC buffered soln 24.3L of pH=4.0, adjust the pH=5.22 of mixed solution, add 95% ethanol 304L, stirred 2 hours down at-5 ℃, left standstill 6 hours, use 142 type continuous centrifuges, centrifugal under 14000r/min, get supernatant 1299L, precipitation 45Kg;
Add water for injection 611L to supernatant, drip pH and be 4.0 HAC/NaAC and palm solution 14L off as, making pH of mixed is 5.1, and this moment, protein concentration was 1.68%, and ionic strength is 0.07, stirred 2 hours down at-6 ℃, left standstill 2 hours, and used 142 type continuous centrifuges, centrifugal under 14000r/min, must precipitate 8.9Kg, abandon supernatant;
Add 30mmol/L sodium chloride solution 18.9L in precipitation, drip pH and be 4.0 HAC/NaAC and palm solution 13.6L off as, making pH is 4.9, stirs 4 hours down at 0 ℃, uses 142 type continuous centrifuges, centrifugal under 14000r/min, abandons supernatant, must precipitate 4.05Kg;
In precipitation, add 160mmol/L sodium-chlor 405L, drip 1mol/LNaHCO
3Solution 375ml makes pH of mixed=6.99, stirs 6 hours down at 8 ℃, filters, and gets filtrate 570L;
Drip the HAC/NaAC buffered soln 130mL of pH=4.0 in filtrate, making pH of mixed is 5.22, stirs 1 hour down at 2 ℃, leaves standstill 6 hours, centrifugal under 14000r/min with 142 type continuous centrifuges, abandons supernatant, must precipitate 2.87Kg;
Add 160mmol/L sodium chloride solution 287L in precipitation, this moment, lysate pH was 5.11, dripped 1mol/LNaHCO
3Solution 340ml, making lysate pH is 7.01, stirs 8 hours down at 5 ℃, filters, and gets filtrate 500L;
Use the ultrafiltration of 8-10KD ultra-filtration membrane, concentrate, get concentrated solution 378L, add 37.8Kg sucrose in the concentrated solution, protein concentration is 18g/L, and ethanol content is 0.01%, and specific conductivity is 0.3Ms; 60 ± 1 ℃, 10 hours inactivation of viruses in the pasteurizing apparatus of microcomputer automatic control; HDL behind the inactivation of viruses, apoAI content are 84%, degerming packing finished product 50ml * 7460 bottles, and in 2 ℃ of preservations.
Embodiment 4
Get raw blood plasma 1000L, drip the HAC/NaAC buffered soln 25L of pH=4.0, adjust the pH=5.25 of mixed solution, add 95% ethanol 320L, stirred 2 hours down at-5 ℃, left standstill 8 hours, use 142 type continuous centrifuges, centrifugal under 14000r/min, get supernatant 1380L, precipitation 50Kg;
Add water for injection 648L to supernatant, drip pH and be 4.0 HAC/NaAC and palm solution 14.3L off as, making pH of mixed is 4.55, and this moment, protein concentration was 1.88%, and ionic strength is 0.15, stirred 2 hours down at-6 ℃, left standstill 2 hours, and used 142 type continuous centrifuges, centrifugal under 14000r/min, must precipitate 9.9Kg, abandon supernatant;
Add 30mmol/L sodium chloride solution 19.8L in precipitation, drip pH and be 4.0 HAC/NaAC and palm solution 15L off as, making pH is 5.0, stirs 4 hours down at 2 ℃, uses 142 type continuous centrifuges, centrifugal under 14000r/min, abandons supernatant, must precipitate 4.32Kg;
In precipitation, add 160mmol/L sodium-chlor 432L, drip 1mol/L NaHCO
3Solution 414ml makes pH of mixed=7.12, stirs 6 hours down at 7 ℃, filters, and gets filtrate 550L;
Drip the HAC/NaAC buffered soln 116mL of pH=4.0 in filtrate, making pH of mixed is 5.12, stirs 1 hour down at 2 ℃, leaves standstill 6 hours, centrifugal under 14000r/min with 142 type continuous centrifuges, abandons supernatant, must precipitate 3.12Kg;
Add 160mmol/L sodium chloride solution 312L in precipitation, this moment, lysate pH was 5.14, dripped 1mol/LNaHCO
3Solution 362ml, making lysate pH is 7.05, stirs 8 hours down at 8 ℃, filters, and gets filtrate 489L;
Use the ultrafiltration of 8-10KD ultra-filtration membrane, concentrate, get concentrated solution 372L, add 37.2Kg sucrose in the concentrated solution, protein concentration is 19.5g/L, and ethanol content is 0.02%, and specific conductivity is 0.2Ms; 60 ± 1 ℃, 10 hours inactivation of viruses in the pasteurizing apparatus of microcomputer automatic control; HDL behind the inactivation of viruses, apo AI content is 79%, degerming packing finished product 50ml * 7240 bottles, and in 2 ℃ of preservations.
Embodiment 5
Get raw blood plasma 1000L, drip the HAC/NaAC buffered soln 24.3L of pH=4.0, adjust the pH=5.15 of mixed solution, add 95% ethanol 331L, stirred 2 hours down at-5 ℃, left standstill 7 hours, use 142 type continuous centrifuges, centrifugal under 14000r/min, get supernatant 1430L, precipitation 57Kg;
Add water for injection 672L to supernatant, drip pH and be 4.0 HAC/NaAC and palm solution 14.3L off as, making pH of mixed is 5.7, and this moment, protein concentration was 2%, and ionic strength is 0.15, stirred 2 hours down at-6 ℃, left standstill 2 hours, and used 142 type continuous centrifuges, centrifugal under 14000r/min, must precipitate 10.4Kg, abandon supernatant;
Add 30mmol/L sodium chloride solution 20.8L in precipitation, drip pH and be 4.0 HAC/NaAC and palm solution 15.1L off as, making pH is 5.39, stirs 4 hours down at 2 ℃, uses 142 type continuous centrifuges, centrifugal under 14000r/min, abandons supernatant, must precipitate 3.98Kg;
In precipitation, add 160mmol/L sodium-chlor 398L, drip 1mol/LNaHCO
3Solution 395ml makes pH of mixed=7.1, stirs 6 hours down at 8 ℃, filters, and gets filtrate 576L;
Drip the HAC/NaAC buffered soln 128mL of pH=4.0 in filtrate, making pH of mixed is 5.16, stirs 1 hour down at 2 ℃, leaves standstill 6 hours, centrifugal under 14000r/min with 142 type continuous centrifuges, abandons supernatant, must precipitate 3.69Kg;
Add 160mmol/L sodium chloride solution 369L in precipitation, this moment, lysate pH was 5.15, dripped 1mol/LNaHCO
3Solution 365ml, making lysate pH is 7.00, stirs 6.5 hours down at 8 ℃, filters, and gets filtrate 560L;
Use the ultrafiltration of 8-10KD ultra-filtration membrane, concentrate, get concentrated solution 400L, add 40Kg sucrose in the concentrated solution, protein concentration is 19.3g/L, and ethanol content is 0.01%, and specific conductivity is 0.4Ms; 60 ± 1 ℃, 10 hours inactivation of viruses in the pasteurizing apparatus of microcomputer automatic control; HDL behind the inactivation of viruses, apo AI content is 77%, degerming packing finished product 50ml * 7910 bottles, and in 2 ℃ of preservations.
Experimental example 1
This experimental example is the calibrating data of product HDL of the present invention.
1, method
1.1 the evaluation of HDL and purity check: adopt immunoelectrophoresis and lipoprotein agarose gel electrophoresis to identify the HDL of preparation, and according to lipoprotein agarose gel electrophoresis its purity of scanning analysis as a result.
1.2 protein content determination: the improvement Lowry method of employing Markwell etc. is measured.
1.3 lipid and lipophorin assay: triglyceride level among the HDL (TG) and total cholesterol (TC) adopt Beijing Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd. enzyme process kit measurement, and phosphatide (PL) adopts improvement xitix reduction method to measure.Lipophorin (apo) AI, AII, B100, CII, CIII, E adopt Sichuan University West China lipophorin research department immunity unidirectional diffusion kit measurement.
1.4 apoAI molecular weight determination: adopt the SDS-PAGE method to measure.
1.5 HDL biological activity determination: adopt the human blood HDL biological activity determination method of foundation such as Xu Yan China to measure.
By per 1 μ g apoAI is 1 HDL activity unit (U).
1.6 sterility test: undertaken by the Pharmacopoeia of the People's Republic of China three appendix of version in 2005 " sterility test method ".
1.7 bacterial endotoxin inspection: undertaken by the Pharmacopoeia of the People's Republic of China 2005 three appendix of version " bacterial endotoxins test ".
1.8 pyrogen test: undertaken by the Pharmacopoeia of the People's Republic of China 2005 three appendix of version " pyrogen test ".
1.9 undue toxicity inspection: undertaken by the Pharmacopoeia of the People's Republic of China three appendix of version in 2005 " undue toxicity test procedure "
2, result
2.1 the evaluation of HDL and purity check
(1) immunoelectrophoresis is seen Fig. 3; As seen from Figure 3, HDL goods and human normal plasma precipitation line occurs in same position.
(2) the lipoprotein agarose gel electrophoresis is seen Fig. 4; As seen from Figure 4, the HDL goods present Yi Tiao district band in the lipoprotein agarose gel electrophoresis, Biorad gel imaging scanning system scanning analysis, and its purity is 100%.
2.2 lipid and lipophorin are formed (%) among the HDL, see the following form; By following table as seen, HDL goods apoAI content of the present invention is higher.
| The HDL goods | The HDL literature value | |||
| Content (mg/dl) | Percentage composition (%) | Percentage composition (%) | ||
| Lipid | ?TG | ?86.9 | ?11 | ?10 |
| ?TC | ?251.1 | ?33 | ?40 | |
| ?PL | ?424.1 | ?56 | ?50 | |
| Lipophorin | ?ApoAI | ?1300.2 | ?89 | ?65-70 |
| ?ApoAII | ?121.0 | ?8 | ?20-25 | |
| ?ApoB100 | Do not detect | ?- | ?- | |
| ?ApoCII | ?15.6 | ?1 | ?1 | |
| ?ApoCIII | ?28.8 | ?2 | ?4 | |
| ?apoE | ?6.8 | ?1 | ?2 | |
2.3 the apoAI molecular weight determination is seen Fig. 5, its molecular weight is 27.7KD;
2.4 HDL preparation biological activity determination sees the following form:
| Embodiment | The pH value | ApoAI content (mg/dL) | Biological activity (U/ml) |
| ????1 | ?6.96 | ????1289 | ????13750 |
| ????2 | ?6.45 | ????1274 | ????12500 |
| ????3 | ?6.41 | ????1304 | ????13750 |
| ????4 | ?6.60 | ????1297 | ????13125 |
| ????5 | ?6.40 | ????1312 | ????13000 |
Experimental example 2
This experimental example is the research of the anti-AS biological activity determination method of human blood HDL, thes contents are as follows:
1, material and reagent
Japan large ear rabbit; The human normal plasma picks up from the blood donor; Human plasma HDL preparation is by the method for the invention preparation; HRP (RZ=3.0), Sigma company product; Purified rabbit liver plasma membrane, goat-anti people apoAI IgG, the anti-apoAI IgG of HRP-, people LDL and VLDL, self-control; TG, TC enzymic measuring reagent box, Beijing Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd.; Apolipoprotein AI, B100, CII, CIII and E immunity unidirectional diffusion test kit, Sichuan University West China lipophorin research department; All the other reagent are homemade analytical pure.
96 hole polystyrene enzyme plates, Corning company product; Model 550 type microplate reader, Bio-RAD company product; L8-55 type ultracentrifuge, Beckman company; The Sebris scanner, Sebris company product.
It is pH 9.6 that bag is cushioned liquid, 50mmol/LNa
2CO
3-NaHCO
3Damping fluid; Washing and dilution buffer liquid are pH 7.4, contain the 20mmol/L PBS of 0.05%Tween-20; Enzyme reaction matrix liquid is pH 5.6, contains 0.04% O-Phenylene Diamine, 0.05%H
2O
20.1mol/L citric acid-Na
2HPO
4Damping fluid (face and use preceding preparation).
2, method
2.1 the preparation of rabbit hepatocyte film
By sucrose density gradient ultracentrifugation method purified rabbit liver plasma membrane.Collect 37%～41% sucrose density interfacial film composition, with damping fluid (50mmol/L Tris-HCl, 100mmol/L NaCl, 0.5mmol/L CaCl
2, pH7.5) after the dilution, in 27000 * g centrifugal 20 minutes.Collecting precipitation adds a small amount of damping fluid, handles 2 * 10 seconds amplitude 14 in ice bath with MSE-150 type ultrasonoscope.The liver plasma membrane of preparation is sub-packed in-70 ℃ of preservations.The film productive rate is the wet hepatic tissue of 1.5～2.0mg/g, with liver homogenate relatively, 5 '-the phosphonuclease vigor improves 6～10 times.
2.2 LDL and VLDL preparation
Collect the LDL component of d=1.030～1.050g/mL and the VLDL component of d=0.95～1.006g/mL by disposable density gradient centrifugation separation of human plasma lipoprotein, to 10mmol/L Tris-HCl, 1mmol/L EDTA, 50mmol/L NaCl, the damping fluid dialysis desalting of pH7.4,4 ℃ of preservations are standby.
Membranin and lipoprotein are all measured protein content with the method for Markwell etc.
2.3 the processing of HDL (embodiment 2)
The human blood HDL preparation that obtains with the embodiment of the invention 2 is a raw material, presses ultracentrifugation preparations such as Havel behind the ultrafiltration and concentration.The HDL preparation transfers density to 1.21g/mL with solid NaBr during preparation, and is centrifugal 24 hours in L8-55 type ultracentrifuge 50000rpm, 10 ℃.Collect upper strata d≤1.21g/mL component, dialysis, the degerming packing, 4 ℃ of preservations are standby.
HDL (embodiment 2) TG and TC content adopt the enzyme process kit measurement; Phosphatide (PL) adopts the xitix reduction method to measure; ApoAI, AII, B100, CII, CIII and E content adopt immune unidirectional diffusion kit measurement.
2.4 HDL biological activity assay test
2.4.1 determination step
Undertaken by HDL acceptor enzyme linked immunosorbent detection method program, see Fig. 6;
During mensuration, the bag of membranin is by the extent of dilution of concentration and the anti-apoAI IgG of HRP-, determines to be respectively 25 μ g membranin/ml and 1: 250 by the chessboard volumetry.
2.4.2 HDL biological activity typical curve
HDL (embodiment 2) with preparation is a standard, by per 1 μ g apoAI is 1 HDL activity unit (U), the HDL reference material is become 80,40,20,10 and 5 (U/mL) with diluted, measure by above-mentioned steps again, draw the bioactive typical curve of absorbance value and HDL, and obtain corresponding double-log regression equation.
2.4.3 sample determination
After the testing sample dilution, in the same enzyme plate of production standard curve, measure its absorbance value.The last activity unit of calculating the HDL preparation with the regression equation and the diluted sample multiple of typical curve.
3, result
3.1 the evaluation of HDL (embodiment 2)
3.1.1 HDL (embodiment 2) lipid and lipophorin are formed, and see the following form:
| HDL | Lipid (%) | Lipophorin (%) | |||||||||
| TC | ?TG | ?PL | ?AI | ?AII | ?B100 | ?CI | ?CII | ?CIII | ?D | ? | |
| Embodiment | |||||||||||
| 2 | 33 | ?12 | ?55 | ?85 | ?8 | Do not detect | Undetermined | ?2 | ?4 | Undetermined | ?0.4 |
| Literature value | 40 | ?10 | ?50 | ?65-70 | ?20-25 | - | ?6 | ?1 | 4 | 3 | ?2 |
As seen from the above table, HDL (embodiment 2) apoAI content is higher than the HDL literature value.
Present Yi Tiao district band 3.1.2 lipoprotein dyes electrophoresis: HDL (embodiment 2) in advance, the Sebris scanner scanning is analyzed, and its purity is 99.8%.
3.1.3 immunoelectrophoresis: see Fig. 3.HDL (embodiment 2) occurs precipitation line with normal human serum in same position.
3.2 HDL biological activity typical curve: the HDL typical curve is the result show, the logarithm of 492nm place absorbance value and HDL activity unit number is in linear relation in 1～16 activity unit's scope, sees Fig. 7.
3.3 the specificity test: as seen from Figure 7, with the enzyme plate of liver plasma membrane bag quilt, the people LDL of purifying and VLDL show people LDL and VLDL and enzyme-antibody linked thing no cross reaction to the enzyme-uncontested property of antibody linked thing effect.
3.4 replica test:
3.4.1 repeatability in the plate: same HDL preparation, dilute 250 and 500 times respectively, record the plate within variance coefficient and be respectively 7.6% and 9.5% (n=10).
3.4.2 repeatability between plate: three enzyme plates are measured same HDL preparation (250 times of dilutions) respectively, have surveyed in 3 days, record that the variation coefficient is 12.3% between plate.
Experimental example 3
This experimental example is the stability test of the anti-AS biological activity of HDL preparation.
At 2～8 ℃, 25 ℃ and the 40 ℃ HDL preparations of placing embodiment 1 preparation respectively, sampling regularly, and measure its pH value, apoAI content and anti-AS biological activity, observe storage temperature and shelf-time to the bioactive influence of the anti-AS of HDL preparation.The results are shown in following table:
| Laying temperature | Storage period | The pH value | ApoAI content (g/dL) | Biological activity (U/ml) |
| ????2-8℃ | 0 month | ????6.96 | ????0.529 | ????6875 |
| January | ????6.45 | ????0.537 | ????6750 | |
| March | ????6.41 | ????0.549 | ????7000 | |
| June | ????6.60 | ????0.519 | ????6750 | |
| September | ????6.40 | ????0.533 | ????6500 | |
| December | ????6.43 | ????0.536 | ????5250 | |
| 18 months | ????6.23 | ????0.533 | ????6750 | |
| ????25℃ | 0 month | ????6.96 | ????0.520 | ????6500 |
| January | ????6.44 | ????0.519 | ????6917 | |
| February | ????6.46 | ????0.509 | ????5908 | |
| March | ????6.40 | ????0.519 | ????7333 | |
| April | ????6.41 | ????0.518 | ????7292 | |
| May | ????6.47 | ????0.522 | ????6375 | |
| ????40℃ | 0 month | ????6.64 | ????0.569 | ????6500 |
| January | ????6.47 | ????0.506 | ????750 | |
| February | ????6.43 | ????0.510 | ????125 | |
| March | ????6.27 | ????0.524 | ????125 |
As seen from the above table, placement was placed 5 months down in 18 months and 25 ℃ under 2～8 ℃, and the anti-AS biological activity of HDL preparation does not all have considerable change, and placement is in the time of one month down at 40 ℃, and its anti-AS biological activity is obviously to reduce; Show that under cold condition (2～8 ℃), the anti-AS biological activity of HDL preparation can keep stable for a long time, and under high temperature (40 ℃) condition, the anti-AS biological activity of HDL preparation can descend obviously; So the suitable envrionment temperature of HDL preparation is 2-8 ℃.
Experimental example 4
This experimental example explanation HDL preparation of the present invention has the effect to treating endotoxemia.
Get normal 200 rabbits, random packet, is divided into experimental group and control group by 100 every group.
Two groups of rabbit injection intracellular toxins (2ng/kg) are caused endotoxemia.
Injecting intracellular toxin preceding 3.5 hours, and giving experimental group intravenous drip embodiment the HDL preparation (10mg/kg is in apoAI content) of 3 preparations earlier, dripping off in 30 minutes.
Find after 10 hours that the release of inflammatory mediators such as experimental group TNF-α, IL-6 is less, and experimental group rabbit infection symptoms is lighter, shows that intravenous drip HDL has the effect to treating endotoxemia, sees the following form:
| Group | The example number | ????IL-6(ng/L) | ?????TNF-α(ng/L) |
| Control group | ????100 | ????280.36±57.29 | ????501.82±99.08 |
| Experimental group | ????100 | ????82.18±7.63 | ????155.61±20.06 |
Compare with control group: P＜0.05
This experimental example confirms that fully HDL preparation of the present invention has the effect to treating endotoxemia.Inventor research thinks that HDL divides the phospholipid layer of subshell to combine closely with the lipoid A in the intracellular toxin molecule, in conjunction with after intracellular toxin no longer with the CD14 receptors bind, thereby blocked intracellular toxin and stimulated Monocytes release inflammatory mediator.
Experimental example 5
This experimental example explanation HDL treatment can effectively reduce multiple organs dysfunction syndromes (MODS) patient inflammatory mediator.
1, purpose: relatively HDL treatment, equipment for continuous renal replacement therapy (CRRT) are to the result of treatment of MODS patient's inflammatory mediator (interleukin-1 ' beta ', interleukin-6 and tumor necrosis factor-alpha etc.).
2, method: choose critical illness patient 40 examples that show as MODS, the MODS diagnosis meets the Case definition that U.S. critical illness association formulated in 1992.The patient is divided into experimental group and control group at random by the sequencing of being admitted to hospital, and odd numbers is an experimental group, 20 examples, and experimental group patient accepts the HDL treatment; Even numbers is a control group, and 20 examples are accepted equipment for continuous renal replacement therapy (CRRT) treatment.Two groups reached treatment at that time at diagnosis MODS respectively and drew blood to be checked after 24 hours.
3, measure: (put exempt from medicine box put available from Science and Technology Development Center of Chinese People's Liberation Army General Hospital exempt from institute) interleukin-1 ' beta ' and interleukin-6 (IL-1 β, IL-6) are measured: extracting vein blood 2ml, after waiting in the injecting tube to solidify, separation of serum, put-20 ℃ of cryopreservation, adopt null readings once to monitor; Tumor necrosis factor-alpha (TNF-α): adopt venous blood 2ml, make antithrombotics, after the separated plasma, put-20 ℃ of cryopreservation, adopt the liquid phase competition law once to monitor with the 10%EDTA disodium.
4, result: after 24 hours, interleukin-1 ' beta ', interleukin-6 and tumor necrosis factor-alpha are starkly lower than control group to experimental group patient through the HDL treatment.See the following form:
| Group | The example number | Time/h | ????IL-1β(ng/L) | ????IL-6(ng/L) | ????TNF-α(ng/L) |
| Control group | ????20 | ????1 | ????20.31±1.34 | ????9.47±1.14 | ????112.62±8.22 |
| ????24 | ????76.11±79.32 | ????80.66±34.27 | ????105.91±136.14 | ||
| Experimental group | ????20 | ????1 | ????19.56±3.09 | ????12.35±4.95 | ????69.77±6.62 |
| ????24 | ????27.72±5.26 | ????33.99±5.74 | ????70.49±4.65 |
Compare with control group: P＜0.01
Systemic inflammatory response (SIR) is the pathogenetic basis of MODS, when body infected or serious blow such as wound after bacterium/toxin or tissue injury will stimulate inflammatory cells such as body scavenger cell, discharge inflammatory mediator (tumor necrosis factor-alpha, interleukin-1 ' beta ' and interleukin-6 etc.), tumour necrosis factor is one of inflammatory mediator that discharges the earliest, can further stimulate and activating macrophage, granulocyte, lymphocyte and endotheliocyte, discharge a large amount of inflammatory mediators, form " waterfall sample " chain reaction of inflammatory mediator mediation, amplify step by step just as domino, make Inflammatory response out of control.If effectively do not block its development, will cause the infull syndromes (MODS) of multi-organ function, even many internal organs system function depletion (MOSF), and the people's death of causing a disease.
This experimental example confirms that the HDL treatment can significantly reduce inflammatory mediator concentration such as multiple organs dysfunction syndromes patient tumors necrosis factor-alpha, interleukin-1 ' beta ' and interleukin-6, effectively reduces the patient death rate.
Claims (10)
1, a kind of human blood high density lipoprotein is characterized in that, the content of apo AI is that the apoAI molecular weight is 27.7KD in 77-89% and/or the human blood high density lipoprotein in this high-density lipoprotein (HDL).
2, a kind of human blood high density lipoprotein product is characterized in that, contains the human blood high density lipoprotein of 0.1-99% in the described human blood high density lipoprotein product.
3, human blood high density lipoprotein product according to claim 2 is characterized in that, described human blood high density lipoprotein product is an intravenous injection, wherein contains 9～11% sucrose and/or 0.5% albumin and/or 150mg ± 1mg/L vitamins C.
According to claim 2 or 3 described human blood high density lipoprotein products, it is characterized in that 4, the content of apo AI is that the apoAI molecular weight is 27.7KD in 77-89% and/or the human blood high density lipoprotein in the described human blood high density lipoprotein.
5, the manufacture method of a kind of human blood high density lipoprotein and products thereof is characterized in that, said method comprising the steps of:
A. get raw blood plasma, dropping buffered soln is 5.0-5.5 to the pH of mixed solution, and 95% ethanol of adding mixed solution 1/4～1/3 volume stirred 1-3 hour down at-5～-2 ℃, left standstill 6-8 hour, the centrifugal supernatant that gets; Add the water for injection of 45-55% supernatant volume to supernatant, drip buffered soln, making pH of mixed is 4.5-6.0, stirs 1-3 hour down at-6～-3 ℃, leaves standstill 1-3 hour, and centrifugal must the precipitation abandoned supernatant;
B. precipitate 0.02～0.1M NaCl solution dissolving, behind accent pH to 5.2 ± 0.5, stirred 2-6 hour centrifugal then must the precipitation down at 0～2 ℃ with 2-3 times of weight; Precipitation is dissolved with 0.1～0.2M NaCl solution of 50-150 times of weight, and regulator solution pH is 7.2 ± 0.5, stirs 6-20 hour down at 2～8 ℃, filters to get filtrate;
C. above-mentioned filtrate adds damping fluid, and making pH is 5.2 ± 0.5, stirs 1-3 hour, under 2～4 ℃, leaves standstill centrifugal collecting precipitation 6-20 hour; Precipitate the 0.1～0.2M NaCl solution dissolving with 50～150 times of amounts, transfer pH to 7.2 ± 0.5, stirring is dissolved precipitation fully, filters, and gets filtrate;
D. filtrate is used the ultra-filtration membrane ultrafiltration, and desalination also concentrates, and must be rich in the solution of HDL, through aftertreatment, gets HDL and products thereof.
6, manufacture method according to claim 5 is characterized in that, regulates pH in the described method and uses 1mol/LNaHCO
3Solution and/or described damping fluid are the HAC/NaAC buffer system of pH=4.
7, manufacture method according to claim 5 is characterized in that, described step C comes again after finishing.
8, manufacture method according to claim 5 is characterized in that, the ultra-filtration membrane among the described step D is the 8-70KD ultra-filtration membrane; Preferred 30～50KD ultra-filtration membrane that uses.
9, manufacture method according to claim 5, it is characterized in that the described aftertreatment of described step D can be: ultrafiltration and concentration liquid adds 9～11% injection sucrose or 0.5% albumin, adds 150mg ± 1mg injection vitamins C by every liter, transfer pH to 7.2 ± 0.5, get HDL solution; Above-mentioned HDL solution is 60 ± 1 ℃, 10 hours inactivation of viruses in the pasteurizing apparatus of microcomputer automatic control; HDL solution behind the inactivation of viruses, the degerming packing, and in 2～8 ℃ of preservations.
10, described human blood high density lipoprotein of claim 1-9 and products thereof is at preparation treatment multiple organ dysfunction syndrome (MODS); Or the preparation cardiovascular system diseases, as the medicine of atherosclerosis (AS); Or in the preparation and the application of acute symptom medicine aspect such as bacteriotoxin.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103833840A (en) * | 2012-11-27 | 2014-06-04 | 上海复星医药（集团）股份有限公司 | Method for extracting high-density lipoprotein and separating and purifying apolipoprotein apoA-I from human plasma |
| CN105367651A (en) * | 2015-12-22 | 2016-03-02 | 郑州莱士血液制品有限公司 | Method for removing pyrogen from human immunoglobulin with low-temperature ethanol method |
| CN105548585A (en) * | 2007-06-08 | 2016-05-04 | 奎斯特诊断投资公司 | Lipoprotein analysis by differential charged-particle mobility |
| CN109096390A (en) * | 2018-07-12 | 2018-12-28 | 安陆恩彼饲料有限公司 | A kind of manufacturing method of human blood high density lipoprotein |
| CN110590937A (en) * | 2018-06-13 | 2019-12-20 | 杨宝田 | Preparation method and application of human apolipoprotein A1 product |
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2005
- 2005-06-21 CN CNB2005100772826A patent/CN1321133C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105548585A (en) * | 2007-06-08 | 2016-05-04 | 奎斯特诊断投资公司 | Lipoprotein analysis by differential charged-particle mobility |
| CN103833840A (en) * | 2012-11-27 | 2014-06-04 | 上海复星医药（集团）股份有限公司 | Method for extracting high-density lipoprotein and separating and purifying apolipoprotein apoA-I from human plasma |
| CN105367651A (en) * | 2015-12-22 | 2016-03-02 | 郑州莱士血液制品有限公司 | Method for removing pyrogen from human immunoglobulin with low-temperature ethanol method |
| CN105367651B (en) * | 2015-12-22 | 2018-11-27 | 郑州莱士血液制品有限公司 | A kind of method of cold ethanol method removal human immunoglobulin(HIg) pyrogen |
| CN110590937A (en) * | 2018-06-13 | 2019-12-20 | 杨宝田 | Preparation method and application of human apolipoprotein A1 product |
| CN109096390A (en) * | 2018-07-12 | 2018-12-28 | 安陆恩彼饲料有限公司 | A kind of manufacturing method of human blood high density lipoprotein |
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