CN101167745B - Small molecular sugar stable buffer used for blood platelet frozen-dried preservation - Google Patents
Small molecular sugar stable buffer used for blood platelet frozen-dried preservation Download PDFInfo
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Abstract
The invention provides a stabilizing and buffering liquid of low molecular weight sugars used for lyophilization preservation of platelet, which is water solution with 35-45mM thickness by adding thelow molecular weight sugars on the basis of basic buffering liquid composing prescriptions (GS), wherein the basic buffering liquid composing prescriptions (GS) comprises 140mMNaCL, 5Mmkcl,12mM sodiumcitrate, 10mM glucose, 12.5mM sucrose and 1mug/mLPGE1. The low molecular weight sugars are trehalose. After the platelet is stabilized and protected by the stabilizing and buffering liquid of low molecular weight sugars of the invention, the platelet is freeze-dried with rehydration. The recovery ratio of a cell is above 70% and accumulating activity evoked by ADP can achieve 50% of fresh platelet. The platelet freeze-dried with rehydration has normal shape and complete structure, thereby a new solution is provided for freeze-drying conservation of the platelet.
Description
Technical field
The invention belongs to blood platelet frozen-dried preservation field, relate to a kind of small molecular sugar stable buffer.
Background technology
The research of blood platelet frozen-dried preservation has great importance, at first, lyophilized platelet has broad application prospects the treatment sick and wounded of hospital, and the thrombocyte demand is in the direct marketable value of China, estimate that indirect benefit can't be estimated more than approximately annual 1000000000 (50～700,000 bags * 1700 yuan).And be applicable to that various large, medium and small hospitals in different areas and area, outlying island store and adjustment mutually.Secondly, the frost drying thrombocyte has important military and is worth, and the soldier can carry, and uses for military first aid, saves wounded's life, plays a role to keeping combat effectiveness of the troops.Once more, can effectively prevent, improve transfusion safety, but thrombocyte freeze-drying prolonged preservation makes blood bank have the competent time that the blood of contributing is chemically examined through the pathophorous propagation of transfusing blood.Lyophilized platelet is convenient to sterilization, deactivation bacterium and virus simultaneously.Lyophilized platelet development helps developing a potential blood products market has wide application prospect as detect at the hemostasis-coagulation factor (as hemophilia, hemorrhage etc.) check, platelet antibody, standard thrombocyte spectrum cell etc.
As far back as nineteen fifties, people have just begun frost drying and have preserved hematoblastic research.Yet research does not at that time obtain desirable result, and the frost drying thrombocyte had not both had the integrity of structure, and infusion does not also show enough anastalsises for aleukia patient or hemorrhage animal model again.The early stage failure of exploring allows people that this store method has been produced suspection, so this research has just lain on the table for many years.Up to the late nineteen seventies early eighties, people such as Read MS. have invented chemical fixation frost drying thrombocyte preservation technology, make frost drying preserve hematoblastic research certain progress has been arranged, the research of frost drying thrombocyte preservation from then on becomes a focus (Richard M.Kaufman of thrombocyte prolonged preservation research again, Platelets:Testing, Dosing and the storage lesion-Recent Advances.American Society of Hematolog are y.2006:492-496.).Nineteen ninety-five, people such as Read MS. adopt 1.8% Paraformaldehyde 96 to handle the fix blood platelet, be suspended in after the cleaning in the human serum albumin protection liquid, frost drying under-20 ℃-40 ℃ condition, be kept at (Read MS in-80 ℃ of refrigerators then, Reddick RL, Bode AP, et al.Preservation of hemostatic and structural properties ofrehydrated lyophilized platelets:potential for long-term storage ofdried platelets for transfusion.Proc Natl Acad Sci USA1995; 92 (2): 397-401.).Electron microscopic observation rehydration frost drying thrombocyte form is normal, and it is similar to orthoplastocyte that flow cytometry is measured the surface receptor expression, and experimentation on animals proof frost drying thrombocyte has the hemostatic function that shortens the bleeding time, but Paraformaldehyde 96 has toxicity.After meanwhile people such as Brry SJ. utilizes and carries out frost drying rehydration behind the glucose incubation thrombocyte, obtained the cell rate of recovery of 75%-85%, but aggregation activity is lower.2003, Wolkers WF. has added trehalose in hematoblastic preservation system, make and reached 85% the cell rate of recovery (Willem F.Wolkers after the frost drying thrombocyte rehydration, NaomiJ.Walker, Fern Tablin, John H.Crowe.Human Platelets Loaded with TrehaloseSurvive Freeze-Drying.Cryobiology, 2001; 42,79-87., Pedrazzli P, Noris P, Perotti C, et al.Transfusion of platelet concentrates cryopreserved withThromboSol plus low-dose dimethylsulphoxide in patients with severethrombocytopenia:a pilot study.Br J Haematol, 2000; 108:653-659.).Yet; the research of blood platelet frozen-dried preservation has been carried out by people's such as Wolkers WF. method in the contriver laboratory, promptly uses aqueous trehalose (10mM KCl, 10mM EGTA; the 10mM imidazoles; 1 μ M PGE1,100mM NaCl, 40mM trehalose) to after the thrombocyte pre-treatment protection; carrying out frost drying preserves; after the rehydration cell rate of recovery be 67.60 ± 14.37% (" a kind of new hematoblastic research of thrombocyte store method-frost drying ", master thesis, Cao Wei).In 5 minutes MA be 10.48 ± 14.37% (" a kind of new hematoblastic research of thrombocyte store method-frost drying ", master thesis, Cao Wei).The experimental result prompting uses this trehalose preprocessing solution very big to hematoblastic aggregation capability damage.
Summary of the invention
After the object of the present invention is to provide a kind of proof load small molecular sugar, normal, the structural integrity of hematoblastic form, aggregation activity is greater than 50% small molecular sugar stable buffer.
Stabilizing buffer of the present invention is to add small molecular sugar on the basis of basic damping fluid (GS) prescription to make its concentration be the aqueous solution of 35～45mM.
The prescription of wherein basic damping fluid (GS) is: 140mMNaCL, 5mMKCL, 12mM Trisodium Citrate, 10mM glucose, 12.5mM sucrose, 1 μ g/mLPGE1.
Wherein small molecular sugar is a trehalose.
Concrete a kind of small molecular sugar stable buffer comprises following component: 140mMNaCL, 5mMKCL, 12mM Trisodium Citrate, 10mM glucose, 12.5mM sucrose, 1 μ g/mLPGE1,35mM trehalose.
Concrete another kind of small molecular sugar stable buffer comprises following component: 140mMNaCL, 5mMKCL, 12mM Trisodium Citrate, 10mM glucose, 12.5mM sucrose, 1 μ g/mLPGE1,40mM trehalose.
Concrete also a kind of small molecular sugar stable buffer comprises following component: 140mMNaCL, 5mMKCL, 12mM Trisodium Citrate, 10mM glucose, 12.5mM sucrose, 1 μ g/mLPGE1,45mM trehalose.
The present invention has used for reference forefathers' research experience; be total to the principle of molten thing according to the low small molecular sugar that waits animal and plant body to contain high density of some freeze proof resist dryings in the bionics; invented small molecular sugar stable buffer; after using small molecular sugar stable liquid of the present invention that thrombocyte is stablized protection; by the freeze-drying rules thrombocyte is carried out rehydration after the freeze-drying; its cell rate of recovery is greater than 70%; ADP inductive aggregation activity can reach in fresh hematoblastic 50%, 5 minute MA even be higher than 60%.Normal, the structural integrity of hematoblastic form after the freeze-drying rehydration.The present invention lays the foundation for the blood platelet frozen-dried preservation research of small molecular sugar stable method.
Description of drawings
Fig. 1 observes fresh hematoblastic form (28000 *) for transmission electron microscope
Fig. 2 a is the hematoblastic form (28000 *) that transmission electron microscope observation GS (T30) handles
Fig. 2 b is the hematoblastic form (28000 *) that transmission electron microscope observation GS (T35) handles
Fig. 2 c is the hematoblastic form (28000 *) that transmission electron microscope observation GS (T40) handles
Fig. 2 d is the hematoblastic form (28000 *) that transmission electron microscope observation GS (T45) handles
Fig. 2 e is the hematoblastic form (28000 *) that transmission electron microscope observation GS handles
Fig. 3 for transmission electron microscope observation freeze-drying aquation after fresh hematoblastic form (28000 *)
Fig. 4 a is the form (28000 *) after the thrombocyte freeze-drying aquation handled of transmission electron microscope observation GS (T30)
Fig. 4 b is the form (28000 *) after the thrombocyte freeze-drying aquation handled of transmission electron microscope observation GS (T35)
Fig. 4 c is the form (28000 *) after the thrombocyte freeze-drying aquation handled of transmission electron microscope observation GS (T40)
Fig. 4 d is the form (28000 *) after the thrombocyte freeze-drying aquation handled of transmission electron microscope observation GS (T45)
Fig. 4 e is the form (28000 *) after the thrombocyte freeze-drying aquation handled of transmission electron microscope observation GS
Embodiment
The present invention adds a certain amount of small molecular sugar (as trehalose) in basic damping fluid, form stabilizing buffer.
Basis damping fluid (GS) prescription: contain 140mMNaCL, 5mMKCL, 12mM Trisodium Citrate, 10mM glucose, 12.5mM sucrose, the aqueous solution of 1 μ g/mLPGE1.
Stabilizing buffer prescription: in basic damping fluid GS, add antifreeze anti-drying dose-trehalose.The final concentration that is mixed with small molecular sugar is the load liquid (stabilizing buffer) of 35～45mM.The present invention experimentizes according to the following steps:
1) collection of thrombocyte sample and preparation:
The thrombocyte sample preparation of hand: the fresh anticoagulated whole blood of mixing is centrifugal under 25 ℃ of-32 ℃ of temperature, and rotating speed is 800 rev/mins, and the time is 10 minutes, and the supernatant liquor of acquisition is platelet rich plasma (PRP) or PC (PC).
Get the PRP or the PC of above acquisition, 22 ℃ of following centrifugal 5min (centrifugal force is 1200g) abandon supernatant.Once abandon supernatant behind the recentrifuge of back with basic damping fluid GS washing, obtain the thrombocyte sample.
2) small molecular sugar load experiment: in the thrombocyte sample for preparing, directly add stabilizing buffer, mixing, resuspended thrombocyte, 37 ℃ of water-bath 4h slightly vibrate every 0.5h, keep thrombocyte to be in suspended state.
3) hematoblastic every index that small molecular sugar load liquid is handled detects:
1. the cell rate of recovery detects: get each the 40 μ L of thrombocyte sample before and after the small molecular sugar load respectively, in CD1200 type blood-counter system counting, being 100% before the load, comparing with it after the load and draw the cell rate of recovery.
2. aggregation activity detects: turbidimetry is adopted in the detection of ADP inductive aggregation activity, promptly gets the sample of 300 μ L, adds the ADP inductor 10 μ L of 1mM, MA in SC-2000 platelet aggregation tester detects 5 minutes.With corresponding platelet poor plasma (PPP) is blank.It is fresh hematoblastic more than 50% (is 100% with fresh hematoblastic MA) that ADP inductive aggregation rate can reach.
3. morphological structure detects: adopt transmission electron microscope observing, thrombocyte sample to be checked is fixed with glutaraldehyde, and dehydration is made ultrathin section(ing) after the Resins, epoxy embedding.Hematoblastic form of observation and microstructure under transmission electron microscope.
4) set by step 2) use stabilizing buffer that thrombocyte stablize protection after, centrifugal removal stable liquid, the resuspended thrombocyte of usefulness frozen-dried protective liquid (1% human serum albumin), the adjustment cell count is 1-2 * 10
11/ L is positioned in the freeze-drying bottle, and every bottle of 2mL carries out frost drying in the LGL-10 freeze drier, and condenser temperature is-50 ℃ ±-10 ℃, vacuum tightness 30Pa ± 20Pa, freeze-drying 18 hours.Carry out rehydration after the freeze-drying, rehydration liquid be the PPP diluent (PPP: sterilized water=1: 1), rehydration lyophilized platelet under the room temperature aseptic condition.
5) the thrombocyte sample that obtains after freeze-drying in the step 4) and the aquation being carried out physical signs detects:
1. the cell rate of recovery detects: getting each 40 μ L of thrombocyte sample of fresh and freeze-drying rehydration respectively, in CD1200 type blood-counter system counting, is 100% with the fresh blood platelet, and the cell counting after the freeze-drying rehydration is compared with it and drawn the cell rate of recovery.
2. aggregation activity detects: turbidimetry is adopted in the detection of ADP inductive aggregation activity, promptly gets the sample of 300 μ L, adds the ADP inductor 10 μ L of 1mM, MA in SC-2000 platelet aggregation tester detects 5 minutes.With corresponding platelet poor plasma (PPP) is blank.It is fresh hematoblastic more than 50% (is 100% with fresh hematoblastic MA) that ADP inductive aggregation rate can reach.
3. morphological structure detects: adopt transmission electron microscope observing, thrombocyte sample to be checked is fixed with glutaraldehyde, and dehydration is made ultrathin section(ing) after the Resins, epoxy embedding.Hematoblastic form of observation and microstructure under transmission electron microscope.
Embodiment
Add trehalose on basic damping fluid GS prescription basis, be mixed with the load liquid of GS (T35), be the stabilizing buffer aqueous solution that contains the 35mM trehalose, its osmotic pressure is 339 ± 8mOMS.Contain in the aqueous solution: trehalose 35mM, glucose 10mM, sucrose 12.5mM, Trisodium Citrate 12mM, KCL 5mM, NaCL 140mM, PGE 1 μ g/mL.
By aforementioned 2) carry out small molecular sugar load experiment and 3) to hematoblastic every index detection of small molecular sugar load, the result:
1. the cell rate of recovery: greater than 70%;
2. aggregation activity: greater than 60%;
3. morphological structure: morphological structure is normal substantially, sees Fig. 2 b.
By aforementioned 4) to thrombocyte freeze-drying and aquation with by 5) the thrombocyte sample that obtains after to freeze-drying and aquation of method carry out physical signs and detect the result:
1. the cell rate of recovery: greater than 70%;
2. aggregation activity: greater than 50%;
3. morphological structure: morphological structure is normal substantially, sees Fig. 4 b.
On thrombocyte form structure, to compare with Fig. 1 fresh blood platelet, the thrombocyte form after GS (T35) stable liquid is handled is normally in the form of annular discs, and no uropodium forms, and membrane structure is complete, and acellular device leaks.
Compare with the thrombocyte after Fig. 3 basis damping fluid GS is stable, thrombocyte form and microstructure after GS (T35) stable liquid is handled normally do not have considerable change.
Above presentation of results, GS (T35) stable liquid is handled the back does not have influence substantially to hematoblastic form and structure.
Other experimental example
Press table 1 prescription and in basic damping fluid, add trehalose preparation stabilizing buffer GS (T30), GS (T40), GS (T45) even load liquid.For example: GS (T40) stabilizing buffer contains the trehalose of 40mM, and its osmotic pressure is 386 ± 6mOMS.
Experiment is the comparison sample with GS simultaneously.In addition with people's such as Wolkers WF. aqueous trehalose (10mM KCl, 10mM EGTA, 10mM imidazoles, 1 μ M PGE1,100mM NaCl, 40mM trehalose) sample in contrast.
Table 1: the stabilizing buffer that contains the different concns trehalose
With with embodiment in identical method hematoblastic every index of handling is detected the result:
One) hematoblastic every index of small molecular sugar load detects:
1. the cell rate of recovery and 2. aggregation activity the results are shown in Table 2, as seen adopt stabilizing buffer GS (30)～GS (45) to handle the back recovery rate of blood platelet all greater than 70%, aggregation activity is all greater than 50%, and uses relatively sample GS aggregation activity less than 40%, uses aggregation activity in the same old way less than 20%.
3. morphological structure: all normal substantially, see Fig. 2 a-e.
Table 2: the hematoblastic cell rate of recovery, aggregation activity detected result
Two) physical signs of the thrombocyte sample that obtains after freeze-drying and the aquation detects:
1. the cell rate of recovery: the results are shown in Table 3, as seen adopt GS (35)～GS (45) stabilizing buffer to handle the back recovery rate of blood platelet all greater than 70%.
2. aggregation activity: concrete outcome sees Table 3, adopt the thrombocyte freeze-drying rehydration after stabilizing buffer GS (T35)～GS (T45) handles, its aggregation activity is all greater than 50%, rehydration is almost lost aggregation activity after the freeze-drying of fresh blood platelet, and the aggregation activity that uses sample GS relatively is less than 4%, uses the aggregation activity only about 10% in the same old way.The stabilizing buffer determined of the present invention comprises GS (35)～GS (45) thus, and these stabilizing buffers can significantly improve the hematoblastic aggregation activity of freeze-drying rehydration.
3. morphological structure: all normal substantially.Sample pictures is referring to Fig. 4 a-e.
Table 3: the cell rate of recovery after the lyophilized platelet rehydration, aggregation activity detected result
Conclusion:
Compare with Fig. 1 fresh blood platelet, the thrombocyte form after stabilizing buffer GS of the present invention (35)～GS (45) handles is normally in the form of annular discs, and no uropodium forms, and membrane structure is complete, and acellular device leaks.It is fresh hematoblastic more than 50% that aggregation activity can reach.
Compare with the fresh blood platelet after Fig. 3 freeze-drying aquation, after the freeze-drying of fresh blood platelet, the platelet membrane structure is imperfect, and organoid leaks, and aggregation activity is lost substantially.And in the form of annular discs with the hematoblastic form of stabilizing buffer GS of the present invention (35)～GS (45) protection back freeze-drying rehydration, no uropodium, membrane structure is complete, and acellular device leaks.It is fresh hematoblastic more than 50% that aggregation activity can reach.
Compare no significant difference with the thrombocyte after Fig. 2 e basis damping fluid GS is stable, the hematoblastic form of stabilizing buffer GS of the present invention (35)～GS (45) protection back freeze-drying rehydration is in the form of annular discs, no uropodium, and membrane structure is complete, and acellular device leaks.Keep the aggregation activity more than 50%.
Compare with the thrombocyte after the stable back freeze-drying of Fig. 4 e basis damping fluid GS aquation; hematoblastic membrane structure disappearance after the stable back freeze-drying of the GS aquation; organoid leaks; aggregation activity is very low; and it is in the form of annular discs with the hematoblastic form of stabilizing buffer GS of the present invention (35)～GS (45) protection back freeze-drying rehydration; no uropodium, membrane structure is complete, and acellular device leaks.It is fresh hematoblastic more than 50% that aggregation activity can reach.
With to compare in the same old way, should preserve carry out frost drying in the same old way, the cell rate of recovery is that MA illustrates and uses preprocessing solution is very big to hematoblastic aggregation capability damage in the same old way for being 10.48 ± 14.37% only in 67.60 ± 14.37%, 5 minutes after the rehydration.Can reach fresh hematoblastic more than 50%, illustrate that thrombocyte is had defencive function and use stabilizing buffer GS of the present invention (35)～GS (45) to handle the hematoblastic aggregation activity in back.
Above presentation of results, use small molecular sugar stable liquid of the present invention pre-treatment after thrombocyte carry out freeze-drying again, still can keep biologically active pdgf after the rehydration, illustrate that stabilizing buffer of the present invention has good defencive function to thrombocyte.
Claims (4)
1. small molecular sugar stable buffer, the aqueous solution that the basic damping fluid GS component of serving as reasons and 35～45mM small molecular sugar trehalose are formed; Described basic damping fluid GS component is: 140mMNaCL, 5mMKCL, 12mM Trisodium Citrate, 10mM glucose, 12.5mM sucrose and 1 μ g/mLPGE1.
2. small molecular sugar stable buffer according to claim 1 is characterized in that, comprises following component: 140mMNaCL, 5mMKCL, 12mM Trisodium Citrate, 10mM glucose, 12.5mM sucrose, 1 μ g/mLPGE1 and 35mM trehalose.
3. small molecular sugar stable buffer according to claim 1 is characterized in that, comprises following component: 140mMNaCL, 5mMKCL, 12mM Trisodium Citrate, 10mM glucose, 12.5mM sucrose, 1 μ g/mLPGE1 and 40mM trehalose.
4. small molecular sugar stable buffer according to claim 1 is characterized in that, comprises following component: 140mMNaCL, 5mMKCL, 12mM Trisodium Citrate, 10mM glucose, 12.5mM sucrose, 1 μ g/mLPGE1 and 45mM trehalose.
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