CN109769797A - A kind of organ preservative fluid - Google Patents
A kind of organ preservative fluid Download PDFInfo
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- CN109769797A CN109769797A CN201711115825.8A CN201711115825A CN109769797A CN 109769797 A CN109769797 A CN 109769797A CN 201711115825 A CN201711115825 A CN 201711115825A CN 109769797 A CN109769797 A CN 109769797A
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- liver
- liquid
- organ
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- hibernation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
The present invention provides a kind of method and preservation liquid used of survival and/or function during maintaining vascular endothelial cell storage in vitro, is suitable for a variety of organs and saves.In vitro liver saves and machine perfusion during can survive more than for 24 hours in organ preservative fluid of the present invention, Microcirculation of Liver structural integrity, have no obvious damage, bile secretion function is good, liver synthesis is good with metabolic function, the time of liver storage in vitro is greatly prolonged, and promotes later period liver-transplantation patients survival rates.
Description
Technical field
It is special the present invention provides the liquid of survival and/or function during a kind of maintenance vascular endothelial cell storage in vitro
There is provided a kind of organ preservative fluids, the storage in vitro suitable for a variety of organs.Belong to field of medical device.
Background technique
Organ transplant at present is the irreplaceable treatment of one kind of terminal phase organ lesion, cancer and acute organ failure
Means, however in China or even world wide, a variety of organ donors are extremely in short supply.And during common organ transplant,
The preservation effect of organ directly affects the success or failure of operation and the postoperative survival of patient.And it is maximum for organ preservation influence, be
The preservation liquid directly contacted with organ.
Since University of Wisconsin solution (UW liquid) appearance in 1987, researcher couple in field
Unremitting research and improvement have been carried out in organ preservative fluid.The goldstandard of organ preservative fluid is UW liquid at present.UW liquid is due to containing
Hydroxyethyl starch leads to liquid high viscosity, extends infusion time, leads to organ microcirculation structural damage, can not answer well
Use now widely used organ machine perfusion.UW liquid is intracellular preservation liquid, has very high potassium content
(125mM), and the potassium ion of high concentration will lead to vessel retraction, influence to save the effect of liquid and the effect of machine perfusion, and
And in use, UW liquid must disposably be washed away substitution by blood, just be avoided that high potassium concentration ion flows into patient body,
The influence to physiology especially heart is caused, and due to the cost of UW liquid valuableness, using being restricted.Current a variety of organ transplants
Donor preservation and organ preservative fluid used in transport, are confined to improve on the basis of UW liquid mostly, wherein protecting without blood
Liquid storage oxygen carrying capacity is low, at high cost, and organ preservative fluid containing blood cell composition is complicated for operation, the easy blood coagulation or haemolysis the defects of,
And it is easily introduced unknown virus.
When carrying out organ transplant, need to separate donor organ and by storage in vitro, ability subsequent transplantation to receptor
In vivo.Organ storage in vitro liquid has that oxygen carrying capacity is low or there are various risks using blood product in existing technology
Problem, the characteristics of rich oxygen content and low-risk can not be had both.For example, disclosed in the patent of Publication No. CN101199273A
Multiple organs storage protection liquid has the function of can reduce ischemia reperfusion injury, but can not provide effective oxygen supply,
Organ can not be saved for a long time.The patent of Publication No. CN104996396A uses whole blood as liquid main body is saved, and there are solidifying
The risks such as blood, haemolysis, thereby increases and it is possible to bring unknown blood disease infection into, it is very risky for receptor sufferer.Publication No.
The patent of CN101199273A discloses a kind of novel multiple organs storage protection liquid, raw with epiphysin, vitamin C and water soluble vitamin
The antioxidant system of plain E composition generates caused oxidative damage to free radical during reducing organ storage；Use mannitol
It maintains solution osmotic pressure and reduces solution viscosity；Using low concentration glucose to adapt to the minimum metabolic demand of organ, but still
It can not so accomplish to supply organ transplantation donor enough oxygen consumption.20090239206 A1 of patent US discloses a kind of New ET-
Kyoto solution organ preservative fluid regard dibutyryl-cAMP as additive, but still can not accomplish to provide enough oxygen
Gas supply.And the patent of Publication No. CN104996396A discloses a kind of preservative fluid for perfusing extracorporeal liver, use whole blood as
Liver supply, easy infection complicated for operation, and easily cause the rough sledding such as haemolysis, blood coagulation, Liver Allograft Preservation risk is high, and liver is protected
Occurs the case where slight swelling after depositing.Publication No. CN103893205A discloses a kind of heart comprising lidocaine and adenosine
Dirty cardioplegic solution and preparation method thereof is to play it cardiac arrest is promoted to act on using lidocaine and adenosine.
Organ hypothermia saves physiological status when substantially simulation animal hibernation.Under hibernation mechanism, hibernation mammal
It survives the several months under extremely low environment temperature, without food or water, but after capable of coming out of hibernation, without apparent organ
Damage.And the mankind do not have this ability then under normal physiological conditions, to reach similar " hibernation " state, and hibernation is needed to protect
The presence for protecting agent, currently, cockstailytic has been used widely in clinical rescue urgent patient as hibernation protective agent.One
To from normal body temperature rat and active and hibernation ground squirrel liver in special protection liquor to the resistance to of low-temperature preservation
Stress studies have shown that 48h it is stored refrigerated after, the liver cell of rat is obviously destroyed, show as liver enzyme release and
Choleresis is reduced；On the contrary, the liver from hibernation ground squirrel shows damage (the Carey H of very little after the refrigeration of 96h
V,Andrews M T,Martin S L.Mammalian hibernation:cellular and molecular
responses to depressed metabolism and temperature[J].Physiological Reviews,
2003, 83(4):1153-1181.).Studies have shown that hibernator can resist ischemia/reperfusion pair compared with non-hibernator
Damage (Frerichs K U, the Hallenbeck J M.Hibernation in ground squirrels of tissue and organ
induces state and species-specific tolerance to hypoxia and aglycemia:an in
vitro study in hippocampal slices[J].Journal of Cerebral Blood Flow&
Metabolism Official Journal of the International Society of Cerebral Blood
Flow&Metabolism.1998,18(2):168)；Separately some researches show that using can be by using class opioid compound
A kind of synthesis opium of DADLE [D-Ala2, D-Leu5]-enkephalin (DADLE) ([D-Ala2, D-Leu5]-enkephalins)
Sample substance saves animal organ 46 hours in the lab, and the store method is that multiple organ saves and quiet with connection jointly
Pulse-phase connects (ED Ratigan, DB Mckay.Exploring principles of hibernation for organ
preservation[J].Transplantation Reviews.2015,30(1):13-19)。
As organ donation becomes the main source of organ transplant donor, organ donation case is likely to occur whenever and wherever possible,
The time that organ saves calculates in hours, and sufferer moment terminal phase will receive the threat of life, and valuable organ transplant supplies
Body every year all can be too long because of the holding time or saves that quality is low causes to be dropped.It is therefore necessary to develop more superior organ to protect
Liquid storage improves the utilization rate of organ donation.
Summary of the invention
In order to solve the above technical problems, the present invention provides survive during a kind of maintenance vascular endothelial cell storage in vitro
And/or the liquid of function, in particular, provide a kind of organ preservative fluid, depositing during maintenance organ transplantation donor storage in vitro
Living and function, the storage in vitro suitable for a variety of organs.
The technical scheme is that:
Provide a kind of method of survival and/or function during maintaining vascular endothelial cell storage in vitro comprising make
The step of cell and preservation liquid contact, it is characterised in that the preservation liquid contains following component: can (a) make described
Cell enters the hibernation protective agent of similar hibernation-like state；(b) energy substrate；(c) scavenger of metabolite is removed；(d) colloid
Osmotic pressure maintains agent and/or colloid osmotic pressure regulator；(e) oxygen.
Preferably, method described above, wherein the preservation liquid is also optionally containing selected from antibiotic, anti-inflammatory agent
One or more of object, immunosuppressor.
Method described above is used for the preservation of organ；Preferably, wherein the organ is liver, heart, kidney
Dirty, pancreas, lung or enteron aisle etc..
A kind of preservation liquid of survival and/or function during maintaining vascular endothelial cell storage in vitro is additionally provided, is contained
Basic salt, pH buffer and deionized water needed for cell, it is characterised in that the preservation liquid also contains following component: (a) energy
The cell is enough set to enter the hibernation protective agent of similar hibernation-like state；(b) energy substrate；(c) removing of metabolite is removed
Agent；(d) colloid osmotic pressure maintains agent and/or colloid osmotic pressure regulator；(e) oxygen.
Preferably, preservation liquid described above, it is characterised in that the preservation liquid also optionally contains selected from antibiotic, resists
One or more of scorching drug, immunosuppressor.
Method described above or the preservation liquid, in which:
The basic salt is to maintain the substance of the balance of the inside and outside salt of cell membrane comprising but be not limited to sodium chloride,
One of potassium chloride, magnesium sulfate etc. are two or more；Preferably, the basic salt is sodium chloride, potassium chloride and sulphur
Sour magnesium.The dosage of basic salt, such as can be that 1000ml saves that contain basic salts substances in liquid be 2.0g-25.0g, preferably
2.0g-23.0g, more preferably 5.0g-10.0g.
The pH buffer is the opposite substance for maintaining pH stable, such as it can be selected from HEPES salt (hydroxyethyl piperazine
Esilate, such as hydroxyethyl piperazineethanesulfonic acid sodium etc.), phosphate (such as sodium phosphate, potassium phosphate etc.), carbonate (such as carbon
Sour sodium, potassium carbonate etc.), hydrophosphate (such as potassium dihydrogen phosphate, dibastic sodium phosphate etc.), bicarbonate (such as sodium bicarbonate, carbonic acid
Hydrogen potassium etc.), one of histidine or two or more；Its suitable amounts of the pH buffer, such as can be protected for 1000ml
Substance containing pH buffer is 0.5g-10.0g, preferably 2.0g-10.0g, more preferably 3.0g-5.0g in liquid storage.
The hibernation protective agent is to inhibit cell metabolism, protection cellular matrix and/or the substance for maintaining cell membrane stability,
Including but not limited to inhibit inhibitor, the neurotransmitter antagonists of cellular informatics access, sodium, potassium or calcium ion channel blocker etc.
One of or it is two or more；Preferably, the hibernation protective agent is selected from chloraldurate, lidocaine, atropine, Pu Lu
One of cacaine, nifedipine, dilantin sodium, Amiodarone Hydrochloride, hyoscine, anisodamine etc. or two kinds.The winter
It sleeps protectant suitable amounts, such as can be that save the substance of protective agent containing hibernation in liquid be 0.05g-5.0g to 1000ml, preferably
For 0.1g-1.0g.
The energy substrate, for example, selected from atriphos, adenosine diphosphate (ADP), adenosine phosphate, acetyl coenzyme A, insulin,
One of glucose is two or more；Preferably, the energy substrate is atriphos or adenosine diphosphate (ADP).Energy
The dosage of substrate, such as can be that energy content substrate is 0.001g-3.0g in 1000ml preservation liquid, preferably 0.1g-1.0g.
The scavenger for removing metabolite is a kind of in the substance for remove the metabolite for causing cell death
Or it is two or more；Preferably, the scavenger be selected from vitamin C, rotenone, the fast alcohol of glycosides difficult to understand, L-NAME,
One of butylated hydroxy anisole, dibutyl hydroxy toluene, propylgallate, sodium isoascorbate, phytic acid, tea polyphenols or
Person is two or more.The dosage of the scavenger, for example, can be 1000ml save liquid in containing the scavenger substance be 0.1g-
5.0g, preferably 0.1g-2.0g.
It is the substance for maintaining the stability of water inside and outside capillary that the colloid osmotic pressure, which maintains agent, such as selected from hydroxyl second
One of base starch, hyaluronate, hyaluronic acid, mannitol are two or more；The colloid osmotic pressure maintains agent
Dosage, such as can be that the maintenance agent substance containing the colloid osmotic pressure is 0.5g-10.0g in 1000ml preservation liquid, preferably
5.0g-10.0g。
The colloid osmotic pressure regulator, such as selected from one of gossypose, dextran, albumin, glucan
Or it is two or more.The dosage of the colloid osmotic pressure regulator can be added in right amount according to the osmotic pressure of needs, such as be adjusted
The preservation liquid osmotic pressure is 290-380mOsm/kg H2O, and wherein colloid osmotic pressure is 1.5-30.0mOsm/kg H2O.
The antibiotic is the substance of infection caused by resisting small pathogen etc., for example, selected from Florfenicol, penicillin,
One of streptomysin, gentamicin, chloramphenicol, cephalosporin, terramycin, erythromycin, colistin, griseofulvin or two kinds
More than；The dosage of the antibiotic, for example, can be 1000ml save liquid in containing antibiotic substance be 0.1g-2.0g, preferably
0.1g-1.0g。
The anti-inflammatory drug is the drug for treating the reaction inflammation occurred after tissue is damaged, such as selected from
Curcumin, Fenbid, Mobic, aspirin, paracetamol, Diclofenac, Indomethacin, brufen, in fenbufen
It is one or two kinds of more than；The dosage of the anti-inflammatory drug, such as can be in 1000ml preservation liquid and be containing anti-inflammatory drug
0.001g-1.0g, preferably 0.001-0.05g.
The immunosuppressor is the substance for inhibiting immune response, such as selected from ciclosporin A, tacrolimus, ring phosphinylidyne
One of amine, rapamycin, sulphur file purine, methotrexate (MTX), glucocorticoid (such as dexamethasone) are two or more.Institute
State the dosage of immunosuppressor, for example, can be 1000ml save liquid in containing immunosuppressor be 0.001g-1.0g, preferably
0.005g-0.05g。
Preferably, the present invention provides a kind of preservation liquid described above, buffers containing basic salt needed for cell, pH
Agent, hibernation protective agent, energy substrate, the scavenger for removing metabolite, colloid osmotic pressure maintain agent and/or colloid osmotic pressure
Regulator, antibiotic, anti-inflammatory drug, immunosuppressor, oxygen and deionized water.
Preferably, the preservation liquid of the preservation liquid, every 1000mL contains:
Preferably, preservation liquid described above, it is characterised in that the pH value for saving liquid is 7.4 ± 0.1, and salinity is
9.0, osmotic pressure is 290-380mOsm/kg H2O, and wherein colloid osmotic pressure is 1.5-30.0 mOsm/kg H2O.
Present invention preservation liquid described above, preferably as maintain to survive during vascular endothelial cell storage in vitro and/
Or the application of the preservation liquid of function；Preferably, the application for saving liquid as organ preservative fluid；It is highly preferred that the device
Official is selected from liver, heart, kidney, pancreas, lung or enteron aisle.
Compared with existing organ preservative fluid, organ preservative fluid technology of the invention is mainly:
1) metabolic rate of tissue and cell is reduced using low temperature.
2) increase of permeability of cell membrane, such as Adenosine triphosphate of DIRECT ENERGY needed for cell is provided are utilized at low ambient temperatures
Glycosides.
3) dissolved oxygen needed for directly providing cell, it is a series of subsequent caused by thoroughly preventing cell and tissue because of anoxic
Damage.
4) neurotransmitter antagonists (such as atropic of antagonism neurotransmitter acetylcholine is utilized in terms of cellular signal transduction
Product) inhibit cellular activity.
5) retarding agent for utilizing sodium, potassium, calcium channel prevents it at low ambient temperatures to intracellular delivery signal, suppression
The activity of cell processed.
6) present invention pays special attention to protect vascular endothelial cell epimatrix, it is made still to be able to maintain life at low ambient temperatures
Reason state is unobstructed to guarantee the small circulation of liver.To ensure that the stabilization of interior environment.
7) Apoptosis when reducing Reperfu- sion from the stability of maintenance organelle mitochondria.
Hibernation protective agent is applied in organ preservative fluid by the present invention, and organ can be made to enter hibernation-like state, is extended and is saved
Time, and organ can be enhanced to ischemic-anoxic resistance, more particularly to improve tolerance of the endothelial cell to low temperature.This hair
In bright, organ induces the state into similar hibernation during preservation, and metabolism is greatly lowered, and various functions are temporarily stopped
Sleep, can armour from adverse environment influence, save activity and function, can not only preferably be protected for more time
It deposits, and can make organ that there is stronger tolerance to ischemical reperfusion injury.
Conventional cryogenic saves organ from being substantially exactly in simulation animal hibernation-like state, and low temperature can reduce metabolism, but
Common cryo-conservation all existing defects cannot such as maintain the shape of organ cell's especially endothelial cell at low temperature well
State, and hibernator allows organ to enter low-temperature condition and does not damage after hibernation terminates rewarming there are number of mechanisms, and
And this process of hibernation-revival-hibernation is repeated during the entire hibernation of animal.Maintenance blood vessel endothelium provided by the invention is thin
The liquid of survival and/or function during born of the same parents' storage in vitro, under Physical temperature-lowering cooperation, hibernation protective agent can be such that organ transplant supplies
Body enters the state of a kind of similar " hibernation ", reduces organ cell's metabolism, can reduce the reaction that organ stimulates various pathology, mention
For various cells to the tolerance of anoxic, the parteriole in abnormal contraction is able to diastole in pathological conditions, micro- in high each organ
Circulation is improved, and is able to extend the time of organ transplant donor storage in vitro and is improved preservation quality.In vitro liver is in this hair
The bright liver organ can survive during saving liquid and machine perfusion more than for 24 hours, and Microcirculation of Liver structural integrity has no
Obvious damage, bile secretion function is good, and liver synthesis is good with metabolic function.The present invention can satisfy clinical liver and fill in vitro
The needs saved are infused, greatly prolong the time of liver storage in vitro, and promote later period liver-transplantation patients survival rates.The present invention
It can meet clinical needs, and have the characteristics that low in cost.
Compared with prior art, organ preservative fluid provided by the invention has the advantages that
(1) organ preservative fluid provided can be used in the cooling, lavation and storage in vitro of a variety of organs, can effectively save,
Liver, heart are more than 24 hours, wherein can be reserved for kidney 72 hours.
(2) hibernation protective agent is applied in organ preservative fluid, organ can be made to enter hibernation-like state, when extending preservation
Between, and organ can be enhanced to ischemic-anoxic resistance, more particularly to improve tolerance of the endothelial cell to low temperature.Although the winter
Dormancy phenomenon is widely present in a variety of mammals, but definite molecular mechanism is not known.It is at present low about the common recognition of hibernation
The animal organ of body temperature and low metabolic rate and hibernation is intact.The present invention is reduced cell metabolism using low temperature and utilized low
The lower permeability of cell membranes increase of temperature come for cell directly feed energy and and oxygen, greatly reduce cold anoxia-induced apoptosis.It is another
Aspect is eliminated to the maximum extent or antagonism increases the signal of cellular activity.Signal as neuron pairing effect cell issues is logical
Cross release neurotransmitters such as acetylcholine.The ingredient for saving antagonism acetylcholine in liquid has blocked neurotransmitter transmitting, in cell
Signal transduction level inhibits the activity of cell.Simultaneously for sodium, the potassium, calcium channel on cell membrane, channel blocking is utilized
The function in its channel has been blocked in agent, prevents the signal of low temperature poor environment from being sent in cell membrane.And save the sodium in liquid
Calcium channel blocker makes tiny blood vessels be in relaxed state.The purpose of these measures be to maintain capilary in entity internal organs it is unobstructed and
Cellular activity is reduced by the method other than low temperature.
(3) organ preservative fluid provided by the invention contains curcumin, can safely and effectively reduce organ because of non-physiology
State bring inflammatory reaction extends the holding time.
(4) contain osmotic pressure regulator in the organ preservative fluid provided, can effectively adjust osmotic pressure, inhibit edema
With space between cells oedema, preservation effect is improved.
(5) contain immunosuppressor in the organ preservative fluid provided, can reduce the activity of immunocyte in organ, mitigate
Immune response, especially has inhibiting effect to the immunological rejection after organ transplant.
In conclusion organ preservative fluid provided by the invention is cheap, efficient, and it is suitable for multiple organ and saves.Institute of the present invention
It states and saves the rat heart after liquid saves 24 hours, liver and the observation of renal tissue perspective Electronic Speculum as the result is shown: cell within a cell
Normally especially Mitochondrial Shape is normal for device form, and mitochondria double membrane structure is complete, and micro-structure especially ridge in mitochondria
It is intact.Optical microscopy shows that cellular morphology is normal, the close structure between cell and cell.Liver passes through institute of the present invention
It is intact to state various functions reservation after being stored at room temperature 24 hours outside preservation liquid, such as bile secretion function, albumin synthesis function etc..
The present invention uses the phenomenon that animal hibernation, combination cell, molecular biology, the branches of learning comprehensive application such as Modern molecular medicine, wound
It makes and has invented this and save liquid.
Detailed description of the invention
Fig. 1 shows that the present invention saves the rat liver HE stained slice tissue examination liver structure that liquid saves
Fig. 2 shows that the present invention saves the rat liver HE stained slice tissue examination bile duct liver Cable Structure that liquid saves
Fig. 3 shows that UW liquid saves rat liver HE stained slice tissue examination liver cell and falls off (40 times of amplification)
Fig. 4 shows that UW liquid saves rat liver HE stained slice tissue examination liver cell and falls off (200 times of amplification)
Fig. 5 has liver cell hyperplasia after cultivating in vitro 6 hours after showing the present invention preservation liquid liver protection 24 hours
Fig. 6 is cultivated 6 hours in vitro after showing UW preservation liquid liver protection 24 hours and is had no liver cell hyperplasia
Fig. 7 shows that the present invention saves the bile duct epithelial cell for the rear new proliferation that liquid saves
Fig. 8 shows that UW saves the biliary tract that liquid saves, and has no the bile duct epithelial cell being newly proliferated
Fig. 9 shows that fresh liver tissue electron microscopic section shows normal mitochondria
Figure 10 shows that UW liquid liver organization electron microscopic section shows that mitochondrial internal structure is destroyed
Figure 11 shows that the present invention saves the liver tissue slices of liquid liver protection 24 hours
Figure 12 shows that UW saves the liquid 24 hours static endothelial denudations for saving heart
Figure 13 shows that UW saves the liquid 24 hours static endothelial denudations for saving heart
Figure 14 shows that the present invention saves the liquid 24 hours endothelial cell integrities for saving heart
Figure 15 shows that the present invention saves the liquid 24 hours endothelial cell integrities for saving heart
Specific embodiment
The preferred embodiments of the present invention will be described in detail with reference to the accompanying drawing, so that advantages and features of the invention energy
It is easier to be readily appreciated by one skilled in the art, more clear explicitly be defined to be made to protection scope of the present invention.
Below in the specific embodiment, if mentioned experimental method is General Experimental Procedures without specified otherwise；Institute
If the use of reagent and material without specified otherwise being that common commercial approach is bought；The present invention is not limited to case study on implementation to apply model
It encloses.
A kind of organ preservative fluid of embodiment 1
The preparation method of the present embodiment organ preservative fluid: under aseptic condition, portions of de-ionized water is taken, substance is firstly added
In ionized water, pH to 7.35-7.45 is adjusted after mixing and using hydrochloric acid and sodium hydroxide, then be finally settled to 1000mL, gone forward side by side
Oxygen is roused in row over-saturation, makes in liquid full of oxygen over-saturation.
It includes each component in following table that every 1000mL, which saves liquid:
2 Rat Liver Transplantation donor preservation effect of embodiment detects (HE dyeing)
Male SD rat 20, it is divided into two groups, every group 10, aseptic operation separates liver, and separation process is rushed with liquid is saved
It washes, warm ischemia time is less than 30min, and the static 4 DEG C of cold preservations of the in vitro liver application UW liquid of first group of 10 rat, second group is answered
The organ preservative fluid low-temperature circulating machine perfusion described in embodiment 1 saves, and perfusion pressure and groundwater increment are physiological amount, Perfusion preservation
Organ preservative fluid inputs in liver from portal vein and arteria hepatica immediately immediately after over-saturation drum oxygen in the process.
After rat liver is saved for 24 hours by the method for the present invention, samples cut according to the progress HE dyeing of routine histologic method immediately
Piece tissue examination.Histotomy result (Fig. 1 and 2) display: after saving liver for 24 hours using organ preservative fluid of the present invention
Liver and bile duct liver Cable Structure are clearly orderly, and central vein inner wall is complete in liver, and bile duct inner wall is completely saved.And it answers
Show that there were significant differences with the liver (Fig. 3 and 4) that UW liquid saves.
3 rat liver of embodiment saves experiment (detection of BrdU cell Proliferation)
Male SD rat 20, it is divided into two groups, every group 10, aseptic operation separates liver, and separation process is rushed with liquid is saved
It washes, warm ischemia time is less than 5min, and the static 4 DEG C of cold preservations of the in vitro liver application UW liquid of first group of 10 rat, second group is answered
The organ preservative fluid low-temperature circulating machine perfusion described in embodiment 1 saves, and perfusion pressure and groundwater increment are physiological amount, Perfusion preservation
Organ preservative fluid inputs in liver from portal vein and arteria hepatica immediately immediately after over-saturation drum oxygen in the process.It saves and opens in vitro
Each experimental group saves in liquid and BrdU (MedChem Express HY-15910) is added when the beginning, and BrdU is saving the starting in liquid
Concentration is 100nM/L, after saving for 24 hours, is sampled immediately according to Super SensitivePolymer-HRP detection
system (932-QDMAN-5X；Biogenex, San Ramon, CA, USA) described in method carry out BrdU specific immunity it is glimmering
Light stained tissue inspection, the BrdU antibody used are that the Purified anti-BrdU Antibody of Biolegend company (is compiled
Number: 339802).
There is liver cell proliferation (figure after saving 6 hours after 24 hours in vitro in preservation liquid culture liver of the invention
5).Then have no that liver cell is proliferated (Fig. 6) after 6 hours of liver that UW liquid saves.Two methods have bright in terms of cell Proliferation
Aobvious difference (Fig. 7 and 8).
Liver bile duct BrdU cell proliferation experiment result (Fig. 7 and 8) display: it is protected using organ preservative fluid of the present invention
In the liver bile duct deposited, there is apparent hepatocyte growth, and the liver for applying UW liquid to save is then without this phenomenon, display
There are more apparent differences under the protection that bile duct epithelial cell saves liquid at two kinds.
The above results show organ preservative fluid of the present invention in the application of organ storage in vitro to rat liver microcirculation
The protective effect of structure, cell Proliferation and liver transfer operation success rate is substantially better than UW liquid.
4 rat liver storage in vitro effect detection (liver mitochondrion) of embodiment
Male SD rat 20, it is divided into two groups, every group 10, aseptic operation separates liver, and separation process is rushed with liquid is saved
It washes, warm ischemia time is less than 30min, and the static 4 DEG C of cold preservations of the in vitro liver application UW liquid of first group of 10 rat, second group is answered
The organ preservative fluid low-temperature circulating machine perfusion described in embodiment 1 saves, and perfusion pressure and groundwater increment are physiological amount, Perfusion preservation
Organ preservative fluid inputs in liver from vena portae hepatica and arteria hepatica immediately immediately after over-saturation drum oxygen in the process.
After saving for 24 hours, samples be sampled film-making Electronic Speculum inspection according to sample method needed for Electronic Speculum immediately.
Histotomy result (Fig. 9-Figure 11) display: after being saved using organ preservative fluid of the present invention, rat liver
Cell mitochondrial inner membrance and outer membrane fusion phenomenon are decreased obviously, and structure especially ridge is completely clear in mitochondria, and apply UW
Rat liver liver cell mitochondrial inner membrane and the outer membrane fusion that liquid saves, show that there were significant differences.
The above results show organ preservative fluid of the present invention in the application of organ storage in vitro to rat liver microcirculation
The protective effect of structure, cell Proliferation and liver transfer operation success rate is substantially better than UW liquid.
5 rat heart storage in vitro effect detection of embodiment (HE dyeing)
Male SD rat 20, it is divided into two groups, every group 10, aseptic operation isolating cardiac, separation process is rushed with liquid is saved
It washes, warm ischemia time is less than 30min, and the static 4 DEG C of cold preservations of the isolated heart application UW liquid of first group of 10 rat, second group is answered
The organ preservative fluid low-temperature circulating machine perfusion described in embodiment 1 saves, and perfusion pressure and groundwater increment are physiological amount, Perfusion preservation
Organ preservative fluid is inputted from aorta intracardiac immediately immediately after over-saturation drum oxygen in the process.
After saving for 24 hours, samples carry out HE stained slice tissue examination according to routine histologic method immediately.Histotomy knot
Fruit (Figure 12-Figure 15) display: the complete of heart arteriovenous endothelial cell can be saved using organ preservative fluid of the present invention
Property, and the heart for applying UW liquid to save, show that there were significant differences.
The experiment of 6 Rat Liver Transplantation of embodiment
Male SD rat 40, it is divided into two groups, every group 20 (10 pairs), using Zheng Shusen works " Rat Liver Transplantation diagram "
(ISBN:9787117198769) the method carries out Rat Liver Transplantation, and wherein liver separation process is rinsed with liquid is saved, and heat lacks
The blood time be less than 30min, after separation first group 10 in rat 10 donor rats in vitro liver application UW liquid static state 4 DEG C it is cold
It saves within 24 hours, saves within organ preservative fluid low-temperature circulating machine perfusion 24 hours described in second group of Application Example 1, temperature 4
DEG C, perfusion pressure and groundwater increment are physiological amount, during Perfusion preservation liver organ save liquid over-saturation drum oxygen after immediately from
Portal vein and arteria hepatica input in liver immediately.
Survival of rats situation is as follows:
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. it is a kind of maintain vascular endothelial cell storage in vitro during survive and/or function method, including make the cell with
The step of saving liquid contact, it is characterised in that the preservation liquid contains following component: the cell can (a) entered similar
The hibernation protective agent of hibernation-like state；(b) energy substrate；(c) scavenger of metabolite is removed；(d) colloid osmotic pressure maintains agent
And/or colloid osmotic pressure regulator；(e) oxygen.
2. according to the method described in claim 1, wherein the preservation liquid is also optionally containing selected from antibiotic, anti-inflammatory agent
One or more of object, immunosuppressor.
3. method according to claim 1 or 2, it is characterised in that the preservation for organ；Preferably, wherein the device
Official is liver, heart, kidney, pancreas, lung or enteron aisle etc..
4. a kind of preservation liquid of survival and/or function during maintaining vascular endothelial cell storage in vitro, contains base needed for cell
This salt, pH buffer and deionized water, it is characterised in that the preservation liquid also contains following component:
(a) cell can be made to enter the hibernation protective agent of similar hibernation-like state；(b) energy substrate；(c) metabolite is removed
Scavenger；(d) colloid osmotic pressure maintains agent and/or colloid osmotic pressure regulator；(e) oxygen.
5. preservation liquid according to claim 4, it is characterised in that the preservation liquid also optionally contains selected from antibiotic, resists
One or more of scorching drug, immunosuppressor.
6. method according to claim 1 to 3 or the preservation liquid according to claim 4-5, in which:
The basic salt is selected from one of sodium chloride, potassium chloride or magnesium sulfate or two or more；
The pH buffer be selected from one of HEPES salt, phosphate, carbonate, hydrophosphate, bicarbonate, histidine or
Person is two or more；
The hibernation protective agent, which is selected from, inhibits cell metabolism class drug, the inhibitor of cellular informatics access, neurotransmitter antagonism
Agent, one of sodium, potassium or calcium ion channel blocker or two or more；Preferably, the hibernation protective agent is selected from water
Close chloral, lidocaine, atropine, procaine, nifedipine, dilantin sodium, Amiodarone Hydrochloride, hyoscine, Tangut Anisodus Radix
One of alkali or two kinds；
The energy substrate is in atriphos, adenosine diphosphate (ADP), adenosine phosphate, acetyl coenzyme A, insulin, glucose
It is one or two kinds of more than；
The scavenger is one or more kinds of in the substance for remove the metabolite for causing cell death；Preferably,
The scavenger is selected from vitamin C, rotenone, the fast alcohol of glycosides difficult to understand, L-NAME, butylated hydroxy anisole, two fourths
One of base hydroxy-methylbenzene, propylgallate, sodium isoascorbate, phytic acid, tea polyphenols are two or more；
The colloid osmotic pressure maintain agent be selected from one of hydroxyethyl starch, hyaluronate, hyaluronic acid, mannitol or
Person is two or more；
The colloid osmotic pressure regulator be selected from one of gossypose, dextran, albumin, glucan or two kinds with
On.
The antibiotic is selected from Florfenicol, penicillin, streptomysin, gentamicin, chloramphenicol, cephalosporin, terramycin, red
One of mycin, colistin, griseofulvin are two or more；
The anti-inflammatory drug is selected from curcumin, Fenbid, Mobic, aspirin, paracetamol, Diclofenac, Yin
Diindyl U.S. is pungent, one of brufen, fenbufen or two or more；
The immunosuppressor be selected from ciclosporin A, tacrolimus, cyclophosphamide, rapamycin, sulphur file purine, methotrexate (MTX),
One of glucocorticoid (such as dexamethasone) is two or more.
7. preservation liquid according to claim 6 contains basic salt, pH buffer, hibernation protective agent, energy needed for cell
Measure substrate, remove the scavenger of metabolite, colloid osmotic pressure maintains agent and/or colloid osmotic pressure regulator, antibiotic, anti-inflammatory
Drug, immunosuppressor, oxygen and deionized water.
8. preservation liquid according to claim 7, wherein the liquid of every 1000mL contains:
9. preservation liquid according to claim 4, it is characterised in that the pH value for saving liquid is 7.4 ± 0.1, and salinity is
9.0, osmotic pressure is 290-380mOsm/kg H2O, and wherein colloid osmotic pressure is 1.5-30.0mOsm/kg H2O.
10. any one of the claim 4-9 preservation liquid survive during vascular endothelial cell storage in vitro as maintaining and/or
The application of the preservation liquid of function；Preferably, the application for saving liquid as organ preservative fluid；It is highly preferred that the organ
Selected from liver, heart, kidney, pancreas, lung or enteron aisle.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711115825.8A CN109769797A (en) | 2017-11-13 | 2017-11-13 | A kind of organ preservative fluid |
| PCT/CN2018/112091 WO2019091290A1 (en) | 2017-11-13 | 2018-10-26 | Organ preservation solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711115825.8A CN109769797A (en) | 2017-11-13 | 2017-11-13 | A kind of organ preservative fluid |
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| CN109769797A true CN109769797A (en) | 2019-05-21 |
Family
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| WO (1) | WO2019091290A1 (en) |
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| CN110226593A (en) * | 2019-07-30 | 2019-09-13 | 葛亮 | A kind of biological tissue's frozen solution |
| CN110663679A (en) * | 2019-10-24 | 2020-01-10 | 青岛瑞思德生物科技有限公司 | Placenta preserving fluid and preparation method thereof |
| CN112425602A (en) * | 2019-08-26 | 2021-03-02 | 无锡市人民医院 | Isolated lung mechanical perfusion liquid and preparation method and application thereof |
| CN112790189A (en) * | 2021-03-10 | 2021-05-14 | 四川大学华西医院 | Organ perfusion and preservation solution and application thereof |
| CN117481629A (en) * | 2023-12-29 | 2024-02-02 | 成都水木医疗科技有限公司 | Multifunctional colloid osmotic pressure instrument and measuring method and application thereof |
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| CA2936650C (en) | 2013-11-21 | 2022-06-21 | The University Of British Columbia | Polymer based transplant preservation solution |
| CN112293411B (en) * | 2020-11-27 | 2021-09-28 | 大连海洋大学 | Low-temperature preservation liquid and preservation method for sperms of echinococcus intermedius |
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