CN106342787A - Organ preservation solution and preparation method thereof - Google Patents
Organ preservation solution and preparation method thereof Download PDFInfo
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- CN106342787A CN106342787A CN201610719416.8A CN201610719416A CN106342787A CN 106342787 A CN106342787 A CN 106342787A CN 201610719416 A CN201610719416 A CN 201610719416A CN 106342787 A CN106342787 A CN 106342787A
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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
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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
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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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
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Abstract
The invention relates to the fields of medicine and biology, in particular to an organ preservation solution and a preparation method thereof. Every 1000mL of the organ preservation solution is prepared from the following components by weight: 3.0-15.0g of sodium citrate, 10.0-30.0g of potassium citrate, 5.0-15.0g of mannitol, 0.5-1.2g of potassium chloride, 0.5-1.8g of magnesium sulfate heptahydrate, 0.5-25.0g of a pH buffer pair, 0.2-25.0g of energy substrate and the balance of water for injection. The organ preservation solution also contains one or more of laminarin, trehalose, laminine and betaine. The organ preservation solution is low in price and efficient, and is suitable for preserving multiple organs.
Description
Technical field
The present invention relates to medical science and field of biology are and in particular to a kind of organ preservative fluid and preparation method thereof.
Background technology
Organ transplantation is described as the great life sciences progress that twentieth century changes human lives, is universally acknowledged treatment
The effective ways of multiple whole Terminal Diseases.According to statistics, the patient of the only annual organ failure of China has 100-150 ten thousand, is badly in need of organ
Transplant patient about 300,000.Therefore, the effective preservation how carrying out isolated organ becomes one of important support technology of organ transplantation.
The basic goal that organ preserves is to maintain the activity of isolated organ, reduces ischemia to greatest extent each to isolated organ
Plant and damage, be allowed to be more applicable for transporting, and can more promptly recover function after surgery.
A kind of effective organ preservative fluid must possess the feature of five aspects: 1, reduces the cellular water leading to because of low temperature
Swollen；2nd, the intercellular substance during preventing lavation from preserving expands；3rd, prevent the acidization of cell；4th, prevent or mitigate Reperfu- sion
Damage；The 5th, the substrate of regeneration high-energy phosphate compound is provided.The organ preservative fluid clinically commonly used at present substantially have 2 kinds of uw liquid and
Htk liquid etc..
Universitywisconsin organ preservative fluid (abbreviation uw liquid) is invented by wisconsin university of the U.S., is recognized
Standard for liver, pancreas, kidney preserves liquid, and clinic confirms that it can preserve kidney 72 hours, liver dirty 20-24 hour.But
This preservation liquid hyperkalemia hyponatremia, easily causes the damage of vascular endothelial cell；Colloid composition hetastarch (hes) is to hemorheology
Impact is larger, and easily causes erythrocyte aggregation；The costs of material such as Raffinose, lactobionic acid are high, greatly increase organ transplantation and suffer from
Person's clinic expense；Viscosity is high, affects perfusion effect.
Contain histidine-histidine salt buffer pair in htk liquid, can substantially suppress Tissue acidification；Its sodium ion and potassium from
Sub- concentration is relatively low；Viscosity low it is easy to expanding.But from the point of view of organ preservation effect, donor liver is dirty to show microcirculation disturbance,
The preservation effect of htk liquid is not as good as uw liquid.
At present, the cell-preservation liquid of China's exploitation has hc-a liquid (hypertonic citric acid purine liquid), by The 2nd Army Medical College head
Levy hospital and Shanghai Central Blood Station develops jointly, it prepares simple, good stability.But hc-a liquid is only limitted to monoblock cutting device
Lavation and kidney being kept in existence in body during official, the organ such as liver, pancreas preserves and still needs to rely on uw liquid.
Additionally, research worker additionally provides the organ preservative fluid of multiple improvement, such as Chinese patent application 200910014423
Disclose a kind of organ preservative fluid and preparation method thereof, be characterized in: contain in every 1000ml organ preservative fluid: potassium citrate
21～29mmol；One hypophosphite monohydrate sodium dihydrogen 6～10mmol；Disodium hydrogen phosphate 30～45mmol；Magnesium sulfate 4～6mmol；Chlorination
Potassium 1～3mmol；Dextran-40 16-24g；Mannitol 110～150mmol；Adenosine 4～6mmol；Acetylcysteine 4～
6mmol；Sodium hydroxide 4～6mmol；Balance of water for injection.But this preservation liquid is dual compartment package, using more bothering, no
Enough simplicity, and then preparation method also relatively complicated complexity, during reply emergency, motility is relatively poor；And this preservation liquid is only right
Kidney has better effects, not good to the preservation effect of other organs.
And for example, Chinese patent application 200810033425 discloses a kind of organ preservative fluid and preparation method thereof, this preservation
Liquid is by sodium citrate, potassium citrate magnesium sulfate, a hypophosphite monohydrate sodium dihydrogen, sodium hydroxide, ribosidoadenine, arginine, color ammonia
Acid, Mannitol and hydrochloric acid Rhizoma Chuanxiong piperazine etc. become to be grouped into.But this preservation liquid also only has better effects to kidney, to other organs
Preservation effect is not good.
In sum, htk liquid and celsior liquid are mainly used in the preservation of heart, and range is restricted；Uw liquid conduct
The multi-organ preservation solution of standard has many advantages, such as, but its viscosity height, potassium ion are too high and expensive, process and inspection
Inconvenience, is not particularly suited for the actual national conditions of China；Domestic hca liquid and its improvement formula are mainly used in kidney being kept in existence, to other organs
Preservation effect is not good.In recent years, as liver is dirty, the big organ transplantation such as heart and pancreas and combined multiple organs trans plantation be mine
Demand improves constantly, and lacking multi-organ preservation solution efficient, cheap, applied widely and adapting to China's national situation becomes restriction China
The major reason of organ transplantation technique development.
In sum, developing one kind new cheap, efficient, and be applied to the organ preservative fluid of multiple organ preservation has very
Important research and clinical value.
Content of the invention
The purpose of the present invention is to overcome the deficiencies in the prior art, provides a kind of new cheap, efficient, and is applied to multiple organ
The organ preservative fluid preserving.
On one side, the invention provides a kind of organ preservative fluid, described organ preservative fluid is, often by weight ratio
1000ml contains:
Preferably, described organ preservative fluid is by weight ratio, and every 1000ml contains:
It is highly preferred that described organ preservative fluid is by weight ratio, every 1000ml contains:
It is further preferred that described organ preservative fluid is by weight ratio, every 1000ml contains:
Energy substrate in organ preservative fluid is used for the energy source adenosine triphosphate of regenerative cell, keeps mitochondrial work(
Can be active, maintain energy metabolism.Energy substrate in organ preservative fluid of the present invention is ribosidoadenine, a phosphoric acid gland
One of glycosides, adenosine diphosphate (ADP), tryptophan, α-ketoglutaric acid, succinic acid, succinyl coenzyme a, acetylcoenzyme a and ubiquinone 10
Or it is several；It is preferably one or more of ribosidoadenine, succinic acid, ubiquinone 10, tryptophan and α-ketoglutaric acid；Enter one
Step is preferably one or more of ribosidoadenine, ubiquinone 10, tryptophan and succinic acid；It is still more preferably adenine
Nucleoside, ubiquinone 10, tryptophan and succinic acid.
Under cryopreservation state, due to hypoxic-ischemic, metabolic pathway is carried out to glycolysiss direction, simultaneously facilitates the life of lactic acid
Become, lead to Tissue acidification.Tissue acidification meeting damaging cells simultaneously lead to lysosome unstable, and activation lysosome simultaneously damages mitochondrial
Function.Acid-base buffer system therefore should be contained in organ preservative fluid, reduce Tissue acidification, maintain intraor extracellular environment relatively steady
Fixed.Ph buffering in organ preservative fluid of the present invention is to for one or more of phosphate, bicarbonate and histidine；
It is preferably one of phosphate and histidine or two kinds；More preferably phosphate and histidine.
One of laminarin, trehalose, laminine and glycine betaine or several is also contained in described organ preservative fluid
Kind.
Laminarin, refers to the macromolecular polysaccharide material in Thallus Laminariae (Thallus Eckloniae).Laminarin has extensive biologic activity, for example
Research shows that laminarin purifies the effect of acidic materials.
Trehalose is a kind of safe and reliable natural saccharide, has medically been successfully applied to trehalose instead blood plasma
As the stabilizer of the bioactive substances such as blood products, vaccine, lymphocyte, cell tissue, this extracellular trehalose also has albumen
There is the function of preventing virus disseminating.
Glycine betaine, for antitumor, blood pressure lowering, anti-peptic ulcer and gastrointestinal dysfunction, treats hepatic disease etc., this
The outer function also with bactericidal antiphlogistic.
In described organ preservative fluid, the content of laminarin is, every 1000ml contains 1.0-10.0g.
In described organ preservative fluid, the content of trehalose is, every 1000ml contains 1.0-16.0g.
In described organ preservative fluid, the content of laminine is, every 1000ml contains 8.0-27.0g.
In described organ preservative fluid, the content of glycine betaine is, every 1000ml contains 4.0-37.0g.
Hydrochloric acid Rhizoma Chuanxiong piperazine, arginine, lactobionic acid, Glutathione, other purine can also be contained in described organ preservative fluid
One or more of alcohol and kapok sugar.
On the other hand, the preparation method of the above-mentioned organ preservative fluid of the present invention also offer, described preparation method include with
Lower step:
(1) weigh the other compositions in addition to energy substrate according to formula consumption, add up the injection of volume 50-80%
After water stirs, add medical activated carbon, after stirring 5-20 minute, using 0.45 μm of membrane filtration decarburization, obtain solution
a；
(2) weigh energy substrate according to formula consumption, after water for injection dissolving, obtain energy substrate solution, by energy
The solution a mixing of amount substrate solution and step (1), benefit adds to the full amount of water for injection, after stirring, using 0.45 μm of filter membrane
Filter, filtrate is organ preservative fluid.
In described preparation method step (1), the addition of medical activated carbon is that every 1000ml water for injection adds 1-5g；
It is preferably every 1000ml water for injection and add 2-4g,；More preferably every 1000ml water for injection adds 3g.
In described preparation method step (1), the mean diameter of medical activated carbon is 2～5 μm, preferably 3～4 μm, enters one
Step is preferably 4 μm.
Compared with prior art, the wound repair liquid that the present invention provides has the advantages that
(1), the organ preservative fluid that the present invention provides can be used in cooling, lavation and the preservation of multiple organs, can effectively protect
Deposit kidney, liver, heart and pancreas more than 48 hours, wherein can preserve kidney 72 hours, liver 48 hours.
(2), laminine is used for organ preservative fluid by the present invention, and laminine has decompression and antibacterial action, anticipates in addition
Other places finds that the addition of laminine can be obviously prolonged the holding time to pancreas, and the holding time of heart is prolonged by less than 24h
Grow to 48h.
(3), the organ preservative fluid that the present invention provides has hyperkalemia hyponatremia characteristic, can reduce the ion exchange of intraor extracellular
And energy expenditure, prevent cellular edema from occurring, effectively extend the holding time.
(4), eliminate hetastarch in the organ preservative fluid that the present invention provides, reduce liquid sticky degree, except containing
Outside Mannitol, also contain the effect that laminarin and trehalose, wherein laminarin and trehalose have stabilate film, by
Mannitol, laminarin and three kinds of compositions of trehalose constitute mixing osmotic pressure regulators, can effectively adjust osmotic pressure, suppression
Cellular edema and gap expansion, effectively extend the holding time.
(5), the present invention provide organ preservative fluid in contain glycine betaine, glycine betaine have protection cell membrane film, protective enzyme,
Maintain the effect of Premeabilisation of cells pressure, the present invention creatively finds, glycine betaine is used for organ preservative fluid can to a certain degree be pressed down
Tissue acidification processed, stablize lysosome；And can to a certain degree suppress cellular edema, can effectively extend to liver, kidney
Holding time.
(6), in freezen protective, anaerobic glycolysises are its unique energy sources to organ, and this process is ph dependency
, ph declines and the accumulation of other metabolites will suppress glycolytic cycle, also leads to energy deficiency.Therefore, ph buffering is right
Function in organ preservative fluid is extremely important, and suitable ph buffering changes to being effective against ph, and carries out across cell
Film transport capacity is weaker, will not cause edema because entering intracellular.Conventional organ preserves liquid and adopts the buffering of single kind right,
And the present invention adopts double ingredient buffer pair of phosphate and histidine, its effect be substantially better than respective single component buffer right,
It is able to maintain that ph is conducive to intracellular h in 8.0 about, ph in 8.0 about organ preservative fluid+By transmembrane transport to outflow
Dynamic, alleviate intracellular acidosis, be liver cell ph when 0-4 DEG C close to 7.7 about, atp can be slowed down and decompose, mitigate to line grain
The infringement of body, eliminates the suppression to glycolysiss key enzyme fructose 1,6-diphosphate kinases for the acidosis, increases the synthesis of atp.
(7), because the transdermal delivery efficiency of different material is different, the effect playing a role hence into cell interior is not yet
With.Conventional organ preserves the energy substrate that liquid adopts single kind, such as ribosidoadenine.And the present invention adopts multiple constituents
Energy substrate, such as ribosidoadenine, ubiquinone 10, tryptophan and succinic acid, because transdermal delivery is imitated between multicomponent energy substrate
Rate is different, therefore can mutually make up the energy synthesis deficiency leading to due to conevying efficiency difference.And the present invention provide contain
The preservation effect of the organ preservative fluid of multicomponent energy substrate is also significantly better than the organ containing single component energy substrate and preserves
Liquid, provides energy for cell under anoxic conditions, and the repair to reperfusion injury is higher, effectively can carry out energy again
Raw.
(8) the organ preservative fluid ph that the present invention provides, in 6.2-8.4, close to physiology ph, can effectively suppress Tissue acidification
And prevent cellular edema, reduce the untoward reaction causing because of ph difference；Osmotic pressure, in 310-380mosm/l, can effectively prevent
Edema, can be obviously prolonged the organ holding time.
(9), the index that mitochondrial respiratory rate (rcr) synthesizes as mitochondrion self-energy being capable of effecting reaction cell and transplanting
The activity of thing, rcr is higher than 3.0 preservation liver, and after transplanting, liver function is preferable.The organ preservative fluid that the present invention provides preserves kidney 48
Hour, be able to maintain that rcr 3.0 about hence it is evident that be higher than prior art.
(10) no incompatibility between the composition that used of organ preservative fluid that, present invention provides, preparation process is simple, property
Matter is stable, cheap.
In sum, the organ preservative fluid that the present invention provides is cheap, efficient, and is applied to multiple organ preservation.
Specific embodiment
The explanation of following examples is only intended to help and understands the method for the present invention and its core concept.It should be pointed out that it is right
For those skilled in the art, under the premise without departing from the principles of the invention, the present invention can also be carried out
Some improvement and modification, these improve and modify and also fall in the protection domain of the claims in the present invention.To disclosed enforcement
The description below of example, makes professional and technical personnel in the field be capable of or uses the present invention.Multiple modifications to these embodiments
Will be apparent from for those skilled in the art, generic principles defined herein can be without departing from this
In the case of the spirit or scope of invention, realize in other embodiments.Therefore, the present invention be not intended to be limited to illustrated herein
These embodiments in, but can apply to meet the broader model consistent with principles disclosed herein and features of novelty
Enclose.
Unless otherwise defined, all technology used herein and scientific terminology have and the technical field of the invention
The same meaning that those of ordinary skill is generally understood that.
Potassium citrate is purchased from Xinning pharmaceutical factory of Taishan, Guangdong city；Sodium citrate is purchased from Shaanxi Xi'an the 4th pharmaceutical factory；Seven
Magnesium sulfate heptahydrate reaches Chemical Co., Ltd. purchased from upper Hypon；Succinic acid, tryptophan, histidine, potassium chloride, a hypophosphite monohydrate dihydro
Sodium, disodium hydrogen phosphate are refined Technological research institute purchased from Shanghai；Ribosidoadenine is purchased from Xing Hu company of Zhaoqing Guangdong city；, trehalose
Purchased from Nanning Zhong Nuo bio-engineering corporation；Laminine is purchased from Shaanxi Ci Yuan Bioisystech Co., Ltd；Laminarin is purchased from west
An Tongze bio tech ltd；Mannitol is purchased from Zhengzhou feudal dynasty chemical products company limited；Glycine betaine is purchased from Jinan gold brightness
Chemical Co., Ltd.；Ubiquinone 10 is purchased from Xi'an Kang Nuo Chemical Co., Ltd.；Optical microscope is purchased from Shanghai optical instrument company；
Centrifuge and constant water bath box are purchased from Shanghai Medical instrument company.
A kind of organ preservative fluid of embodiment 1
Formula, every 1000ml contains:
Preparation method:
(1) weigh the other compositions in addition to ribosidoadenine according to formula consumption, add up the injection of volume 600ml
After water stirs, add the medical activated carbon 1g that mean diameter is 2 μm, after stirring 5 minutes, using 0.45 μm of membrane filtration
Decarburization, obtains solution a；
(2) weigh ribosidoadenine according to formula consumption, after the dissolving of 50ml water for injection, obtain energy substrate molten
Liquid, the solution a mixing of energy substrate solution and step (1) is mended and is injected water to 1000ml, after stirring, uses
0.45 μm of membrane filtration, filtrate is organ preservative fluid.
A kind of organ preservative fluid of embodiment 2
Formula, every 1000ml contains:
Preparation method:
(1) weigh the other compositions in addition to ribosidoadenine, ubiquinone 10 and succinic acid according to formula consumption, add up
After the water for injection of volume 800ml stirs, add the medical activated carbon 5g that mean diameter is 3 μm, after stirring 20 minutes, make
With 0.45 μm of membrane filtration decarburization, obtain solution a；
(2) ribosidoadenine, ubiquinone 10 and succinic acid are weighed according to formula consumption, using the dissolving of 100ml water for injection
Afterwards, obtain energy substrate solution, the solution a mixing of energy substrate solution and step (1) mended and injected water to 1000ml,
After stirring, using 0.45 μm of membrane filtration, filtrate is organ preservative fluid.
A kind of organ preservative fluid of embodiment 3
Formula, every 1000ml contains:
Preparation method:
(1) weigh to become except other in addition to succinic acid of ribosidoadenine, ubiquinone 10, tryptophan according to formula consumption
Point, after the water for injection of totalling volume 700ml stirs, add the medical activated carbon 3g that mean diameter is 4 μm, stir 15 points
Zhong Hou, using 0.45 μm of membrane filtration decarburization, obtains solution a；
(2) ribosidoadenine, ubiquinone 10, tryptophan and succinic acid are weighed according to formula consumption, using 200ml injection
After water dissolution, obtain energy substrate solution, by the solution a mixing of energy substrate solution and step (1), benefit injects water to
1000ml, after stirring, using 0.45 μm of membrane filtration, filtrate is organ preservative fluid.
A kind of organ preservative fluid of embodiment 4
Formula, every 1000ml contains:
Preparation method:
(1) weigh the other compositions in addition to ribosidoadenine and succinic acid according to formula consumption, add up volume 700ml
Water for injection stir after, add mean diameter to be 4 μm of medical activated carbon 3g, after stirring 15 minutes, using 0.45 μm
Membrane filtration decarburization, obtain solution a；
(2) weigh ribosidoadenine and succinic acid according to formula consumption, after the dissolving of 200ml water for injection, obtain energy
Amount substrate solution, the solution a mixing of energy substrate solution and step (1) is mended and is injected water to 1000ml, stirs
Afterwards, using 0.45 μm of membrane filtration, filtrate is organ preservative fluid.
A kind of organ preservative fluid of embodiment 5
Formula, every 1000ml contains:
Preparation method:
(1) weigh the other compositions in addition to ribosidoadenine and ubiquinone 10 according to formula consumption, add up volume
After the water for injection of 600ml stirs, add the medical activated carbon 2g that mean diameter is 5 μm, after stirring 8 minutes, use
0.45 μm of membrane filtration decarburization, obtains solution a；
(2) weigh ribosidoadenine and ubiquinone 10 according to formula consumption, after the dissolving of 200ml water for injection, obtain energy
Amount substrate solution, the solution a mixing of energy substrate solution and step (1) is mended and is injected water to 1000ml, stirs
Afterwards, using 0.45 μm of membrane filtration, filtrate is organ preservative fluid.
A kind of organ preservative fluid of embodiment 6
Formula, every 1000ml contains:
Preparation method:
(1) weigh the other compositions in addition to ribosidoadenine, tryptophan and succinic acid according to formula consumption, add up body
After the water for injection of long-pending 600ml stirs, add the medical activated carbon 2g that mean diameter is 5 μm, after stirring 10 minutes, use
0.45 μm of membrane filtration decarburization, obtains solution a；
(2) ribosidoadenine, tryptophan and succinic acid are weighed according to formula consumption, after the dissolving of 200ml water for injection,
Obtain energy substrate solution, the solution a mixing of energy substrate solution and step (1) is mended and injected water to 1000ml, stirring
After uniformly, using 0.45 μm of membrane filtration, filtrate is organ preservative fluid.
A kind of organ preservative fluid of comparative example 1
Being distinguished as without laminarin and trehalose of formula and embodiment 3；
Preparation method is with embodiment 3.
A kind of organ preservative fluid of comparative example 2
Being distinguished as without laminine of formula and embodiment 3；
Preparation method is with embodiment 3.
A kind of organ preservative fluid of comparative example 3
Being distinguished as without glycine betaine of formula and embodiment 3；
Preparation method is with embodiment 3.
A kind of organ preservative fluid of comparative example 4
Being distinguished as without histidine of formula and embodiment 3；
Preparation method is with embodiment 3.
A kind of organ preservative fluid of comparative example 5
Being distinguished as without disodium hydrogen phosphate and a hypophosphite monohydrate sodium dihydrogen of formula and embodiment 3；
Preparation method is with embodiment 3.
A kind of organ preservative fluid of comparative example 6
Being distinguished as without ubiquinone 10, tryptophan and succinic acid of formula and embodiment 3, and the content of ribosidoadenine is
11.0g；
Preparation method is with embodiment 3.
Morphological change during experimental example 1 Ren sus domestica cryopreservation
Laboratory animal: healthy adult experiment Ba-Ma mini pig, male and female are random, body weight 25-30kg.
Experimental technique:
1st, Preoperative Method and anesthesia: pre-operative anxiety 16h, 12h of intaking.Donor adopts basal anesthesia, ketamine IM 1mg/
Kg induced anesthesia, propofol maintains.
2nd, cut for kidney: technology is quickly cut using general holonephros.
3rd, preserve: after obtaining for kidney, in the organ preservative fluid of embodiment 1-6 and the preparation of comparative example 1-6, low temperature is protected
Deposit, with hca- liquid (being provided by the second medical officer big Long March hospital) for comparison.
4th, pathologic finding: at the 1st, 24,48,72 hours of kidney being kept in existence, take nephrolithotomy specimen, make paraffin section,
Carry out om observation；Make electron microscopic section and carry out transmission electron microscope morphological observation.
Experimental result:
Hca- liquid group: preserve 24 hours, light microscopic finding Renal Structure slightly changes, kidney bead, kidney Minute Tubule Structures
Clear-cut, interstitial Mild edema；Preserve 48 hours, most of kidney bead blood capillary profile is still clear, kidney tubular epithelial
Arrive severe cavity sample degeneration in extensively, interstitial edema substantially, brush border infringement serious it is seen that kidney tubular epithelial multiple special mess shape
Necrosis；Preserve 72 hours, kidney bead capillary vessel member no normal profile, kidney tubular epithelial extensively and serious degenerative simultaneously
Lamellar necrosis, iuntercellular dissociates, and interstitial edema is obvious.Electronic Speculum result is consistent with light microscopic.
Embodiment of the present invention 1-6 group: in 48 preserving, the morphological change of embodiment 1-6 group and hca- liquid group are no
Significant difference；But preserve 72 hours, renal tissue profile does not relatively have obvious deterioration for 48 hours, cell breakage and lamellar is bad
Extremely, deterioration degree is by being followed successively by weak sequence by force: embodiment 5,1,4,2,6,3.
Comparative example 1-6 group of the present invention: preserve 24 hours and occurred compared with 1 hour substantially deteriorating, cell breakage and lamellar occur
Necrosis.
Morphological change during the small-sized pig liver cold preservation-reperfusion of experimental example 2
Laboratory animal: with experimental example 1
Experimental technique:
1st, Preoperative Method and anesthesia: with experimental example 1.
2nd, pulmonary preservation: technology is quickly cut using general full liver.
3rd, preservation-Reperfu- sion: carry out cryopreservation after obtaining for liver, preserve liquid and implement 1-5 and comparative example 1-6 for the present invention
The organ preservative fluid of preparation, behind 1,12 hours preserving and Reperfu- sion after 1 hour, takes specimen to carry out om observation, with hca-
Liquid and uw liquid (being provided by the second medical officer big Long March hospital) are comparison.
Experimental result:
Hca- liquid group: preserve 1 hour, blood lipid structure is normal, slight hepatic cell edema；Preserve 12 little
When, lobuli hepatis structure is still clear, part liver plate disintegrate, and hepatocyte universal mild to moderate balloon sample becomes, a small amount of stem cell necrosis；Again
After perfusion one hour, hepatic sinusoid is substantially expanded, swelling of liver cell, the major part disintegrate of liver plate, is dispersed in a small amount of steatosis and a small amount of
Necrosis.
Uw liquid group: preserve 1 hour, blood lipid structure is normal, slight hepatic cell edema；Preserve 12 hours, liver
Leaflet structure is still clear, swelling of liver cell, has a small amount of hepatocyte to become balloon sample to become, lobule peripheral part hepatocyte has point-like bad
Extremely.
Embodiment of the present invention 1-6 group: preserve 1 hour, blood lipid structure is normal；Preserve 12 hours, hepatic tissue
Morphosiss are normal, slight hepatic cell edema, and other does not have a significant change, and liver morphology changes degree by by force to weak
It is followed successively by: 1,4,2,5,6,3.
Comparative example 1-6 group of the present invention: preserve 1 hour, lobuli hepatis structure is still clear, swelling of liver cell, has a small amount of hepatocyte
Balloon sample is become to become, lobule peripheral part hepatocyte has spotty necrosis.
Experimental example 3 renal transplantation is tested
Animal model: female experimental dog, body weight 13-15kg.
Organ preservative fluid: matched group: hca- liquid and uw liquid；Experimental group: embodiment of the present invention 1-6 and comparative example 1-6 system
Standby organ preservative fluid.
Experimental technique: fasting in preoperative 12 hours, anaesthetized with 3% pentobarbital 30mg/kg intraperitoneal injection.Anaesthetize successfully
Afterwards, take median abdominal incision, be about 12cm downwards from xiphoid-process, cut into abdomen.Dissociate left kidney and kidney arteriovenous, ureter, and cut-out is defeated
Urinary catheter, is about 7cm, is not cut off in renal veinss and deep tremulous pulse root portion.Take out kidney immediately and preserve liquid (0 DEG C) as each group
200ml is through analysis of renal artery infusion kidney, until kidney is in pale asphyxia, kidney is preserved liquid together with 500ml and is placed in kidney bag, in 0-4
Store under the conditions of DEG C.Constriction experiment animal kidney arteriovenous and ureter remnant respectively, closes abdomen.Postoperative give ceftriaxone sodium immediately
1g intravenous drip.After 72 hours, same experimental dog is anaesthetized in the same way, opens abdominal cavity by former otch, and extends to downwards shame
Above bone, the outer arteriovenous of right side ilium of dissociating, take out kidney, renal veinss and external iliac vein are with 7-0prelene vascular anastomosiss line end side
It coincide, renal artery and external iliac artery end to end anastomosis, thoroughly stop blooding after open blood flow.Cut bladder, indwelling catheter.In ureter
Put double j pipes of f4.5, coincide with bladder with 5-0 catgut.Subsequently cut the right kidney of experimental dog, close abdomen, operation terminates.Postoperative give benefit
Liquid (daily 10% glucose 500ml, balance liquid 500ml) and ceftriaxone sodium 1g are until laboratory animal starts voluntarily to take food.Art
Renal function index change before and after detection transplanting afterwards.60 days clinical follow phases, the animal reaching 60 days is considered as long-term surviving.
Experimental result:
1st, apoptosis situation during renal cortex cryopreservation, apoptotic index is as follows:
2nd, mitochondria respiratoring control rate (rcr) measurement result is as follows:
3rd, the time-to-live: using the time-to-live of organ preservative fluid and the survival of uw liquid of the preparation of embodiment of the present invention 1-6
Time is suitable, obviously higher than comparative example 1-6 in hca- liquid and the present invention；Using the preparation of embodiment of the present invention 1-6
The long-term surviving dog quantity of organ preservative fluid is also significantly more than comparative example 1-6 in hca- liquid and the present invention.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Within god and principle, any modification, equivalent substitution and improvement made etc., should be included within the scope of the present invention.
Claims (9)
1. a kind of organ preservative fluid it is characterised in that: described organ preservative fluid is that every 1000ml contains by weight ratio:
2. organ preservative fluid as claimed in claim 1 it is characterised in that: described ph buffering is to for phosphate, bicarbonate
One or more of with histidine, preferably one of phosphate and histidine or two kinds, more preferably phosphate
And histidine.
3. organ preservative fluid as claimed in claim 1 it is characterised in that: described energy substrate be ribosidoadenine, a phosphorus
In adenosine monophosphate, adenosine diphosphate (ADP), tryptophan, α-ketoglutaric acid, succinic acid, succinyl coenzyme a, acetylcoenzyme a and ubiquinone 10
One or more；It is preferably one or more of ribosidoadenine, succinic acid, ubiquinone 10, tryptophan and α-ketoglutaric acid；
More preferably one or more of ribosidoadenine, ubiquinone 10, tryptophan and succinic acid；It is still more preferably gland
Purine nucleosides, ubiquinone 10, tryptophan and succinic acid.
4. the organ preservative fluid as described in claim 1-3 any one it is characterised in that: described organ preservative fluid also contains
One or more of laminarin, trehalose, laminine and glycine betaine.
5. organ preservative fluid as claimed in claim 4 it is characterised in that: the content of described laminarin is, every 1000ml
Containing 1.0-10.0g.
6. organ preservative fluid as claimed in claim 4 it is characterised in that: the content of described trehalose is that every 1000ml contains
There is 1.0-16.0g.
7. organ preservative fluid as claimed in claim 4 it is characterised in that: the content of described laminine is, every 1000ml
Containing 8.0-27.0g.
8. organ preservative fluid as claimed in claim 4 it is characterised in that: the content of described glycine betaine is that every 1000ml contains
There is 4.0-37.0g.
9. the method for the organ preservative fluid described in preparation claim 1-8 any one, carries and being characterised by: described preparation method
Comprise the following steps:
(1) other compositions in addition to energy substrate are weighed according to formula consumption, the water for injection adding up volume 50-80% stirs
After mixing uniformly, add medical activated carbon, after stirring 5-20 minute, using 0.45 μm of membrane filtration decarburization, obtain solution a；
(2) weigh energy substrate according to formula consumption, after water for injection dissolving, obtain energy substrate solution, by energy bottom
The solution a mixing of thing solution and step (1), benefit adds to the full amount of water for injection, after stirring, using 0.45 μm of filter membrane mistake
Filter, filtrate is organ preservative fluid.
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| CN109258624A (en) * | 2018-09-20 | 2019-01-25 | 中国人民解放军第二军医大学第二附属医院 | A kind of in vitro amputation saves liquid and preparation method thereof |
| CN109430251A (en) * | 2018-12-18 | 2019-03-08 | 广州康琪莱生物科技有限公司 | A kind of store method of the preservation liquid of adipose tissue and preparation method thereof with adipose tissue |
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