CN109511649B - Normal-temperature mechanical perfusion system capable of expanding liver supply source - Google Patents
Normal-temperature mechanical perfusion system capable of expanding liver supply source Download PDFInfo
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- CN109511649B CN109511649B CN201811392862.8A CN201811392862A CN109511649B CN 109511649 B CN109511649 B CN 109511649B CN 201811392862 A CN201811392862 A CN 201811392862A CN 109511649 B CN109511649 B CN 109511649B
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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
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
- A01N1/0247—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components for perfusion, i.e. for circulating fluid through organs, blood vessels or other 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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
The invention provides a normal-temperature mechanical perfusion system capable of expanding a liver supply source, which comprises a central processing unit assembly, a power assembly, a liver storage assembly, a liquid circulation assembly, a monitoring assembly and a liquid collecting assembly, wherein the liquid circulation assembly comprises a liver perfusate storage device, liver perfusate is stored in the liver perfusate storage device, and the liver perfusate contains naringenin derivatives and does not contain erythrocytes.
Description
Technical Field
The invention belongs to the technical field of design and manufacture of medical instruments, and particularly relates to a normal-temperature mechanical perfusion system capable of amplifying a liver supply source.
Background
China is a country with high incidence of liver diseases, and according to '2013 China health statistics yearbook' and 'China tumor registration annual newspaper', the number of new annual diseases of viral hepatitis is 137 ten thousands, and the first infectious disease of hepatitis B is the first place; the number of new-onset cases of liver cancer is 35.5 ten thousand, the annual death rate is up to 26.26/10 ten thousand, and the liver cancer occupies the second place of malignant tumor; the medical care cost for liver diseases is measured in billions of yuan every year, and the medical care cost becomes a serious problem affecting the health, economy and social development of the nation. With the continuous progress of liver transplantation operation technology, perioperative monitoring and high-efficiency immunosuppressive drugs, liver transplantation has become a reliable and effective means for saving patients with end-stage liver diseases. However, with the proliferation of the number of cases of liver transplantation, the problem of relative shortage of liver supplies is becoming more prominent. Statistically, about 15% of patients are excluded from the liver transplant waiting list each year due to exacerbation or death. The shortage of liver supplies has forced organ transplant physicians to continuously expand the liver supply standards for safe and effective transplantation, and therefore, some liver supplies that do not reach the ideal donors are increasingly used for liver transplantation. A feeder that does not reach an ideal donor is also referred to as an "enlarged ideal feeder" or an "marginally feeder". Such livers include DCD for liver, advanced age for liver, fatty liver for liver, incompatible blood type for liver and infectious disease for liver.
DCD is supplied to the liver, namely dead liver donation, and is one of effective ways for solving the serious shortage of supplied liver. The international general classification standard of Maastrichit in the Netherlands divides DCD donors into two major types of controllable (M-III) and uncontrollable (M-1, M-II, M-IV and M-V), wherein the controllable type means serious irreversible injury, but does not reach the complete set of medical standard of brain death, and the life support is removed in a planned way by the consent of family members to wait for death. The "working guideline for donation of dead organs of liver" classifies the donation of organs into 3 categories: C-I is an international standardized donation of brain death organs; C-II is the international standard DCD, including M-I to M-V; C-III, i.e., donation of double death organs of heart and brain, means that although the DBD standard is met, the donation is still implemented according to the DCD program in view of the deletion of the brain death legislation. The new idea is that warm ischemia time should be calculated from the appearance of clinical hypotension rather than liver arrest. It follows that any DCD donor type is likely to suffer from prolonged warm ischemia, especially if the donor is cardiopulmonary resuscitated or uncontrolled, and will experience prolonged hypotensive attacks, severely damaging the donor function. Research shows that the warm ischemia time and the cold preservation time have important influence on the liver supplying quality of DCD and the receptor prognosis. Disruption of oxygen and nutrient sources and accumulation of metabolites may be the most important factors causing thermal ischemic injury, with the inevitable consequences of cellular energy metabolism disorders, severe reduction or even depletion of hepatic Adenosine Triphosphate (ATP), dramatic decrease in total adenine nucleotide, further leading to oxygen free radical production, intracellular calcium overload, dysfunction of cells and organelles, and apoptosis.
There are 3 essential stages in the liver transplantation process, (1) pre-storage stage including donor management and liver acquisition; (2) the preservation stage comprises liver washing and in-vitro preservation; (3) a reperfusion phase. Each stage can damage the graft and affect the prognosis of the liver transplant recipient. The injury in the pre-preservation stage is called pre-preservation injury, and refers to the basic disease existing in the donor and the injury caused by the donor operation to the graft, and the injury can be avoided as much as possible by reasonable liver donation standard and careful and skilled operation. The damage during the preservation phase is the damage that is experienced before blocking hepatic blood flow to reperfusion, and this damage can only be reduced but not avoided. Cold storage injury is the major form of injury during the storage phase, mainly due to endothelial cell edema and acidosis caused by low temperature and hypoxia during storage. The reperfusion injury belongs to post-storage injury, and occurs in two stages, stage 1 is characterized by immune system activation and oxidative stress; stage 2 is characterized by disorders of inflammatory cells, cytokines and adhesion factors. Compared with the liver supply of normal sources such as DBD liver supply and the like, the DCD liver supply is subjected to long-term thermal ischemia, so that ischemia-reperfusion injury is more likely to occur in the inhibition engineering, irreversible injury is caused to the liver supply, and the requirement of clinical transplantation cannot be met. At present, various scholars respectively try to improve the liver supply quality of DCD from the aspects of donor pretreatment, improvement of liver supply cutting operation technology (injury in the low blood pressure stage cannot be avoided), optimization of an organ preservation method, receptor drug intervention (influencing other system functions of an organism) and the like, shorten the warm/cold ischemia time, prolong the liver ischemia tolerance time limit, and improve the liver transplantation effect of DCD, wherein the optimization of the organ preservation method becomes a breakthrough point with the most application prospect due to the advantages of wide applicability, strong feasibility, remarkable effect and the like.
The normal temperature mechanical perfusion is that in the normal temperature (about 37 ℃), a machine provides power to form a closed loop so as to continuously pump perfusion liquid into the liver supply, metabolic substrates are provided, and metabolic waste is removed at the same time, so that the damage in the process of obtaining and storing the liver supply is reduced, the quality of the liver supply is improved, and the storage time of the in vitro liver is prolonged. The low-temperature mechanical perfusion has the advantages of simple equipment, low cost and the like. The normal-temperature mechanical perfusion is mainly characterized in that the perfusion temperature is consistent with the body temperature, and the perfusion fluid continuously supplements metabolic substrates and supplies oxygen to simulate liver perfusion in a physiological state, so that tissue damage is reduced as much as possible. At present, most scholars think that the perfusion fluid for normal-temperature mechanical perfusion needs blood which is based on whole blood, diluted and heparinized and has balanced pH, and the perfusion fluid can have the function of transporting oxygen after red blood cells are added, can maximally imitate the properties of the perfusion fluid in a physiological state, and can delay the occurrence of interstitial edema in the mechanical perfusion process. Dries et al first determined the components and proportions of perfusate mechanically perfused at room temperature, i.e., all nutrients, oxygen and protective substances required for normal liver metabolism, including concentrated red blood cells, plasma, albumin based formulations, and the addition of nutrient solutions, various trace elements, antibiotics, etc. Meanwhile, in order to further reduce the influence of thermal ischemia reperfusion injury on liver function in the liver transplantation process, many scholars try to add corresponding pharmaceutical active substances into the perfusion solution to restore the normal physiological function of the liver in the perfusion process and reduce the damage of the thermal ischemia reperfusion injury on liver tissues, such as protease inhibitors, epidermal growth factors and albumin, and some plant active ingredients, such as ligustrazine, red paeony root total glycosides, angelica polysaccharide and the like. However, although the normal temperature mechanical perfusion system combined with the corresponding perfusate can reduce the influence of the thermal ischemia reperfusion injury on the liver function to a certain extent and expand the liver supply source, the perfusion system still has many disadvantages, such as the perfusion system still needs to adopt the perfusate containing blood for perfusion, and the blood needs to come from the donor, which also limits the scope of material taking. Meanwhile, the use of a perfusion fluid containing blood increases the risk of rejection and infection, and the tolerance of the perfusion fluid to warm ischemia is to be further improved.
Therefore, it is an urgent problem to obtain a normal temperature mechanical perfusion system which can enhance the tolerance of the liver to thermal ischemia, expand the liver supply source and recover the physiological activity of the liver as soon as possible in the mechanical perfusion process.
Disclosure of Invention
The invention provides a normal-temperature mechanical perfusion system suitable for liver storage, aiming at solving the problems of enlarging a liver supply source and enabling a liver to recover physiological activity as soon as possible in a mechanical perfusion process.
In one embodiment, the central processor unit is connected to the monitoring unit for processing the data information collected by the monitoring unit.
In one embodiment, the central processor unit is connected to the power unit and the liquid circulation unit, and is configured to control the power output by the power unit to control the perfusion mode, perfusion pressure, perfusion flow rate, perfusion temperature, and perfusion oxygenation efficiency of the perfused perfusate.
In one embodiment, the central processor assembly is connected to the liver storage assembly for controlling the temperature during perfusion.
In one embodiment, the monitoring assembly is coupled to the fluid circulation assembly and the liver storage assembly for monitoring and collecting data information of temperature, perfusion pattern of the perfusion fluid, perfusion pressure and perfusion flow rate during perfusion.
In one embodiment, the power assembly is coupled to the fluid circulation assembly for powering the perfusate.
In one embodiment, the fluid circulation assembly is connected to a liver storage assembly for delivering perfusate to the liver ex vivo for storage and filtering the perfusate for recycling.
In one embodiment, the fluid collection assembly is connected to a liver storage assembly for collecting secretions, e.g., bile, produced by the liver during perfusion; the liquid collecting assembly is connected with the monitoring assembly and collects the quantity data of the secretion generated in the perfusion process together with the monitoring assembly.
In one embodiment, the power assembly comprises an infusion power device, preferably a peristaltic or roller pump, or an artificial blood pump commonly used in the art, such as a pneumatic blood pump, an electro-hydraulic blood pump, or the like. The perfusion motive device may provide a continuous perfusion motive force, or a pulsed perfusion motive force.
In one embodiment, the liver storage assembly comprises a liver storage device comprising an inner layer and an outer layer, the interlayer between the inner and outer layers comprising a temperature regulating device, preferably the temperature regulating device is a thermo-regulating tube in the interlayer. The temperature control device is provided with a temperature monitoring device, and the temperature monitoring device is connected with the central processing unit and used for collecting temperature information of the liver storage device and controlling and collecting the temperature in the liver storage device.
In one embodiment, the fluid circulation assembly includes an oxygen supply device, preferably a membrane oxygenator, an artificial lung or a membrane lung, operative to supply oxygen to the perfusate to turn the perfusate into an oxygenated perfusate, and to provide the liver cells with oxygen to maintain physiological function during perfusion.
In one embodiment, the fluid circulation assembly further comprises a perfusate filtration device and a perfusate delivery channel. The perfusate filter device is used for filtering impurities in the perfusate and metabolic waste generated by the liver, and the perfusate is recycled after being filtered.
In one embodiment, the fluid circulation assembly further comprises a perfusion fluid delivery channel. The perfusion conveying channel comprises an input conduit and an output conduit and is used for inputting fresh liver perfusion fluid to the liver preserved in vitro and outputting the perfusion fluid from the liver preserved in vitro. The input conduit is a Y-shaped conduit, one end of the input conduit is provided with two conduits which are respectively used for inputting the hepatic artery perfusate and the portal vein perfusate, preferably, the liver perfusate storage in the liquid circulation component is connected with the liver storage device in the liver storage component through the input conduit.
In one embodiment, the monitoring assembly includes a temperature sensor, a pressure sensor, a flow rate sensor and a volume meter for monitoring the temperature during perfusion, the perfusion pressure of the perfusate, the perfusion flow rate and the amount of secretions produced by the liver during perfusion.
In one embodiment, the central processor assembly includes a control assembly and a data processing assembly for processing the monitored items of data of the monitoring assembly and controlling the temperature, perfusion pressure, perfusion flow rate and oxygenation efficiency of the perfusion fluid during perfusion. Furthermore, the central processing unit also comprises an information transmission device which can wirelessly transmit the data parameters monitored by the monitoring component to a corresponding external receiving device.
In one embodiment, the fluid circulation assembly further comprises a liver perfusate reservoir comprising a temperature regulating device, preferably the liver perfusate reservoir comprises an inner layer and an outer layer, and a temperature regulating device is included in an interlayer between the inner layer and the outer layer, preferably the temperature regulating device is a cold and hot regulating tube in the interlayer. The liver perfusate storage device is used for storing a liver perfusate, and the liver perfusate contains naringenin derivatives and does not contain erythrocytes.
In one embodiment, the naringenin derivatives are naringenin-7-O-acetate and naringenin-7-O-propionate, and preferably, the naringenin derivatives are naringenin-7-O-acetate. Naringenin, which is a aglycone of naringin widely existing in plants and traditional Chinese medicines, belongs to flavanone compounds, has the effects of resisting bacteria, resisting inflammation, scavenging free radicals, resisting oxidation, relieving cough, eliminating phlegm, reducing blood fat, resisting cancer, resisting tumors, relieving spasm, benefiting gallbladder, preventing and treating liver diseases, inhibiting platelet coagulation, resisting atherosclerosis and the like, is widely applied to the fields of medicines, foods and the like at present, and research shows that naringenin can inhibit inflammatory factor storm caused by TNF-a and the like and reduce tissue injury at present. However, most of flavonoids such as naringenin and the like have poor fat solubility and water solubility and low bioavailability, and the fat solubility or the water solubility can be improved and the bioavailability can be improved on the premise of ensuring the physiological activity by modifying the structure of the naringenin or other flavonoids and introducing fat soluble or water soluble groups. naringenin-7-O-acetate and naringenin-7-O-propionate are synthesized by four-step chemical synthesis method of "benzylation-hydrolysis-acylation-hydrogenation reduction", the solubility of two derivatives in water is 637.34 + -53.23 μ g/mL and 59.74 + -4.81 μ g/mL respectively, both are higher than the solubility of naringenin ("preparation of naringenin-7-O-acetate and naringenin-7-O-propionate and anti-platelet aggregation activity", Schumann et al, natural products research and development, Dev 2016, 28: 1273-1278).
In one embodiment, the liver perfusate further contains a biological enzyme, preferably exoglycosidase, more preferably α -galactosidase and α -N-acetylgalactosaminidase, which removes blood group antigens from the cell surface, e.g., A, B antibody, prevents acute rejection during liver transplantation and reduces chronic rejection.
In one embodiment, the liver perfusion fluid comprises artificial blood, and the artificial blood is preferably artificially synthesized fluorocarbon with oxygen carrying function. The fluorocarbon blood is colloid ultramicro emulsion composed of perfluor compound, and has good oxygen carrying capacity. Under certain concentration and oxygen partial pressure, the oxygen solubility is 20 times of that of water and 2 times higher than that of blood. The fluorocarbon compound preferably used as artificial blood includes perfluoro-n-butyl furan, perfluorotributylamine, freon E4, perfluorodecalin, perfluoromethyldecalin, perfluorotripropylamine, etc. The artificial blood carries oxygen and maintains the physiological function of cells. Preferably, the artificial blood is Perfluorocarbon (PFC).
In one embodiment, the liver perfusate further comprises a colloid, lecithin, adenosine triphosphate, insulin, compound amino acid injection 18AA, antibiotics, glucose, normal saline, 10% potassium chloride solution, 5% sodium bicarbonate solution, 10% calcium chloride solution, vitamin B12, vitamin E, vitamin C, dexamethasone, alprostadil. The colloid is preferably PEG, hydroxyl starch or 20% albumin. The antibiotic is preferably penicillin, ampicillin, cefuroxime sodium or the commonly used cephalosporin antibiotics known in the art.
The physiological saline, potassium chloride, calcium chloride and sodium bicarbonate solution are main electrolyte components in a human body, can maintain the osmotic pressure of the perfusate to be close to the plasma of the human body, and provides a good liquid environment for the liver, wherein 5% of sodium bicarbonate can neutralize a large amount of acidic substances generated by anaerobic metabolism of organs in the process of obtaining and before perfusion, and stabilize the pH value in the perfusate; the physiological saline can increase the volume of the perfusate, is used as a main solvent of the perfusate, and can provide stable osmotic pressure of the crystal so as to ensure that the perfusate is in an isotonic state.
The lecithin plays an auxiliary role in emulsifying or dispersing the artificial blood. The PEG, the hydroxyl starch or the 20% albumin can be used as colloid to maintain the osmotic pressure of the perfusate and prevent the liver cells from generating edema caused by long-time perfusion. The Adenosine Triphosphate (ATP) is an energy substance of the cell, which can provide the corresponding energy required for physiology to the liver cells. The vitamin B12, the vitamin E and the vitamin C provide corresponding substances for metabolism of liver cells, and the vitamin C and the vitamin E have an antioxidant function and can reduce oxidative damage caused by free radicals generated by the liver in a perfusion process.
The compound amino acid injection consists of a plurality of different types of amino acids, wherein the amino acids are important components of proteins and peptides and provide metabolic substrates for in vitro liver protein synthesis. The antibacterial drugs penicillin, ampicillin or cefuroxime sodium and the like can prevent infection to a certain extent.
The insulin promotes the transfer of glucose into cells and promotes protein synthesis; the glucose provides a substrate for energy metabolism of an isolated organ and is a main energy source; the dexamethasone can stabilize cell membrane and organelle membrane, and relieve organ inflammation reaction. The alprostadil can expand blood vessels in organs and improve microcirculation of perfusate in the liver.
In one embodiment, the liver perfusate preferably consists of per 1000ml perfusate: 400mg of naringenin-7-O-acetate, 2-6g of hydroxyl starch, 20-40g of adenosine triphosphate, 15-45g of artificial blood, 2-6g of lecithin, 1-2mg of galactosidase, 1-2mg of acetylgalactosamine enzyme, 30-60u of insulin, 300ml of compound amino acid injection 18AA 100, 1-5g of antibiotic, 5-30g of glucose, 300ml of physiological saline, 5-15ml of 10% potassium chloride, 10-30ml of 5% sodium bicarbonate, 10-20ml of 10% calcium chloride, 123-8 mg of vitamin B, 2-6mg of vitamin E, 1-5mg of vitamin C, 1-6mg of dexamethasone and 5-15 mu g of alprostadil; further preferably 325mg of naringenin-7-O-acetate, 2.6g of hydroxyl starch, 30g of adenosine triphosphate, 36g of artificial blood, 4g of lecithin, 1.25mg of galactosidase, 1.25mg of acetylgalactosaminidase, 43u of insulin, 18AA 150ml of compound amino acid injection, 2.5g of antibiotic, 28g of glucose, 200ml of normal saline, 15ml of 10% potassium chloride, 25ml of 5% sodium bicarbonate, 20ml of 10% calcium chloride, vitamin B124.2mg, 3.6mg of vitamin E, 2.5mg of vitamin C, 4.6mg of dexamethasone and 10 mu g of alprostadil, wherein the solvent is water.
In one embodiment, the normothermic mechanical perfusion system suitable for liver preservation can be used for preserving a liver ex vivo, preferably, the preservation temperature is normothermic preservation, for example, 25-37 ℃. More preferably, the donor of the ex vivo liver has experienced cardiac arrest or warm ischemia, e.g., warm ischemia or cardiac arrest for a period of 30-120 min.
Compared with the prior art, the invention has the following advantages: the normal-temperature mechanical perfusion system suitable for liver preservation adopts special liver perfusion liquid, the perfusion liquid has good effects of resisting oxidation, removing free radicals, inhibiting inflammatory factor storm in the perfusion process and the like, so that the liver can be prevented from ischemia reperfusion injury in the in vitro preservation process, and the liver can keep a good physiological activity state in the in vitro preservation process. The normal-temperature mechanical perfusion system suitable for liver preservation can greatly expand the source of a liver donor, can well solve the problem of insufficient source of the liver donor, and has good market prospect and social public value.
Drawings
FIG. 1: the invention discloses a schematic diagram of a mechanical perfusion system for liver preservation, wherein 1: a central processor assembly, 2: peristaltic pump, 3: liver perfusate reservoir, 4: input catheter a, 5: membrane oxygenator, 6: input catheter B, 7: input catheter C, 8: liver storage device, 9: output duct a, 10: perfusate filter equipment, 11: an output conduit B; 12: liquid collecting device, 13: liquid meter, 14: liver normal atmospheric temperature mechanical perfusion system, 15: monitoring data collection device, 16: temperature sensor, 17: pressure sensor, 18: flow rate sensor, 19: data processing apparatus, 20: control assembly, 21: an information transmission device;
FIG. 2: a Bcl-xl immunohistochemical result graph, wherein FIG. 1A is the immunohistochemical result of the Bcl-xl expression of the perfusate A group, and FIG. 1B is the immunohistochemical result of the Bcl-xl expression of the perfusate B group;
FIG. 3: fig. 2A is a graph of immunohistochemistry results for perfusate group a Bax expression, and fig. 2B is a graph of immunohistochemistry results for perfusate group B Bax expression.
Detailed Description
The structure and effect of the mechanical perfusion system for liver preservation according to the present invention will be described in detail below with reference to the preferred embodiments of the present invention, but the following description should not be construed as limiting the scope of the present invention.
Example 1 construction and connection of Normal temperature mechanical perfusion System for liver preservation
As shown in fig. 1, in the perfusion system (14), a central processor assembly (1) is connected with a peristaltic pump (2), the output power of the peristaltic pump (2) is controlled by a control assembly (20) in the central processor assembly (1), the peristaltic pump (2) controls the perfusion pressure and flow rate of the perfusate through the change of the output power, further, the peristaltic pump (2) is connected with a liver perfusate storage (3), the liver perfusate storage (3) is an inner layer and an outer layer, a cold and hot adjusting tube is arranged between the inner layer and the outer layer, and the cold and hot adjusting tube is controlled by the control assembly (20) and is used for adjusting the temperature of the perfusate; a temperature sensor (16) is arranged in the liver perfusate storage (3) and is used for monitoring the temperature of the liver perfusate; the device comprises a liver perfusate storage (3), a membrane oxygenator (5), a flow velocity sensor (18) and a central processing unit (1), wherein the liver perfusate storage (3) is connected with the membrane oxygenator (5) through an input conduit A (4), the flow velocity sensor is arranged between the liver perfusate storage (3) and the membrane oxygenator (5) and used for monitoring the total flow velocity of the liver perfusate, and the oxygenation efficiency of the membrane oxygenator (5) is controlled through a control assembly (20) in the central processing unit (1); the membrane oxygenator (5) is connected with a liver storage device (8) through an input catheter B (6) and an input catheter C (7), the input catheter B (6) is a hepatic artery perfusion catheter, and a flow rate sensor (18) and a pressure sensor (17) are arranged on the input catheter B (6) and are used for monitoring the perfusion flow rate of the hepatic artery and the pressure of perfusion liquid; the input catheter C (7) is a portal vein input catheter, and is also provided with a flow rate sensor (18) and a pressure sensor (17) for monitoring the flow rate of portal vein perfusion and the pressure of perfusate; an opening is arranged in the liver storage device (8), and the input catheter B (6) and the input catheter C (7) can enter the liver storage device (8) and are connected with the liver preserved in vitro. The liver storage device (8) is divided into an inner layer and an outer layer, a cold and hot adjusting pipe is arranged in an interlayer between the inner layer and the outer layer, and the cold and hot adjusting pipe is controlled by a control assembly (20) and is used for adjusting the internal environment temperature of the liver storage device (8); a temperature sensor (16) is arranged in the liver storage device (8) and used for monitoring the internal environment temperature.
The liver storage device (8) is connected with the perfusate filtering device (10) through an output conduit A (9), an opening is formed in the liver storage device (8), and the output conduit A (9) can enter the interior of the liver storage device to be connected with a liver stored in vitro; the perfusate filtering device (10) is connected with the liver perfusate storage (3) and returns the filtered perfusate to the liver perfusate storage (3); the liver storage device 8 is connected with a liquid collecting device (12) through an output conduit B (11) and is used for collecting bile secreted by the liver in the perfusion process; the liquid collecting device (12) is internally provided with a liquid meter (13) used for measuring the bile amount secreted by the liver in the perfusion process.
Further, the perfusion system (14) is provided with a monitoring data collecting device (15), the monitoring data collecting device (15) collects monitoring data generated by the temperature sensor (16), the pressure sensor (17), the flow rate sensor (18) and the liquid meter (12), and transmits the collected data to the central processor assembly (1). The central processing unit assembly (1) is provided with a data processing device (19) for processing monitoring data collected by the monitoring data collection device (15), and the central processing unit assembly (1) further comprises an information transmission device (21) which can transmit data parameter processing results of the data processing device (19) to a corresponding receiving device outside.
Further, liver perfusate is stored in the liver perfusate storage (3), and the liver perfusate consists of per 1000ml of perfusate: 400mg of naringenin-7-O-acetate, 2-6g of hydroxyl starch, 20-40g of adenosine triphosphate, 15-45g of artificial blood, 2-6g of lecithin, 1-2mg of galactosidase, 1-2mg of acetylgalactosamine enzyme, 30-60u of insulin, 300ml of compound amino acid injection 18AA 100, 1-5g of antibiotic, 5-30g of glucose, 300ml of physiological saline, 5-15ml of 10% potassium chloride, 10-30ml of 5% sodium bicarbonate, 10-20ml of 10% calcium chloride, 123-8 mg of vitamin B, 2-6mg of vitamin E, 1-5mg of vitamin C, 1-6mg of dexamethasone and 5-15 mu g of alprostadil.
Example 2 harvesting and perfusion of donor liver
1 laboratory animal
The male Chinese miniature pig has 20-25 heads and 30 + -3 Kg body weight. All experimental animals are treated by humanity, and the experimental animal treatment method accords with the guide for experimental animal management and use issued by the national institutes of health, and the experimental scheme obtains the permission of the ethical committee of the relevant experimental animals.
2. Method of producing a composite material
(1) The experimental animals are fasted and water is forbidden for 8 hours before operation. Pentobarbital sodium is injected into muscles for induction anesthesia, and the weight is weighed and recorded. Fixing the supine four limbs on an operating table, absorbing oxygen at high flow, preparing skin, connecting with precordial lead ECG monitor, and connecting pigtail end with oxyhemoglobin saturation probe. Establishing peripheral venous access through ear vein by scalp needle, fully fixing, inducing anesthesia with 1mg/kg of succinylcholine chloride injection before trachea intubation, connecting an anesthesia machine for ventilation after judging that the scalp needle is inserted into a correct position, and keeping the ratio of inhalation to exhalation of 1: 2. Through the peripheral venous access, vecuronium bromide is interrupted to maintain muscle relaxation, and propofol is given to maintain anesthesia.
(2) Liver acquisition: the crisscross incision enters the abdominal cavity layer by layer, the liver duodenal ligament is dissected, the ligamentum duodenale is ligated after dissociating out of the common bile duct, the catheter is inserted at the near end, the hepatic artery and the portal vein are separated and suspended, and the whole hepatic artery is completely dissociated. And the spleen vein is dissociated, the inferior hepatic vena cava is dissociated after the spleen vein dissociation is completed, and then the superior hepatic vena cava is dissociated. After successful dissociation, intubation is carried out from the spleen vein to the portal vein, the abdominal aorta is dissociated, intubation is carried out after the abdominal aorta is dissociated, 12500 units of intravenous heparin are used for whole body heparinization, after the heparinization is completed, liver perfusion is carried out, the perfusion volume of UW liquid through the portal vein is about 500ml, the perfusion volume of the abdominal aorta is about 1000ml, and the biliary tract is flushed by about 150 ml. And (3) continuously covering sterile ice chips on the surface of the liver in the perfusion process to help the liver to cool and perfuse, then cutting the liver, and putting the liver into a sterile basin filled with a 4 ℃ protective solution for trimming.
(3) Preheating of the mechanical perfusion system: the mechanical perfusion system described in example 1 was used to start the system to maintain the perfusate temperature and the liver storage compartment temperature at 36.5 ℃ and the perfusion preservation system was run to await access to the donor liver.
(4) In vitro liver access perfusion system: when the device is used for cutting the liver, the relatively abundant length of the blood vessel is reserved, and the input catheter C (7) and the input catheter B (6) are quickly inserted into the portal vein and the artery cannula to avoid damage. The output catheter A (9) is connected to the liver, and the perfusate enters the filtering device (10) through the output catheter to be filtered and flows back to the liver perfusate device (3). One end of an output catheter B (11) is connected with a bile duct of the liver, and the other end is connected with a liquid collecting device (12).
(5) And (3) perfusion preservation: after the connection is finished, the hepatic artery pressure is adjusted to be 80-120 mm Hg, the portal vein pressure is adjusted to be 10-20 mm Hg, the total perfusion flow is kept at 400-. The flow and pressure of portal vein and hepatic artery are respectively monitored in real time through a pressure sensor (17) and a flow velocity sensor (18), the output power of the peristaltic pump is adjusted to maintain the set perfusion pressure and flow velocity, and the portal vein and arterial blood flow is maintained in the design range. 400ml of perfusate was drained and 400ml of fresh perfusate was added every 3h by means of an equivalent displacement.
Example 3 Effect of naringenin-7-O-acetate on mechanical perfusion of liver preserved ex vivo
Naringenin (naringenin) has the effects of resisting bacteria, resisting inflammation, scavenging free radicals, resisting oxidation, relieving cough, eliminating phlegm, reducing blood fat, resisting cancer, resisting tumor, relieving spasm, benefiting gallbladder, preventing and treating liver diseases, inhibiting platelet coagulation, resisting atherosclerosis and the like, but whether naringenin-7-O-acetate which is a derivative of naringenin can be used for preserving isolated liver or not, and whether naringenin-7-O-acetate can be used as a perfusate component or not is beneficial to mechanical perfusion preservation of liver or not is unknown at present. To explore the effect of naringenin-7-O-acetate on mechanical perfusion of the liver ex vivo, two different liver perfusates were designed in this example, with the specific components as shown in table 1:
TABLE 1 composition of perfusate A and perfusate B (1000mL)
1. Perfusion method
The male Chinese miniature pig has 8 heads and the weight of 30 plus or minus 3 Kg. Two groups, group a and group B, of 4 were randomly grouped. Group A was perfused with perfusate A and group B was perfused with perfusate B, donor liver was harvested and perfused as in example 2, and donor liver was mechanically perfused in vitro for 9 hours. The experimental set-up was a control group of male chinese minipigs without any experimental treatment.
2. Method for detecting liver state
2.1 aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) in perfusate
And (3) respectively taking the perfusate in the output catheter when the perfusate is perfused for 3h, 6h and 9h, and detecting aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) by using a full-automatic biochemical analyzer. Whole blood of the male China miniature pigs in the control group is taken, and aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) are detected by using a full-automatic biochemical analyzer.
2.2TUNEL method for detecting hepatocyte apoptosis
After the perfusion of each group of experiments is stopped for 9 hours, taking the liver tissue for formaldehyde fixation; after the control group was sacrificed, liver tissue was fixed with formaldehyde. After the conventional paraffin embedding treatment is carried out in the pathology department, a wax block is made into thick slices, the slices are gently searched by a polylysine glass slide, the slices are placed in a 37 ℃ baking machine for baking, the slices are placed in a 60 ℃ oven for baking about 2 hours before detection, the apoptosis condition of liver cells is determined by adopting a TdT-mediated dUTP nick end labeling (TUNEL) method, each tissue slice is observed under a light microscope, 5 visual fields are randomly selected for each tissue slice, the wavelength of 520 +/-20 nm is applied to each same visual field for observation and counting of green fluorescence, and the average value of the green fluorescence is taken to represent the number of apoptotic cells. The red fluorescence was observed and counted using a wavelength of 620nm, and the average thereof was taken as the total number of cells in the field. Apoptosis Index (AI) ═ number of apoptosis/total number of cells in the field × 100%.
2.3 detection of hepatocytes Bax and Bcl-2
After the perfusion of each group of experiments is stopped for 9 hours, taking the liver tissue for formaldehyde fixation; after the control group was sacrificed, liver tissue was fixed with formaldehyde. After the conventional paraffin embedding treatment is carried out in the pathology department, the wax block is made into a thick section, the section is gently searched by a polylysine glass slide, the section is placed in a baking machine at 37 ℃ for baking, the section is placed in an oven at 60 ℃ for baking for about 2 hours before detection, the content of cell Bax and Bcl-xl is determined by adopting the conventional immunohistochemical method in the field, and the Bax/Bcl-xl ratio is calculated.
2.4 detection of inflammatory factors in perfusate
When the perfusion is respectively taken for 3h, 6h and 9h, the perfusate in the catheter is output, and the whole blood of the male Chinese miniature pig in the control group is taken and measured strictly according to the instruction of a commercial Elisa kit. The detection items comprise: the contents of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the whole blood of the perfusate and the control group.
2.5 detection of bile yield
The status and yield of bile in the fluid collection device 12 were examined at 3h, 6h, and 9h of perfusion, respectively.
2.6 statistical treatment
SPSS 13.0 statistical software is used for data processing, and single-factor variance is adopted for mean comparison among multiple groups. P < 0.05 is statistically significant.
3. Results of the experiment
The contents of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in groups A and B after 3h, 6h and 9h of perfusion are shown in Table 2, and the data of the contents are expressed as an average value.
TABLE 2 AST, ALT, TNF-. alpha.and IL-6 content changes at different time points in groups A and B
As can be seen from the results shown in table 2, although the content of the infusion solution containing naringenin-7-O-acetate, aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), tumor necrosis factor- α (TNF- α), and interleukin-6 (IL-6) increased at the initial stage of infusion, all the indicators fell back after a period of infusion, while the infusion solution containing no naringenin-7-O-acetate had a significant tendency of increasing all the indicators after 9 hours of infusion, which indicates that the liver tissue was damaged to some extent during the infusion process. It can be seen that naringenin-7-O-acetate has positive effects in scavenging free radicals, inhibiting inflammatory factors, reducing reperfusion injury of liver tissue, and maintaining good condition of isolated liver.
The apoptosis index of liver cells and the content of Bax/Bcl-xl ratio after 9 hours of perfusion in the A group and the B group are shown in Table 3, and the data are expressed by taking the average value.
TABLE 3 liver apoptosis index and Bax/Bcl-xl ratio content
As can be seen from Table 3, the liver tissues of group A and group B both undergo apoptosis after being perfused for 9h, but the apoptosis ratio of group B is obviously higher than that of group A, and Bax and Bcl-xl are closely related to the apoptosis of liver cells, and the higher the Bax/Bcl-xl ratio is, the higher the apoptosis degree is. As can be seen from Table 3 and FIGS. 2-3, liver cells in group A had higher Bcl-xl expression and relatively lower Bax expression, and the ratio was significantly lower than that in group B, indicating that liver cells in group A had less apoptosis. Therefore, naringenin-7-O-acetate also plays a positive role in reducing the apoptosis of liver cells.
Through observing the state and the yield of the bile in the liquid collecting device 12, the drainage quantity of the bile in the group A is gradually increased within 3 hours after the beginning of perfusion, and more floccules can be seen; the bile is relatively stable within 3h to 8h, about 4.25ml/h, and the floccule is reduced; after 8h of perfusion, the bile volume is reduced, but the yellow color and the clear bile are still presented. In 3h after the group B begins to be perfused, no obvious bile secretion occurs, a small amount of bile appears after 3h-8h, and the bile is darker and more floccules can be seen. This suggests that naringenin-7-O-acetate also plays a positive role in preventing the lesions of intrahepatic bile ducts during perfusion.
Example 4 Effect of naringenin-7-O-acetate content in liver perfusate on liver preservation
In order to explore the influence of the content of naringenin-7-O-acetate in the liver perfusate on liver preservation, 3 different liver perfusates were designed in this example, and the specific components are shown in table 4:
TABLE 4 composition of perfusate C, perfusate D, perfusate E (1L)
1. Perfusion method
The male Chinese miniature pig has 12 heads and the weight of 30 plus or minus 3 Kg. Three groups, C group, D group and E group, 4 of each group were randomly grouped. Group C was perfused with perfusate C and group D was perfused with perfusate D, donor liver was harvested and perfused as in example 2, and donor liver was mechanically perfused in vitro for 9 hours. The experimental set-up was a control group of male chinese minipigs without any experimental treatment.
2. Method for detecting liver state
When the samples are respectively perfused for 3h, 6h and 9h, the perfusate in the output catheter and the whole blood of the male Chinese miniature pig of the control group are taken to detect the contents of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), and the specific detection method is shown in example 3.
3. Results of the experiment
After 3h, 6h and 9h of perfusion, the contents of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in groups C, D and E are shown in Table 5, and the contents are expressed by averaging.
TABLE 5 variation of AST, ALT, TNF-. alpha.and IL-6 content at different time points in groups C, D and E
From the results shown in table 5, it can be seen that, compared with the control group, in the liver perfused mechanically in vitro, each detection index is increased at the initial stage of perfusion, but, as the perfusion time goes on, each detection index falls back to a level close to normal under the action of the perfusion system and the perfusate of the present application, wherein the effect of the perfusate E is most obvious, which shows that the effect of adding 325mg/L of naringenin-7-O-acetate in the perfusate is the best.
Example 5 Effect of different warm ischemia time on Ex vivo liver preservation
In order to investigate the liver function repairing effect of the perfusate E after different periods of thermal ischemia, the liver 1h and 2h after cardiac arrest was used as the liver supply in this example, and perfused with the perfusate E for 9 hours.
1. Harvesting of donor liver
The male Chinese miniature pig has 8 heads and the weight of 30 plus or minus 3 Kg. Two groups, F and G, were randomly grouped, 8 per group.
Placing the anesthetized miniature pig on a cardiopulmonary resuscitation machine, injecting 1G of KCl to induce cardiac arrest, and giving cardiopulmonary resuscitation (CPR) for 30min after the cardiac arrest, wherein death is declared without a life reaction within 5min after the CPR is finished, the liver is picked up in the F group within 1 hour after the death, and the liver is picked up in the G group within 2 hours after the death. Donor liver harvest and perfusion see example 2.
2. Method for detecting liver state
And (3) respectively taking perfusate in the output catheter when the perfusate is perfused for 3h, 6h and 9h, and detecting the contents of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), wherein the specific detection method is shown in example 3.
3. Results of the experiment
The contents of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in groups F and G after 3h, 6h and 9h of perfusion are shown in Table 6, and the data of the contents are expressed as an average value.
TABLE 6 variation of AST, ALT, TNF-. alpha.and IL-6 content at different time points in groups F and G
Based on the results in table 6, it can be seen that, as the ischemia time increases, the indexes of the liver at the initial stage of perfusion increase significantly, which indicates that, the longer the warm ischemia time, the damage of the liver tissue and the decrease of the liver function are significant in the same month, and after perfusate E is perfused for 9 hours, the indexes of the liver drop back significantly, which indicates that perfusate E has a repairing effect on the liver which has undergone warm ischemia. By comparing the indexes of the group F and the group G at the end of perfusion, although there is a certain difference, the difference is not very large, which shows that the perfusate E still has a good repairing effect on the liver tissue which is subjected to long-term warm ischemia.
Example 6 transplantation of ex vivo preserved liver
In order to further improve the effect of transplantation into the body after perfusion with perfusate E, the liver subjected to warm ischemia was transplanted using the group F liver and the group G liver in example 5.
1. Laboratory animal
The male Chinese miniature pig has 8 heads and the weight of 30 plus or minus 3 Kg. Two groups of 4 per group were randomly grouped as donor transplant donors, with H groups: receiving a group F liver transplant; group I: group G liver transplants were received.
2. Transplantation method
Fixing a receptor pig, anesthetizing, preparing skin, disinfecting, paving a towel, enabling a herringbone incision to enter an abdominal cavity layer by layer, dissecting a common bile duct, a hepatic artery and a portal vein, suspending after the dissection is finished, beginning to dissect the superior and inferior hepatic vena cava, dissociating the hepatic artery, sequentially blocking and cutting off the hepatic artery (entering a no-liver stage), the portal vein, the inferior and superior hepatic vena cava and the inferior hepatic vena cava, and quickly implanting a donor liver after the liver is removed; the anastomosis of the superior vena cava and the inferior vena cava of the liver is started immediately, after the anastomosis is finished, the portal vein is flushed and anastomosed, the portal vein and the semi-open superior vena cava of the liver are opened, the superior vena cava and the inferior vena cava of the liver are fully opened after the vital signs are monitored to be relatively stable (no liver period is finished), the inferior vena cava and the hepatic artery are opened after the anastomosis is finished, the bile secreted by the liver is observed (the new liver function is recovered), the common bile duct is anastomosed, the bile is drained by a retention T-shaped tube, the abdomen is closed layer by layer, the abdomen is closed, the bile is drained by the T-shaped tube, the vital signs are relatively stable, the operation is finished, the vital signs are monitored, appropriate antibiotics and hormones are added during the period, the treatment is carried out gradually.
3. Method for detecting liver state after transplantation
3. Results of the experiment
Acute infection and death occur after 1 case of operation in the H group; it was successfully taken off line, survived and no acute rejection occurred with 3 cases.
Group I1 cases died due to surgical operation errors; it was successfully taken off line, survived and no acute rejection occurred with 3 cases.
The contents of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in blood on day 14 after liver transplantation in groups H and I are shown in Table 7, and the contents are expressed by averaging.
TABLE 7 AST, ALT, TNF-. alpha.and IL-6 levels in blood 14 days after group H and group I transplants
As can be seen from the data in Table 7, the AST, ALT, TNF-. alpha.and IL-6 levels in the blood of the recipients approached normal values 14 days after the transplantation in the livers of groups H and I. From the above results, it can be seen that the perfusate E can still meet the requirement of liver transplantation after the perfusion repair of the thermally ischemic liver. Therefore, the perfusate E can greatly enhance the tolerance degree of the liver to the thermal ischemia and expand the source of the liver supply.
The invention has been described in detail with respect to a general description and specific embodiments thereof, but it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (14)
1. A mechanical perfusion system for liver preservation, characterized in that the mechanical perfusion system (14) comprises a central processor assembly (1), a peristaltic pump (2), a liver perfusate reservoir (3), a membrane oxygenator (5), a liver storage device (8), a liquid collection device (12), a monitoring data collection device (15), the liver perfusate reservoir (3) having stored therein a liver perfusate, the composition of each 1000ml of liver perfusate being: 400mg of naringenin-7-O-acetate, 2-6g of hydroxyl starch, 20-40g of adenosine triphosphate, 15-45g of artificial blood, 2-6g of lecithin, 1-2mg of galactosidase, 1-2mg of acetylgalactosamine enzyme, 30-60u of insulin, 300ml of compound amino acid injection 18AA 100, 1-5g of antibiotic, 5-30g of glucose, 300ml of physiological saline, 5-15ml of 10% potassium chloride, 10-30ml of 5% sodium bicarbonate, 10-20ml of 10% calcium chloride, 123-8 mg of vitamin B, 2-6mg of vitamin E, 1-5mg of vitamin C, 1-6mg of dexamethasone and 5-15 mu g of alprostadil.
2. The mechanical perfusion system for liver preservation according to claim 1, wherein the liver perfusate reservoir (3) is formed by an inner layer and an outer layer, and a cold and hot adjusting pipe is arranged between the inner layer and the outer layer for adjusting the temperature of the perfusate; a temperature sensor (16) is arranged in the liver perfusate storage (3) and used for monitoring the temperature of the liver perfusate.
3. The mechanical perfusion system for liver preservation according to claim 1 or 2, wherein the liver storage device (8) is formed by an inner layer and an outer layer, and a heat and cold regulating tube is arranged between the inner layer and the outer layer for regulating the internal environment temperature of the liver storage device (8); a temperature sensor (16) is arranged in the liver storage device (8) and used for monitoring the internal environment temperature.
4. Mechanical perfusion system for liver preservation according to any of claims 1-2, wherein the liquid collection device (12) is provided with a liquid meter (13) for metering the amount of bile secreted by the liver during perfusion.
5. Mechanical perfusion system for liver preservation according to any of claims 1-2, wherein the mechanical perfusion system (14) further comprises a temperature sensor (16), a pressure sensor (17), a flow rate sensor (18).
6. Mechanical perfusion system for liver preservation according to any of claims 1-2, wherein the central processor unit (1) comprises a control unit (20) and data processing means (19), the central processor unit (1) further comprising information transmission means (21).
7. The mechanical perfusion system for liver preservation according to any one of claims 1-2, wherein the central processor unit (1) is connected to a peristaltic pump (2), the peristaltic pump (2) is connected to the liver perfusate reservoir (3), the liver perfusate reservoir (3) is connected to a membrane oxygenator (5) through an input conduit a (4), the membrane oxygenator (5) is connected to a liver storage device (8) through an input conduit B (6) and an input conduit C (7), the liver storage device (8) is connected to a perfusate filtering device (10) through an output conduit a (9), and the perfusate filtering device (10) is connected to the liver perfusate reservoir (3); the monitoring data collecting device (15) collects monitoring data generated by the temperature sensor (16), the pressure sensor (17), the flow rate sensor (18) and the liquid meter (13), and transmits the collected data to the central processor assembly (1).
8. Mechanical perfusion system for liver preservation according to any of claims 1-2, wherein each 1000ml of perfusate consists of: 325mg of naringenin-7-O-acetate, 2.6g of hydroxyl starch, 30g of adenosine triphosphate, 36g of perfluorocarbon, 4g of lecithin, 1.25mg of galactosidase, 1.25mg of acetylgalactosaminidase, 43u of insulin, 18AA 150ml of compound amino acid injection, 2.5g of antibiotic, 28g of glucose, 200ml of normal saline, 15ml of 10% potassium chloride, 25ml of 5% sodium bicarbonate, 20ml of 10% calcium chloride, vitamin B124.2mg, 3.6mg of vitamin E, 2.5mg of vitamin C, 4.6mg of dexamethasone and 10 mu g of alprostadil.
9. Use of the mechanical perfusion system for liver preservation according to any one of claims 1-8, wherein the normothermic mechanical perfusion system for liver preservation can be used for preserving a liver ex vivo.
10. The mechanical perfusion system for liver preservation of claim 9, wherein the preservation temperature of the liver is normothermic preservation.
11. The mechanical perfusion system for liver preservation according to claim 10, wherein the preservation temperature of the liver is between 25 ℃ and 37 ℃.
12. The mechanical perfusion system for liver preservation of claim 9, wherein the donor of the liver has experienced cardiac arrest or warm ischemia.
13. The mechanical perfusion system for liver preservation according to claim 12, wherein the warm ischemia or cardiac arrest is for a period of 30-120 min.
14. The mechanical perfusion system for liver preservation according to claim 13, wherein the warm ischemia or cardiac arrest is performed for a period of 120 min.
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