CN106342787A - Organ preservation solution and preparation method thereof - Google Patents

Abstract

The invention relates to the fields of medicine and biology, in particular to an organ preservation solution and a preparation method thereof. The composition of the organ preservation solution is as follows, per 1000mL: 3.0-15.0g sodium citrate, 10.0-30.0g potassium citrate, 5.0-15.0g mannitol, 0.5-1.2g potassium chloride , Magnesium sulfate heptahydrate 0.5-1.8g, pH buffer pair 0.5-25.0g, energy substrate 0.2-25.0g, and the balance is water for injection. The organ preservation solution also contains one or more of laminarin, trehalose, laminin and betaine. The organ preservation solution provided by the invention is cheap and efficient, and is suitable for multi-organ preservation.

Description

A kind of organ preservation solution and preparation method thereof

technical field

The invention relates to the fields of medicine and biology, in particular to an organ preservation solution and a preparation method thereof.

Background technique

Organ transplantation is hailed as a major life science development that changed human life in the 20th century, and is an effective method recognized around the world to treat a variety of terminal diseases. According to statistics, in my country alone, there are 1-1.5 million patients with organ failure each year, and about 300,000 patients in urgent need of organ transplantation. Therefore, how to effectively preserve isolated organs has become one of the important supporting technologies for organ transplantation.

The fundamental purpose of organ preservation is to maintain the viability of isolated organs, minimize various damages to isolated organs caused by ischemia, make them more suitable for transportation, and restore their functions more quickly after surgery.

An effective organ preservation solution must have five characteristics: 1. Reduce cell edema caused by low temperature; 2. Prevent cell space expansion during lavage preservation; 3. Prevent acidification of cells; 4. Prevent or Reduce reperfusion injury; 5. Provide substrates for regeneration of high-energy phosphate compounds. At present, there are roughly two kinds of UW solution and HTK solution commonly used in clinical practice for organ preservation.

University Wisconsin Organ Preservation Solution (UW solution for short) was invented by the University of Wisconsin in the United States. It is recognized as the standard preservation solution for liver, pancreas and kidney. It has been clinically proven that it can preserve the kidney for 72 hours and the liver for 20-24 hours. However, the preservation solution is high in potassium and low in sodium, which can easily cause damage to vascular endothelial cells; the colloidal component hydroxyethyl starch (HES) has a great influence on hemorheology and can easily cause red blood cell aggregation; raw materials such as raffinose and lacturonic acid The high cost will greatly increase the clinical cost of organ transplant patients; the high viscosity will affect the perfusion effect.

HTK solution contains histidine-histidine salt buffer pair, which can significantly inhibit tissue acidification; its concentration of sodium ions and potassium ions is low; its viscosity is low, and it is easy to spread. However, judging from the effect of organ preservation, the liver of the donor showed microcirculation disturbance, and the preservation effect of HTK solution was not as good as that of UW solution.

At present, the cell preservation solution developed in my country is HC-A solution (hypertonic citrate purine solution), which is jointly developed by Changzheng Hospital of Second Military Medical University and Shanghai Central Blood Station. It is simple to prepare and has good stability. However, HC-A solution is limited to in vivo lavage and kidney preservation when organs are harvested en bloc, and the preservation of liver, pancreas and other organs still depends on UW solution.

In addition, researchers have also provided a variety of improved organ preservation solutions. For example, Chinese patent application 200910014423 discloses an organ preservation solution and its preparation method, which is characterized in that every 1000ml of organ preservation solution contains: potassium citrate 21 ～29mmol; sodium dihydrogen phosphate monohydrate 6～10mmol; disodium hydrogen phosphate 30～45mmol; magnesium sulfate 4～6mmol; potassium chloride 1～3mmol; dextran-4016-24g; mannitol 110～150mmol; adenosine 4～ 6mmol; 4-6mmol of acetylcysteine; 4-6mmol of sodium hydroxide; the balance is water for injection. However, the preservation solution is packaged in two chambers, which is cumbersome to use and not convenient enough, and the preparation method is also relatively cumbersome and complicated, and the flexibility is relatively poor when dealing with emergencies; and the preservation solution only has a good effect on the kidney, and has a good effect on other organs. Doesn't save well.

As another example, Chinese patent application 200810033425 discloses a kind of organ preservation solution and its preparation method. The preservation solution is composed of sodium citrate, potassium citrate magnesium sulfate, sodium dihydrogen phosphate monohydrate, sodium hydroxide, adenosine , arginine, tryptophan, mannitol and trabenzine hydrochloride and other ingredients. However, this preservation solution only has a good effect on the kidney, and has a poor effect on the preservation of other organs.

In summary, HTK solution and Celsior solution are mainly used for the preservation of the heart, and the range of application is limited; UW solution has many advantages as a standard multi-organ preservation solution, but its viscosity is high, potassium ions are too high, and it is expensive. It is inconvenient to test and is not applicable to the actual situation of our country; the domestic HCA solution and its improved formula are mainly used for kidney preservation, and the preservation effect on other organs is not good. In recent years, as the demand for large organ transplants such as the liver, heart and pancreas, and multi-organ joint transplantation in China continues to increase, the lack of efficient, cheap, wide-ranging application and adaptation to my country's national conditions has become a limitation of my country's organ transplantation technology. important reason for development.

To sum up, it is of great research and clinical value to develop a new type of organ preservation solution that is cheap, efficient and suitable for preservation of multiple organs.

Contents of the invention

The object of the present invention is to overcome the deficiencies of the prior art, and provide a new type of organ preservation solution which is cheap, efficient and suitable for the preservation of multiple organs.

In one aspect, the present invention provides an organ preservation solution, the organ preservation solution contains by weight, per 1000mL:

Preferably, the organ preservation solution contains by weight, per 1000mL:

More preferably, the organ preservation solution contains by weight, per 1000mL:

Further preferably, the organ preservation solution contains by weight, per 1000mL:

The energy substrate in the organ preservation solution is used to regenerate adenosine triphosphate, the energy source of cells, maintain the functional activity of mitochondria, and maintain energy metabolism. The energy substrates in the organ preservation solution of the present invention are adenosine, adenosine monophosphate, adenosine diphosphate, tryptophan, α-ketoglutarate, succinic acid, succinyl-CoA, acetyl-CoA One or more of A and coenzyme Q10; preferably one or more of adenosine, succinic acid, coenzyme Q10, tryptophan and α-ketoglutarate; more preferably adenosine , coenzyme Q10, tryptophan and succinic acid; more preferably adenosine, coenzyme Q10, tryptophan and succinic acid.

Under cryopreservation, due to ischemia and hypoxia, the metabolic pathway moves towards glycolysis, and at the same time promotes the production of lactic acid, resulting in tissue acidification. Tissue acidification damages cells and destabilizes lysosomes, activates lysosomes and impairs mitochondrial function. Therefore, the organ preservation solution should contain an acid-base buffer system to reduce tissue acidification and maintain a relatively stable internal and external environment. The pH buffer pair in the organ preservation solution of the present invention is one or more of phosphate, bicarbonate and histidine; preferably one or both of phosphate and histidine; more preferably For phosphate and histidine.

The organ preservation solution also contains one or more of laminarin, trehalose, laminin and betaine.

Laminaria polysaccharide refers to the macromolecular polysaccharides in kelp. Laminarin has a wide range of biological activities, for example, studies have shown that laminarin can purify acidic substances.

Trehalose is a safe and reliable natural sugar. In medicine, trehalose has been successfully used instead of plasma protein as a stabilizer for biologically active substances such as blood products, vaccines, lymphocytes, and cell tissues. In addition, trehalose also has the ability to prevent The function of viral transmission.

Betaine is used for anti-tumor, lowering blood pressure, anti-peptic ulcer and gastrointestinal dysfunction, treatment of liver diseases, etc. In addition, it also has the function of sterilization and anti-inflammation.

The content of laminarin in the organ preservation solution is 1.0-10.0g per 1000mL.

The content of trehalose in the organ preservation solution is 1.0-16.0g per 1000mL.

The content of laminin in the organ preservation solution is 8.0-27.0g per 1000mL.

The content of betaine in the organ preservation solution is 4.0-37.0g per 1000mL.

The organ preservation solution may also contain one or more of trabutazine hydrochloride, arginine, lactobionic acid, glutathione, allopurinol and kapolose.

On the other hand, the present invention also provides a method for preparing the above-mentioned organ preservation solution, the preparation method comprising the following steps:

(1) Weigh other ingredients except the energy substrate according to the dosage of the formula, add 50-80% of the total volume of water for injection and stir evenly, add medical activated carbon, stir for 5-20 minutes, and filter with a 0.45 μm filter membrane Decarburization to obtain solution A;

(2) Weigh the energy substrate according to the dosage of the formula, and dissolve it with water for injection to obtain the energy substrate solution, mix the energy substrate solution with the solution A of step (1), add water for injection to the full amount, and stir evenly, Use a 0.45 μm filter membrane to filter, and the filtrate is the organ preservation solution.

The amount of Chinese activated carbon added in step (1) of the preparation method is 1-5g per 1000mL of water for injection; preferably 2-4g per 1000mL of water for injection; more preferably 3g per 1000mL of water for injection.

In step (1) of the preparation method, the average particle size of the activated carbon for Chinese medicine is 2-5 μm, preferably 3-4 μm, more preferably 4 μm.

Compared with the prior art, the wound repair liquid provided by the invention has the following beneficial effects:

(1) The organ preservation solution provided by the present invention can be used for cooling, lavage and preservation of various organs, and can effectively preserve the kidney, liver, heart and pancreas for more than 48 hours, among which the kidney can be preserved for 72 hours and the liver for 48 hours.

(2), the present invention uses laminin for organ preservation solution. Laminin has decompression and antibacterial effects. In addition, it is unexpectedly found that the addition of laminin can significantly prolong the storage time of the pancreas, and the storage time of the heart is changed from Less than 24h extended to 48h.

(3) The organ preservation solution provided by the present invention has the characteristics of high potassium and low sodium, which can reduce ion exchange and energy consumption inside and outside the cells, prevent cell edema, and effectively prolong the storage time.

(4), hydroxyethyl starch is removed in the organ preservation solution provided by the present invention, and the viscosity of the liquid is reduced. In addition to containing mannitol, it also contains laminarin and trehalose, wherein laminarin and trehalose have stable biofilm The role of the mixed osmotic pressure regulator is composed of three components: mannitol, laminarin, and trehalose, which can effectively adjust the osmotic pressure, inhibit cell edema and gap expansion, and effectively prolong the storage time.

(5) The organ preservation solution provided by the present invention contains betaine. Betaine has the functions of protecting cell membranes, protecting enzymes, and maintaining cell osmotic pressure. Inhibit tissue acidification, stabilize lysosomes; and inhibit cell edema to a certain extent, and effectively prolong the storage time for liver and kidney.

(6) When the organ is cryopreserved, anaerobic glycolysis is its only source of energy. This process is pH-dependent. The drop in pH and the accumulation of other metabolites will inhibit the glycolysis process and will also lead to energy loss. lack. Therefore, the function of the pH buffer pair in the organ preservation solution is very important. A suitable pH buffer pair can effectively resist pH changes, and has a weak ability to transport across the cell membrane, and will not cause edema due to entering the cell. Conventional organ preservation solutions use a single type of buffer pair, but the present invention uses a two-component buffer pair of phosphate and histidine, which is significantly better than the respective single-component buffer pair, and can maintain a pH of about 8.0, and a pH of about 8.0 The organ preservation solution is conducive to the outward flow of H ⁺ in the cells through transmembrane transport, and relieves intracellular acidosis. The pH of liver cells is close to 7.7 at 0-4°C, which can slow down the decomposition of ATP and reduce the damage to mitochondria. Eliminate the inhibition of acidosis on the key enzyme of glycolysis, 1,6-bisphosphate fructokinase, and increase the synthesis of ATP.

(7) Since the transmembrane transport efficiency of different substances is different, the effect of entering the cell is also different. Conventional organ preservation solutions use a single type of energy substrate, such as adenosine. However, the present invention adopts energy substrates of various components, such as adenine nucleoside, coenzyme Q10, tryptophan and succinic acid. Due to the difference in transmembrane transport efficiency between the multi-component energy substrates, they can complement each other due to the difference in transport efficiency. resulting in insufficient energy synthesis. The preservation effect of the organ preservation solution containing multi-component energy substrates provided by the present invention is also significantly better than that of organ preservation solutions containing single-component energy substrates. It provides energy for cells under hypoxic conditions and has a repairing effect on reperfusion injury. Stronger and capable of efficient energy regeneration.

(8) The pH of the organ preservation solution provided by the present invention is 6.2-8.4, which is close to physiological pH, can effectively inhibit tissue acidification and prevent cell edema, and reduce adverse reactions caused by pH differences; the osmotic pressure is 310-380mOsm/L, which can effectively Preventing edema can significantly prolong the preservation time of organs.

(9) Mitochondrial respiration rate (RCR), as an indicator of energy synthesis in mitochondria, can effectively reflect the activity of cells and grafts. Liver preservation with RCR higher than 3.0 has better liver function after transplantation. The organ preservation solution provided by the present invention preserves the kidney for 48 hours, and can maintain the RCR at about 3.0, which is significantly higher than that of the prior art.

(10) There is no incompatibility between the components used in the organ preservation solution provided by the present invention, the preparation process is simple, the property is stable, and the price is low.

In summary, the organ preservation solution provided by the present invention is cheap, efficient and suitable for multi-organ preservation.

detailed description

The descriptions of the following embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to these embodiments shown herein, but can be applied in a wider scope consistent with the principles and novel features disclosed herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Potassium citrate was purchased from Xinning Pharmaceutical Factory, Taishan City, Guangdong; sodium citrate was purchased from Xi’an Fourth Pharmaceutical Factory, Shaanxi; magnesium sulfate heptahydrate was purchased from Shanghai Baoda Chemical Co., Ltd.; succinic acid, tryptophan, histamine Acid, potassium chloride, sodium dihydrogen phosphate monohydrate, and disodium hydrogen phosphate were purchased from Shanghai Institute of Fine Chemical Technology; adenosine was purchased from Xinghu Company, Zhaoqing City, Guangdong; trehalose was purchased from Nanning Zhongnuo Bioengineering Company; Lamininin was purchased from Shaanxi Ciyuan Biotechnology Co., Ltd.; laminarin was purchased from Xi’an Tongze Biotechnology Co., Ltd.; mannitol was purchased from Zhengzhou Dynasty Chemical Products Co., Ltd.; betaine was purchased from Jinan Jinhui Chemical Co., Ltd.; coenzyme Q10 was purchased from Xi'an Kangnuo Chemical Co., Ltd.; the optical microscope was purchased from Shanghai Optical Instrument Company; the centrifuge and constant temperature water bath were purchased from Shanghai Medical Instrument Company.

Embodiment 1 A kind of organ preservation solution

Formula, each 1000mL contains:

Preparation:

(1) Weigh other ingredients except adenosine according to the dosage of the formula, add 600mL of water for injection and stir evenly, add 1g of medical activated carbon with an average particle size of 2μm, stir for 5 minutes, use 0.45μm Decarburization by membrane filtration to obtain solution A;

(2) Weigh adenosine according to the dosage of the formula, dissolve it in 50mL of water for injection to obtain an energy substrate solution, mix the energy substrate solution with solution A of step (1), add water for injection to 1000mL, and stir evenly Finally, use a 0.45 μm filter membrane to filter, and the filtrate is the organ preservation solution.

Embodiment 2 A kind of organ preservation solution

Formula, each 1000mL contains:

Preparation:

(1) Weigh other ingredients except adenosine, coenzyme Q10 and succinic acid according to the dosage of the formula, add water for injection with a total volume of 800mL and stir evenly, then add 5g of medical activated carbon with an average particle size of 3μm, and stir for 20 minutes Finally, use a 0.45 μm filter membrane to filter and decarburize to obtain solution A;

(2) Weigh adenosine, coenzyme Q10 and succinic acid according to the dosage of the formula, and dissolve them in 100mL of water for injection to obtain the energy substrate solution, mix the energy substrate solution with solution A in step (1), and add additional injection Make up to 1000mL with water, stir evenly, and filter with a 0.45μm filter membrane, and the filtrate is the organ preservation solution.

Embodiment 3 A kind of organ preservation solution

Formula, each 1000mL contains:

Preparation:

(1) Weigh other ingredients except adenosine, coenzyme Q10, tryptophan and succinic acid according to the dosage of the formula, add up to 700mL of water for injection and stir evenly, then add 3g of medical activated carbon with an average particle size of 4μm , after stirring for 15 minutes, use a 0.45 μm filter membrane to filter and decarburize to obtain solution A;

(2) Weigh adenosine, coenzyme Q10, tryptophan and succinic acid according to the dosage of the formula, and dissolve them in 200mL of water for injection to obtain the energy substrate solution, and mix the energy substrate solution with the solution A of step (1) , add water for injection to 1000mL, stir evenly, and filter with a 0.45μm filter membrane, and the filtrate is the organ preservation solution.

Embodiment 4 A kind of organ preservation solution

Formula, each 1000mL contains:

Preparation:

(1) Weigh other components except adenosine and succinic acid according to the dosage of the formula, add water for injection with a total volume of 700mL and stir evenly, add 3g of medical activated carbon with an average particle size of 4μm, stir for 15 minutes, use 0.45μm membrane filtration decarburization to obtain solution A;

(2) Weigh adenosine and succinic acid according to the dosage of the formula, and dissolve them in 200mL of water for injection to obtain an energy substrate solution, mix the energy substrate solution with solution A of step (1), and add water for injection to 1000mL , after stirring evenly, use a 0.45μm filter membrane to filter, and the filtrate is the organ preservation solution.

Embodiment 5 A kind of organ preservation solution

Formula, each 1000mL contains:

Preparation:

(1) Weigh other ingredients except adenosine and coenzyme Q10 according to the dosage of the formula, add up to 600mL of water for injection and stir evenly, add 2g of medical activated carbon with an average particle size of 5μm, stir for 8 minutes, use 0.45μm membrane filtration decarburization to obtain solution A;

(2) Weigh adenosine and coenzyme Q10 according to the dosage of the formula, and dissolve them in 200mL of water for injection to obtain an energy substrate solution, mix the energy substrate solution with solution A of step (1), and add water for injection to 1000mL , after stirring evenly, use a 0.45μm filter membrane to filter, and the filtrate is the organ preservation solution.

Embodiment 6 A kind of organ preservation solution

Formula, each 1000mL contains:

Preparation:

(1) Weigh the other components except adenosine, tryptophan and succinic acid according to the formula dosage, add the water for injection with a total volume of 600mL and stir evenly, then add 2g of medical activated carbon with an average particle size of 5 μm, and stir for 10 Minutes later, use a 0.45 μm filter membrane to filter and decarburize to obtain solution A;

(2) Weigh adenosine, tryptophan and succinic acid according to the formula dosage, and after dissolving in 200mL water for injection, obtain the energy substrate solution, mix the energy substrate solution with the solution A of step (1), add Add water for injection to 1000mL, stir evenly, and filter through a 0.45μm filter membrane, and the filtrate is the organ preservation solution.

Comparative Example 1 A kind of organ preservation solution

The difference between the formula and Example 3 is that it does not contain laminarin and trehalose;

The preparation method is the same as in Example 3.

Comparative example 2 a kind of organ preservation solution

The difference of formula and embodiment 3 is not containing laminin;

The preparation method is the same as in Example 3.

Comparative example 3 a kind of organ preservation solution

The difference of formula and embodiment 3 is not containing betaine;

The preparation method is the same as in Example 3.

Comparative example 4 a kind of organ preservation solution

The difference between formula and embodiment 3 is not to contain histidine;

The preparation method is the same as in Example 3.

Comparative example 5 a kind of organ preservation solution

The difference of formula and embodiment 3 is not containing disodium hydrogen phosphate and sodium dihydrogen phosphate monohydrate;

The preparation method is the same as in Example 3.

Comparative example 6 a kind of organ preservation solution

The difference between the formula and Example 3 is that it does not contain coenzyme Q10, tryptophan and succinic acid, and the content of adenosine is 11.0g;

The preparation method is the same as in Example 3.

Experimental Example 1 Morphological changes of pig kidneys during cryopreservation

Experimental animals: healthy adult Bama miniature pigs for experiments, male and female randomly, weighing 25-30kg.

experimental method:

1. Preoperative preparation and anesthesia: Fasting for 16 hours and water intake for 12 hours before operation. The donor was given basic anesthesia, induced by intramuscular injection of ketamine 1 mg/kg, and maintained with propofol.

2. Donor kidney extraction: adopt the general whole kidney rapid extraction technique.

3. Preservation: After the donor kidneys were obtained, they were stored at low temperature in the organ preservation solutions prepared in Examples 1-6 and Comparative Examples 1-6 respectively, with HCA-II solution (provided by the Second Military Medical University Changzheng Hospital) as a control.

4. Pathological examination: At the 1st, 24th, 48th, and 72nd hour of kidney preservation, kidney parenchyma samples were taken, paraffin sections were made, and light microscope observations were made; electron microscope sections were made for transmission electron microscope morphology observation.

Experimental results:

HCA-Ⅱ liquid group: After 24 hours of storage, the structure of the kidneys showed slight changes under the light microscope, the structure of renal glomerulus and renal tubules was clear, and the interstitium was slightly edema; after 48 hours of storage, most of the renal glomerular capillaries were still clear. Extensive moderate to severe vacuolar degeneration of the renal tubular epithelium, obvious interstitial edema, severe brush border damage, and multiple small focal necrosis of the renal tubular epithelium; after 72 hours of storage, the glomerular capillary part of the kidney has no normal outline, and the renal tubular epithelium Extensive and severe degeneration and sheet necrosis, intercellular dissociation, interstitial edema. The results of the electron microscope are consistent with those of the light microscope.

Example 1-6 groups of the present invention: within 48 hours of storage, the morphological changes of Example 1-6 groups were not significantly different from those of the HCA-II liquid group; but after 72 hours of storage, the outline of kidney tissue did not deteriorate significantly compared with 48 hours , no cell fragmentation and flaky necrosis, and the order of deterioration from strong to weak is: Examples 5, 1, 4, 2, 6, and 3.

Comparative example 1-6 group of the present invention: after being stored for 24 hours, compared with 1 hour, the obvious deterioration occurred, and cell fragmentation and flaky necrosis occurred.

Experimental example 2 Morphological changes of minipig liver during cryopreservation-reperfusion

Experimental animals: same as Experiment 1

experimental method:

1. Preoperative preparation and anesthesia: same as experimental example 1.

2. Donor liver harvesting: the general whole liver rapid harvesting technique is adopted.

3. Preservation-reperfusion: low-temperature preservation is carried out after the donor liver is obtained. The preservation solution is the organ preservation solution prepared in Embodiment 1-5 of the present invention and Comparative Example 1-6. After 1, 12 hours of preservation and 1 hour after reperfusion , Take samples for light microscope observation, with HCA-II solution and UW solution (provided by Changzheng Hospital of the Second Military Medical University) as controls.

Experimental results:

HCA-Ⅱ solution group: After 1 hour of storage, the morphology and structure of liver tissue were basically normal, with mild edema of liver cells; after 12 hours of storage, the structure of liver lobules was still clear, some liver plates disintegrated, and liver cells were generally mild to moderate ballooning. A small amount of stem cell necrosis; one hour after reperfusion, hepatic sinusoids dilated significantly, hepatocytes swelled, most of the liver plates disintegrated, scattered a small amount of fatty degeneration and a small amount of necrosis.

UW solution group: After being stored for 1 hour, the morphology and structure of the liver tissue are basically normal, and the liver cells are slightly edema; after being stored for 12 hours, the structure of the liver lobules is still clear, the liver cells are swollen, a small amount of liver cells become balloon-like, and some liver cells around the lobules are a little bit necrosis.

Groups 1-6 of Examples of the present invention: preserved for 1 hour, the morphology and structure of liver tissue were basically normal; preserved for 12 hours, the morphology and structure of liver tissue were basically normal, with mild edema of liver cells, and no other significant changes, the degree of liver tissue morphology change was determined by The order from strong to weak is: 1, 4, 2, 5, 6, 3.

Comparative example 1-6 group of the present invention: preserved for 1 hour, the structure of the hepatic lobules was still clear, the hepatic cells were swollen, a small amount of hepatic cells became balloon-like, and some hepatic cells around the lobules were punctately necrotic.

Experimental Example 3 Kidney Transplantation Experiment

Animal model: female mongrel dog, weighing 13-15kg.

Organ preservation solution: control group: HCA-II solution and UW solution; experimental group: organ preservation solution prepared in Examples 1-6 and Comparative Examples 1-6 of the present invention.

Experimental method: fasting for 12 hours before operation, anesthetized by intraperitoneal injection of 3% pentobarbital 30mg/kg. After successful anesthesia, an incision was made in the middle of the abdomen, about 12 cm long from the xiphoid process, and cut into the abdomen. Free the left kidney, renal artery and vein, and ureter, cut off the ureter, which is about 7 cm long, and cut them at the root of the renal vein and deep artery respectively. Immediately take out the kidneys and perfuse the kidneys with 200mL of preservation solution (0°C) for each group through the renal artery until the kidneys are pale, put the kidneys together with 500mL of preservation solution in a kidney bag and store them at 0-4°C. Ligate the renal arteriovenous and ureteral stumps of experimental animals respectively, and close the abdomen. Immediately after the operation, ceftriaxone sodium 1g was administered intravenously. After 72 hours, the same experimental dog was anesthetized in the same way, the abdominal cavity was opened according to the original incision, and extended downward to the upper part of the pubic bone, the right external iliac artery and vein were freed, the kidney was taken out, and the renal vein and external iliac vein were anastomosed with 7-0 prelene End-to-side anastomosis, end-to-end anastomosis of the renal artery and external iliac artery, and complete hemostasis after opening the blood flow. The bladder was cut open and a urinary catheter placed. The ureter has a built-in F4.5 double J tube, which is anastomosed with the bladder with 5-0 catgut. Then the right kidney of the experimental dog was cut open, the abdomen was closed, and the operation was completed. After the operation, rehydration (500 mL of 10% glucose and 500 mL of balance liquid per day) and 1 g of ceftriaxone sodium was given until the experimental animals began to eat on their own. The changes of renal function indexes before and after transplantation were detected after operation. The postoperative observation period was 60 days, and animals that reached 60 days were regarded as long-term survival.

Experimental results:

1. The apoptosis of kidney cortex during cryopreservation, the apoptosis index is as follows:

2. The measurement results of mitochondrial respiratory control ratio (RCR) are as follows:

3. Survival time: The survival time of the organ preservation solution prepared by using the examples 1-6 of the present invention is equivalent to that of the UW solution, which is significantly higher than that of the HCA-II solution and the comparative examples 1-6 of the present invention; The number of long-term surviving dogs in the organ preservation solution prepared in Examples 1-6 of the invention is also significantly more than that in HCA-II solution and Comparative Examples 1-6 in the present invention.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (9)

1. An organ preservation solution, characterized in that: the organ preservation solution is proportioned by weight, and every 1000mL contains: 2. The organ preservation solution according to claim 1, characterized in that: the pH buffer pair is one or more of phosphate, bicarbonate and histidine, preferably phosphate and histidine One or both of them, more preferably phosphate and histidine. 3. The organ preservation solution according to claim 1, wherein the energy substrate is adenosine, adenosine monophosphate, adenosine diphosphate, tryptophan, α-ketoglutarate, One or more of succinic acid, succinyl-CoA, acetyl-CoA and coenzyme Q10; preferably one or more of adenosine, succinic acid, coenzyme Q10, tryptophan and α-ketoglutarate several; more preferably one or more of adenosine, coenzyme Q10, tryptophan and succinic acid; more preferably adenosine, coenzyme Q10, tryptophan and succinic acid. 4. The organ preservation solution according to any one of claims 1-3, characterized in that: the organ preservation solution also contains one or more of laminarin, trehalose, laminin and betaine. 5. The organ preservation solution according to claim 4, characterized in that: the content of the laminarin is 1.0-10.0 g per 1000 mL. 6. The organ preservation solution according to claim 4, wherein the content of the trehalose is 1.0-16.0 g per 1000 mL. 7. The organ preservation solution according to claim 4, characterized in that: the content of the laminin is 8.0-27.0g per 1000mL. 8. The organ preservation solution according to claim 4, characterized in that: the content of betaine is 4.0-37.0g per 1000mL. 9. The method for preparing the organ preservation solution described in any one of claims 1-8, characterized in that: the preparation method comprises the following steps: (1) Weigh other ingredients except the energy substrate according to the dosage of the formula, add 50-80% of the total volume of water for injection and stir evenly, add medical activated carbon, stir for 5-20 minutes, and filter with a 0.45 μm filter membrane Decarburization to obtain solution A; (2) Weigh the energy substrate according to the dosage of the formula, and dissolve it with water for injection to obtain the energy substrate solution, mix the energy substrate solution with the solution A of step (1), add water for injection to the full amount, and stir evenly, Use a 0.45 μm filter membrane to filter, and the filtrate is the organ preservation solution.