WO2023236132A1

WO2023236132A1 - Method for constructing immune tolerance induction scheme for orthotopic liver transplantation

Info

Publication number: WO2023236132A1
Application number: PCT/CN2022/097775
Authority: WO
Inventors: 周绍棠
Original assignee: 周绍棠
Priority date: 2022-06-09
Filing date: 2022-06-09
Publication date: 2023-12-14

Abstract

Provided are an immune tolerance scheme for orthotopic liver transplantation and a method for constructing an animal model thereof. The animal model features proper and reasonable design and good reproducibility, which meets the requirements of orthotopic liver transplantation in rats. The present invention also possesses similarities to liver transplantation in humans, a high postoperative survival rate, good reproducibility, and a tolerance rate of up to 100%. The postoperative liver function assay and pathological analysis demonstrate that the present invention can solve the problems of inefficiency, high mortality rate, and high requirements on personnel and microscopic devices, and can be applied to the basic research of tolerance induction in donor-recipient combinations.

Description

A method for constructing an immune tolerance induction protocol for orthotopic liver transplantation

Technical field

The present invention relates to animal model construction technology and its tolerance induction scheme, specifically refers to the formation of reverse chimerism after the mobilization of recipient bone marrow stem cells after orthotopic liver transplantation and the determination of the immune tolerance induction scheme.

Background technique

Liver transplantation is recognized as the only effective method to treat end-stage liver disease, but big data research results report that there is almost no progress in the long-term survival of liver transplant recipients. Patients did not experience better long-term survival (survival rate of 2 years or more). The main reason is the long-term use of immunosuppressants. Life-long use of immunosuppressants brings a series of complications: renal insufficiency occurs in 25% of recipients; neurotoxicity 10-30%; diabetes 9-18%; kidney 20-33 %, 26-40% of liver, and 29% of lung recipients develop cardiovascular disease; the fourth cause of death after transplantation is infection, which occurs in 2/3 of recipients; new tumors are 11 times that of ordinary people, and digestive system diseases: 12% Recipients, hyperlipidemia: 46%, blood diseases: 30%; development and quality of life are not satisfactory, and the economic cost is huge: if a liver transplant recipient survives for 15 years, it will cost 60,000 to 70,000 yuan per year, totaling 1 million above.

Immune (or transplantation) tolerance refers to good graft function without pathologically confirmed chronic rejection (Banff criteria) when immunosuppressants are completely stopped. For human organ transplantation, this state should be maintained for at least 1 year, and for small animals such as rats, 2 months is enough. Inducing tolerance to organ transplants has been called another holy grail in the field. The methods are summarized as follows: 1) elimination of T cells; 2) blocking of costimulatory pathways; 3) induction of mixed chimeras; 4) enhancement of regulatory T cells. The above-mentioned programs target the recipient's immune system and have serious side effects. The current plan is to eliminate the hematopoietic ability of the recipient's bone marrow and reconstruct the chimera formed after the donor's bone marrow hematopoietic system, focusing on donor-specific immune unresponsiveness and continued maintenance of mixed chimerism.

However, the current general tolerance model uses whole liver transplantation. The aforementioned regimen is highly toxic and prone to refractory complications. There are no plans for clinical application worldwide. Therefore, a solution for studying immune tolerance in liver transplantation is urgently needed.

Contents of the invention

Finding immune tolerance regimens for clinical use is the most important goal in the field of organ transplantation. To solve this problem, we started with donor grafts and invented new models and new solutions for building immune tolerance. Its construction method includes the following steps:

Donor surgery

(1): Select SPF rats (SD, Lewis) with a body weight of 200-350g raised at a room temperature of 22±2°C and a humidity of 50±10%, and fast for 12 hours before surgery, but water is not allowed;

(2): Select rats with a weight difference of no more than 30g as donors, anesthetize the rats with isoflurane inhalation, trim and disinfect the abdomen;

(3): Fix the rat in a supine position on the operating table, continue to inhale isoflurane to maintain anesthesia, make a transverse incision to open the abdominal cavity of the rat, and pull the xiphoid process to expose the liver;

(4): Pull the intestine out of the left side of the abdominal cavity, wrap the intestine with wet gauze, and free the abdominal aorta below the level of the right renal vein;

(5): Expose the abdominal aorta on the celiac trunk and block it with a vascular clamp;

(6): Use a No. 1 needle to puncture the abdominal aorta below the level of the right renal vein and inject 5 ml of normal saline containing low molecular weight heparin 625IU/Kg, systemic heparinization, and retrograde perfusion of the liver;

(7): Cut an oblique opening in the superior mesenteric vein, inject 10 ml of lactated Ringer's solution, perfuse the liver again, cut the inferior vena cava at the level of the renal vein and bleed until the donor rat dies, stop anesthesia, and inject the liver several times. The liver is poured with 0-40°C normal saline to prevent liver rewarming;

(8): Ligate the superior mesenteric vein branches and splenic vein, and free the superior mesenteric vein. The celiac trunk was freed, and the splenic artery and left gastric artery were ligated. Preserve 5 mm of the extrahepatic bile duct, insert a support tube into the bile duct and fix it. The pyloric vein and gastroduodenal artery were ligated together. The left and right adrenal veins were freed and ligated, the right renal vein was ligated with 10-0 silk suture, and the inferior vena cava was cut off at the level of the left renal vein;

(9) Separate the tissues around the liver counterclockwise, ligate the left inferior diaphragmatic vein close to the liver, cut off the hepatic vein close to the diaphragm, remove the donor liver, and insert lactated Ringer's solution at 0-4°C; in the background, in the portal vein and inferior The vena cava is sleeved separately, such as a hemi-liver (50%) graft, and the left halves of the left lobe, caudate lobe, and middle lobe of the liver are ligated and cut off. For 70% grafts, the left lobe of the liver is ligated and cut off.

recipient surgery

(1) Select SPF grade rats (BN, Lewis) with a body weight of 200 to 350g raised at a room temperature of 22±2°C and a humidity of 50±10%. They should fast for 12 hours before surgery, but not water;

(2) Select rats with a weight difference of no more than 30g as recipients, anesthetize the rats with isoflurane inhalation, trim and disinfect the abdomen;

(3) The rat was fixed on the operating table in a supine position, and anesthesia was maintained by continuous inhalation of isoflurane. The abdominal cavity of the rat was opened through a longitudinal incision in the middle, and the xiphoid process was pulled to expose the liver; the left and right costal arches were pulled with homemade paperclip retractors;

(4) Separate and cut off the perihepatic tissue counterclockwise from the hepatogastric ligament, ligate the left inferior diaphragmatic vein near the diaphragm, ligate the left and right adrenal veins, skeletonize the inferior vena cava to the level of the right renal vein, ligate the proper hepatic artery, and ligate the proper hepatic artery near the common bile duct. Cut off the end, use mosquito clamps to clamp the diaphragm ring, and vascular clamps to block the portal vein and inferior vena cava respectively, cut off the portal vein, infrahepatic inferior vena cava, and suprahepatic inferior vena cava, and remove the original liver of the recipient; stop inhaling isoflurane;

(5) Place the donor liver in situ, using a one-point method, suture the posterior and anterior walls of the suprahepatic and inferior vena cava with 8-0 vascular suture from left to right, remove the mosquito clamp, and use a pug vascular clamp to block the suprahepatic and inferior vena cava. . After the portal vein is connected by the sleeve method, the portal vein clamp is removed, and the suprahepatic and inferior vena cava mosquito clamps are used to restore portal vein blood flow. The sleeve method was used to connect the inferior vena cava to restore blood flow, and 10 ml of normal saline was injected through the dorsal vein of the penis to help restore blood circulation. On the right side of the portal vein, block the common hepatic artery with a vascular clamp, ligate the gastroduodenal artery, cut an oblique opening in the common hepatic artery with scissors, insert the hepatic artery and vascular support tube into the common hepatic artery, ligate and fix it, and open it. To restore hepatic artery blood flow, use the same method to connect to the bile duct;

(6) After continuously suturing the midline incision with 5-0 suture, apply lidocaine glue to reduce pain, and suture the skin layer intermittently. During the operation, 4 mg/kg of cyclosporine was given by subcutaneous injection, and 1.2 ml of ceftizoxime sodium was injected intraperitoneally.

(7) After the operation, after waking up from anesthesia, keep the rats warm by indirect irradiation with infrared lamp for 12 hours, and continue to raise them at a room temperature of 22±2°C and a humidity of 50±10% to observe the survival of the rats;

(8) After surgery, the experimental group was injected subcutaneously with granulocyte colony-stimulating factor 100u/kg, once a day, 5 times in total; ceftizoxime sodium 100mg/kg, once a day, 3 times in total; and cyclosporine 4mg/kg, once a day. Once, 9 times in total. The control group received subcutaneous injection of ceftizoxime sodium 100 mg/kg once a day for a total of 3 times; cyclosporine 4 mg/kg once a day for a total of 9 times;

Observe the rat's activities, drinking water, urine volume, urine color, wounds, etc. every day.

After adopting the above method, the present invention has the following advantages: the design of the present invention is reasonable and ingenious, and the 50% liver transplant model is widely used in clinical practice. The operation is simple, reproducible, and can well simulate clinical practice. It is consistent with the characteristics of immune tolerance after orthotopic liver transplantation in rats: the function of the liver graft is normal, the graft has no acute or chronic rejection, and the structure is normal. High survival rate. Tolerance rate 100%. It can effectively solve the existing research goals of completely replicating the liver transplantation process, which requires a long time, high mortality rate, and high personnel and technical requirements; as well as the effectiveness, simplicity, repeatability, and operability of the tolerance program, etc.

Description of the drawings

Figure 1A,B,C shows the whole liver graft and after implantation (control group 1).

Figure 1D, E, and F show the half-liver graft after processing and implantation ((control group 2, tolerance group)).

Figure 2 is the survival situation of immune-tolerant rats after rat orthotopic liver transplantation according to the present invention. Chronic graft rejection or related death, which was pathologically confirmed, was recorded as the survival endpoint, and the survival time from transplantation to the survival endpoint was calculated.

Figure 3 shows the liver function of three groups of experimental rats after rat orthotopic liver transplantation.

Figure 4 is a comparison chart of HE staining of immune-tolerant grafts after orthotopic liver transplantation in rats of the present invention.

Detailed ways

Example 1: Liver transplantation

Donor surgery

(4): Pull the intestinal tube out of the left side of the abdominal cavity, and use wet gauze to free the abdominal aorta below the level of the right renal vein;

(7): Cut an oblique opening in the superior mesenteric vein, inject 10 ml of lactated Ringer's solution, perfuse the liver again, cut the inferior vena cava at the level of the renal vein and bleed until the donor rat dies, stop anesthesia, and inject the liver several times. The liver is poured with 0-4°C normal saline to prevent liver rewarming;

(8): Ligate the superior mesenteric vein branches and splenic vein, and free the superior mesenteric vein. The celiac trunk is freed, the splenic artery and the left gastric artery are ligated, and 5 mm of the extrahepatic bile duct is preserved. A support tube is inserted into the bile duct and ligated and fixed. The pyloric vein and gastroduodenal artery were ligated together. The left and right adrenal veins were freed and ligated, the right renal vein was ligated with 10-0 silk suture, and the inferior vena cava was cut off at the level of the left renal vein;

(9) Separate the tissue around the liver counterclockwise, ligate the left inferior diaphragmatic vein close to the liver, cut off the hepatic vein close to the diaphragm, remove the donor liver, and insert 0-40C lactated Ringer's solution; in the background, on the portal vein and inferior The vena cava is sleeved separately, such as a hemi-liver (50%) graft, and the left halves of the left lobe, caudate lobe, and middle lobe of the liver are ligated and cut off. For 70% grafts, the left lobe of the liver is ligated and cut off.

recipient surgery

(3) Fix the rat in the supine position on the operating table, continue to inhale isoflurane to maintain anesthesia, open the abdominal cavity of the rat through a longitudinal incision in the middle, and pull the xiphoid process to expose the liver; the left and right costal arches are pulled with homemade retractors made of paper clips;

(4) Separate and cut off the perihepatic tissue counterclockwise from the hepatogastric ligament, ligate the left inferior diaphragmatic vein near the diaphragm, ligate the left and right adrenal veins, skeletonize the inferior vena cava to the level of the right renal vein, ligate the proper hepatic artery, and cut off at the proximal end of the common bile duct , use mosquito clamps to clamp the diaphragm ring, and vascular clamps to block the portal vein and inferior vena cava respectively, cut off the suprahepatic inferior vena cava, infrahepatic inferior vena cava, and portal vein near the liver, and remove the original liver of the recipient; stop inhaling isoflurane;

(6) After continuously suturing the midline incision with 4-0 suture, apply lidocaine glue to reduce pain, and suture the skin layer intermittently.

(7) After the operation, after anesthesia, the rats were kept warm by indirect irradiation with infrared lamp for 12 hours before waking up from anesthesia. They were then kept at a room temperature of 22±2°C and a humidity of 50±10% to observe the survival of the rats;

(8) After surgery, the experimental group was injected subcutaneously with granulocyte colony-stimulating factor 100u/kg, once a day, 5 times in total; ceftizoxime sodium 100mg/kg, once a day, 3 times in total; and cyclosporine 4mg/kg, once a day. Once, 9 times in total. The control group received subcutaneous injection of ceftizoxime sodium 100 mg/kg once a day for a total of 3 times; cyclosporine 4 mg/kg once a day for a total of 9 times (Table 1).

(9) Observe the rat's activities, drinking water, urine output, urine color, wounds, etc. every day.

Embodiment 2

1. Experimental materials: transplanted rats after liver transplantation, isoflurane, a set of surgical instruments, etc.;

2. Preparation method:

(1): Select transplanted rats raised at room temperature of 25±2°C and humidity of 50±10%;

(2): The recipient rats were weighed and divided into a whole-liver control group (6 rats), a half-liver control group (6 rats), and an experimental group (16 rats). The rats were anesthetized by isoflurane inhalation and their abdomens were disinfected. ;

(3): Open the abdominal cavity of the rat through a midline incision, pull the intestinal tube outward to the left, expose the abdominal aorta, insert the blood collection needle into the test tube containing EDTA anticoagulant, and stand upside down several times to prevent blood coagulation;

(4): Adjust the centrifuge speed to 2000 and centrifuge for 10 minutes;

(5): Transfer the serum to a cryovial and label the specimen.

(6) After perfusing the liver with physiological saline through the mesenteric vein, free the perihepatic tissue, obtain liver specimens, and soak them in 4% neutral paraformaldehyde liquid.

(7) Set the corresponding parameters on the fully automatic biochemical instrument. Plasma loading. The fully automatic biochemistry instrument automatically measures and outputs the results.

Table 1. Study groups, treatments and results. Cyclosporine: CSA; Granulocyte colony-stimulating factor: GSF. Updated on 2022.05.25.

Embodiment 3

(1) Fixed tissue cutting

Remove the tissue from the fixative and cut it with a scalpel in a fume hood until the tissue thickness is about 3 mm. Place the trimmed tissue and corresponding labels in the dehydration box;

(2) Dewax the paraffin sections to water: place the sections in xylene I for 20 min - xylene II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min, and wash with tap water;

(3) Hematoxylin staining: Stain the sections with hematoxylin dye for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, return to blue with blue-returning solution, and rinse with running water;

(4) Eosin staining: The sections were dehydrated in 85% and 95% gradient alcohol for 5 minutes each, and then stained in eosin staining solution for 5 minutes;

(5) Dehydration and sealing: the sections are placed in absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min for transparency, and the sections are sealed with neutral gum;

(6) Microscopic examination, image collection and analysis.

3. Interpretation of results:

The nucleus is blue and the cytoplasm is red.

Figure 4A, B. Whole liver graft (control group). A small number of lymphocyte infiltrates can be seen in most portal areas in the liver tissue, as well as mild interlobular cholangitis in individual portal areas. At the same time, lymphocyte stasis and infiltration can be seen in some liver sinusoids in the liver lobules, indicating very mild or suspected acute rejection. reaction; at the same time, some false bile duct hyperplasia can be seen in some portal areas, indicating that there are very few false bile duct hyperplasia in the portal area caused by extreme ischemic damage factors in liver tissue. Overall, the degree of acute rejection is also very mild. If the acute rejection score of human liver transplantation is RAI (Rejection activity index = 2), it is suspected of acute rejection or uncertain acute rejection.

Figure 4C,D. Half-liver graft (control group). It can be seen that there is a lot of lymphocyte infiltration in the portal area of the liver tissue, and mild proliferation of fibrous tissue and false bile ducts can be seen. Some of the proliferated fibrous tissue extends to the adjacent portal area and liver tissue, causing slight disorder of the local liver tissue structure, but no further pseudo bile ducts are seen. Leaflets form.

Figure 4E, F. Tolerance group (experimental group). The structure of the liver lobules was regular, and there was no obvious lymphocyte infiltration in the portal area. The structure of the interlobular bile ducts was present and good, and no edema, necrosis, or cholestasis was found in the liver cells.

Claims

A method for constructing an immune tolerance induction scheme for liver transplantation, with features including the following:

Donor surgery

(1): Select SPF rats (Lewis) with a body weight of 200 to 350g raised at a room temperature of 22±2°C and a humidity of 50±10%. They should fast for 12 hours before surgery, but do not withhold water;

(2): Select rats with a weight difference of no more than 40g as donors, anesthetize the rats with isoflurane inhalation, trim and disinfect the abdomen;

(3): Fix the rat in a supine position on the operating table, continue to inhale isoflurane to maintain anesthesia, make a transverse incision to open the abdominal cavity of the rat, and pull the xiphoid process to expose the liver;

(4): Pull the intestinal tube out of the left side of the abdominal cavity, and use wet gauze to free the abdominal aorta below the level of the right renal vein;

(5): Expose the abdominal aorta on the celiac trunk and block it with a vascular clamp;

(6): Use a fine needle to puncture the abdominal aorta at the level of the right renal vein and inject 5 ml of normal saline containing low molecular weight heparin 625IU/Kg, systemic heparinization, and retrograde perfusion of the liver;

(7): Cut an oblique opening in the superior mesenteric vein, inject 10 ml of lactated Ringer's solution, perfuse the liver again, cut the inferior vena cava at the level of the renal vein and bleed until the donor rat dies, stop anesthesia, and inject the liver several times. The liver is poured with 0-4°C normal saline to prevent liver rewarming;

(8): Ligate the superior mesenteric vein branches and splenic vein, and free the superior mesenteric vein. The celiac trunk was freed, and the splenic artery and left gastric artery were ligated. Preserve 5 mm of the extrahepatic bile duct, insert a support tube into the bile duct, and ligate the pyloric vein and gastroduodenal artery. The left and right adrenal veins were freed and ligated, the right renal vein was ligated with 10-0 silk suture, and the inferior vena cava was cut off at the level of the left renal vein;

(9) Separate the tissues around the liver counterclockwise, ligate the left inferior diaphragmatic vein close to the liver, cut off the hepatic vein close to the diaphragm, remove the donor liver, and insert lactated Ringer's solution at 0-4°C; in the background, in the portal vein and The inferior vena cava was sleeved separately, such as a hemi-liver (50%) graft, and the left halves of the left lobe, caudate lobe, and middle lobe of the liver were ligated and cut off. For 70% grafts, ligate and cut off the left lobe of the liver;

recipient surgery

(1) Select SPF grade rats (BN, Lewis) with a body weight of 200 to 350g raised at a room temperature of 22±2°C and a humidity of 50±10%. They should fast for 12 hours before surgery, but not water;

(2) Select rats with a weight difference of no more than 30g as recipients, anesthetize the rats with isoflurane inhalation, trim and disinfect the abdomen;

(3) Fix the rat in the supine position on the operating table, continue to inhale isoflurane to maintain anesthesia, open the abdominal cavity of the rat through a longitudinal incision in the middle, and pull the xiphoid process to expose the liver; the left and right costal arches are pulled with homemade retractors made of paper clips;

(4) Separate and cut off the perihepatic tissue counterclockwise from the hepatogastric ligament, ligate the left inferior diaphragmatic vein near the diaphragm, ligate the left and right adrenal veins, skeletonize the inferior vena cava to the level of the right renal vein, ligate the proper hepatic artery, and cut off at the proximal end of the common bile duct , use mosquito clamps to clamp the diaphragm ring, and vascular clamps to block the portal vein and inferior vena cava respectively, cut off the suprahepatic inferior vena cava, infrahepatic inferior vena cava, and portal vein near the liver, and remove the original liver of the recipient; stop inhaling isoflurane;

(5) Place the donor liver in situ, use a one-point method, and suture the posterior and anterior walls of the suprahepatic and inferior vena cava with 8-0 vascular suture from left to right. Remove the mosquito clamp and replace it with a pug vascular clamp to block the suprahepatic and inferior vena cava. . After connecting the portal vein with the cuff method, remove the portal vein clamp and the suprahepatic and inferior vena cava pug clamp to restore portal blood flow. The cuff method was used to connect the subhepatic inferior vena cava to restore blood flow, and 10 ml of normal saline was injected through the dorsal vein of the penis to help restore blood circulation. On the right side of the portal vein, the common hepatic artery is blocked with a vascular clamp, and the gastroduodenal artery is ligated. An oblique opening is cut in the common hepatic artery with scissors. The support tube for the hepatic artery is inserted into the common hepatic artery. After ligation and fixation, it is opened and recovered. Hepatic artery blood flow, in the same way, connects to the biliary tract;

(6) After continuously suturing the midline incision with 4-0 suture, apply lidocaine glue to reduce pain, and suture the skin layer intermittently. During the operation, 4 mg/kg of cyclosporine (CSA) was injected subcutaneously and 100 mg/kg of ceftizole was injected intraperitoneally;

(7) After waking up from anesthesia, irradiate the rats indirectly with an infrared lamp to keep them warm for 12 hours, and continue to raise them at a room temperature of 22±2°C and a humidity of 50±10% to observe the survival of the rats;

(8) After surgery, the experimental group was injected subcutaneously with granulocyte colony-stimulating factor 100u/kg, once a day, 5 times in total; ceftizoxime sodium 100mg/kg, once a day, 3 times in total; and cyclosporine 4mg/kg, once a day. Once, 9 times in total. The control group received subcutaneous injection of ceftizoxime sodium 100 mg/kg once a day for a total of 3 times; cyclosporine 4 mg/kg once a day for a total of 9 times.
The method for constructing an animal model of liver transplantation immune tolerance according to claim 1, characterized in that the obtained rat recipient acquires tolerance with a tolerance rate of 100% and the time is more than 10 months.
The present invention can be extended to other basic studies on tolerance induction in different combinations of donor-recipient rats, as well as all experimental large animals that may be used, such as pigs, dogs, sheep, horses, monkeys, and humans.