阅读说明：本技术 一种原位肝移植免疫耐受诱导方案的构建方法 (Construction method of in-situ liver transplantation immune tolerance induction scheme ) 是由 周绍棠 陈国勇 刘方洲 汤高枫 赵会博 魏思东 于 2021-07-16 设计创作，主要内容包括：本发明公开一种原位肝移植免疫耐受方案及其动物模型的构建方法。其所述动物模型设计巧妙、合理、重复性好,符合大鼠原位肝移植术要求,和人肝移植相似。术后存活率高,重复性好,耐受率高达100％,在术后经肝功能检测,病理分析证实。能有效解决研究目标和肝移植完整复制过程所涉及时间久、死亡率高、人员和显微技术要求高等问题。同时可延伸到其他供-受体不同组合的耐受诱导的基础研究。(The invention discloses an in-situ liver transplantation immune tolerance scheme and a construction method of an animal model thereof. The animal model is ingenious and reasonable in design and good in repeatability, meets the requirements of rat orthotopic liver transplantation and is similar to human liver transplantation. The survival rate after operation is high, the repeatability is good, the tolerance rate is up to 100 percent, and the liver function detection and the pathological analysis after operation prove that. Can effectively solve the problems of long time, high death rate, high requirements on personnel and microtechnology, and the like in the complete replication process of research targets and liver transplantation. While extending to other basic studies of tolerance induction for different combinations of donor-receptor.)

1. A method for constructing an immune tolerance induction scheme for orthotopic liver transplantation is characterized by comprising the following steps:

donor operation

(1): selecting SPF rat Lewis with the weight of 200-350 g and fed at the room temperature of 22 +/-2 ℃ and the humidity of 50 +/-10 percent, and fasting for 12 hours before operation without water prohibition;

(2): selecting rats with the weight difference of not more than 40g as donors, carrying out isoflurane inhalation anesthesia on the rats, picking hairs on the abdomen of the rats and disinfecting the rats;

(3): fixing the rat in a supine position on an operating table, continuously inhaling isoflurane to maintain anesthesia, transversely cutting to open the abdominal cavity of the rat, and drawing the xiphoid process to expose the liver;

(4): pulling out the intestinal canal from the left side of the abdominal cavity, and dissociating the abdominal aorta at the level of the right renal vein by using the intestinal tract of the wet gauze;

(5): exposing the abdominal aorta above the abdominal trunk, blocking it with a vascular clamp;

(6): puncturing abdominal aorta at right renal vein level with fine needle, injecting physiological saline containing low molecular heparin 625IU/Kg 5ml, heparinizing whole body, and perfusing liver in reverse direction;

(7): cutting an oblique opening on the superior mesenteric vein, injecting 10ml of lactated ringer's solution, perfusing the liver again, cutting open the inferior vena cava at the renal vein level to bleed until the donor rat dies, stopping anesthesia, and pouring 0-4 ℃ physiological saline into the liver for multiple times to prevent the liver from rewarming:

(8): superior mesenteric vein branches and splenic veins were ligated and freed from superior mesenteric veins. The abdominal trunk is dissociated, and the splenic artery and the left gastric artery are ligated. The 5mm position of the extrahepatic bile duct is reserved, a supporting tube is inserted into the biliary tract, and the pyloric vein and the gastroduodenal artery are ligated together. Dissociating and ligating the left and right adrenal veins, ligating the right renal vein with 10-0 silk thread, and cutting off the inferior vena cava at the level of the left renal vein;

(9) separating the tissues around the liver anticlockwise, ligating the left subphragmatic vein close to the liver, cutting off the hepatic vein close to the diaphragm, removing the donor liver, and putting into the lactating ringer's solution at 0-4 ℃; on the posterior aspect, suprasleeve tubes, such as a hemihepatic (50%) graft, are placed over the portal and inferior vena cava, respectively, and left lobe, caudate lobe, and left half of the middle lobe of the liver are ligated and trimmed. As 70% of the grafts, ligate and prune the liver left lobe;

recipient surgery

(1) Selecting SPF rats (BN, Lewis) with the weight of 200-350 g and fed at the room temperature of 22 +/-2 ℃ and the humidity of 50 +/-10%, and fasting for 12 hours before operation without water prohibition;

(2) selecting rats with the weight difference of not more than 30g as receptors, carrying out isoflurane inhalation anesthesia on the rats, picking hairs on the abdomen, and disinfecting;

(3) fixing the rat in a supine position on an operating table, continuously inhaling isoflurane to maintain anesthesia, opening the abdominal cavity of the rat through a middle longitudinal incision, and drawing the xiphoid process to expose the liver; the left and right costal arches are pulled by a self-made draw hook by a clip;

(4) separating and cutting perihepatic tissue from a liver and stomach ligament anticlockwise, ligating a left infradiaphragmatic vein near a diaphragm, ligating a left adrenal vein and a right adrenal vein, forming an inferior vena cava skeletonized to the level of a right renal vein, ligating an inherent hepatic artery, cutting off the proximal end of a common bile duct, clamping a diaphragm ring by using mosquito forceps, and respectively blocking an portal vein and an inferior vena cava by using vascular clamps, cutting off the upper and lower hepatic veins, the lower hepatic vena cava and the portal vein near the liver, and removing a receptor primitive liver; stopping the inhalation of isoflurane;

(5) placing the donor liver in situ, performing one-point method, suturing the posterior wall and the anterior wall of the hepatic superior and inferior vena cava from left to right by using an 8-0 blood vessel wire, removing the mosquito clamp, and blocking the hepatic superior and inferior vena cava by using a pug vascular clamp. After the cuff procedure was performed to connect the portal vein, the portal vein clamps were removed and the hepatic superior and inferior vena cava pug clamps were removed to restore portal vein blood flow. The oversleeve method connects the inferior hepatic vena cava to restore blood flow, and 10ml of normal saline is injected into the dorsal vein of the penis to help restore blood circulation. On the right side of the portal vein, a vascular clamp blocks the hepatic artery, ligates the gastroduodenal artery, cuts an oblique opening on the hepatic artery by scissors, inserts a support tube for the hepatic artery into the hepatic artery, ligates and fixes the hepatic artery, opens the hepatic artery after ligation, and connects the biliary tract by the same method;

(6) after the median incision was continuously sutured with 4-0 thread, lidocaine mucilage was applied to relieve pain, and the skin layer was sutured intermittently. In the operation, 4mg/kg of Cyclosporine (CSA) is subcutaneously injected, and 100mg/kg of ceftizole is intraperitoneally injected;

(7) after the rats are anaesthetized and awake, the rats are not directly irradiated by an infrared lamp for keeping warm for 12 hours, and the rats are continuously raised at the room temperature of 22 +/-2 ℃ and the humidity of 50 +/-10 percent, and the survival condition of the rats is observed;

(8) injecting granulocyte colony stimulating factor 100u/kg subcutaneously in the postoperative experimental group once a day for 5 times; subcutaneously injecting 100mg/kg of ceftizoxime sodium once a day for 3 times; cyclosporin 4mg/kg once a day for 9 times. A control group is injected with 100mg/kg of ceftizoxime sodium subcutaneously for 3 times once a day; cyclosporin 4mg/kg once a day for 9 times.

2. The method of claim 1, wherein the obtained rat receptor is tolerant, the tolerance rate is 100%, and the time is more than 10 months.

3. The invention extends to other rats for the basic study of tolerance induction of different combinations of receptors, and all experimental large animals such as pigs, dogs, sheep, horses, monkeys, and humans, etc. that may be used.

Technical Field

The invention relates to an animal model construction technology and a tolerance induction scheme thereof, in particular to a construction method of an animal model of in-situ liver transplantation and determination of an immune tolerance induction scheme.

Background

Liver transplantation is the only accepted effective means for treating end-stage liver disease, but the results of big data research report that the long-term survival of liver transplant recipients has little progress. Patients do not have the benefit of better long-term survival (survival rate of 2 years or more). The main reason is the long-term use of immunosuppressive agents, which are used throughout the life with a series of complications: renal insufficiency occurs in 25% of recipients; neurotoxins 10% and 30% of recipients; 9-18% of diabetes; cardiovascular disease occurs in 20-33% of kidney, 26-40% of liver, and 29% of lung recipients; the 4 th cause of death for transplantation, infection, occurred in 2/3 recipients; the new tumor is 11 times that of a normal person, and digestive system diseases: 12% of recipients, hyperlipidemia: 46% of recipients, hematological disorders: 30% of recipients; the development and the life quality are not satisfactory, and the economic consumption is huge: if one liver transplant recipient lives for 15 years, it takes 6 to 7 thousands per year, and a total of 100 thousands or more. The implementation of the tolerance scheme applied to human beings can not only greatly reduce the related complications, but also avoid huge cost of 100 ten thousand yuan per person, wherein the number of liver transplantation cases per year in China is 5000 persons, and the cost is saved by 50 hundred million yuan per year. About 20000 people are transplanted to the liver worldwide, which amounts to about 200 hundred million, one liver receives two herbs, and theoretically, the number of transplanted recipients is increased by one time, which is 400 hundred million.

Immune (transplant) tolerance refers to a good graft function without pathologically confirmed chronic rejection (Banff criteria) with complete withdrawal of immunosuppressive agents. For human organ transplantation, this state is maintained for at least 1 year. For rats, it is administered for 4-6 months. Induction of organ transplant tolerance, is known as another holy grail in the field. The method is summarized as follows: 1) elimination of T cells; 2) blockade of co-stimulatory pathways; 3) induction of mixed chimeras; 4) increasing regulatory T cells. The above scheme intervenes in the immune system of the receptor, and has great side effects. Current protocols focus on donor-specific immune unresponsiveness and sustained mixed chimera maintenance.

The current general tolerance model employs a whole liver graft, but no clinically applicable protocol exists worldwide.

Therefore, a scheme for researching the immunological tolerance of liver transplantation needs to be researched and solved urgently.

Disclosure of Invention

The search for immune tolerance protocols for research and possible clinical applications is the most important goal in the field of organ transplantation. To solve this problem, we began with donor grafts and invented a new model, a new protocol for immune tolerance. The construction method comprises the following steps:

donor operation

(1): selecting SPF rats (SD, Lewis) with the weight of 200-350 g and fed at the room temperature of 22 +/-2 ℃ and the humidity of 50 +/-10%, and fasting for 12 hours before operation without water prohibition;

(2): selecting rats with the weight difference of not more than 30g as donors, carrying out isoflurane inhalation anesthesia on the rats, picking hairs on the abdomen of the rats and disinfecting the rats;

(3): fixing the rat in a supine position on an operating table, continuously inhaling isoflurane to maintain anesthesia, transversely cutting to open the abdominal cavity of the rat, and drawing the xiphoid process to expose the liver;

(4): pulling out the intestinal canal from the left side of the abdominal cavity, and wrapping the intestinal canal with wet gauze to free the abdominal aorta at the level of the right renal vein;

(5): exposing the abdominal aorta above the abdominal trunk, blocking it with a vascular clamp;

(6): puncture abdominal aorta under the level of right renal vein with No. 1 needle and inject 5ml of physiological saline containing 625IU/Kg of low molecular heparin, heparinize whole body, pour liver in retrograde motion;

(7): cutting an oblique opening on the superior mesenteric vein, injecting 10ml of lactated ringer's solution, perfusing the liver again, cutting open the inferior vena cava at the renal vein level to bleed until the donor rat dies, stopping anesthesia, and repeatedly pouring 0-40 ℃ physiological saline into the liver to prevent the liver from rewarming;

(8): superior mesenteric vein branches and splenic veins were ligated and freed from superior mesenteric veins. The abdominal trunk is dissociated, and the splenic artery and the left gastric artery are ligated. Keeping the extrahepatic bile duct at the position of 5mm, inserting the supporting tube into the biliary tract and fixing. The pyloric vein and the gastroduodenal artery were ligated together. Dissociating and ligating the left and right adrenal veins, ligating the right renal vein with 10-0 silk thread, and cutting off the inferior vena cava at the level of the left renal vein;

(9) separating the tissues around the liver anticlockwise, ligating the left subphragmatic vein close to the liver, cutting off the hepatic vein close to the diaphragm, removing the donor liver, and putting into the lactating ringer's solution at 0-4 ℃; in the posterior aspect, suprasleeve tubes, such as a hemihepatic (50%) graft, are placed over the portal and inferior vena cava, respectively, and left lobe, caudate lobe, and left half of the middle lobe of the liver are ligated and trimmed. For example, 70% of the grafts, the left lobe of the liver is ligated and trimmed.

Recipient surgery

(1) Selecting SPF rats (BN, Lewis) with the weight of 200-350 g and fed at the room temperature of 22 +/-2 ℃ and the humidity of 50 +/-10%, and fasting for 12 hours before operation without water prohibition;

(2) selecting rats with the weight difference of not more than 30g as receptors, carrying out isoflurane inhalation anesthesia on the rats, picking hairs on the abdomen, and disinfecting;

(3) fixing the rat in a supine position on an operating table, continuously inhaling isoflurane to maintain anesthesia, opening the abdominal cavity of the rat through a middle longitudinal incision, and drawing the xiphoid process to expose the liver; the left and right costal arches are drawn by a self-made clip retractor;

(4) separating and cutting perihepatic tissue from a liver and stomach ligament anticlockwise, ligating a left infradiaphragmatic vein near a diaphragm, ligating a left adrenal vein and a right adrenal vein, forming an inferior vena cava skeletonized to the level of a right renal vein, ligating an inherent hepatic artery, cutting off the proximal end of a common bile duct, clamping a diaphragm ring by using mosquito forceps, and respectively blocking a portal vein, an inferior vena cava, a portal vein, a inferior vena cava, an upper vena cava and a lower vena cava of a liver by using vascular clamps, and removing a receptor primitive liver; stopping the inhalation of isoflurane;

(5) the liver is placed in situ, and one-point method is adopted, 8-0 blood vessel suture is used from left to right, the posterior wall and the anterior wall of the upper and lower vena cava of the liver are removed, the mosquito clamp is removed, and the upper and lower vena cava of the liver are blocked by the hamaboard vascular clamp. After the sleeve method is connected with the portal vein, the portal vein clamp is removed, and the mosquito clamp for the upper and lower vena cava of the liver recovers the portal vein blood flow. The sleeve method is used for connecting the inferior vena cava to restore blood flow, and 10ml of normal saline is injected into the dorsal vein of the penis to help restore blood circulation. On the right side of the portal vein, a vascular clamp blocks the hepatic artery, the gastroduodenal artery is ligated, an oblique opening is cut out on the hepatic artery by scissors, a blood vessel supporting tube for the hepatic artery is inserted into the hepatic artery, the hepatic artery supporting tube is opened after ligation and fixation, the blood flow of the hepatic artery is recovered, and the biliary tract is connected by the same method;

(6) after the median incision was continuously sutured with 5-0 thread, lidocaine mucilage was applied to relieve pain, and the skin layer was sutured intermittently. Cyclosporine 4mg/kg is given during operation, subcutaneous injection and intraperitoneal injection of 1.2ml of ceftizoxime sodium are given.

(7) After the operation, after the anesthesia is clear, the rat is not directly irradiated by an infrared lamp for keeping warm for 12 hours, the rat is continuously raised at the room temperature of 22 +/-2 ℃ and the humidity of 50 +/-10 percent, and the survival condition of the rat is observed;

(8) injecting granulocyte colony stimulating factor 100u/kg subcutaneously in the postoperative experimental group once a day for 5 times; subcutaneously injecting 100mg/kg of ceftizoxime sodium once a day for 3 times; cyclosporin 4mg/kg once a day for 9 times. A control group is injected with 100mg/kg of ceftizoxime sodium subcutaneously for 3 times once a day; cyclosporin 4mg/kg once a day for 9 times;

the rats were observed daily for activity, water intake, urine volume, urine color, wound, etc.

After the method is adopted, the invention has the following advantages: the invention has reasonable and ingenious design, 50 percent of liver transplantation models and wide clinical application. The method is simple to operate, good in repeatability, and good in clinical simulation, and accords with the immune tolerance characteristics of rats after in-situ liver transplantation: the liver transplant has normal function, no acute and chronic rejection reaction and normal structure. The survival rate is high. The tolerance rate is 100%. The method can effectively solve the problems of long time, high death rate and high personnel technical requirement that the existing research target must completely replicate the liver transplantation process; and the problems of effectiveness, simplicity, repeatability, operability, etc. of the tolerance protocol.

Drawings

FIGS. 1A, B, C are the whole liver graft and post-implantation (control 1).

Fig. 1D, E, F are half liver graft treatment and post implantation ((control group 2, tolerant group).

FIG. 2 is a graph showing the survival of an immune-tolerant rat after in situ liver transplantation in a rat according to the present invention. The graft was found to have chronic rejection or associated death and was pathologically confirmed as the survival endpoint, and the survival time from transplantation to the survival endpoint was calculated.

FIG. 3 is the liver function of 3 experimental rats after rat orthotopic liver transplantation.

FIG. 4 is a graph comparing HE staining of an immune tolerant graft after orthotopic liver transplantation in rats of the invention.

Detailed Description

The first embodiment is as follows: liver transplantation

Donor operation

(1): selecting SPF rats (SD, Lewis) with the weight of 200-350 g and fed at the room temperature of 22 +/-2 ℃ and the humidity of 50 +/-10%, and fasting for 12 hours before operation without water prohibition;

(2): selecting rats with the weight difference of not more than 30g as donors, carrying out isoflurane inhalation anesthesia on the rats, picking hairs on the abdomen of the rats and disinfecting the rats;

(3): fixing the rat in a supine position on an operating table, continuously inhaling isoflurane to maintain anesthesia, transversely cutting to open the abdominal cavity of the rat, and drawing the xiphoid process to expose the liver;

(4): pulling out the intestinal canal from the left side of the abdominal cavity, and dissociating the abdominal aorta at the level of the right renal vein by using the intestinal tract of the wet gauze;

(5): exposing the abdominal aorta above the abdominal trunk, blocking it with a vascular clamp;

(6): puncture abdominal aorta under the level of right renal vein with No. 1 needle and inject 5ml of physiological saline containing 625IU/Kg of low molecular heparin, heparinize whole body, pour liver in retrograde motion;

(7): cutting an oblique opening on the superior mesenteric vein, injecting 10ml of lactated ringer's solution, perfusing the liver again, cutting open the inferior vena cava at the renal vein level to bleed until the donor rat dies, stopping anesthesia, and repeatedly pouring 0-4 ℃ physiological saline into the liver to prevent the liver from rewarming;

(8): superior mesenteric vein branches and splenic veins were ligated and freed from superior mesenteric veins. Dissociating the celiac trunk, ligating the splenic artery and the left gastric artery, reserving the extrahepatic bile duct at a position of 5mm, inserting a supporting tube into the biliary tract, and ligating and fixing. The pyloric vein and the gastroduodenal artery were ligated together. Dissociating and ligating the left and right adrenal veins, ligating the right renal vein with 10-0 silk thread, and cutting off the inferior vena cava at the level of the left renal vein;

(9) separating tissues around the liver anticlockwise, ligating a left subphragmatic vein tightly to the liver, cutting off the hepatic vein tightly to the diaphragm, removing a donor liver, and putting 0-40C lactated ringer's solution; on the posterior aspect, suprasleeve tubes, such as a hemihepatic (50%) graft, are placed over the portal and inferior vena cava, respectively, and left lobe, caudate lobe, and left half of the middle lobe of the liver are ligated and trimmed. For example, 70% of the grafts, the left lobe of the liver is ligated and trimmed.

Table 1 study grouping, processing and results. Cyclosporin (2): CSA; granulocyte colony stimulating factor: GSF

Recipient surgery

(1) Selecting SPF rats (BN, Lewis) with the weight of 200-350 g and fed at the room temperature of 22 +/-2 ℃ and the humidity of 50 +/-10%, and fasting for 12 hours before operation without water prohibition;

(2) selecting rats with the weight difference of not more than 30g as receptors, carrying out isoflurane inhalation anesthesia on the rats, picking hairs on the abdomen, and disinfecting;

(3) fixing the rat in a supine position on an operating table, continuously inhaling isoflurane to maintain anesthesia, opening the abdominal cavity of the rat through a middle longitudinal incision, and drawing the xiphoid process to expose the liver; the left and right costal arches are pulled by a self-made draw hook by a clip;

(4) separating and cutting perihepatic tissue from a liver and stomach ligament anticlockwise, ligating a left infradiaphragmatic vein near a diaphragm, ligating a left adrenal vein and a right adrenal vein, forming an inferior vena cava skeletonized to the level of a right renal vein, ligating an inherent hepatic artery, cutting off the proximal end of a common bile duct, clamping a diaphragm ring by using mosquito forceps, and respectively blocking an portal vein and an inferior vena cava by using vascular clamps, cutting off the upper and lower hepatic veins, the lower hepatic vena cava and the portal vein near the liver, and removing a receptor primitive liver; stopping the inhalation of isoflurane;

(5) the liver is placed in situ, and one-point method is adopted, 8-0 blood vessel suture is used from left to right, the posterior wall and the anterior wall of the upper and lower vena cava of the liver are removed, the mosquito clamp is removed, and the upper and lower vena cava of the liver are blocked by the hamaboard vascular clamp. After the sleeve method is connected with the portal vein, the portal vein clamp is removed, and the mosquito clamp for the upper and lower vena cava of the liver recovers the portal vein blood flow. The sleeve method is used for connecting the inferior vena cava to restore blood flow, and 10ml of normal saline is injected into the dorsal vein of the penis to help restore blood circulation. On the right side of the portal vein, a vascular clamp blocks the hepatic artery, the gastroduodenal artery is ligated, an oblique opening is cut out on the hepatic artery by scissors, a blood vessel supporting tube for the hepatic artery is inserted into the hepatic artery, the hepatic artery supporting tube is opened after ligation and fixation, the blood flow of the hepatic artery is recovered, and the biliary tract is connected by the same method;

(6) after the median incision was continuously sutured with 4-0 thread, lidocaine mucilage was applied to relieve pain, and the skin layer was sutured intermittently.

(7) After the operation, the rats are anaesthetized to be sober, the rats are not directly irradiated by an infrared lamp for keeping warm for 12 hours, the rats are continuously raised at the room temperature of 22 +/-2 ℃ and the humidity of 50 +/-10 percent, and the survival condition of the rats is observed;

(8) injecting granulocyte colony stimulating factor 100u/kg subcutaneously in the postoperative experimental group once a day for 5 times; subcutaneously injecting 100mg/kg of ceftizoxime sodium once a day for 3 times; cyclosporin 4mg/kg once a day for 9 times. A control group is injected with 100mg/kg of ceftizoxime sodium subcutaneously for 3 times once a day; cyclosporin 4mg/kg once a day for 9 times (Table 1).

(9) The rats were observed daily for activity, drinking water, urine volume, urine color, wound, etc.

Example two

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

2. the preparation method comprises the following steps:

(1): selecting transplanted rats raised at room temperature of 25 + -2 deg.C and humidity of 50 + -10%;

(2): weighing receptor mice, dividing the mice into a whole liver control group (6 mice), a half liver control group (6 mice) and an experimental group (16 mice), and carrying out isoflurane inhalation anesthesia and abdominal disinfection on the rats;

(3): opening abdominal cavity of rat at median incision of rat, pulling intestinal canal to left and outside, exposing abdominal aorta, inserting blood taking needle into main artery and connecting to test tube containing EDTA anticoagulant, standing upside down for several times to prevent blood coagulation;

(4): regulating the rotating speed of the centrifuge to 2000, and centrifuging for 10 minutes;

(5): transferring the serum to a freezing tube and marking the specimen.

(6) After the liver was perfused with normal saline through the mesenteric vein, the perihepatic tissue was dissociated to obtain a liver specimen, which was immersed in 4% neutral paraformaldehyde solution.

(7) Corresponding parameters are set on the full-automatic biochemical analyzer. And (4) loading plasma. The full-automatic biochemical analyzer automatically measures and outputs results.

EXAMPLE III

(1) Fixed tissue cutting

The tissue was taken out of the fixed solution and cut with a scalpel in a fume hood to a tissue thickness of about 3 mm. Placing the trimmed tissues and the corresponding labels in a dehydration box;

(2) paraffin section dewaxing to water: sequentially placing the slices in xylene I20 min-xylene II 20 min-absolute ethanol I5 min-absolute ethanol II5 min-75% ethanol 5min, and washing with tap water;

(3) and hematoxylin staining: staining the slices in hematoxylin staining solution for 3-5min, washing with tap water, differentiating the differentiation solution, washing with tap water, returning blue to the blue solution, and washing with running water;

(4) eosin staining: the slices are dehydrated for 5min respectively by adding 85 percent and 95 percent gradient alcohol in sequence, and are dyed for 5min in eosin dye solution;

(5) dewatering and sealing: placing the slices in anhydrous ethanol I5 min-anhydrous ethanol II5 min-anhydrous ethanol III5 min-dimethyl I5 min-xylene II5min, sealing with neutral gum;

(6) microscopic examination and image acquisition and analysis.

Thirdly, interpretation of results:

the nucleus was blue and the cytoplasm was red.

Fig. 4A, b whole liver grafts (control). In liver tissue, a plurality of portal areas can be seen to have a few lymphocyte infiltrations and slight lobular cholangitis in individual portal areas, and meanwhile, part of liver lobules can be seen to have lymphocyte siltation and infiltration in liver sinuses, which indicates very slight or suspected acute rejection; meanwhile, the hyperplasia of the pseudo bile duct in a few portal areas can be seen, which indicates that the hyperplasia of the pseudo bile duct in the portal areas is very little caused by the factors of ischemia injury of liver tissues. The acute Rejection degree is also very slight in general, and acute Rejection or uncertain acute Rejection is suspected if the acute Rejection index (Rejection activity index) of the transplanted liver of a human body is scored as RAI (2).

Fig. 4C, d. It can be seen that there is much lymphocyte infiltration in the portal area of liver tissue, and that the fibrous tissue and the slightly hyperplastic fibrous tissue of the pseudobile duct extend into the adjacent portal area and liver tissue to cause slight disorder of local liver tissue structure, but no further pseudolobule formation is seen.

Fig. 4E, f. The hepatic lobule structure is regular, obvious lymphocyte infiltration is not seen in the portal area, the structure of the bile duct between the hepatic lobules is good, and the hepatic cells do not show any edema, necrosis and cholestasis.