恒河猴肝移植模型急性排斥反应时T细胞亚群的变化

doi:10.3969/j.issn.2095-4344.2014.49.014

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (49): 7948-7954.doi: 10.3969/j.issn.2095-4344.2014.49.014

• 器官移植动物模型 organ transplantation and animal model • 上一篇下一篇

恒河猴肝移植模型急性排斥反应时T细胞亚群的变化

冉江华，刘静，张熙冰，张升宁，吴淑媛，李来邦，李望，李立

昆明医科大学附属甘美医院肝胆胰外科，云南省器官移植研究所肝移植研究中心，云南省昆明市 650011

修回日期:2014-09-15 出版日期:2014-11-30 发布日期:2014-11-30
通讯作者: 李立，博士，教授，博士生导师，昆明医科大学附属甘美医院肝胆胰外科，云南省器官移植研究所肝移植研究中心，云南省昆明市 650011
作者简介:冉江华，男，1965年生，重庆市人，汉族，2006年昆明医科大学毕业，博士，教授，硕士生导师，主要从事器官移植、免疫耐受、肝胆胰外科等方面的研究。
基金资助:
昆明市科技局重大项目(08S100304)

Variation of T cell subset during acute rejection after liver transplantation in rhesus monkeys

Ran Jiang-hua, Liu Jing, Zhang Xi-bing, Zhang Sheng-ning, Wu Shu-yuan, Li Lai-bang, Li Wang, Li Li

Department of Hepatobiliary and Pancreatic Surgery, the Affiliated Ganmei Hospital of Kunming Medical University, Liver Transplantation Center of Organ Transplantation Institute of Yunnan Province, Kunming 650011, Yunnan Province, China

Revised:2014-09-15 Online:2014-11-30 Published:2014-11-30
Contact: Li Li, M.D., Professor, Doctoral supervisor, Department of Hepatobiliary and Pancreatic Surgery, the Affiliated Ganmei Hospital of Kunming Medical University, Liver Transplantation Center of Organ Transplantation Institute of Yunnan Province, Kunming 650011, Yunnan Province, China
About author:Ran Jiang-hua, M.D., Professor, Master’s supervisor, Department of Hepatobiliary and Pancreatic Surgery, the Affiliated Ganmei Hospital of Kunming Medical University, Liver Transplantation Center of Organ Transplantation Institute of Yunnan Province, Kunming 650011, Yunnan Province, China
Supported by:
the Major Program of Kunming Science and Technology Bureau, No. 08S100304

摘要/Abstract

摘要：

背景：寻找肝移植急性排斥反应的早期诊断指标，可快速而有效的评估移植后排斥反应的风险，而T淋巴细胞在急性排斥反应中发挥主要作用。

目的：观察恒河猴肝移植后血液T淋巴细胞亚群的变化与急性排斥反应的关系。

方法：采用改良血管袖套+胆道支撑管+动脉吻合法建立16例稳定的恒河猴肝移植模型，并将模型随机分为2组：实验组围手术期不给予免疫抑制治疗，对照组围手术期给予免疫抑制治疗，分别在移植后6，12，24，72 h共4个时间点收集血标本和肝组织，全自动生化分析仪测定丙氨酸氨基转移酶、天冬氨酸氨基转移酶、总胆红素，用流式细胞仪检测血CD4⁺/CD8⁺水平，然后取移植后肝脏组织进行苏木精-伊红染色以观察肝脏组织形态结构的改变，并根据Banff评分系统判断排斥反应程度。

结果与结论：实验组恒河猴肝移植后12，24，72 h均有急性排斥反应发生，其中实验组丙氨酸氨基转移酶、天冬氨酸氨基转移酶和总胆红素在移植后24，72 h表达水平明显高于对照组(P < 0.05)，实验组和对照组CD4⁺/CD8⁺的表达在移植后6 h即开始增高，实验组增高最明显，实验组移植后24，72 h CD4⁺/CD8⁺的表达明显高于对照组(P < 0.05)，实验组移植后72 h肝脏组织形态病理改变程度重于对照组，Banff评分高于对照组(P < 0.05)。说明在肝移植后急性排斥反应早期，CD4⁺/CD8⁺的表达变化早于肝组织病理学及肝功能的改变。肝移植后血CD4⁺/CD8⁺水平的升高提示人体细胞免疫功能增强，对肝移植后急性排斥反应的早期诊断具重要意义。

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

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关键词: 实验动物, 组织工程, 肝移植, 急性排斥反应, 恒河猴, T细胞亚群, 器官移植, CD4⁺；CD8⁺, 早期

Abstract:

BACKGROUND: Looking for the early diagnosis of acute rejection indicators after liver transplantation can assess the risk after liver transplantation quickly and effectively, and T lymphocytes play the significant role in acute rejection.

OBJECTIVE: To observe the relationship between acute rejection and variation of expression of T cell subset in blood after liver transplantation in rhesus monkey.

METHODS: The sixteen liver transplant models in rhesus monkey which were constructed successfully by the method of “double-cuff and one support tube” were divided into two groups randomly: experiment group (no treated by immunosuppressant in perioperative period) and control group (treated by immunosuppressant in perioperative period). Then the blood specimen and liver tissue respectively were collected at 6, 12, 24 and 72 hours after operation. The levels of alanine transferase, aspartate aminotransferase, and total bilirubin were detected with the fully automatic biochemical analyser. The levels of CD4⁺/CD8⁺were tested by flow cytometry. The liver tissue in rhesus monkey after liver transplantation was detected by hematoxylin-eosin staining. The degree of acute rejection was evaluated by Banff Score System.

RESULTS AND CONCLUSION: Acute rejection appeared in the experiment group at 12, 24, and 72 hours after liver transplantation. Levels of alanine transferase, aspartate aminotransferase, and total bilirubin were significantly higher in the experimental group than in the control group at 24 and 72 hours after transplantation (P < 0.05). The expression of CD4⁺/CD8⁺of the experiment group and control group began to rise at 6 hours after surgery, but the experiment group increased the most obvious. CD4⁺/CD8⁺ expression was significantly greater in the experimental group than in the control group at 24 and 72 hours after transplantation (P < 0.05). Morphological pathology was severer, and Banff score was higher in the experiment group than in the control group at 72 hours (P < 0.05). These data suggested that the variation of expression of CD4⁺/CD8⁺was earlier than the change of liver tissue pathology and the change of liver function in the early acute rejection after liver transplantation. The rise of level of CD4⁺/CD8⁺ after liver transplantation indicated the increase of cellular immunity in body, which had an important role in the early diagnosis of acute rejection after liver transplantation.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

Key words: liver transplantation, macaca mulatta, T-lymphocyte subsets, graft rejection, acute rejection

中图分类号:

R318

冉江华，刘静，张熙冰，张升宁，吴淑媛，李来邦，李望，李立. 恒河猴肝移植模型急性排斥反应时T细胞亚群的变化[J]. 中国组织工程研究, 2014, 18(49): 7948-7954.

Ran Jiang-hua, Liu Jing, Zhang Xi-bing, Zhang Sheng-ning, Wu Shu-yuan, Li Lai-bang, Li Wang, Li Li. Variation of T cell subset during acute rejection after liver transplantation in rhesus monkeys[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(49): 7948-7954.

图/表（结果） 2

Variation of T-lymphocyte subsets

In the experiment group without treatment of immunosuppressant, the amounts of CD4⁺ and CD8⁺ cells were significantly more than that of control group on the scatter diagram (Figure 1). Regarding the amount of CD4⁺ and CD8⁺ cells and the ratio of CD4⁺/CD8⁺, there was no significant differences between two groups (P > 0.05) at 6 and 12 hours, but the significant differences were found at 24 and 72 hours (P < 0.05). With the extension of time, the variation trend of CD4⁺T cell amount in peripheral blood of experiment group and control group since 6 hours to 72 hours after operation was as follows: CD4⁺ T cell amount in peripheral blood of experiment group and control group increased with the extension of time; the increasing extent of experiment group was far greater than that of control group; CD8⁺T cells amount in peripheral blood of experiment group and control group presented a decreasing trend with the extension of time, the decreasing extent of experiment group was significantly superior to that of control group (Table 1).

Variation of biochemical indices of liver damage

Comparison of serum alanine transaminase, aspartate aminotransferase and total bilirubin of rhesus monkeys in two groups after liver transplantation: alanine transaminase, aspartate aminotransferase and total bilirubin of experiment group were significantly higher than that of control group at 24 and 72 hours after operation (P < 0.05). The animals in experiment group did not take anti-rejection drugs or hormone after operation, so the damage of hepatic cells caused by the attack of inflammatory cells was more severe than that of control group. Alanine transaminase, aspartate aminotransferase and total bilirubin of two groups presented increasing trend with the extension of time. These three detection indices of two groups were close at 6 and 12 hours after operation, and the differences between two groups had no statistical significance (P > 0.05). Hepatic function presented increasing trend with the extension of time after liver transplantation, the differences of the interior-group in two groups had statistical significance (P > 0.05). Furthermore, the variation of experiment group was greater than of control group, especially from 24 to 72 hours after operation (Table 2).

Histopathology of rhesus monkey hepatic tissue after liver transplantation

Hepatic tissue of experiment group and control group was basically normal at 6 hours after operation, and no rejection feature was found under microscope (Figure 5, A1 and B1). At the end of 12 hours after operation, hematoxylin-eosin staining indicated the hepatic tissue of experiment group had a mild rejection, Banff score was I-II; the rejection grade of control group was undefined, Banff score was 0-I (Figure 2, A2 and B2). At the end of 24 hours after operation, hematoxylin-eosin staining indicated the hepatic tissue of experiment group had a mediate rejection, Banff score was II-III; control group met the diagnostic criteria of mild acute rejection, Banff score was 0-I (Figure 2, A3 and B3). At the end of 72 hours after operation, hematoxylin-eosin staining indicated the hepatic tissue of experiment group had a severe rejection and Banff score was III; control group presented the mild rejection, and Banff score was 0-I; rejection of the experimental group was severer than in the control group. Significant difference in Banff score was detected between

the two groups (P < 0.05) (Figure 5, A4 and B4).

Complications

All receptors were noted, venous thrombosis and biliary complications. No severe complications of abdominal abscess. Two receptors were visible in hepatic artery thrombosis patients, but not directly killing receptors.

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引言

材料方法

Design

Randomized controlled animal experiment.

Time and setting

The experiment was performed from September 2009 to September 2010 in the Experiment Animal Center of Kunming Medical University and the Key Laboratory of Second Affiliated Hospital of Kunming Medical University, China.

Materials

Animals

Both the donors and receptors were healthy Rhesus monkeys (provided by Experiment Animal Center of Kunming Medical University), totally 16 monkeys, Animal Permit No. SCXK(Dian)2009-0004. The receptors were male, weighing 7.2-11.5 kg and the donors were male and female, weighing 5.3-8.1 kg, keeping them in clean animal laboratory at Experiment Animal Center of Kunming Medical University in China, eating and drinking freely. The donors were not limited eating or drinking before the operation, but the amount of the food was restricted. The receptors fasted from food at 12 hours before the operation and fasted from water at 6 hours before the operation. The receptors were injected with cefazolin sodium at 0.1 g/kg. The treatment for animals during the experiment was in accordance with Guidance Suggestions for the Care and Use of Laboratory Animals, issued by State Department of Science and Technology in 2006^[11].

Methods

Establishment of liver transplantation models

Allograft models of rhesus monkey undergoing liver transplantation were established with the method developed by our team. Briefly, a large, cross-shaped incision was made in the abdominal wall. The perihepatic ligaments and inferior vena cava above and below the

liver were separated from adjacent structures. The hepatic artery, portal vein and biliary tract were separated at the porta hepatis. Tissues surrounding the inferior vena cava below the liver between the right renal and right adrenal veins were separated over 0.5-1.0 cm. The right suprarenal and lumbar veins were ligated adjacent to the inferior vena cava using a 4-0 suture. The donor liver was transplanted using standard orthotopic liver transplantation techniques (double-cuff and one support tube). The inferior vena cava above the liver was anastomosed using 5-0 prolene, the cuff of the portal vein was anastomosed and the portal vein was declamped. When blood was observed flowing from the inferior vena cava below the liver, the cuff was anastomosed. The inferior vena cava above and below the liver were declamped to terminate the anhepatic phase. The liver and gastrointestinal tract were perfused with 0.9% sodium chloride injection at 40-50 ℃ for rewarming until the color of the liver was restored. The common hepatic artery was anastomosed and a supporting tube was placed in the common bile duct. The abdominal cavity was washed with warm saline. If no hemorrhage or bile leakage was detected, the abdominal wall was closed^[12-13]. Receptor rhesus monkeys after 1.0-2.0 hours were awake and live, and more than 24 hours were regarded as successful operation.

Experimental groups

A total of 16 experiment animals were assigned to two groups randomly. Each group had four donors and four receptors. (1) Experiment group, the animals did not undergo anti-rejection treatment after the allograft models were established successfully. (2) Control group, the animals were treated with the intravenous injection of 250 mg urbason during the operation and FK506 anti-rejection drugs at 0.1 mg/(kg•d) after operation.

Acquirement of the specimens

The specimens were obtained respectively under narcosis at the end of 6, 12, 24 and 72 hours after operation (if the animal died in 30 minutes before or after the time point, this animal was counted as the sample of the corresponding time point). 3-6 mL of venous blood was taken. A part of blood sample was centrifuged (4 ℃, 3 000 r/min, 10 minutes). The supernatant was stored at -70 ℃ in an ultra low temperature freezer. Hepatic function was examined until all the specimens were collected. A part of blood sample was put into the tube with anticoagulant for the detection of T cell subset by flow cytometry. A small incision was incised at the abdominal wall. 0.5 cm × 0.5 cm × 1.0 cm liver tissue was taken from the edge of liver. The specimens were fixed in 10% neutral formalin, paraffin section was prepared and hematoxylin-eosin staining was conducted.

Biochemical indices after liver damage

The serum specimens were taken and kept under -70 ℃ and the detection of hepatic function was performed. Automatic biochemical analyzer was utilized to measure the expression variation of alanine transaminase and total bilirubin by directing activity determination.

Flow cytometry assay

The blood samples for flow cytometry assay described in the acquirement of the specimens were delivered to flow cytometry laboratory of Kunming Institute of Animal Sciences, Chinese Academy of Sciences and Assay by flow cytometry. We (1) separated(1-2)×10⁶ cells into two specific centrifuge tubes for flow cytometry, added appropriate amount of PBS, centrifuged at 1 500 r/min for 5 minutes, and removed the supernatant. (2) After washing the cells with pre-cold PBS, the concentration of the cells was adjusted to 1×10⁵/mL, and 1 μL PE or FITC-labeled anti-monkey CD4 monoclonal antibody was added (0.1 g/L) into 10 μL cell suspension. (3) After incubating for 30 minutes under 4 ℃, the cells were washed twice with PBS. (4) Finally, the cells were washed with 0.5 mL of PBS, and the cells were detected with flow cytometry.

Histopathological assay

The hepatic tissue stained in formaldehyde was made into paraffin section. The acute rejection was observed under microscope. The pathological change of acute rejection was sorted to four grades according to Banff Score System^[14-15]: undefined (0 grade), the infiltration of inflammatory cells of three bodies at portal area (hepatic artery, portal vein and bile duct) did not meet the diagnostic criteria of acute rejection; mild (I grade): the injection inflammatory cells invaded a small part of bile duct and blood vessel at portal area. There was a small amount of inflammatory cells, and inflammatory cells were limited at the gap of portal area; mediate (II grade): the rejection inflammatory cells invaded most or entire bile duct and blood vessel at portal area; severe (III grade): based on the mediate variation, the rejection inflammatory cells invaded the periphery of portal area, combined with mediate or severed phlebitis and extended to liver parenchyma, caused necrosis of hepatic cells that located at the periphery of veins.

Main outcome measures

There were the changes of liver function after transplantation, degree of rejection of transplantation of liver tissue after hematoxylin eosin staining, and the expression of CD4⁺/CD8⁺ after transplantation by using flow cytometry.

Statistical analysis

All data were analyzed by SPSS 17.0 software. Measurement data were expressed as mean ± SD. The differences among different groups were compared using measurement data of two independent samples t-test. The same group at different time points was compared using paired t-test. Pathology of acute rejection and Banff scores in each group were compared using two-sample comparison rank-sum test. A value of P < 0.05 was considered statistically significant.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

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讨论

Flow cytometry is an analytic method for studying the physical, chemical and biological properties of biomone (such as cells) with flow cytometer. It combines monoclonal antibody technique, laser technique, computer technique, cytochemical and immunochemical technique. Flow cytometer can make the quantitative, objective and multiparameter correlative measurement for the physical, chemical and biological properties of biomone (such as cells). Lymphocyte is the most important cell mass of normal immune system. T cells carry out the important function in human immune system, so the quantity of T cells and the variation of T cell subset will sensitively reflect the immunologic status. Therefore, we detected the quantity of CD4⁺ T cells in peripheral blood of experiment group and control group after liver transplantation with flow cytometry and explored the initiative status of immunity. The results indicated the quantity of CD4⁺ T cells of experiment group that was not treated with immunosuppressant was significantly more than that of control group on the scatter diagram. The quantities of CD8⁺ T cells of experiment group were less than of control group; that was to say, CD4⁺ T cells probably played a role in the acute rejection after liver transplantation in rhesus monkeys.

T cell is the focus of immunological rejection and immune regulation, T cell subset is the important factor of immune system maintaining, it needs the coregulation of multiple costimulatory molecules or other factors to keep the dynamic balance^[16-19]. The probable mechanism of the acute rejection is similar to the immune response to the general antigens. However, the difference is that the target antigen is allogeneic tissue antigen. The rough generating process of the acute rejection is as follows: (1) Antigen presenting cells of the graft moves to the peripheral lymphatic tissue of the recipient and activate CD4⁺ T cells; (2) the activated CD4⁺ T cells activated CD8⁺ CTL and proliferated to effector cells; (3) Th1 cells and CTL migrate to the local area and exert immunological effect. It is universally acknowledged that major histocompatibility complex and its outcomes are the important mark in the rejection of allotransplantation^[20-22]. Other reporter believe T cells identify major histocompatibility complex-I transplantation antigen by two pathways: (1) direct pathway: CD8⁺ T cells directly identify major histocompatibility complex-I antigens presented by antigen presenting cells of the donors; (2) indirect pathway: CD4⁺ T cells identify major histocompatibility complex-I antigens presented by the recipient antigen presenting cells, the activated CD4⁺ T cells further activate CD8⁺CTL and proliferate to effector cells. A plenty of in vitro experiments demonstrated CD4⁺ T cells could identify major histocompatibility complex-I antigens combined with major histocompatibility complex-II molecules^[23-35]. CD4⁺ T cell identify the enthetic antigens, sensitize, activate, become effector T cells and generate immune response. It is the most important cell that initiates the transplantation rejection. Under the normal physiologic condition, most effector cells become apoptotic cells after the infection is controlled or the antigen stimulation decline, only a small part of effector cells transfer to anamnestic CD4⁺ T cells. They can keep living in the body for a long time. They distribute in non-lymphoid organs and keep splitting at a low speed so that the organism can keep a certain level of anamnestic T cells. The genealogical relationship of anamnestic T cells is undefined currently; there is a lot of hypothesis, and the traditional view is that most effector cells are induced to the apoptotic cells after the primary response finishes. Only a few cells survive and turn into the quiescent anamnestic T cells, which is the classical linear differentiation theory. Later the parallel differentiation theory of anamnestic T cells is advanced by some researcher. According to this point of view, based on some causes, a part of primary T cells differentiate to effector T cells under the stimulation of antigen or other factors; another part of T cells differentiate to anamnestic T cells. The characters of anamnestic T cells play a very effective role in anti-infection of exotic etiology, but they play a disadvantageous role in transplantation. It is reported that there is a positive correlation between the ratio of anamnestic T cells in peripheral blood and the incidence rate of allotransplantation rejection^[36-37], multi kinds of factors involve in the rejection medicated by CD4⁺ T cells and CD8⁺ T cells^[38-40].

The results of this experiment indicate that CD4⁺T cell amount in peripheral blood of experiment group and control group increased with the extension of time, the increasing extent of experiment group is far greater than that of control group. CD8⁺T cell amount in peripheral blood of experiment group and control group presents a decreasing trend with the extension of time; the decreasing extent of experiment group is significantly superior to that of control group. There is difference of CD4⁺/CD8⁺ between experiment group and control group. The co-elevation of CD4⁺ and CD4⁺/CD8⁺ has a certain significant to the diagnosis of acute rejection after liver transplantation.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程
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恒河猴肝移植模型急性排斥反应时T细胞亚群的变化

Variation of T cell subset during acute rejection after liver transplantation in rhesus monkeys

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