人肝细胞—乳鼠原位移植模型构建中细胞免疫机制的研究

发布时间：2018-04-19 11:23

本文选题：肝细胞 + 甲胎蛋白　；参考：《山东大学》2009年博士论文

【摘要】： 背景和目的近年来通过异种肝细胞移植的手段,解决同种供体短缺的问题已成为全球研究的热点。虽然异种肝细胞移植在短期内改善肝脏先天性酶缺陷和血液系统病变及急、慢性肝衰竭等方面的有效性已在动物实验模型中得到证实,但其后发生的免疫排斥问题仍然成为阻碍异种肝细胞移植在临床中得以广泛应用研究的最大难题。目前,有关异种肝细胞移植的免疫排斥机制的研究主要存在以下几个问题:一、异种移植物会面临来自受体更激烈,更迅速的免疫排斥,在几小时内就可能完全丧失功能或坏死,因而有关异种细胞移植中免疫排斥的动态研究不能很好地开展。二、相对于同种肝细胞移植,T淋巴细胞介导免疫排斥在异种肝细胞移植过程中的作用尚存在争议,有关异种肝细胞原位移植的免疫排斥机制鲜有报道。三、异种移植中的免疫排斥反应常因移植物种类的不同,所带抗原数量、强度以及受者的免疫功能状态的差异,特别是具体到某一器官如肝脏,其免疫排斥反应将各具其特殊性。针对上述问题,我们根据发育免疫学原理,对课题设计进行了可行性分析:首先,肝脏在移植领域被成为“免疫特惠器官”,长期临床研究发现肝脏移植后急性排斥反应的发生率及严重程度远远低于其它器官,具有独特的耐受原性;同时,肝脏是甲胎蛋白的主要合成及分泌器官,甲胎蛋白是一种胚胎性肿瘤相关蛋白,免疫应答中的AFP主要表现为免疫抑制的生物学特征,而小鼠出生后一段时间内AFP在肝脏仍保持着较高水平的表达;因此,我们认为该发育阶段的肝脏免疫微环境具有适合异种移植的特殊性。另外,针对“外来细胞抗原所致免疫耐受”的现象,研究认为免疫系统接触外来抗原所产生的免疫耐受程度与免疫系统发育未成熟有关。如果我们在受体免疫系统发育的早期给予异种抗原的移植,将会减缓受体的异种免疫排斥,或者直接形成耐受。综上所述,我们选用新生小鼠肝脏作为移植受体,设想该发育阶段的肝脏微环境会缓解或者推迟机体的免疫排斥的强度或速度,将有利于免疫排斥发生的动态过程及其相关机制的研究。同时,鉴于主要组织相容性抗原为移植排斥反应发生的主要移植抗原,它由种系基因构成,代表了某个物种在进化上的特异性。肝癌细胞不仅具有与人类正常肝细胞相同的移植抗原,同时作为正常肝细胞的替代物在肝脏相关疾病的实验性治疗中被证明有良好的效果。因此,本实验选用3株人肝癌细胞,1株人正常肝细胞及1株人肺癌细胞作为待选异种移植物,通过对比筛选合适的受一供体组合,力图建立一种理想的人肝细胞一乳鼠异种原位移植模型,并系统地对移植过程中细胞免疫排斥的时程变化及其相关机制进行研究。研究方法 1、人肝细胞—乳鼠原位移植模型的探索性构建 1)根据动物模型构建方法,体外肝部直接注射进行五种人相关细胞的原位移植; 2)活体荧光染料DiI标记技术对移植的人细胞进行受体内的定位示踪; 3)解剖观察移植细胞在乳鼠肝内的移植成功率及生长状况; 4)常规病理技术分析各组人细胞在乳鼠肝内的免疫病理发展过程; 5)免疫组化检测移植物在受体内AFP及Suvivin蛋白的表达状况。 2、乳鼠血清AFP表达特征与肝细胞移植微环境的关系 1)等数量级的人肝癌细胞HepG2分别进行乳鼠皮下及肝内原位移植,成鼠原位移植不同数量级的HepG2细胞; 2)活体荧光染料DiI标记技术对移植细胞进行受体内定位示踪; 3)常规病理切片对比分析移植细胞在各移植部位免疫病理学过程; 4)定量ELISA检测移植前后乳鼠血清AFP的动态变化特征。 3、人肝细胞—乳鼠原位移植中细胞免疫排斥机制的研究 1)利用免疫调节剂(CsA,PolyI:C)分别设置免疫激活及免疫抑制两个参照状态; 2)不同免疫状态下人肝细胞—乳鼠原位移植模型的构建; 3)不同免疫状态下移植细胞免疫病理过程的变化; 4)NK细胞杀伤活性的检测; 5)刀豆蛋白诱导的T淋巴细胞活性的检测; 6)流式分析T淋巴细胞亚群分布的变化; 7)定量ELISA检测小鼠IL-2,IL-10血清水平的变化特征。研究结果第一部分解剖观察移植情况肝癌细胞移植组均具有较高的移植成功率,个案伴有一定程度的肝内转移。人肺癌细胞移植成功率较低,并伴有消退趋势。人正常肝细胞移植组仅2个案例肝脏观察到异常部位。活体荧光染料Dil的定位分析荧光定位分析确定解剖观察到的结节组织为移植或转移的人细胞,各组肝癌细胞在肝内占位明显,荧光信号均匀稳定;人肺癌细胞占位较小;人正常肝细胞组解剖观察到的异常部位为荧光染料非特异性着色,无细胞性结构。常规病理切片分析原位移植初期(0-5天),人肝癌细胞保持着良好的肿瘤形态特征,8天前后移植区域逐渐出现淋巴细胞的浸润,移植物的相继坏死,发生肝内转移的人肝癌细胞至实验结束未出现淋巴细胞的浸润及坏死。原位移植的人肺癌细胞于移植后5天出现明显的淋巴细胞浸润及严重坏死。免疫组织化学检测原位移植及转移的各组人癌细胞均保持着原有的蛋白表达特征:Bel7402与HepG2:AFP~+;SK-Hep-1与A549:AFP~-;Survivin全部阳性表达。第二部分人肝癌细胞HepG2的乳鼠皮下移植皮下移植物于移植第2天出现淋巴细胞的浸润及后续的严重坏死,并于移植后2周完全消退。人肝癌细胞HepG2的成鼠肝内移植仅有10%的个案形成类似乳鼠原位移植的移植物结节,移植物于移植后24小时出现明显的淋巴细胞浸润,或被炎症细胞包裹,人肝癌细胞空泡化继而坏死,移植细胞数量级的增大,并未缓解免疫排斥反应出现的强度与速度。 AFP血清浓度变化正常昆明种小鼠:AFP血清浓度出生后三天内保持较高水平的表达,并于出生后两周恢复至成体水平,痕量。人肝癌细胞的移植引起乳鼠肝脏AFP的特异性再表达,于移植后的5-9天保持高水平的血清浓度,与正常小鼠有显著差异。人正常细胞与人肺癌细胞的移植,及成鼠肝内的移植未引起血清水平的显著变化。乳鼠皮下移植未引起AFP血清浓度的变化。第三部分免疫调节剂的影响 CsA组:移植物占位偏大,形态不规则,移植细胞呈侵袭性生长;于实验第14天移植区域出现微弱的淋巴细胞浸润,发展趋势缓慢,移植后35d内移植物未出现明显坏死。 PolyI:C组:移植物占位明显小于正常移植组,移植后第2天就出现明显的淋巴细胞浸润,并于移植后第5天出现大面积坏死。 NK细胞杀伤活性移植组:移植后第4天NK细胞杀伤活性明显升高,CsA组:NK细胞杀伤于移植后呈缓慢上升趋势,总体水平低于移植组,PolyI:C组NK细胞杀伤活性一直保持较高水平,与其它两组差异显著。 T淋巴细胞的转化活性移植组:移植第7天T淋巴细胞转化活性由缓慢上升转为明显升高,具有显著性;CsA组与PolyI:C组的T细胞转化活性皆缓慢上升,无大幅度变化,PolyI:C组最高,CsA组最低。 T淋巴细胞亚群的分布出生后小鼠T细胞分化尚未完善,CD4~+,CD8~+T细胞数量随时间不断增加。移植组CD4~+与CD8~+T细胞在各时间点皆高于正常小鼠,CD4~+T细胞数量增长较快,于移植后11天CD4~+/CD8~+比值达到最高值,显著高于其它组。CsA组CD4~+T细胞百分含量位于各组最低,CD4~+/CD8~+比值出现紊乱直至逆转。PolyI:C组CD4~+与CD8~+T细胞百分率增长速率明显高于其它组,CD4~+/CD8~+比值略高于正常小鼠。细胞因子IL-2,IL-10的表达特征移植组小鼠于移植后第3天IL-2血清浓度降至最低,IL-10达到最高浓度,随后逆转,第7天IL-2浓度诱生至最高点,IL-10浓度降至最低点;CsA组IL-2受到明显的抑制,IL-10浓度较其它组最高;PolyI:C组IL-10浓度低于移植组,IL-2浓度显著高于其它实验组。研究结论 1、首次对昆明种小鼠出生后血清甲胎蛋白的表达特征进行了定量分析,与BALB/c/J,BALB/c/BOM and C3H/He等种系小鼠具有相似的表达模式。 2、乳鼠肝脏内AFP高水平的表达可保证异种移植物的存活,推迟T细胞诱导的异种移植免疫排斥反应的发生。 3、针对异种肝细胞移植中细胞免疫排斥反应的动态研究,本实验构建的人肝细胞—乳鼠异种原位移植模型是一种良好的研究平台。 4、异种移植细胞免疫排斥过程中,NK细胞活性的诱发早于T细胞,但是对移植物的存活未形成明显的影响。 5、CD4+淋巴细胞是介导免疫排斥反应中的主要T细胞亚群,CD4+/CD8+T淋巴细胞比率的失调甚至逆转导致个体免疫排斥的发生。 6、激活的CD4~+T0淋巴细胞经历了向T1诱导分化的免疫偏移,高水平表达的TH1类细胞因子(IL-2)介导了细胞免疫排斥的效应期,可以作为T细胞引发的免疫排斥反应的评价指标。 7、单纯使用免疫抑制剂CSA并未能完全抑制异种肝细胞移植中细胞免疫排斥的发生,只起到了延缓的作用。
[Abstract]:Background and Purpose

In recent years , the problem of homologous donor shortage has become the focus of global research by means of xenogeneic hepatocyte transplantation . Although the effectiveness of xenogeneic hepatocyte transplantation in improving liver congenital enzyme defects and blood system pathological changes and acute and chronic liver failure has been confirmed in animal experiment model , the problem of immune rejection has become the biggest problem which prevents xenogeneic hepatocyte transplantation from being widely used in clinic .

At present , the study of immune rejection mechanism related to xenogeneic liver transplantation mainly has the following problems : firstly , xenotransplantation may face more intense and more rapid immune rejection , which may completely lose function or necrosis within a few hours , so the mechanism of immune rejection in xenotransplantation can not be well carried out .

In view of the above - mentioned problems , we have carried out the feasibility analysis on the subject design according to the developmental immunology principle : firstly , the liver is regarded as an " immune special organ " in the field of transplantation .

Research Methods

1 . Exploratory Construction of Orthotopic Transplantation Model for Human Hepatocytes

1 ) carrying out in - situ transplantation of five human - related cells by direct injection of the liver part in vitro according to the construction method of the animal model ;

2 ) the living fluorescent dye DiI labeling technique is used for carrying out positioning and tracing on transplanted human cells in vivo ;

3 ) transplanting success rate and growth condition of transplanted cells in rat liver ;

4 ) Immunopathological development of human cells in rat liver was analyzed by routine pathological technique .

5 ) The expression of AFP and Suvivin protein was detected by immunohistochemistry .

2 . The relationship between the expression of AFP in serum and the microenvironment of liver transplantation

1 ) carrying out in - situ transplantation of human liver cancer cells HepG2 on the order of magnitude and the like to obtain HepG2 cells in different orders of magnitude in situ transplantation ;

2 ) the living fluorescent dye DiI labeling technique is used for carrying out in vivo positioning and tracing on the transplanted cells ;

3 ) the pathological process of the transplanted cells in each transplantation site was analyzed by routine pathological sections ;

4 ) Quantitative ELISA was used to detect the dynamic changes of serum AFP level before and after transplantation .

Study on the Mechanism of Cell Immune Rejection in Orthotopic Transplantation of Human Hepatocytes

1 ) respectively setting two reference states of immune activation and immunosuppression by using an immune modulator ( CsA , PolyI : C ) ;

2 ) Construction of orthotopic liver transplantation model of human hepatocytes in different immune states ;

3 ) the changes of the immune pathological process of the transplanted cells in different immune states ;

4 ) detection of NK cell killing activity ;

5 ) detection of T lymphocyte activity induced by knife and bean protein ;

6 ) Flow analysis of T lymphocyte subsets distribution ;

7 ) The serum levels of IL - 2 and IL - 10 were detected by quantitative ELISA .

Results of the study

The first part of anatomy observation and transplantation

The success rate of transplantation of human lung cancer cells was higher than that in liver transplantation group . The success rate of human lung cancer cell transplantation was lower , with the tendency of regression .

The localization and analysis of live fluorescent dye Dil

Fluorescence localization analysis determined that the observed nodules were transplanted or transferred into human cells . All groups of liver cancer cells were occupying lesions in the liver , the fluorescence signals were stable , the cells of human lung cancer were small , and the abnormal spots observed in normal liver cells were non - specific coloring of fluorescent dyes and no cellular structure .

Routine pathological section analysis

In the early stage of orthotopic transplantation ( 0 - 5 days ) , the human liver cancer cells maintained a good tumor morphology . After 8 days , the transplantation of human liver cancer cells gradually showed the infiltration of lymphocytes , the subsequent necrosis of the graft , and the invasion and necrosis of lymphocytes were not observed at the end of the experiment . The human lung cancer cells transplanted in situ appeared to have obvious lymphocyte infiltration and severe necrosis on 5 days after transplantation .

immunohistochemical detection

All groups of human cancer cells in situ transplantation and metastasis maintained the original expression of the protein : Bel7402 and HepG2 : AFP + ; SK - Hep- 1 and A549 : AFP ~ - ; Survivin was all positive .

Subcutaneous Transplantation of Human Hepatocellular Carcinoma HepG2 Cells in Vitro

The infiltration and subsequent severe necrosis of lymphocytes appeared on the second day after transplantation , and completely disappeared 2 weeks after transplantation .

Intrahepatic transplantation of human hepatocellular carcinoma cell line HepG2

There were only 10 % of cases forming graft nodules similar to in - situ transplantation of milk - like mice , which showed significant lymphocytic infiltration 24 hours after transplantation , or by inflammatory cells , vacuolation of human hepatoma cells , necrosis , and an increase in the magnitude of transplanted cells , which did not alleviate the intensity and speed of the immune rejection reaction .

AFP serum concentration change

Normal Kunming mice : AFP serum concentration remained high level in three days after birth , and recovered to adult level in two weeks after birth , trace amount .

The specific re - expression of AFP in the rat liver was induced by transplantation of human liver cancer cells , which maintained a high level of serum concentration on 5 - 9 days after transplantation , which was significantly different from normal mice .

The transplantation of human normal cells with human lung cancer cells and transplantation of rat liver did not result in significant changes in serum levels .

The Effect of the Third Part of Immunomodulator

CsA group : graft occupation was large , irregular shape , invasive growth of transplanted cells , weak lymphocyte infiltration appeared in the transplantation area on 14th day of experiment , the development trend was slow , and no obvious necrosis appeared in the graft after transplantation .

PolyI : C group : graft occupation was significantly smaller than that in normal transplantation group , and obvious lymphocyte infiltration appeared on the second day after transplantation , and large - area necrosis appeared on the fifth day after transplantation .

NK cell killing activity

The NK cell killing activity of the transplanted group was significantly higher than that in the transplantation group and the total level was lower than that in the transplantation group . The cytotoxicity of NK cells in the group of PolyI : C was kept at a high level , and the difference was significant with the other two groups .

T lymphocyte transformation activity

The T cell transformation activity of CsA group and PolyI : C group increased slowly without significant change . PolyI : C was the highest in group C and lowest in CsA group .

distribution of T lymphocyte subsets

The percentage of CD4 + / CD8 ~ + in the CD4 ~ + and CD8 ~ + T cells was significantly higher than that in other groups . The percentage of CD4 ~ + and CD8 ~ + T cells in CsA group was significantly higher than that in other groups . The ratio of CD4 ~ + / CD8 ~ + in group C was higher than that in other groups .

Expression characteristics of cytokines IL - 2 and IL - 10

The concentration of IL - 2 decreased to the lowest at the third day after transplantation . The concentration of IL - 10 in CsA group decreased to the lowest point . The concentration of IL - 10 in CsA group decreased to the lowest point . The concentration of IL - 10 in CsA group was higher than that in other groups .

Conclusions of the study

1 . The expression characteristics of serum AFP were analyzed quantitatively for the first time in Kunming mice , which was similar to BALB / c / J , BALB / c / BOM and C3H / He .

2 . The high level expression of AFP in the rat liver can guarantee the survival of the xenotransplantation and delay the occurrence of the immune rejection of xenotransplantation induced by T cells .

3 . Aiming at the dynamic study of cellular immune rejection in xenogeneic hepatocyte transplantation , the human hepatocyte - mouse xenograft model established in this experiment is a good platform for research .

4 . In the process of immune rejection of xenografted cells , NK cell activity induced early in T cells , but there was no significant effect on the survival of the graft .

5 . CD4 + lymphocytes are the main T - cell subsets in the immune rejection reaction , and the imbalance of CD4 + / CD8 + T - lymphocyte ratio even reverses the occurrence of individual immune rejection .

6 . The activated CD4 ~ + T0 lymphocytes underwent an immune shift to the T1 - induced differentiation , and the high - level expression of TH1 - type cytokines ( IL - 2 ) mediated the effector phase of cell immune rejection , which could be used as an evaluation index for the immune rejection reaction induced by T cells .

7 . The use of immune inhibitor CSA could not completely inhibit the occurrence of cellular immune rejection in xenogeneic liver transplantation , and only played a delayed role .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392.4

【参考文献】