本文選題：肝細(xì)胞 + 甲胎蛋白��； 參考：《山東大學(xué)》2009年博士論文

【摘要】： 背景和目的 近年來通過異種肝細(xì)胞移植的手段,解決同種供體短缺的問題已成為全球研究的熱點(diǎn)。雖然異種肝細(xì)胞移植在短期內(nèi)改善肝臟先天性酶缺陷和血液系統(tǒng)病變及急、慢性肝衰竭等方面的有效性已在動物實(shí)驗(yàn)?zāi)Ｐ椭械玫阶C實(shí),但其后發(fā)生的免疫排斥問題仍然成為阻礙異種肝細(xì)胞移植在臨床中得以廣泛應(yīng)用研究的最大難題。 目前,有關(guān)異種肝細(xì)胞移植的免疫排斥機(jī)制的研究主要存在以下幾個問題:一、異種移植物會面臨來自受體更激烈,更迅速的免疫排斥,在幾小時內(nèi)就可能完全喪失功能或壞死,因而有關(guān)異種細(xì)胞移植中免疫排斥的動態(tài)研究不能很好地開展。二、相對于同種肝細(xì)胞移植,T淋巴細(xì)胞介導(dǎo)免疫排斥在異種肝細(xì)胞移植過程中的作用尚存在爭議,有關(guān)異種肝細(xì)胞原位移植的免疫排斥機(jī)制鮮有報道。三、異種移植中的免疫排斥反應(yīng)常因移植物種類的不同,所帶抗原數(shù)量、強(qiáng)度以及受者的免疫功能狀態(tài)的差異,特別是具體到某一器官如肝臟,其免疫排斥反應(yīng)將各具其特殊性。 針對上述問題,我們根據(jù)發(fā)育免疫學(xué)原理,對課題設(shè)計進(jìn)行了可行性分析:首先,肝臟在移植領(lǐng)域被成為“免疫特惠器官”,長期臨床研究發(fā)現(xiàn)肝臟移植后急性排斥反應(yīng)的發(fā)生率及嚴(yán)重程度遠(yuǎn)遠(yuǎn)低于其它器官,具有獨(dú)特的耐受原性;同時,肝臟是甲胎蛋白的主要合成及分泌器官,甲胎蛋白是一種胚胎性腫瘤相關(guān)蛋白,免疫應(yīng)答中的AFP主要表現(xiàn)為免疫抑制的生物學(xué)特征,而小鼠出生后一段時間內(nèi)AFP在肝臟仍保持著較高水平的表達(dá);因此,我們認(rèn)為該發(fā)育階段的肝臟免疫微環(huán)境具有適合異種移植的特殊性。另外,針對“外來細(xì)胞抗原所致免疫耐受”的現(xiàn)象,研究認(rèn)為免疫系統(tǒng)接觸外來抗原所產(chǎn)生的免疫耐受程度與免疫系統(tǒng)發(fā)育未成熟有關(guān)。如果我們在受體免疫系統(tǒng)發(fā)育的早期給予異種抗原的移植,將會減緩受體的異種免疫排斥,或者直接形成耐受。綜上所述,我們選用新生小鼠肝臟作為移植受體,設(shè)想該發(fā)育階段的肝臟微環(huán)境會緩解或者推遲機(jī)體的免疫排斥的強(qiáng)度或速度,將有利于免疫排斥發(fā)生的動態(tài)過程及其相關(guān)機(jī)制的研究。同時,鑒于主要組織相容性抗原為移植排斥反應(yīng)發(fā)生的主要移植抗原,它由種系基因構(gòu)成,代表了某個物種在進(jìn)化上的特異性。肝癌細(xì)胞不僅具有與人類正常肝細(xì)胞相同的移植抗原,同時作為正常肝細(xì)胞的替代物在肝臟相關(guān)疾病的實(shí)驗(yàn)性治療中被證明有良好的效果。因此,本實(shí)驗(yàn)選用3株人肝癌細(xì)胞,1株人正常肝細(xì)胞及1株人肺癌細(xì)胞作為待選異種移植物,通過對比篩選合適的受一供體組合,力圖建立一種理想的人肝細(xì)胞一乳鼠異種原位移植模型,并系統(tǒng)地對移植過程中細(xì)胞免疫排斥的時程變化及其相關(guān)機(jī)制進(jìn)行研究。 研究方法 1、人肝細(xì)胞—乳鼠原位移植模型的探索性構(gòu)建 1)根據(jù)動物模型構(gòu)建方法,體外肝部直接注射進(jìn)行五種人相關(guān)細(xì)胞的原位移植; 2)活體熒光染料DiI標(biāo)記技術(shù)對移植的人細(xì)胞進(jìn)行受體內(nèi)的定位示蹤; 3)解剖觀察移植細(xì)胞在乳鼠肝內(nèi)的移植成功率及生長狀況; 4)常規(guī)病理技術(shù)分析各組人細(xì)胞在乳鼠肝內(nèi)的免疫病理發(fā)展過程; 5)免疫組化檢測移植物在受體內(nèi)AFP及Suvivin蛋白的表達(dá)狀況。 2、乳鼠血清AFP表達(dá)特征與肝細(xì)胞移植微環(huán)境的關(guān)系 1)等數(shù)量級的人肝癌細(xì)胞HepG2分別進(jìn)行乳鼠皮下及肝內(nèi)原位移植,成鼠原位移植不同數(shù)量級的HepG2細(xì)胞; 2)活體熒光染料DiI標(biāo)記技術(shù)對移植細(xì)胞進(jìn)行受體內(nèi)定位示蹤; 3)常規(guī)病理切片對比分析移植細(xì)胞在各移植部位免疫病理學(xué)過程; 4)定量ELISA檢測移植前后乳鼠血清AFP的動態(tài)變化特征。 3、人肝細(xì)胞—乳鼠原位移植中細(xì)胞免疫排斥機(jī)制的研究 1)利用免疫調(diào)節(jié)劑(CsA,PolyI:C)分別設(shè)置免疫激活及免疫抑制兩個參照狀態(tài); 2)不同免疫狀態(tài)下人肝細(xì)胞—乳鼠原位移植模型的構(gòu)建; 3)不同免疫狀態(tài)下移植細(xì)胞免疫病理過程的變化; 4)NK細(xì)胞殺傷活性的檢測; 5)刀豆蛋白誘導(dǎo)的T淋巴細(xì)胞活性的檢測; 6)流式分析T淋巴細(xì)胞亞群分布的變化; 7)定量ELISA檢測小鼠IL-2,IL-10血清水平的變化特征。 研究結(jié)果 第一部分解剖觀察移植情況 肝癌細(xì)胞移植組均具有較高的移植成功率,個案伴有一定程度的肝內(nèi)轉(zhuǎn)移。人肺癌細(xì)胞移植成功率較低,并伴有消退趨勢。人正常肝細(xì)胞移植組僅2個案例肝臟觀察到異常部位。 活體熒光染料Dil的定位分析 熒光定位分析確定解剖觀察到的結(jié)節(jié)組織為移植或轉(zhuǎn)移的人細(xì)胞,各組肝癌細(xì)胞在肝內(nèi)占位明顯,熒光信號均勻穩(wěn)定;人肺癌細(xì)胞占位較小;人正常肝細(xì)胞組解剖觀察到的異常部位為熒光染料非特異性著色,無細(xì)胞性結(jié)構(gòu)。 常規(guī)病理切片分析 原位移植初期(0-5天),人肝癌細(xì)胞保持著良好的腫瘤形態(tài)特征,8天前后移植區(qū)域逐漸出現(xiàn)淋巴細(xì)胞的浸潤,移植物的相繼壞死,發(fā)生肝內(nèi)轉(zhuǎn)移的人肝癌細(xì)胞至實(shí)驗(yàn)結(jié)束未出現(xiàn)淋巴細(xì)胞的浸潤及壞死。原位移植的人肺癌細(xì)胞于移植后5天出現(xiàn)明顯的淋巴細(xì)胞浸潤及嚴(yán)重壞死。 免疫組織化學(xué)檢測 原位移植及轉(zhuǎn)移的各組人癌細(xì)胞均保持著原有的蛋白表達(dá)特征:Bel7402與HepG2:AFP~+;SK-Hep-1與A549:AFP~-;Survivin全部陽性表達(dá)。 第二部分人肝癌細(xì)胞HepG2的乳鼠皮下移植 皮下移植物于移植第2天出現(xiàn)淋巴細(xì)胞的浸潤及后續(xù)的嚴(yán)重壞死,并于移植后2周完全消退。 人肝癌細(xì)胞HepG2的成鼠肝內(nèi)移植 僅有10%的個案形成類似乳鼠原位移植的移植物結(jié)節(jié),移植物于移植后24小時出現(xiàn)明顯的淋巴細(xì)胞浸潤,或被炎癥細(xì)胞包裹,人肝癌細(xì)胞空泡化繼而壞死,移植細(xì)胞數(shù)量級的增大,并未緩解免疫排斥反應(yīng)出現(xiàn)的強(qiáng)度與速度。 AFP血清濃度變化 正常昆明種小鼠:AFP血清濃度出生后三天內(nèi)保持較高水平的表達(dá),并于出生后兩周恢復(fù)至成體水平,痕量。 人肝癌細(xì)胞的移植引起乳鼠肝臟AFP的特異性再表達(dá),于移植后的5-9天保持高水平的血清濃度,與正常小鼠有顯著差異。 人正常細(xì)胞與人肺癌細(xì)胞的移植,及成鼠肝內(nèi)的移植未引起血清水平的顯著變化。乳鼠皮下移植未引起AFP血清濃度的變化。 第三部分免疫調(diào)節(jié)劑的影響 CsA組:移植物占位偏大,形態(tài)不規(guī)則,移植細(xì)胞呈侵襲性生長;于實(shí)驗(yàn)第14天移植區(qū)域出現(xiàn)微弱的淋巴細(xì)胞浸潤,發(fā)展趨勢緩慢,移植后35d內(nèi)移植物未出現(xiàn)明顯壞死。 PolyI:C組:移植物占位明顯小于正常移植組,移植后第2天就出現(xiàn)明顯的淋巴細(xì)胞浸潤,并于移植后第5天出現(xiàn)大面積壞死。 NK細(xì)胞殺傷活性 移植組:移植后第4天NK細(xì)胞殺傷活性明顯升高,CsA組:NK細(xì)胞殺傷于移植后呈緩慢上升趨勢,總體水平低于移植組,PolyI:C組NK細(xì)胞殺傷活性一直保持較高水平,與其它兩組差異顯著。 T淋巴細(xì)胞的轉(zhuǎn)化活性 移植組:移植第7天T淋巴細(xì)胞轉(zhuǎn)化活性由緩慢上升轉(zhuǎn)為明顯升高,具有顯著性;CsA組與PolyI:C組的T細(xì)胞轉(zhuǎn)化活性皆緩慢上升,無大幅度變化,PolyI:C組最高,CsA組最低。 T淋巴細(xì)胞亞群的分布 出生后小鼠T細(xì)胞分化尚未完善,CD4~+,CD8~+T細(xì)胞數(shù)量隨時間不斷增加。移植組CD4~+與CD8~+T細(xì)胞在各時間點(diǎn)皆高于正常小鼠,CD4~+T細(xì)胞數(shù)量增長較快,于移植后11天CD4~+/CD8~+比值達(dá)到最高值,顯著高于其它組。CsA組CD4~+T細(xì)胞百分含量位于各組最低,CD4~+/CD8~+比值出現(xiàn)紊亂直至逆轉(zhuǎn)。PolyI:C組CD4~+與CD8~+T細(xì)胞百分率增長速率明顯高于其它組,CD4~+/CD8~+比值略高于正常小鼠。 細(xì)胞因子IL-2,IL-10的表達(dá)特征 移植組小鼠于移植后第3天IL-2血清濃度降至最低,IL-10達(dá)到最高濃度,隨后逆轉(zhuǎn),第7天IL-2濃度誘生至最高點(diǎn),IL-10濃度降至最低點(diǎn);CsA組IL-2受到明顯的抑制,IL-10濃度較其它組最高;PolyI:C組IL-10濃度低于移植組,IL-2濃度顯著高于其它實(shí)驗(yàn)組。 研究結(jié)論 1、首次對昆明種小鼠出生后血清甲胎蛋白的表達(dá)特征進(jìn)行了定量分析,與BALB/c/J,BALB/c/BOM and C3H/He等種系小鼠具有相似的表達(dá)模式。 2、乳鼠肝臟內(nèi)AFP高水平的表達(dá)可保證異種移植物的存活,推遲T細(xì)胞誘導(dǎo)的異種移植免疫排斥反應(yīng)的發(fā)生。 3、針對異種肝細(xì)胞移植中細(xì)胞免疫排斥反應(yīng)的動態(tài)研究,本實(shí)驗(yàn)構(gòu)建的人肝細(xì)胞—乳鼠異種原位移植模型是一種良好的研究平臺。 4、異種移植細(xì)胞免疫排斥過程中,NK細(xì)胞活性的誘發(fā)早于T細(xì)胞,但是對移植物的存活未形成明顯的影響。 5、CD4+淋巴細(xì)胞是介導(dǎo)免疫排斥反應(yīng)中的主要T細(xì)胞亞群,CD4+/CD8+T淋巴細(xì)胞比率的失調(diào)甚至逆轉(zhuǎn)導(dǎo)致個體免疫排斥的發(fā)生。 6、激活的CD4~+T0淋巴細(xì)胞經(jīng)歷了向T1誘導(dǎo)分化的免疫偏移,高水平表達(dá)的TH1類細(xì)胞因子(IL-2)介導(dǎo)了細(xì)胞免疫排斥的效應(yīng)期,可以作為T細(xì)胞引發(fā)的免疫排斥反應(yīng)的評價指標(biāo)。 7、單純使用免疫抑制劑CSA并未能完全抑制異種肝細(xì)胞移植中細(xì)胞免疫排斥的發(fā)生,只起到了延緩的作用。
[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 .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392.4

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