【作者】 齐紫平；

【导师】 田志刚；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2012， 博士

【摘要】 某些重大疾病的研究需要在体内探究免疫系统对病原体感染、转化以及破坏过的局部组织产生免疫应答的过程,而直接对疾病个体进行深入研究却存在一定风险,MHC分子限制性的嵌合体小鼠将是一个最实用的模型。目前认为造血干细胞能在免疫缺陷或是致死照射的小鼠体内发育分化成免疫系统,常见的受体鼠有NOD-scid Il2r γ-/-(NSG)小鼠及BALB/c背景的Rag2-/-Il2r γ-/-小鼠。同样,外源肝细胞或是胚胎干细胞也可以在某些特殊的受体小鼠体内扩增成为肝脏嵌合小鼠,比如尿激酶型血纤维蛋白溶酶原激活剂转基因小鼠模型(uPA模型)及延胡索酰乙酰乙酸脱氢酶缺陷小鼠模型(FAH模型)。然而,上述两种模型都不是研究免疫系统与病原体感染组织相互作用的最佳模型。人体生物学的研究受限于伦理因素及技术难度,人源化小鼠的发展却为其提供了便利。免疫系统人源化(HIS)小鼠在研究人类免疫反应及嗜人免疫系统病原菌感染方面表现出极大的应用前景；而肝脏人源化小鼠也在人类嗜肝病原菌感染研究和抗肝炎病毒药物的临床评判方面发挥了重要的作用。但是,进一步研究某些特殊疾病(如HBV,HCV感染引发的肝炎以及疟疾的肝脏阶段)的病理发生,免疫关系,以及发病机制需要一种更好的小鼠模型,即免疫系统和病原体靶向组织同时实现人源化,并能通过MHC分子相互识别。最近,研究者们建立了AFC8-hu HSC/Hep小鼠模型,他们从15-18周的胎肝组织中分离得到肝脏祖细胞,并跟CD34+人造血干细胞一起转输给BALB/c背景的Rag2-/-Il2r γ-/-小鼠,从而成功建立了免疫系统和肝脏同时人源化的小鼠模型。尽管该模型能较好满足研究的需要,但是胎肝组织的获取难度限制了该模型的广泛应用。近年来,也有报道称通过骨髓转输的方法,造血干细胞能在体内转化成肝细胞。在本论文中,我们用延胡索酰乙酰乙酸脱氢酶缺陷的小鼠进行骨髓重建,以探求造血干细胞能否在受体小鼠体内同时发育成免疫系统和肝脏细胞。该模型的成功建立将极大地促进疾病状态下免疫系统与肝细胞的相互作用的研究。本研究中,我们首先通过体重监测,血清ALT检测,以及肝损伤的观测等手段观察了FAH缺陷鼠在NTBC停药后的生长情况；接着,我们将小鼠肝细胞或人源肝细胞系转入FAH缺陷鼠体内,观察外源肝细胞能否成功植入受体鼠肝脏并实现扩增；然后,我们尝试运用同基因,异基因,甚至异种骨髓移植的方法在FAH缺陷鼠体内实现免疫系统及肝脏的双重建。在转输骨髓后,给受体鼠停止NTBC用药,并观察其体重及血清ALT变化。为评判免疫系统的重建,我们每两周检测一次外周血。重建8周后,我们分离检测嵌合鼠的骨髓,胸腺,外周血,肝脏以及淋巴结中供体淋巴细胞分布情况,并用组织学染色的方法分析小鼠脾脏和淋巴结中的免疫重建状况。与此同时,我们给嵌合鼠免疫HBV疫苗或是OVA蛋白,然后用RIA或ELISA的方法检测小鼠血清中特异抗体的产生情况。为评价肝细胞的重建,我们分离停药后长期存活鼠的肝细胞并用流式分析表型。同时,我们也用H&E染色及免疫组化来分析肝组织切片。通过上述实验,我们取得了以下一些主要结果：1.FAH小鼠在NTBC停药后自发肝损伤首先,我们对比了FAH缺陷鼠在NTBC停药与不停药时的生长情况。我们发现,停药后缺陷鼠的体重逐渐下降,当体重损失达到30%时,小鼠就会死亡。血清ALT也在逐周增加提示肝损伤在逐渐加重。分离停药鼠的肝细胞体外培养,发现停药后肝细胞更易凋亡。肝脏组织病理学分析发现淋巴细胞浸润到肝脏局部,进一步研究发现,T,B淋巴细胞都可能参与了停药后小鼠肝损伤的过程。总之,上述结果表明,NTBC能够保护FAH缺陷鼠免于肝损伤,NTBC的撤除可作为启动FAH缺陷鼠肝细胞坏死的开关。2.外源肝细胞向FAH移植后的体内扩增接着,我们将新鲜分离的EGFP-Tg鼠的肝细胞转入FAH缺陷鼠体内,然后给小鼠停药。与不转输的停药对照组相比,转输组小鼠的体重及血清ALT水平能较快的恢复到正常水平,并且转输组小鼠的存活时间大大延长。同时,我们在转输鼠的肝脏内检测到EGFP阳性的供体肝细胞,它们能提供FAH来恢复受体鼠的肝脏功能。但是,当我们用人源肝细胞系L02细胞进行转输时,受体小鼠会发生肝脏肿瘤而死亡。我们在小鼠的肝脏中观察到许多肿瘤结节,可能是L02细胞系在体内扩增太快导致的。3.同基因骨髓移植后FAH小鼠体内免疫系统及肝脏的双重建接下来,我们将同基因型的骨髓细胞转入到FAH小鼠体内观察重建情况。我们选取了两种供体小鼠,分别是EGFP-Tg小鼠和HBs-Tg小鼠。我们在转输28天后给小鼠停药,结果发现,绝大部分骨髓转输小鼠在停药后能存活至少5个月以上。骨髓转输小鼠表现出稳定的造血重建能力,髓系及淋巴系的造血发育都和正常供体鼠一样。嵌合鼠外周血中NK,B,CD4T,CD8T细胞的数量也能逐渐发育正常,8周后受体鼠各脏器中免疫细胞就能重建完全,脾脏及淋巴结的组织学染色也进一步证明免疫重建的成功。为检测重建免疫系统的功能,我们用HBV疫苗免疫小鼠2次,结果发现,所有嵌合鼠都能产生跟供体鼠相类似的HBV特异性的抗体。我们用停药后长期存活的嵌合小鼠来评判肝细胞重建状况。肝组织染色结果发现供体来源的肝细胞成簇存在,进一步用流式分析肝细胞的表型发现髓样单个核细胞(CD45+F4/80+Gr-1+CD11b+CD11c-)可能是体内骨髓来源肝细胞的前体。综上结果可知,同基因型小鼠来源的造血干细胞是能够在FAH缺陷鼠体内同时重建免疫系统及肝脏系统的。4.异基因骨髓移植后FAH小鼠体内免疫系统及肝脏的双重建确定同基因型的骨髓能在FAH缺陷鼠体内成功实现免疫系统及肝细胞的双重建之后,我们又进行了异基因型的骨髓移植。我们同样选取了两种供体小鼠,分别是C3H/HeJ小鼠(H-2k)and HBV-Tg小鼠(H-2d)。大约60%的异基因骨髓移植小鼠能在停药后存活5个月以上。通过检测嵌合鼠外周血单个核细胞的比例,我们发现NK,B,CD4T,CD8T淋巴细胞能逐渐发育正常,脾脏及淋巴结的组织学染色也进一步证明免疫系统重建成功。跟供体鼠相似,嵌合鼠在OVA蛋白免疫后能产生OVA特异性的抗体。同时,从嵌合鼠中分离出的肝细胞部分表达供体鼠MHC-I类分子,并且这种肝细胞也表达受体鼠的MHC-I分子及CD45抗原,提示骨髓来源的肝细胞可能与受体原有的肝细胞发生了细胞融合。5.异种骨髓移植后FAH小鼠体内免疫系统及肝脏的双重建接下来,我们想探明异种骨髓细胞能否在FAH缺陷鼠体内实现免疫系统及肝脏的双重建。我们将S.D.大鼠(RT1A,Fah+/+)的骨髓细胞转入FAH缺陷鼠体内,结果发现约有50%的嵌合鼠停药后能存活5个月以上,血清转氨酶也维持在正常水平。嵌合小鼠表现出稳定的造血重建能力,髓系及淋巴系的造血发育也很正常,外周血中供体来源的单个核细胞(RT1A+)比例接近100%。嵌合鼠外周血中NK,B,CD4T,CD8T淋巴细胞逐渐发育正常,8周后受体鼠各脏器中T细胞比例接近于供体大鼠。与此同时,从嵌合鼠中分离出的肝细胞部分表达大鼠RT1A分子,肝脏组织学分析也显示FAH阳性肝细胞成团分布。总之,异种来源的造血干细胞同样能在FAH缺陷鼠体内完成双重建。6.人源造血干细胞转输后FAH-rag2双缺陷小鼠体内免疫系统及肝脏的双重建基于之前同基因,异基因,异种骨髓转输所取得的结果,我们将纯化的人脐血干细胞转入FAH-rag2双缺陷小鼠体内。结果发现,停药后转输鼠体重先是由于亚致死照射下降10%左右,然后迅速恢复正常并稳步上升。血清ALT也一直维持在较低水平,暗示着人源造血干细胞的移植部分恢复了嵌合鼠体内的肝脏功能。同时我们注意到,与非转输对照鼠相比,大多数嵌合鼠的存活时间更长。另外,嵌合鼠的肝脏功能恢复与人造血干细胞的植入程度有关,比较不同的受体小鼠,我们发现FRG受体鼠在接受转输后存活时间长于FAH单缺陷鼠。我们也确实在FRG受体鼠体内检测到更多的人源细胞。肝脏H&E染色能检测到人源肝细胞,它的细胞更大,嗜酸性染色较浅。由于B6背景的小鼠HSC植入较少,我们在肝组织中只检测到少量FAH阳性的人源肝细胞。总之,人源HSC转输后,受体鼠肝脏内确实有部分肝细胞被人源肝细胞取代,它们产生FAH来减轻肝损伤。小结：本研究主要发现来自一个个体的造血干细胞能在免疫缺陷或是照射过的FAH缺陷鼠体内成功发育分化成免疫细胞和肝细胞。我们第一次比较系统地逐步通过同基因,异基因,异种骨髓移植,证明了单种造血干细胞可以在小鼠体内实现免疫系统及肝细胞的双重建。同时,我们也发现给FAH-rag2双缺陷小鼠转输人源造血干细胞能部分恢复受体小鼠的肝脏功能。上述结果提示,将来运用更加适合人源化构建的免疫缺陷型FAH小鼠,如Fah-/-NSG小鼠,有可能构建出一种更理想的人源化小鼠,这种小鼠具备免疫系统及肝细胞的双重人源化,并具备HLA分子识别的一致性。尽管我们的模型还有许多有待改进的地方,但我们相信这种双重建小鼠模型将给临床前检测及免疫系统与病变器官的相互作用研究提供更好的机会!更多还原

【Abstract】 A MHC-restricted chimeras mouse will be a most practical tool for studying donor’s immune response against donor’s non-immune cells from organ with pathogen infection, transformation and damage in recipient mice, without putting individuals at risk. It was extensively accepted hematopoietic stem cells (HSC) may engraft and develop into immune system in lethal irradiated or immunodeficient mice, such as NOD-scid112r γ-/-(NSG) recipients or BALB/c-Rag2-/-Il2r γ-/-recipients. Liver chimeric mice were also reproted to be developed by exogenous hepatocytes or embryonic stem cells transplantation, like the uroplasminogen-activator (uPA) transgenic model or fumarylacetoacetate hydrolase (FAH) deficient model. However, neither of the chimeric mice are suitable for further investigation of the interaction between the immune system and pathogen infected organs.Development of humanized mice provides insights into in vivo human biology that would be severely limited by ethical and/or technical constraints. Human immune system (HIS) mice are already established, showing a potential as the available model for the study of human immune response and human lymphotropic pathogens in mice, and human liver chimeric mice were developed for study of human hepatotropic pathogens or preclinical evaluation of anti-hepatitis virus drug candidates. However, further investigation of the pathology, immune correlates, and mechanisms of highly specialized pathogens like HBV, HCV and malaria (at liver stage) needs an excellent mouse model engrafted with MHC-restricted human immune system and pathogen-targeting organs. Recently, AFC8-hu HSC/Hep mice model was developed by meeting this requirement through co-implantation of human CD34+HSCs and hepatocyte progenitor cells from a15-18weeks old fetal liver tissue into BALB/c-Rag2-/-Il2r γ-/-mice. Although this approach successfully provides immune system and liver cells together in recipients, its extensive utilization is limited by obtaining human fetal liver tissues.It was recently reported that HSC may also differentiate into hepatocytes in bone marrow transplanted (BMT) mice. Here, using a strain of mice deficient in tyrosine catabolic enzyme fumarylacetoacetate hydrolase (fah-/-), we tried to see whether donor’s HSC may concurrently differentiate into immune cells and hepatocytes in recipient, which will greatly benefit exploiting the donor’s MHC-restricted interaction between immune cells and hepatocytes.In this study, we firstly assessed the bodyweight, serum ALT and liver failure in fah-/-mice after NTBC withdrawal. Then, we transplanted mouse or human hepatocytes into fah-/-mice or FRG mice to see whether exogenous hepatocytes could repopulate in the recipient liver. After that, we tried to established chimeric mice model with a dual immunologic and hepatic reconstitution by syngeneic, allogeneic or even xenogeneic bone marrow transplantation. Bodyweight and serum ALT was measured in BMT animals after NTBC withdrawal. To confirm immunologic reconstitution in recipients, PBMC subset were monitored every two weeks in BMT mice, and the mononuclear cells from bone marrow, thymus, spleen, blood, liver and lymph node were detected using flow cytometry after8weeks rebuild time. The histopathology of spleen and lymph node was aslo assessed by H&E staining and immunohistochemistry (IHC). Further more, BMT mice were immunized by HBV vaccine or OVA protain, and antigen-specific antibody was examined by RIA or ELISA. To evaluate the hepatic reconstitution, hepatocytes from long-term survivors were isolated and analyzed using flow cytometry. Liver histology of sections of BMT mice was aslo detected by H&E staining and IHC. The major results of our studies are shown as follows:1. Fah-/-mice suffer from progressive liver failure after NTBC withdrawal.Firstly, we compared the life status of fah-/-mice with or without NTBC. We found that the body weight of fah-/-mice reduced gradually and finally died when the loss of bodyweight exceed30%when NTBC was off. Serun ALT increased week by week indicated progressive liver failure in these mice. Hepatocytes from fah-/-mice without NTBC feeding were more prone to apoptosis. Liver histology of these mice also showed lymphocytes inflitration, and further analysis shown that T, B lymphocytes may play a important role in the progressive liver failure. Together, these results implied that NTBC protect fah-/-mice from death of liver injury, and NTBC cut off could be a switch to initiate hepatocytes necrosis in fah-/-mice.2. Liver repopulation with exogenous hepatocytes in fah-/-mice.Next, we transplanted fresh isolated hepatocytes (EGFP-positive) into fah-/- mice, then we stopped NTBC feeding. In contrast to congtrol fah-/-mice, the body weight and serum ALT from recipient mice with hepatocytes transplantation could soon recovered to normal. Transplanted mice survived far more longer than control mice after NTBC withdrawal. We also detected EGFP-positive hepatocytes in these mice, and such hepatocytes could provide FAH to to restore liver function in recipient. However, when we changed the donor cells to human hepatocytes cell line (LO2), the recipient mice died of hepatoma. The uncontrolled expansion of LO2cells in the liver maybe cause the death, because we found many tumor nodules in the liver from LO2transplanted mice.3. A dual immunologic and hepatic reconstitution in recipient fah-/-mice after syn-BMTFurther, we transplanted syngeneic bone marrow cells into fah-/-/129SvvJ (haplotype H-2b) recipients. We choose two different mice as donor, EGFP-Tg mice and HBs-Tg mice. Most BMT mice survived at least5months after NTBC withdrawal beginning at day28. Surviving recipients also show stable mutilineage hematopoietic reconstitution after syn-BMT. And there was little difference in myeloid and lymphoid development between chimeras and donor mice. NK, B, CD4and CD8T cells could reconstitute normally in chimeras’ peripheral blood, and there was a similar ratio of T cells in all immune organs of recipient mice to that of donor mice. Spleen and inguinal lymph node histology of serial sections further confirmed successful immunologic reconstitution in BMT mice. To directly test the immune response after reconstitution, mice were immunized2times with HBV vaccine. All recipient mice produced specific anti-HBsAg antibodies in serum, similar to the donor mice. To evaluate the hepatic reconstitution, long-term survivors were sacrificed and analyzed. Liver histology showed that donor-derived hepatocytes (FAH-positive) were organized in a cell cluster. Further study demonstrated that myelomonocytic cells (CD45+F4/80+Gr-1+CD11b+CD11c-) were the progenitors for the bone marrow-derived hepatocytes. Together, these data imply that HSC from a syngeneic mouse may concurrently reconstitute immunologic and hepatic system in recipient fah-/-mice.4. A dual immunologic and hepatic reconstitution in recipient fah-/-mice after allo-BMT. After successful liver and immune reconstitution after syn-BMT in fah-/-mice, we carried out allo-BMT. We also choose two allogeneic mice as donor, C3H/HeJ mice (haplotype H-2k) and HBV-Tg mice (haplotype H-2d). About60%of BMT mice survived at least5months after NTBC withdrawal. We monitored PBMC subset from BMT mice, and found contents of NK, B, CD4and CD8T cells became normal in chimeras. Meanwhile, spleen and inguinal lymph node histology of serial sections further confirmed successful immunologic reconstitution in BMT mice. BMT mice produced specific anti-OVA antibodies in serum, similar to donor mice after immunization with OVA protein. Meanwhile, hepatocytes from BMT mice partially expressed donor MHC class Ⅰ antigen, and such hepatocytes were partially positive for recipient MHC class Ⅰ antigen and CD45antigen, implying cellular fusion between donor BMC and resident hepatocytes.5. A dual immunologic and hepatic reconstitution in recipient fah-/-mice after xeno-BMT.We next attempted to define whether xenogeneic HSC could successfully reconstitute immunologic and hepatic system in fah-/-mice. We transplanted S.D. rat (RT1A,fah+/+) bone marrow into fah-/-/129SvJ (haplotype H-2b) recipients. About50%of BMT mice survived at least5months after NTBC withdrawal, and serum ALT almost kept in a normal level. Surviving recipients also show stable mutilineage hematopoietic reconstitution after Xeno-BMT. Nearly100%of the PBMCs from the chimeras were RT1A+similar to donor cells, and there was little difference in contents of myeloid and lymphoid cells between chimeras and recipient mice. NK, CD4and CD8T cells could reconstitute normally in chimeras’peripheral blood, and there was a similar ratio of T cells in all immune organs of recipient mice to that of donor rat. Meanwhile, hepatocytes from Rat-BMT mice were partially positive for donor MHC class I antigen (RT1A+), implying the generation of bone marrow derived hepotocytes. Liver histology also showed that FAH-positive cells were organized in a cell cluster. Altogether, HSC from xenogeneic donor could indeed reconstitute immunologic and hepatic system in fah-/-mice.6. Immunologic and hepatic reconstitution in recipient fah-/-rag2-/-mice after human HSC transplantation.Based on the inspiring results from syn/allo/xeno-BMT, we then transplanted purified human hematopoietic stem cells (CD34+cord blood cells) into immunodeficient fah-/-/129SvJ.B6recipients (fah-/-rag2-/-). After NTBC withdrawal, the body weight of most chimeras was about10%loss at the beginning because of sub lethal irradiation, but soon they recovered and kept getting on body weight gradually. Serum ALT from these mice also kept in a normal level, implying that human HSC transplantation partially restored liver function in chimeras. Meanwhile, most chimeras survived longer than no cell transferred mice. Additionally, the liver function restoration was relative to the engraftment of human HSC. We compared different recipient mice, and found that chimeras in fah-/-rag2-/-112r γ-/-mice survived better than that in fah-/-mice, and we indeed detected the most human cells in these mice. H&E staining of the liver section clearly distinguished human hepatocyte clusters, which were larger and less eosinophilic and hence appear paler than mouse cells. Since the poor engraftment of human HSC in B6recipients (fah-/-rag2-/-), we could only detected few FAH-positive hepatocytes in liver tissue. Thus, after HSC transplantation, recipient mice could be partially repopulated with human origin hepatocytes, which provided FAH to alleviate live injury.Conclusion:The major finding of the current study was that HSCs from one individual were successfully differentiated into both immune cells and hepatocytes by using an irradiated or imunodeficient fah-/-mouse. We, for the first time, developed a mouse model with immune and liver reconstitution from one donor’s HSCs, which was carried out by systematically performing syngeneic, allogeneic or even xenogeneic BMT. We further showed that the purified human hematopoietic stem cells transplantation could partially restore liver function in fah-rag2double knockout mice. These finding indicated that a better humanized mouse model, with a HLA-identity between human immune cells and human hepatocytes, would be developed by using a more imunodeficient fah-/-mouse, such as fah-/-NSG in future. Although further improvements of our model need to be done, we expect that such model provide a new opportunity to perform pre-clinical testing and to investigate many human biological processes which are happened between donor immune cells and organs with MHC-identity.更多还原