HLA-G抑制人NK、T细胞介导的异种移植排斥反应的研究
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摘要
第一部分HLA-G1抑制人自然杀伤细胞杀伤猪内皮细胞的研究
【目的】研究HLA-G1基因抑制人NK细胞系NK92和外周血单个核细胞(hPBMC)对猪血管内皮细胞(PEC)杀伤的作用。
【方法】利用脂质体介导的基因转染技术将pcDNA3-HLA-G1质粒转入原代培养的PEC，用流式细胞仪及间接免疫荧光显微镜在蛋白质水平上检测HLA-G1分子在PEC上的表达；以NK细胞系(NK92)和hPBMC为效应细胞，用四甲基偶氮唑盐(MTT)法检测转染有HLA-G1基因的PEC对NK92和hPBMC杀伤活性的抵御作用。
【结果】利用脂质体转染技术成功将pcDNA3-HLA-G1转入原代培养的PEC；NK92和hPBMC对转染有HLA-G1的PEC的杀伤效率(41.5％±14.0％；45.4％±12.1％)与对照组(75.3％±10.5％；74.6％±11.2％)相比，均有明显降低(P＜0.05)。
【结论】HLA-G1分子可以明显抑制NK92细胞以及hPBMC对PEC的杀伤作用。
第二部分建立表达可溶性HLA-G1真核细胞系并获取可溶性HLA-G1
【目的】为了研究可溶性HLA-G1(sHLA-G1)在抑制NK细胞以及T细胞介导的异种排斥反应中的作用，在本实验中建立表达sHLA-G1的真核细胞系，获取纯化的sHLA-G1蛋白。
【方法】利用核转染技术将pcDNA3-sHLA-G1质粒转入LCL721.221细胞；流式细胞仪和荧光显微镜对转染率进行判定；G418筛选阳性细胞；应用RT-PCR和Dot-ELISA方法分别在基因水平和蛋白水平对表达sHLA-G1的LCL721.221细胞进行鉴定；收集LCL721.221-sHLA-G1细胞培养液；将HB-95细胞注入Balb／c小鼠腹腔，产生含抗体的腹水后采用正辛酸-硫酸氨沉淀法纯化W6／32抗体，再用免疫亲和层析提纯sHLA-G1蛋白。
【结果】核转染技术可以高效将pcDNA3-sHLA-G1质粒转入LCL721.221细胞，转染率约为14％，RT-PCR和Dot-ELISA的结果显示转染和筛选均获得成功；G418筛选出稳定表达sHLA-G1的LCL721.221细胞，应用免疫亲和层析方法提纯LCL721.221-sHLA-G1培养液中sHLA-G1纯化蛋白约1.3mg。
【结论】核转染技术可以高效将pcDNA3-sHLA-G1转入LCL721.221细胞。免疫亲和层析方法成功提纯sHLA-G1纯化蛋白。
第三部分可溶性HLA-G1抑制NK92的生物学功能的研究
【目的】研究sHLA-G1对NK细胞的粘附功能、胞吐、杀伤活性以及释放细胞因子等功能的影响作用。
【方法】利用细胞粘附实验研究sHLA-G1对NK92细胞在静止状态和滚动状态对猪血管内皮细胞系SV-40-PED粘附功能的抑制作用；借助β-hexosaminidase释放实验间接反映NK92细胞杀伤SV-40-PED时所释放的穿孔素、颗粒酶水平以及sHLA-G1对此的抑制作用；利用ELISA方法检测sHLA-G1对NK92所释放的IFN-γ、TNF-α水平的抑制作用；利用MTT方法检测sHLA-G1抑制NK92对SV-40-PED的杀伤作用。
【结果】sHLA-G1无论是在静止状态下还是滚动状态下都能显著抑制NK92的粘附功能(P＜0.01)；β-hexosaminidase释放实验结果显示，NK细胞与靶细胞SV-40-PED相互作用2h，即有明显的胞吐效应，酶释放率达14.5％，随着时间延长，酶释放率增加，6h时达高峰64.5％，sHLA-G1组中NK92细胞的胞吐维持在较低水平，二者间有显著差异(P＜0.05)；ELISA实验结果显示对照组中NK92所释放的IFN-γ、TNF-α因子维持在一个较高的水平，sHLA-G1组中NK92所释放的IFN-γ、TNF-α因子较对照组有显著降低(P＜0.05)；MTT杀伤实验结果显示sHLA-G1组NK92对SV-40-PED的杀伤效率(25.5±2.1％)显著低于对照组(71.2±2.6％，P＜0.01)。
【结论】sHLA-G1可以显著抑制NK92细胞的生物学功能，从而抑制NK92介导的异种移植排斥反应。
第四部分人外周血单个核细胞移植给SCID小鼠异种GVHD模型的建立
【目的】建立一种能直接观测异种器官移植给人的异种移植所发生的各种免疫反应的动物模型。
【方法】阳性对照组：SCID小鼠经尾静脉注射5×10~7 C57BL／6小鼠脾细胞；人外周血单核细胞(hPBMC)组：SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)、尾静脉注射5×10~7hPBMC；NK92组：根据预处理因素分为3组，A组SCID小鼠在实验前1d腹腔注射30μl生理盐水，实验当天给予Co~(60)照射(3.5Gy)和尾静脉注射5×10~7NK92细胞；B组SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)和尾静脉注射5×10~7 NK92细胞；C组SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)和尾静脉注射5×10~7NK92细胞(NK92细胞悬液加入终浓度为200u／ml的rhlL-2)。观察各组SCID小鼠体重、体型、体位、毛发、腹泻和死亡时间；ELISA方法检测SCID小鼠(PBMC组和NK92组)IFN-γ、TNF-α的分泌水平；对死亡SCID小鼠行常规病理学检查。
【结果】SCID小鼠在输注5×10~7 C57BL／6脾细胞5d后出现弓背、消瘦、皮毛紊乱无光泽、体重减轻等GVHD症状，7只SCID小鼠在5～8d内死亡，肝脏出现大面积肝细胞坏死，肝脏失去正常的结构，肝窦中有大量的淋巴细胞浸润；hPBMC组SCID小鼠在2周后开始出现GVHD症状，肝脏出现大面积肝细胞坏死，肝窦中有大量的淋巴细胞浸润，分别在15～25d内死亡；NK92组中A、B、C组SCID小鼠均未出现GVHD；ELISA检测结果显示hPBMC组中在发生异种GVHD时TNF-α的分泌水平明显升高，IFN-γ的水平也有一定程度上的升高，而在NK92组，IFN-γ、TNF-α的分泌水平较低，并且显现出持续降低趋势。
【结论】成功建立人外周血单个核细胞移植给SCID小鼠异种GVHD模型，为研究异种移植排斥反应以及今后建立人移植物对猪的异种GVHD提供一定的启示。
第五部分可溶性HLA-G1抑制外周血单个核细胞移植给SCID小鼠的异种GVHD
【目的】研究sHLA-G1在体内环境中对T细胞活性的影响作用，并且抑制人外周血单个核细胞(hPBMC)移植给SCID小鼠发生的异种GVHD。
【方法】采用单向混合淋巴细胞培养实验检测sHLA-G1对T细胞增殖能力的抑制作用；SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)、尾静脉注射5×10~7 hPBMC细胞，其中实验组1、2中SCID小鼠分别经尾静脉注射2ng和4ng的sHLA-G1治疗(实验当天、3、6、9、12和15d)，对照组SCID小鼠注射等量的生理盐水；ELISA方法检测各组SCID小鼠IFN-γ、TNF-α的分泌水平；取死亡SCID小鼠行常规病理学检查。
【结果】对照组中hPBMC的增殖强度比实验组1和实验组2均有显著性增强(P＜0.05)；实验组1和实验组2中SCID小鼠存活时间比对照组有显著延长(P＜0.01)；对照组中SCID小鼠血清中的IFN-γ、TNF-α水平随着GVHD症状的出现，IFN-γ、TNF-α水平显著升高，实验组1，2中SCID小鼠血清中的IFN-γ、TNF-α水平一直维持在较低的水平；病理学检查对照组SCID小鼠肝脏出现大面积肝细胞坏死，肝脏失去正常的结构，肝窦中有大量的淋巴细胞浸润，而实验组中SCID小鼠肝脏病理学检查均为肝脏轻微可逆性病变，肝窦中仅有散在淋巴细胞浸润。
【结论】sHLA-G1可以在体内环境中抑制T细胞的生物学功能，并且可以明显抑制hPBMC移植给SCID小鼠发生的异种GVHD。
Part I A Study of HLA-G1 Protection of Porcine Endothelial Cells Against Human NK Cell Cytotoxicity
[Objective] To study the effect of protecting porcine aortic endothelial cells (PEC) transfected with HLA-G1 from human NK cell lysis.
[ Methods ] The recombinant expression vector pcDNA3.0-HLA-G1 was transfected into primary cultured PEC by lipofection. Surface expression of HLA-G1 in transected PEC was confirmed by an immunofluoresence technique. Human peripheral blood mononuclear cells (hPBMC) and NK cell line (NK92) were used as NK effect cells with pcDNA3-HLA-G1-transfected PEC as targets in a MTT method using pcDNA3 transfection as a negative control.
[Results] Expression of HLA-G1 on PEC conferred significant protection against NK-mediated lysis. The rate of NK92 cytotoxicity was reduced to 41.5% ± 14.0% from 75.3% ±10.5% in the control group (P<0.01). Similarly the rate of the hPBMC cytotoxicity among different donors (n =7) was reduced to 45.4% ± 12.1% in contrast to 74.6% ±11.2% in the control group (P<0.05).
[Conclusion] HLA-G1 molecules can directly protect xenogeneic PEC against attack by human NK cells. These results indicate that the expression of HLA-G1 on the porcine cell surface may provide a new approach to overcome NK-mediated immunity to xenografts. Part II Establishment of cell line stably expressing soluble HLA-G1 and purification of soluble HLA-G1 protein
[Objective] To establish the LCL721.221 cell line stably expressing sHLA-G1 and purify the sHLA-G1 protein.
[Methods] The recombinant plasmid pcDNA3.0-sHLA-G1 was transfected by a novel nonviral, electroporation-based gene transfer method termed nucleofection into the host cell lymphoblastoid cell line LCL721.221 which does not express any HLA-classical I molecules. After selection by G418, the cell line stably expressing sHLA-G1 is identified by RT-PCR and Dot-ELISA with HLA-G specific monoclonal antibody MEM-G/9. The W6/32 antibody (anti HLA-G monoclonal) was produced by injecting the hybridoma cell line HB-95 cell into Balb/c mice. Then the sHLA-G1 protein is purified by affinity chromatograpHy using the monoclonal antibody.
[Results] After analysis by RT-PCR and Dot-ELISA, it is confirmed that the eukaryotic cell line expressing sHLA-G1 has been established successfully. And about 1.3mg sHLA-G1 protein was purified by affinity chromatography using the monoclonal antibody W6/32.
[Conclusion] In this study, we have established the LCL721.221 cell line expressing sHLA-G1 and get the sHLA-G1 protein successfully.
Part III Soluble HLA-G1 inhibit the biological function of NK92
[Objective] To study the function of sHLA-G1 inhibition the biological function of NK92.
[Methods] The effect of sHLA-G1 on the interaction between SV-40-PED and NK92 cells was assessed in terms of adhesion and cytotoxicity. The inhibitive effect of the releasing of porforin and granzyme by NK92 was analyzed by the β-hexosaminidase release assay. The IFN-γ、 TNF-α secretion by NK92 wsa detect by ELISA methods. And the cytotoxicity of NK92 was analyzed by MTT methods.
[Results] sHLA-G1 conferred a significant degrading the role of NK92 to adhere to SV-40-PED (P<0.01) and degrading the releasing of porforin and granzyme by NK92, the IFN-γ、 TNF-α secretion by NK92 was inhibition significant by sHLA-G1, and sHLA-G1 protected SV-40-PED against NK2 medicated lysis, the rate of NK92 cell cytotoxicity was reduced to 25.5±2.1% in contrast to 71.2±2.6% in the control group (P<0.05).
[Conclusion] sHLA-G1 can inhibit the biological function of NK92. These results indicate that sHLA-G1 may be useful to prevent human NK cells to be responsed to porcine xenografts.
Part IV Establishment A Xenograft Model for graft-versus-host Disease in SCID Mice Engrafted With Human Peripheral Blood Mononuclear Cells
[Objective] To Establishment the xenograft model for graft-versus-host disease (XGVHD) by transfer of human peripheral blood mononuclear cells (hPBMCs) and NK92 cells into severe combined immunodeflcient (SCID) mice.
[Methods] SCID mice were injected i.v 5×10~7hPBMCs and NK92 cells under sterile conditions. SCID mice were pretreated 1 day prior to hPBMCs and NK92 cells injection with 30μl anti-asialo GM1 antibodies. Immediately prior to hPBMCs and NK92 cells engraftment SCID mice were irradiated with a dose of γ-radiation (3.5 Gy). NK92 cells with 200u/ml of rIL-2 were injected into SCID mice in one of NK92 group. IFN-γ and TNF-α ELISA kits were used for IFN-γ and TNF-α assay.
[Results] The high level of hPBMCs engraftment achieved in SCID mice induces severe XGVHD with concomitant weight loss, hunched back, and ruffled fur. Histological analysis of hPBMCs-SCID tissues showed massive leukomonocyte infiltration in liver. The IFN-γ、 TNF-α secretion were increased significantly when XGVHD occurred. Almost 100% of the SCID mice engraft with hPBMCs died within 25 days postengraftment. Inversely, the XGVHD didn't occur in SCID mice in NK92 group.
[Conclusion] Successfully development a human model of xenogeneic graft-versus-host disease in SCID mice engrafted with hPBMCs. Part V Soluble HLA-G1 inhibit the XGVHD disease induce by engraft Human Peripheral Blood mononuclear cells into SCID mice
[Objective] To study the whether sHLA-G1 can inhibit the biological function of T cells in vivo, inhibit the XGVHD disease induce by engraft hPBMCs into SCID mice.
[Methods] The effects of sHLA-G1 inhibition the proliferation of T cell was analyzed by mixed lymphocyte culture method. SCID mice were injected i.v 5×10~7 hPBMCs under sterile conditions. SCID mice were pretreated 1 day prior to hPBMCs injection with 30μl anti-asialo GM1 antibodies. Immediately prior to hPBMCs engraftment SCID mice were irradiated with a dose of γ-radiation (3.5 Gy). In the two experiment groups, SCID mice were injected i.v with a dose of 2ng and 4ng sHLA-G1 (0、3、 6、 9、 12and15d).
[Results] sHLA-G1 can inhibitive the proliferation of T cell markedly. SCID mice in control group induce severe XGVHD with concomitant weight loss, hunched back, and ruffled fur. Histological analysis of hPBMCs-SCID tissues showed massive leukomonocyte infiltration in liver. The IFN-γ、 TNF-α secretion were increased significantly when XGVHD occurred. Almost 100% of the SCID mice engraft with hPBMCs died within 25 days postengraftment. Inversely, SCID mice in experiment groups didn't suffer from XGVHD disease, The IFN-γ TNF-α secretion were kept in a low level. Histological analysis of hPBMCs-SCID- sHLA-G1 tissues showed only a few leukomonocyte infiltrated in liver.
[Conclusion] sHLA-G1 can inhibit the biological function of T cells in vivo, and inhibit the XGVHD disease induce by engraft hPBMCs into SCID mice.
【目的】研究HLA-G1基因抑制人NK细胞系NK92和外周血单个核细胞(hPBMC)对猪血管内皮细胞(PEC)杀伤的作用。
【方法】利用脂质体介导的基因转染技术将pcDNA3-HLA-G1质粒转入原代培养的PEC，用流式细胞仪及间接免疫荧光显微镜在蛋白质水平上检测HLA-G1分子在PEC上的表达；以NK细胞系(NK92)和hPBMC为效应细胞，用四甲基偶氮唑盐(MTT)法检测转染有HLA-G1基因的PEC对NK92和hPBMC杀伤活性的抵御作用。
【结果】利用脂质体转染技术成功将pcDNA3-HLA-G1转入原代培养的PEC；NK92和hPBMC对转染有HLA-G1的PEC的杀伤效率(41.5％±14.0％；45.4％±12.1％)与对照组(75.3％±10.5％；74.6％±11.2％)相比，均有明显降低(P＜0.05)。
【结论】HLA-G1分子可以明显抑制NK92细胞以及hPBMC对PEC的杀伤作用。
第二部分建立表达可溶性HLA-G1真核细胞系并获取可溶性HLA-G1
【目的】为了研究可溶性HLA-G1(sHLA-G1)在抑制NK细胞以及T细胞介导的异种排斥反应中的作用，在本实验中建立表达sHLA-G1的真核细胞系，获取纯化的sHLA-G1蛋白。
【方法】利用核转染技术将pcDNA3-sHLA-G1质粒转入LCL721.221细胞；流式细胞仪和荧光显微镜对转染率进行判定；G418筛选阳性细胞；应用RT-PCR和Dot-ELISA方法分别在基因水平和蛋白水平对表达sHLA-G1的LCL721.221细胞进行鉴定；收集LCL721.221-sHLA-G1细胞培养液；将HB-95细胞注入Balb／c小鼠腹腔，产生含抗体的腹水后采用正辛酸-硫酸氨沉淀法纯化W6／32抗体，再用免疫亲和层析提纯sHLA-G1蛋白。
【结果】核转染技术可以高效将pcDNA3-sHLA-G1质粒转入LCL721.221细胞，转染率约为14％，RT-PCR和Dot-ELISA的结果显示转染和筛选均获得成功；G418筛选出稳定表达sHLA-G1的LCL721.221细胞，应用免疫亲和层析方法提纯LCL721.221-sHLA-G1培养液中sHLA-G1纯化蛋白约1.3mg。
【结论】核转染技术可以高效将pcDNA3-sHLA-G1转入LCL721.221细胞。免疫亲和层析方法成功提纯sHLA-G1纯化蛋白。
第三部分可溶性HLA-G1抑制NK92的生物学功能的研究
【目的】研究sHLA-G1对NK细胞的粘附功能、胞吐、杀伤活性以及释放细胞因子等功能的影响作用。
【方法】利用细胞粘附实验研究sHLA-G1对NK92细胞在静止状态和滚动状态对猪血管内皮细胞系SV-40-PED粘附功能的抑制作用；借助β-hexosaminidase释放实验间接反映NK92细胞杀伤SV-40-PED时所释放的穿孔素、颗粒酶水平以及sHLA-G1对此的抑制作用；利用ELISA方法检测sHLA-G1对NK92所释放的IFN-γ、TNF-α水平的抑制作用；利用MTT方法检测sHLA-G1抑制NK92对SV-40-PED的杀伤作用。
【结果】sHLA-G1无论是在静止状态下还是滚动状态下都能显著抑制NK92的粘附功能(P＜0.01)；β-hexosaminidase释放实验结果显示，NK细胞与靶细胞SV-40-PED相互作用2h，即有明显的胞吐效应，酶释放率达14.5％，随着时间延长，酶释放率增加，6h时达高峰64.5％，sHLA-G1组中NK92细胞的胞吐维持在较低水平，二者间有显著差异(P＜0.05)；ELISA实验结果显示对照组中NK92所释放的IFN-γ、TNF-α因子维持在一个较高的水平，sHLA-G1组中NK92所释放的IFN-γ、TNF-α因子较对照组有显著降低(P＜0.05)；MTT杀伤实验结果显示sHLA-G1组NK92对SV-40-PED的杀伤效率(25.5±2.1％)显著低于对照组(71.2±2.6％，P＜0.01)。
【结论】sHLA-G1可以显著抑制NK92细胞的生物学功能，从而抑制NK92介导的异种移植排斥反应。
第四部分人外周血单个核细胞移植给SCID小鼠异种GVHD模型的建立
【目的】建立一种能直接观测异种器官移植给人的异种移植所发生的各种免疫反应的动物模型。
【方法】阳性对照组：SCID小鼠经尾静脉注射5×10~7 C57BL／6小鼠脾细胞；人外周血单核细胞(hPBMC)组：SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)、尾静脉注射5×10~7hPBMC；NK92组：根据预处理因素分为3组，A组SCID小鼠在实验前1d腹腔注射30μl生理盐水，实验当天给予Co~(60)照射(3.5Gy)和尾静脉注射5×10~7NK92细胞；B组SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)和尾静脉注射5×10~7 NK92细胞；C组SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)和尾静脉注射5×10~7NK92细胞(NK92细胞悬液加入终浓度为200u／ml的rhlL-2)。观察各组SCID小鼠体重、体型、体位、毛发、腹泻和死亡时间；ELISA方法检测SCID小鼠(PBMC组和NK92组)IFN-γ、TNF-α的分泌水平；对死亡SCID小鼠行常规病理学检查。
【结果】SCID小鼠在输注5×10~7 C57BL／6脾细胞5d后出现弓背、消瘦、皮毛紊乱无光泽、体重减轻等GVHD症状，7只SCID小鼠在5～8d内死亡，肝脏出现大面积肝细胞坏死，肝脏失去正常的结构，肝窦中有大量的淋巴细胞浸润；hPBMC组SCID小鼠在2周后开始出现GVHD症状，肝脏出现大面积肝细胞坏死，肝窦中有大量的淋巴细胞浸润，分别在15～25d内死亡；NK92组中A、B、C组SCID小鼠均未出现GVHD；ELISA检测结果显示hPBMC组中在发生异种GVHD时TNF-α的分泌水平明显升高，IFN-γ的水平也有一定程度上的升高，而在NK92组，IFN-γ、TNF-α的分泌水平较低，并且显现出持续降低趋势。
【结论】成功建立人外周血单个核细胞移植给SCID小鼠异种GVHD模型，为研究异种移植排斥反应以及今后建立人移植物对猪的异种GVHD提供一定的启示。
第五部分可溶性HLA-G1抑制外周血单个核细胞移植给SCID小鼠的异种GVHD
【目的】研究sHLA-G1在体内环境中对T细胞活性的影响作用，并且抑制人外周血单个核细胞(hPBMC)移植给SCID小鼠发生的异种GVHD。
【方法】采用单向混合淋巴细胞培养实验检测sHLA-G1对T细胞增殖能力的抑制作用；SCID小鼠在实验前1d腹腔注射30μl anti-asialo GM1抗体，实验当天给予Co~(60)照射(3.5Gy)、尾静脉注射5×10~7 hPBMC细胞，其中实验组1、2中SCID小鼠分别经尾静脉注射2ng和4ng的sHLA-G1治疗(实验当天、3、6、9、12和15d)，对照组SCID小鼠注射等量的生理盐水；ELISA方法检测各组SCID小鼠IFN-γ、TNF-α的分泌水平；取死亡SCID小鼠行常规病理学检查。
【结果】对照组中hPBMC的增殖强度比实验组1和实验组2均有显著性增强(P＜0.05)；实验组1和实验组2中SCID小鼠存活时间比对照组有显著延长(P＜0.01)；对照组中SCID小鼠血清中的IFN-γ、TNF-α水平随着GVHD症状的出现，IFN-γ、TNF-α水平显著升高，实验组1，2中SCID小鼠血清中的IFN-γ、TNF-α水平一直维持在较低的水平；病理学检查对照组SCID小鼠肝脏出现大面积肝细胞坏死，肝脏失去正常的结构，肝窦中有大量的淋巴细胞浸润，而实验组中SCID小鼠肝脏病理学检查均为肝脏轻微可逆性病变，肝窦中仅有散在淋巴细胞浸润。
【结论】sHLA-G1可以在体内环境中抑制T细胞的生物学功能，并且可以明显抑制hPBMC移植给SCID小鼠发生的异种GVHD。
Part I A Study of HLA-G1 Protection of Porcine Endothelial Cells Against Human NK Cell Cytotoxicity
[Objective] To study the effect of protecting porcine aortic endothelial cells (PEC) transfected with HLA-G1 from human NK cell lysis.
[ Methods ] The recombinant expression vector pcDNA3.0-HLA-G1 was transfected into primary cultured PEC by lipofection. Surface expression of HLA-G1 in transected PEC was confirmed by an immunofluoresence technique. Human peripheral blood mononuclear cells (hPBMC) and NK cell line (NK92) were used as NK effect cells with pcDNA3-HLA-G1-transfected PEC as targets in a MTT method using pcDNA3 transfection as a negative control.
[Results] Expression of HLA-G1 on PEC conferred significant protection against NK-mediated lysis. The rate of NK92 cytotoxicity was reduced to 41.5% ± 14.0% from 75.3% ±10.5% in the control group (P<0.01). Similarly the rate of the hPBMC cytotoxicity among different donors (n =7) was reduced to 45.4% ± 12.1% in contrast to 74.6% ±11.2% in the control group (P<0.05).
[Conclusion] HLA-G1 molecules can directly protect xenogeneic PEC against attack by human NK cells. These results indicate that the expression of HLA-G1 on the porcine cell surface may provide a new approach to overcome NK-mediated immunity to xenografts. Part II Establishment of cell line stably expressing soluble HLA-G1 and purification of soluble HLA-G1 protein
[Objective] To establish the LCL721.221 cell line stably expressing sHLA-G1 and purify the sHLA-G1 protein.
[Methods] The recombinant plasmid pcDNA3.0-sHLA-G1 was transfected by a novel nonviral, electroporation-based gene transfer method termed nucleofection into the host cell lymphoblastoid cell line LCL721.221 which does not express any HLA-classical I molecules. After selection by G418, the cell line stably expressing sHLA-G1 is identified by RT-PCR and Dot-ELISA with HLA-G specific monoclonal antibody MEM-G/9. The W6/32 antibody (anti HLA-G monoclonal) was produced by injecting the hybridoma cell line HB-95 cell into Balb/c mice. Then the sHLA-G1 protein is purified by affinity chromatograpHy using the monoclonal antibody.
[Results] After analysis by RT-PCR and Dot-ELISA, it is confirmed that the eukaryotic cell line expressing sHLA-G1 has been established successfully. And about 1.3mg sHLA-G1 protein was purified by affinity chromatography using the monoclonal antibody W6/32.
[Conclusion] In this study, we have established the LCL721.221 cell line expressing sHLA-G1 and get the sHLA-G1 protein successfully.
Part III Soluble HLA-G1 inhibit the biological function of NK92
[Objective] To study the function of sHLA-G1 inhibition the biological function of NK92.
[Methods] The effect of sHLA-G1 on the interaction between SV-40-PED and NK92 cells was assessed in terms of adhesion and cytotoxicity. The inhibitive effect of the releasing of porforin and granzyme by NK92 was analyzed by the β-hexosaminidase release assay. The IFN-γ、 TNF-α secretion by NK92 wsa detect by ELISA methods. And the cytotoxicity of NK92 was analyzed by MTT methods.
[Results] sHLA-G1 conferred a significant degrading the role of NK92 to adhere to SV-40-PED (P<0.01) and degrading the releasing of porforin and granzyme by NK92, the IFN-γ、 TNF-α secretion by NK92 was inhibition significant by sHLA-G1, and sHLA-G1 protected SV-40-PED against NK2 medicated lysis, the rate of NK92 cell cytotoxicity was reduced to 25.5±2.1% in contrast to 71.2±2.6% in the control group (P<0.05).
[Conclusion] sHLA-G1 can inhibit the biological function of NK92. These results indicate that sHLA-G1 may be useful to prevent human NK cells to be responsed to porcine xenografts.
Part IV Establishment A Xenograft Model for graft-versus-host Disease in SCID Mice Engrafted With Human Peripheral Blood Mononuclear Cells
[Objective] To Establishment the xenograft model for graft-versus-host disease (XGVHD) by transfer of human peripheral blood mononuclear cells (hPBMCs) and NK92 cells into severe combined immunodeflcient (SCID) mice.
[Methods] SCID mice were injected i.v 5×10~7hPBMCs and NK92 cells under sterile conditions. SCID mice were pretreated 1 day prior to hPBMCs and NK92 cells injection with 30μl anti-asialo GM1 antibodies. Immediately prior to hPBMCs and NK92 cells engraftment SCID mice were irradiated with a dose of γ-radiation (3.5 Gy). NK92 cells with 200u/ml of rIL-2 were injected into SCID mice in one of NK92 group. IFN-γ and TNF-α ELISA kits were used for IFN-γ and TNF-α assay.
[Results] The high level of hPBMCs engraftment achieved in SCID mice induces severe XGVHD with concomitant weight loss, hunched back, and ruffled fur. Histological analysis of hPBMCs-SCID tissues showed massive leukomonocyte infiltration in liver. The IFN-γ、 TNF-α secretion were increased significantly when XGVHD occurred. Almost 100% of the SCID mice engraft with hPBMCs died within 25 days postengraftment. Inversely, the XGVHD didn't occur in SCID mice in NK92 group.
[Conclusion] Successfully development a human model of xenogeneic graft-versus-host disease in SCID mice engrafted with hPBMCs. Part V Soluble HLA-G1 inhibit the XGVHD disease induce by engraft Human Peripheral Blood mononuclear cells into SCID mice
[Objective] To study the whether sHLA-G1 can inhibit the biological function of T cells in vivo, inhibit the XGVHD disease induce by engraft hPBMCs into SCID mice.
[Methods] The effects of sHLA-G1 inhibition the proliferation of T cell was analyzed by mixed lymphocyte culture method. SCID mice were injected i.v 5×10~7 hPBMCs under sterile conditions. SCID mice were pretreated 1 day prior to hPBMCs injection with 30μl anti-asialo GM1 antibodies. Immediately prior to hPBMCs engraftment SCID mice were irradiated with a dose of γ-radiation (3.5 Gy). In the two experiment groups, SCID mice were injected i.v with a dose of 2ng and 4ng sHLA-G1 (0、3、 6、 9、 12and15d).
[Results] sHLA-G1 can inhibitive the proliferation of T cell markedly. SCID mice in control group induce severe XGVHD with concomitant weight loss, hunched back, and ruffled fur. Histological analysis of hPBMCs-SCID tissues showed massive leukomonocyte infiltration in liver. The IFN-γ、 TNF-α secretion were increased significantly when XGVHD occurred. Almost 100% of the SCID mice engraft with hPBMCs died within 25 days postengraftment. Inversely, SCID mice in experiment groups didn't suffer from XGVHD disease, The IFN-γ TNF-α secretion were kept in a low level. Histological analysis of hPBMCs-SCID- sHLA-G1 tissues showed only a few leukomonocyte infiltrated in liver.
[Conclusion] sHLA-G1 can inhibit the biological function of T cells in vivo, and inhibit the XGVHD disease induce by engraft hPBMCs into SCID mice.
引文
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8. Arnaiz-Villena A, Martinez-Laso J, Alvarez M, et al. Primate MHC-E and G alleles. Immunogenetics. 1997; 46: 251-266.
9. Bainbridge DR, Ellis SA, Sargent IL, et al. Little evidence of HLA-G mRNA polymorphism in Caucasian or Arrocaribbean population. J Immunol. 1999; 163: 2023-2037.
10. Carosella ED, et al: HLA-G and -E: fundalnental and physiopathological aspects. Immunol Today. 2000; 21: 532-534.
11. Mallet V, Blaschitz A, Crisa L, et al. HLA-G in the human thymus: a subpopulation of medullary epithelial but not CD83 (+) dendritic cells expresses HLA-G as a membrane-bound and soluble protein. Int Immunol. 1999; 11(6): 889-898.
12. Yaping Yang, Wenjiang Chu, Daniel E. Geraghty,et al. Expression of HLA-G in human mononuclear phaglcytes and selective induction by IFN- T J.Immunol.1996; 156:4224-4231
13. Philippe Moreau, Edgardo Carosella, Magali Teyssier, et al. Soluble HLA-G molecule: an alternatively spliced HLA-G mRNA form candidate to encode it in peripheral blood mononuclear cells and human trophoblasts. Human Immunol.1995; 43:231-236
14. Moreau P, Mouillot G, Rousseau P, et al. HLA-G gene repression is reversed by demethylation.Proc Natl Acad Sci U S A. 2003; 100(3): 1191-1196.
15. Thellin O, Coumans B, Zorzi W, et al. Tolerance to foeto-placental 'graft': ten ways to support a child for nine months. Curr. Opin. Immunol. 2000; 12: 731-737.
16. Davis DM, Reyburn HT, Pazmany L, et al. Impaired spontaneous endocytosis of HLA - G. Eur Immunl. 1997; 27: 2714 -2719
17. Diehl M, Munz C, Keilholz W, et al. Nonclassical HLA-G molecules are classical peptide presenters. Curr Biol. 1996; 6(3): 305-314.
18. Gobin SJ P , Wilson L , Keijser V , et al. Antigen processing and presentation by human trophoblst - derived cell lines. J Immunol. 1997; 158:3587-3592.
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29. Burt D, Johnson D, Rinke de Wit T, et al: Cellular immune recognition of HLA-G-expressing choriocarcinoma cell line Jeg-3. Int J Cancer. 1991; Suppl, 6: 117-122.
30. Ritrau B, et al: HLA-G inhibits the allogeneic proliferative response. J Reprod Immunol. 1999; 43: 203-211.
31. Khalil-Daher I, Rouas-Freiss N, Carosella ED, et al. Human leukocyte antigen-G: immunotolerant major histocompatibility complex molecule in transplantation. World J Surg. 2000; 24(7): 819-822.
32. Fournel S, Aguerre-Girr M, Huc X, et al. Cutting Edge: Soluble HLA-G1 Triggers CD95/CD95 Ligand- Mediated Apoptosis in Activated CD8+ Cells by Interacting with CD8. J. Immunol. 2000; 164: 6100-6104.
33. Bainbridge D, Ellis S, S argent I. HLA-G suppresses proliferation of CD4+ T lympHocytes. J. Reprod. Immunol. 2000;48:17-26.
34. Riteau B, Menier C, Khalil-Daher I, et al. HLA-G inhibits the allogeneic proliferative response. J. Repors. Immunol. 1999; 43:203-211.
35. Kapasi K, Albert S, Yie S, et al. HLA-G has a concentration-dependent effect on the generation of an allo-CTL response. Immunology. 2000; 101:191-200.
36. Le Gal FA, Riteau B, Sedlik C, et al. HLA-G-mediated inhibition of antigen-specific cytotoxic T lymphocytes. Int Immunol. 1999; 11 (8): 1351-1356.
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58. Forte P, Pazmany L, Matter-Reissmann UB, et al: HLA-G inhibits rolling adhesion of activated human NK cells on porcine endothelial cells. J Immunol.2001; 167(10): 6002-6008.
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4. Bainbridge D, Ellis S, Le Bouteiller P, et al. HLA-G remains a mystery. Trends Immunol. 2001; 22: 548-552.
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3. Forte P, Pazmany L, Matter-Reissmann UB, et al. HLA2Ginhibits rolling adhesion of activated human NK cells on porcine endothelial cells. J Immunol. 2001; 167: 6002-6008.
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10. Ishitani A, Geraghty DE. Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class Ⅰ and class Ⅱantigens. Proc. Natl. Acad. Sci. USA. 1992; 89: 3947-3951.
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5. Igaz p. Recent strategies to overcome the hyperacute rejection in pig to human xenotransplantation. Yale J Biol Med. 2001; 74(5): 329-340.
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10. Lin Y, Soares MP, Sato K., et al. Rejection of cardiac xenografls by CD4+ or CD8+ T cells. Immunol. 1999; 162(2): 1206-1214.
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14. Igaz P. Recent strategies to overcome the hyperacute rejection in pig to human xenotransplantation. Yale J Biol Med. 2001; 74(5): 329-340.
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21. Forte P, Pazmany L, Matter2Reissmann UB, et al. HLA2Ginhibits rolling adhesion of activated human NK cells on porcine endothelial cells. J Immunol. 2001; 167: 6002-6008.
1．王坤杰，李幼平，杨宇如。异种移植中的移植物抗宿主病，中国修复重建外科杂志，2000；14(3)：181-185。
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12. Mollnes TE, Fiane AE. Perspectives on complement in xenotransplantation. Mol Immunol. 2003; 40(2-4): 135-143.
13. Lin Y, Soares MP, Sato K., et al. Rejection of cardiac xenografts by CD4+ or CD8+ T cells. Immunol. 1999; 162 (2): 1206-1214.
14. Candinas D, Bach FH, Hancock WW. Delayed xenograft rejection in complement-depleted T-cell-deficient rat recipients of guinea pig cardiac grafts. Transplant Proc. 1996; 28(2): 678
15. Igaz P. Recent strategies to overcome the hyperacute rejection in pig to human xenotransplantation. Yale J Biol Med. 2001; 74(5): 329-340.
16. Hancock WW. Beyond hyperacute rejection: strategies for development of pig-->primate xenotransplantation. Kidney Int Suppl. 1997; 58: S36-40.
1. Carosella ED, et al: HLA-G and -E: fundamental and physiopathological aspects. Immunol Today. 2000; 21: 532-534.
2. Thellin O, Coumans B, Zorzi W, et al. Tolerance to foeto-placental 'graft': ten ways to support a child for nine months. Curr. Opin. Immunol. 2000; 12: 731-737.
3. Rebmarm V, Pfeiffer K, Passler M, et al. Detection of soluble HLA-Gmolecules in plasma and amniotic fluid. Tissue antigens. 1999; 53: 14.
4. Puppo F, Costa P, Contini S, et al. Determination of soluble HLA-G and HLA-A, B, and C molecules in pregnancy. Transplant. Proc. 1999; 31: 1841.
5. Hiby S, King A, Sharkey A, et al. Molecular studies of trophoblast HLA-G: polymorphism, isoforms, imprinting and expression in preimplantation embryo. Tissue Antigens. 1999; 53: 1.
6. Hoffmann-Fezer G, Gall C, Zengerle U, et al. Immunohistology and immunocytology of human T-cell chimerism and graft-versus-host disease in SCID mice. Blood. 1993; 81(12): 3440-3448.
7. Murphy WJ, Bennett M, Anver MR, et al. Human-mouse lymphoid chimeras: host-vs. -graft and graft-vs. -host reactions. Eur J Immunol. 1992; 22(6): 1421-1427.
8. Huppes W, De Geus B, Zurcher C, et al. Acute human vs. mouse graft vs. host disease in normal and immunodeficient mice. Eur J Immunol. 1992; 22(1): 197-206.
9. Sandhu JS, Gorczynski R, Shpitz B, et al. A human model of xenogeneic graft-versus-host disease in SCID mice engrafted with human peripheral blood lymphocytes. Transplantation. 1995; 60(2): 179-184.
10. Burt D, Johnson D, Rinke de Wit T, et al: Cellular immune recognition of HLA-G-expressing choriocarcinoma cell line Jeg-3. Int J Cancer. 1991; Suppl, 6: 117-122.
11. Ritrau B, et al: HLA-G inhibits the allogeneic proliferative response. J Reprod Immunol. 1999; 43: 203-211.
12. Khalil-Daher I, Rouas-Freiss N, Carosella ED, et al. Human leukocyte antigen-G: immunotolerant major histocompatibility complex molecule in transplantation. World J Surg. 2000; 24(7): 819-822.
13. Fournel S, Aguerre-Girr M, Hue X, et al. Cutting Edge: Soluble HLA-G1 Triggers CD95/CD95 Ligand- Mediated Apoptosis in Activated CD8+ Cells by Interacting with CD8. J. Immunol. 2000; 164: 6100-6104.
14. Bainbridge D, Ellis S, S argent I. HLA-G suppresses proliferation of CD4+ T lympHocytes. J. Reprod. Immunol. 2000; 48: 17-26.
15. Riteau B, Menier C, Khalil-Daher I, et al. HLA-G inhibits the allogeneic proliferative response. J. Repors. Immunol. 1999; 43: 203-211.
16. Kapasi K, Albert S, Yie S, et al. HLA-G has a concentration-dependent effect on the generation of an allo-CTL response. Immunology. 2000; 101: 191-200.
17. Carosella ED, Moreau P, Aractingi S, et al: HLA-G: a shield against inflammatory aggression. Trends in Immunology. 2001; 22(10): 553-555.
1. Sunderland CA, Redman CWG, Stirrat GM. HLA-A, B, C antigens are expressed on nonvillous trophoblasts of the early human placenta. J Immunol. 1981; 127: 2614-2615.
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4. Park B, Ahn K. An essential function of tapasin in quality control of HLA-G molecules. J Biol Chem. 2003; 278(16): 14337-14345.
5. Bainbridge D, Ellis S, Bouteiller P, et al: HLA-G remains a mystery. Trends in Immunology. 2001; 22(10): 548-552.
6. Tomoyuki Fujii, Akiko Ishitani, and Daniel E. Geraghty. A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4. J. Immunol. 1994; 153: 5516-5524
7. LeMaoult J, Le Discorde M, Rouas-Freiss N, et al. Biology and functions of human leukocyte antigen-G in health and sickness. Tissue Antigens. 2003; 62(4): 273-284.
8. Arnaiz-Villena A, Martinez-Laso J, Alvarez M, et al. Primate MHC-E and G alleles. Immunogenetics. 1997; 46: 251-266.
9. Bainbridge DR, Ellis SA, Sargent IL, et al. Little evidence of HLA-G mRNA polymorphism in Caucasian or Arrocaribbean population. J Immunol. 1999; 163: 2023-2037.
10. Carosella ED, et al: HLA-G and -E: fundalnental and physiopathological aspects. Immunol Today. 2000; 21: 532-534.
11. Mallet V, Blaschitz A, Crisa L, et al. HLA-G in the human thymus: a subpopulation of medullary epithelial but not CD83 (+) dendritic cells expresses HLA-G as a membrane-bound and soluble protein. Int Immunol. 1999; 11(6): 889-898.
12. Yaping Yang, Wenjiang Chu, Daniel E. Geraghty,et al. Expression of HLA-G in human mononuclear phaglcytes and selective induction by IFN- T J.Immunol.1996; 156:4224-4231
13. Philippe Moreau, Edgardo Carosella, Magali Teyssier, et al. Soluble HLA-G molecule: an alternatively spliced HLA-G mRNA form candidate to encode it in peripheral blood mononuclear cells and human trophoblasts. Human Immunol.1995; 43:231-236
14. Moreau P, Mouillot G, Rousseau P, et al. HLA-G gene repression is reversed by demethylation.Proc Natl Acad Sci U S A. 2003; 100(3): 1191-1196.
15. Thellin O, Coumans B, Zorzi W, et al. Tolerance to foeto-placental 'graft': ten ways to support a child for nine months. Curr. Opin. Immunol. 2000; 12: 731-737.
16. Davis DM, Reyburn HT, Pazmany L, et al. Impaired spontaneous endocytosis of HLA - G. Eur Immunl. 1997; 27: 2714 -2719
17. Diehl M, Munz C, Keilholz W, et al. Nonclassical HLA-G molecules are classical peptide presenters. Curr Biol. 1996; 6(3): 305-314.
18. Gobin SJ P , Wilson L , Keijser V , et al. Antigen processing and presentation by human trophoblst - derived cell lines. J Immunol. 1997; 158:3587-3592.
19. Munz C, Nickolaus P, Lammert E, et al. The role of peptide presentation in the physiological function of HLA-GSemin Cancer Biol. 1999; 9(1): 47-54.
20. Fournel S, Aguerre-Girr M, Huc X, et al. Cutting Edge: Soluble HLA-G1 Triggers CD95/CD95 Ligand- Mediated Apoptosis in Activated CD8+ Cells by Interacting with CD8. J Immunol. 2000; 164: 6100-6104.
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