KIR和MHC Ⅰ类分子基因共转移建立异基因骨髓移植双向免疫耐受
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摘要
移植物抗宿主病(graft versus host disease，GVHD)和移植排斥严重影响异基因骨髓移植(allogenic bone marrow transplantation，allo-BMT)的成功率，是临床亟待解决的问题。杀伤抑制受体(killer inhibitory receptor，KIR)分子存在于NK、T细胞以及某些单核巨噬细胞、B细胞表面，能识别自身组织细胞特定MHC Ⅰ类分子，建立抑制信号传导通路，消除T、NK细胞毒活性，减少炎性细胞因子的分泌并降低TCR分子的表达。移植排斥发生的部分原因是供者组织细胞不表达与受者KIR分子相适应的MHC Ⅰ类分子，导致受者NK、T细胞的活化不能被抑制，而引发对供者的攻击。同理，GVHD的发生是由于供者的免疫细胞缺乏与受者MHC Ⅰ类分子相配的KIR，不能在被活化的供者免疫细胞和受者组织细胞之间建立抑制性信号传导通路，供者淋巴细胞必然攻击受者，从而发生GVHD。在不能完全改变受者体细胞遗传表型的情况下，本研究拟将受鼠KIR分子和MHC Ⅰ类分子基因共转移入供鼠的骨髓和脾细胞中，使其有效表达，令供鼠造血免疫细胞携带受者KIR和MHC Ⅰ类分子后，进行异基因骨髓移植。通过KIR与MHC Ⅰ类分子双向特异性结合，建立杀伤细胞的抑制性信号传递通路，诱导个体特异性免疫耐受，达到预防GVHD及移植排斥的双重目的。
方法：(1)利用RT-PCR技术克隆BALB/c(H-2~d)小鼠的KIR分子——Ly49A和MHC Ⅰ类分子——H-2D~d的编码基因，构建Ly49A和基因重组逆转录病毒表达载体pMSCV-Ly49A(puro~+)和pMSCV-H-2D~d(neo~+)。(2)将重组病毒表达载体分别转染入PT67细胞，在PT67细胞中包装成具有感染能力的病毒颗粒，将两种重组逆转录病毒共同感染C_(57)BL/6小鼠骨髓细胞和脾细胞，免疫荧光和流式细胞仪检测病毒感染细胞外源Ly49A和H-2D~d基因的表达。(3)采用CFU-Mix克隆培养、淋巴细胞增殖实验、混合淋巴细胞培养、测定CTL杀伤和NK细胞杀伤活性等方法，体外研究转基因细胞的造血功能、免疫功能及杀伤瘤细胞作用。(4)将转基因细胞分别用于C_(57)BL/6(H-2~b)→BALB/c(H-2~d)
异基因骨髓移植GVHD和排斥反应动物模型中，记录动物生存期并观察
GVHD症状，测定移植后血清IL一2水平和骨髓细胞嵌合体形成的比率，分析
受鼠来源的KIR和MHCI类分子基因转移对骨髓移植小鼠GVHD和排斥反
应的作用。
结果:(l)限制性内切酶鉴定、DNA测序分析表明，成功克隆并构建了
BALB/e(H一Zd)鼠的Ly49A和H一ZDd的重组逆转录病毒表达质粒pMsev-H一Znd
(neo+)和pMscv-Ly49A印uro+)，构建的重组表达质粒中的外源基因方向、序
列均正确。(2)两种重组表达质粒在PT67细胞中包装成具有感染能力的病毒
颗粒，产生病毒滴度在5.8又105一3.4xl06cfi对ml之间。两种重组逆转录病毒共同
感染C57BL场骨髓细胞和脾细胞后，免疫荧光显示细胞表面两种外源分子均得
到表达，流式细胞仪检测病毒感染2天后同时表达外源切49A和H一ZDd基因
的脾细胞和骨髓细胞的比例分别达到48.3土3.7%和13.肚2.3%，病毒感染后经
抗生素筛选4周后则为62.4习.9%和18.1=1=2.6%。(3) CFU一Mix培养证明，病
毒感染的C57BL16鼠骨髓细胞的造血克隆形成能力与未感染的C57BL/6鼠脾细
胞相比，无统计学差异(P>0.05)。病毒感染后淋巴细胞对Co叭的刺激反应
无影响。混合淋巴细胞培养结果表明，Ly49A和H一ZDd双基因逆转录病毒共感
染的Cs7BL/6鼠淋巴细胞对BALB/c鼠脾细胞的刺激强度或应答程度，与未感
染感染的C57BL/6鼠骨髓细胞相比，均减弱。双基因转移的C57BL/6鼠脾细胞
与BALB/c鼠脾细胞之间的相互CTL和NK细胞杀伤作用亦减弱，而经病毒
感染的C57BL/6小鼠对BALB/c鼠肿瘤细胞WEHI3的CTL杀伤活性并不改变，
对YAC一1细胞的NK活性亦无变化。(4)在动物模型中，与GVHD阳性对照
组(12.4士1.33天)相t匕，Ly49A单基因转移组(27.6士4.27天)、H一Znd单基
因转移组(19.0士x.77天)及切49A联合H一Znd双基因转移组(33.7士5.22天)
均能显著延长移植小鼠的存活期(P值分别为0.0006、0.0072、0.0001)。转基
因治疗有效组小鼠的一般状况如精神、活动等表现较好，体重减轻较少，外周
血白细胞低下的时间更短。移植后14天，血清IL一2检测显示，H一ZDd单基因
转移组(3.01士o.32ng/ml)、切49A+H一Znd双基因转移组(2.4狂o.42ng/ml)小
鼠IL一2水平较对照组(4.48士0.028n创ml)显著降低(p=0.000、0.000)。组织
病理检查及骨髓细胞嵌合体的检测均证实了Ly49A和H一ZDd双基因转移对骨
髓移植后GVHD和排斥反应的防治效果。
结论:(l)采用逆转录病毒介导，将BALB/。小鼠Ly49A和H一ZDd基因
共转移入C57CU6鼠脾细胞和骨髓细胞中，并在细胞膜表面得到表达。(2)
切49A十H一ZDd双基因转移能降低体外培养的Cs7BL/6鼠免疫细胞与BALB/c
小鼠免疫细胞间的刺激作用和应答强度，减弱细胞之间的相互CTL和NK细
胞杀伤作用，但不影响Cs7BL/6鼠免疫细胞对有丝分裂原的反应及对瘤细胞的
杀伤。(3)在异基因骨髓移植动物模型(Cs7BL/6~BALB/c)中，Ly49A+H一ZDd
双基因转移可延长移植小鼠的生存期，减轻GVHD组织损伤，降低血清IL一2
水平，利于移植后嵌合体的形成。(4)将受者的KIR及M
The graft versus host disease (GVHD) and graft rejection is the major lethal complications associated with allogenic bone marrow transplantation (allo-BMT). Killer inhibitory receptor(KIR), which expressed on the surface of NK, some T cell, mononuclear phagocyte or B cell, can recognize the specific major histocompatibility complex class I (MHC I) molecular of self tissue to conduct negative signals and inhibit T cell and NK cell activation. And then, the secretion of inflaming factors and expression of TCR molecular decreased. Some reason of rejection is the donor cells don't express the proper MHC I molecule matching the receptor and the NK cell and T cell are activated to attack the donor cell. In the same way, without the KIR matched with receptor MHC I, the donor immunocyte couldn't establish the negative signal between the donor and receptor. As a result, the lymphocytes of donor are attacked and the GVHD occurs.
In this research, the genes of MHC I and KIR from receptor mice (B ALB/c) were transferred into the bone marrow and spleen cells of donor mice (Cs7BL/6) and expressed on the hematopoietic and immunologic cells of donor. And then the allo-BMT was carried out on BALB/c mice. The bi-directional specific combination of KIR and MHC I molecule would establish the negative signals to killer cells, induce the specific immunologic tolerance of individual and prevent the GVHD and the graft rejection.
Method
1. The coding gene of Ly49A and H-2Dd, which were respectively the KIR and MHC I of BALB/c, were cloned by RT-PCR, and the retrovirus expressing vectors of pMSCV-Ly49A (puro+) and pMSCV-H-2Dd (neo+) were constructed.
2. The two reconstructed retrovirus expressing vectors were respectively transfected into PT67 cell, packed to infective virus. The Cs7BL/6 mouse bone marrow cell and spleen cell were infected by the two kinds of reconstructed retrovirus, and the gene expression of extracecullar Ly49A and H-2Ddwere detected by immunofluorescence and flow cytometry.
3. The hematopoietic function, immune function and tumor-killing function of gene-transferred cells were evaluated with CPU-Mix culture, lymphocyte proliferation, mixed lymphocyte culture and killing activities of CTL and NK.
4. The gene-transferred cells were transfused respectively in the Cs7BL/6 (H-2b ) -BALB/c(H-2d) allograft GVHD model and graft rejection model. The mean survival time (MST) and the symptoms of GVHD were observed and the IL-2 level of serum after transplantation, the ratio of bone marrow chimera were measured in order to analyze the effect of genes transfer of KIR and MHC I molecule on GVHD and rejection.
Results
1. The restriction endonuclease digestion and DNA sequencing analysis showed the reconstruction of retrovirus expressing plasmids of pMSCV-H-2Dd (neo+) and pMSCV-Ly49A (puro+) were successful.
2. The two reconstructed plasmids were packed to infective virus particles in incasing cells PT67 with a tite of about 5.8xl05-3.4xl06cfu/ml. These two kinds of retrovirus were co-infected in Cs7BL/6 bone marrow cell and spleen cell, and the two kinds of external molecules on cell surface all did express proved by immunofluorescence. The percentage of spleen cells of expressing both Ly49A and H-2Dd is 48.3+3.7% at 2 days after infection, and that of bone marrow cells is 13.6+2.3 %. After antibiotics screening of 4 weeks, they reached 62.4+3.9% and 18.1+2.6% respectively.
3. The mixed colony-forming unit (CPU-Mix ) culture showed that the hematopoietic function of virus infected Cs7BL/6 mice bone marrow cells had no statistical difference with the non-infected C57BL/6 cells. The virus infection had no effect on the response ability of lymphocytes to the ConA stimulation. The mixed lymphocyte culture showed the reconstructed retrovirus infected Cs7BL/6 mice
bone marrow cells had a remarkably weaker effect to BALB/c mice spleen cells on the stimulation intensity or the response ability extensity than the non-infected and empty-vector-infected Cj7BL/6 cells. The i
方法：(1)利用RT-PCR技术克隆BALB/c(H-2~d)小鼠的KIR分子——Ly49A和MHC Ⅰ类分子——H-2D~d的编码基因，构建Ly49A和基因重组逆转录病毒表达载体pMSCV-Ly49A(puro~+)和pMSCV-H-2D~d(neo~+)。(2)将重组病毒表达载体分别转染入PT67细胞，在PT67细胞中包装成具有感染能力的病毒颗粒，将两种重组逆转录病毒共同感染C_(57)BL/6小鼠骨髓细胞和脾细胞，免疫荧光和流式细胞仪检测病毒感染细胞外源Ly49A和H-2D~d基因的表达。(3)采用CFU-Mix克隆培养、淋巴细胞增殖实验、混合淋巴细胞培养、测定CTL杀伤和NK细胞杀伤活性等方法，体外研究转基因细胞的造血功能、免疫功能及杀伤瘤细胞作用。(4)将转基因细胞分别用于C_(57)BL/6(H-2~b)→BALB/c(H-2~d)
异基因骨髓移植GVHD和排斥反应动物模型中，记录动物生存期并观察
GVHD症状，测定移植后血清IL一2水平和骨髓细胞嵌合体形成的比率，分析
受鼠来源的KIR和MHCI类分子基因转移对骨髓移植小鼠GVHD和排斥反
应的作用。
结果:(l)限制性内切酶鉴定、DNA测序分析表明，成功克隆并构建了
BALB/e(H一Zd)鼠的Ly49A和H一ZDd的重组逆转录病毒表达质粒pMsev-H一Znd
(neo+)和pMscv-Ly49A印uro+)，构建的重组表达质粒中的外源基因方向、序
列均正确。(2)两种重组表达质粒在PT67细胞中包装成具有感染能力的病毒
颗粒，产生病毒滴度在5.8又105一3.4xl06cfi对ml之间。两种重组逆转录病毒共同
感染C57BL场骨髓细胞和脾细胞后，免疫荧光显示细胞表面两种外源分子均得
到表达，流式细胞仪检测病毒感染2天后同时表达外源切49A和H一ZDd基因
的脾细胞和骨髓细胞的比例分别达到48.3土3.7%和13.肚2.3%，病毒感染后经
抗生素筛选4周后则为62.4习.9%和18.1=1=2.6%。(3) CFU一Mix培养证明，病
毒感染的C57BL16鼠骨髓细胞的造血克隆形成能力与未感染的C57BL/6鼠脾细
胞相比，无统计学差异(P>0.05)。病毒感染后淋巴细胞对Co叭的刺激反应
无影响。混合淋巴细胞培养结果表明，Ly49A和H一ZDd双基因逆转录病毒共感
染的Cs7BL/6鼠淋巴细胞对BALB/c鼠脾细胞的刺激强度或应答程度，与未感
染感染的C57BL/6鼠骨髓细胞相比，均减弱。双基因转移的C57BL/6鼠脾细胞
与BALB/c鼠脾细胞之间的相互CTL和NK细胞杀伤作用亦减弱，而经病毒
感染的C57BL/6小鼠对BALB/c鼠肿瘤细胞WEHI3的CTL杀伤活性并不改变，
对YAC一1细胞的NK活性亦无变化。(4)在动物模型中，与GVHD阳性对照
组(12.4士1.33天)相t匕，Ly49A单基因转移组(27.6士4.27天)、H一Znd单基
因转移组(19.0士x.77天)及切49A联合H一Znd双基因转移组(33.7士5.22天)
均能显著延长移植小鼠的存活期(P值分别为0.0006、0.0072、0.0001)。转基
因治疗有效组小鼠的一般状况如精神、活动等表现较好，体重减轻较少，外周
血白细胞低下的时间更短。移植后14天，血清IL一2检测显示，H一ZDd单基因
转移组(3.01士o.32ng/ml)、切49A+H一Znd双基因转移组(2.4狂o.42ng/ml)小
鼠IL一2水平较对照组(4.48士0.028n创ml)显著降低(p=0.000、0.000)。组织
病理检查及骨髓细胞嵌合体的检测均证实了Ly49A和H一ZDd双基因转移对骨
髓移植后GVHD和排斥反应的防治效果。
结论:(l)采用逆转录病毒介导，将BALB/。小鼠Ly49A和H一ZDd基因
共转移入C57CU6鼠脾细胞和骨髓细胞中，并在细胞膜表面得到表达。(2)
切49A十H一ZDd双基因转移能降低体外培养的Cs7BL/6鼠免疫细胞与BALB/c
小鼠免疫细胞间的刺激作用和应答强度，减弱细胞之间的相互CTL和NK细
胞杀伤作用，但不影响Cs7BL/6鼠免疫细胞对有丝分裂原的反应及对瘤细胞的
杀伤。(3)在异基因骨髓移植动物模型(Cs7BL/6~BALB/c)中，Ly49A+H一ZDd
双基因转移可延长移植小鼠的生存期，减轻GVHD组织损伤，降低血清IL一2
水平，利于移植后嵌合体的形成。(4)将受者的KIR及M
The graft versus host disease (GVHD) and graft rejection is the major lethal complications associated with allogenic bone marrow transplantation (allo-BMT). Killer inhibitory receptor(KIR), which expressed on the surface of NK, some T cell, mononuclear phagocyte or B cell, can recognize the specific major histocompatibility complex class I (MHC I) molecular of self tissue to conduct negative signals and inhibit T cell and NK cell activation. And then, the secretion of inflaming factors and expression of TCR molecular decreased. Some reason of rejection is the donor cells don't express the proper MHC I molecule matching the receptor and the NK cell and T cell are activated to attack the donor cell. In the same way, without the KIR matched with receptor MHC I, the donor immunocyte couldn't establish the negative signal between the donor and receptor. As a result, the lymphocytes of donor are attacked and the GVHD occurs.
In this research, the genes of MHC I and KIR from receptor mice (B ALB/c) were transferred into the bone marrow and spleen cells of donor mice (Cs7BL/6) and expressed on the hematopoietic and immunologic cells of donor. And then the allo-BMT was carried out on BALB/c mice. The bi-directional specific combination of KIR and MHC I molecule would establish the negative signals to killer cells, induce the specific immunologic tolerance of individual and prevent the GVHD and the graft rejection.
Method
1. The coding gene of Ly49A and H-2Dd, which were respectively the KIR and MHC I of BALB/c, were cloned by RT-PCR, and the retrovirus expressing vectors of pMSCV-Ly49A (puro+) and pMSCV-H-2Dd (neo+) were constructed.
2. The two reconstructed retrovirus expressing vectors were respectively transfected into PT67 cell, packed to infective virus. The Cs7BL/6 mouse bone marrow cell and spleen cell were infected by the two kinds of reconstructed retrovirus, and the gene expression of extracecullar Ly49A and H-2Ddwere detected by immunofluorescence and flow cytometry.
3. The hematopoietic function, immune function and tumor-killing function of gene-transferred cells were evaluated with CPU-Mix culture, lymphocyte proliferation, mixed lymphocyte culture and killing activities of CTL and NK.
4. The gene-transferred cells were transfused respectively in the Cs7BL/6 (H-2b ) -BALB/c(H-2d) allograft GVHD model and graft rejection model. The mean survival time (MST) and the symptoms of GVHD were observed and the IL-2 level of serum after transplantation, the ratio of bone marrow chimera were measured in order to analyze the effect of genes transfer of KIR and MHC I molecule on GVHD and rejection.
Results
1. The restriction endonuclease digestion and DNA sequencing analysis showed the reconstruction of retrovirus expressing plasmids of pMSCV-H-2Dd (neo+) and pMSCV-Ly49A (puro+) were successful.
2. The two reconstructed plasmids were packed to infective virus particles in incasing cells PT67 with a tite of about 5.8xl05-3.4xl06cfu/ml. These two kinds of retrovirus were co-infected in Cs7BL/6 bone marrow cell and spleen cell, and the two kinds of external molecules on cell surface all did express proved by immunofluorescence. The percentage of spleen cells of expressing both Ly49A and H-2Dd is 48.3+3.7% at 2 days after infection, and that of bone marrow cells is 13.6+2.3 %. After antibiotics screening of 4 weeks, they reached 62.4+3.9% and 18.1+2.6% respectively.
3. The mixed colony-forming unit (CPU-Mix ) culture showed that the hematopoietic function of virus infected Cs7BL/6 mice bone marrow cells had no statistical difference with the non-infected C57BL/6 cells. The virus infection had no effect on the response ability of lymphocytes to the ConA stimulation. The mixed lymphocyte culture showed the reconstructed retrovirus infected Cs7BL/6 mice
bone marrow cells had a remarkably weaker effect to BALB/c mice spleen cells on the stimulation intensity or the response ability extensity than the non-infected and empty-vector-infected Cj7BL/6 cells. The i
引文
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17． Chan PY, Takei F. Expression of a T cell receptor-like molecule on normal and malignant murine T cells detected by rat monoclonal antibodies to nonclonotypic determinants. J Immunol, 1986; 136: 1346-1353．
18． Chan PY, Takei F. Characterization of a murine T cell surface disulfide-linked dimer of 45-kDa glycopeptides (YE1/48 antigen). Comparison with T cell receptor, purification, and partial amino acid sequences. J Immunol, 1988;140: 161-169．
19． Chan PY, Takei F. Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48． J Immunol. 1989; 142:1727-1736
20． Margulies DH, Evens GA, Ozato K, et al. Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer. J of Immunol, 1983, 130:463-470
21． Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning-a Laboratory Manual. 2nd Ed. New York: CSH, 1989．
22． Young NT. KIR genes, Killer cells and clinical transplantation. Transplantation, 1999;68) : 1626-1628
23． Tanaka J, Tutumi Y, Mori A, et al. sequential analysis of HLA-C-specific killer cell inhibitory receptor (CD158b) expressing peripheral blood mononuclear cells during chronic graft-versus-host disease. Bone Marrow Transplant, 2000;26:287-290
24． Germann UA, Gottesman MM, Pastan I. Expression of multidrug resistance-adenosine deaminase fusion gene. J Biol Chem, 1989;264:7418-7424
25． Bowtell DD, Cory S, Johnson GR, et al. Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes. J Virol, 1988; 62:2464-2473
26． Emerman M, Temin HH. Genes with promoter in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell, 1984;39:459-467
27． Ghattas IR, Sanes JR, Majors JE. The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in the culture cells and in embros. Mol Cell Biol, 1991; 11:5548-5859
28． Karger BD, Feigner PL, Gadek TR, et al. Evaluation of procedures for transfection using lipofectin regeagent. Focus, 1990; 12: 25-29
29． Lu L, Ge Y, Li ZH, et al. CD34 stem/progenitor cells purified from cryopreserved normal cord blood can be transduced with high efficiency by a retroviral vector and expanded ex vivo with stable integration and expression of Fanconi anemia complementation C gene. Cell Transplant, 1995; 4:493-503
30． 彭秀玲，袁汉英．基因工程实验技术．第一版．湖南科学技术山版社，1987，57
31． Hawley RG, Hawley TS, Fong AZ, et al. Thrombopoietic potential and serial repopulating
ability of murine hematopoietic stem cells constitutively expressing intedeukin 11. Proc Natl Acad Sci USA, 1996; 93:10297-10302
32. Keller G, Wall C, Fong AZ, et al. Overexpression of HOX11 leads to the immortalization of embryonic precursors with both primitive and definitive hematopoietic potential. Blood, 1998; 92:877-897
33. Nienhuis AW, McDomagh KT, Bodine DM, et al. Gene transfer into hematopoietic stem cell. Cancer, 1991; 67(10 Suppl):2700-2704
34. Robbins PD, Ghivizzani SC. Viral vectors for gene therapy. Pharmacol Ther, 1998;80:35-47
35． 李家增，王鸿利，韩忠朝主编．血液实验学．上海科学技术出版社，1997，1st Ed： 126-128
36. Niks M, Otto M, Busova B, Stefanovic J. Quantification of proliferative and suppressive responses of human T lymphocytes following ConA stimulation.J Immunol Methods, 1990; 126: 263-271
37. Van Buskirk AM, Adams PW, Orosz GC. Nonradioactive alternative to clinical mixed lymphocyte reaotion. Hum Immumol, 1995; 43:38
38． 刘海鹰，周玲，曾毅．检测Epstein-Barr病毒特异性细胞毒性T淋巴细胞方法的建立及其初步研究．中华实验和临床病毒学杂志，1998；12：357-360
39． 何金生，宗庭益．LDH释放法检测NK细胞活性的方法研究．中国实验临床免疫学杂志，1996；8：10-12
40. Bellone G, Valiante NM, Viale O, et al. Regulation of hematopoiesis in vitro by alloreactive natural killer cell clones. J Exp Med, 1993; 177:1117-1120
41. Moretta A. A major histocompatibility complex class 1 specific receptors on human natural killer and T lymphocytes. Immunol Rev, 1997; 155: 105-106
42. Loung NT, Uhrberg M. KIR expression shapes cytotoxic repertoires: a developmental program of survival. Trends Immumol, 2002;23:71-75
43. Colonna M. Natural killer cell receptors specific for MHC class 1 molecules. Curt Opin Immunol, 1996; 8:101
44. Warner NL, Moore MA, Metcalf D. A transplantable myeiomonocytic leukemia in BALB-c mice: cytology, karyotype, and muramidase content. J Natl Cancer Inst. 1969; 43(4): 963-982
45. Tsukada N, Kobata T, Aizawa Y, et al. Graft versus leukemia effected graft versus host
disease can be differentiated by cytotoxic mechanisms in a murine model of allogeneic bone marrow transplantation. Blood, 1999;93: 2738-2747
46． 李家增，王鸿利，韩忠朝主编．血液实验学．上海科学技术出版社，1997，1st Ed：440-441
47． Daniel HF, Kazuhiro K, Rhett S, et al. Donor CD4 enriched cells of Th2 cytokine phenotype regulate graft versus host disease without impaired allo-genetic graft in sublethally irradiated mice. Blood, 1994; 84:3540-3543
48． Daniel HF, Bernard W, Michael L, et al. Non-host-reactive donor CD8+ T cells of Tc2 phenotype potently inhibit marrow graft rejection. Blood, 1998;91:4045-4050
49． Schmidt H, Ehninger G, Dopfer R, et al. Correlation between low CSA plasma concentration and severity of acute GvHD in bone marrow transplantation. Blut, 1988;57: 139-142
50． Norin AJ, Brewer RJ, Lui Q, et al. Donor antigen-independent effector mechanisms of graft rejection: a novel cell surface target of IL-2-activated natural killer cells. Transplant Proc, 1999;31:787-788
2． Theobald M. Allorecognition and graft-versus-host disease. Bone Marrow Transplant, 1995; 15:489-498
3． Rossini AA, Greiner DL, Mordes JP. Induction of immunologic tolerance for transplantation. Physiol Rev, 1999;79:99-141
4． Nakamura H, Gress RE. Interleukin 2 enhancement of veto suppressor cell function in T-cell-depleted bone marrow in vitro and in vivo. Transplantation, 1990;49:931-937
5． Krenger W, Hill GR, Ferrara JL. Cytokine cascades in acute graft-versus-host disease. Transplantation, 1997;64:553-558
6． Hoglund P, Sundback J, Olsson-Alheim MY, et al. Host MHC class I gene control of NK-cell specificity in the mouse. Immunol Rev, 1997;155:11-28
7． Adachi T, Wakabayashi C, Nakayama T, et al. CD72 negatively regulates signaling through the antigen receptor of B cells. J Immunol, 2000;164:1223-1229
8． Selvakumar A, Steffens U, Dupont B, et al. Polymorphism and domain variability of human killer cell inhibitory receptors. Immunol Rev, 1997;155:183-196
9． Ortaldo JR, Winkler PR, Willette BJ, et al. Structure/function relationship of activating Ly-49D and inhibitory Ly-49G2 NK receptors. J Immunol, 1999; 163:5269-5277
10． Lopez-Botet M, Perez-Villar JJ, Carretero M, et al. Structure and function of the CD94 C-type lectin receptor complex involved in recognition of HLA class I molecules. Immunol Rev, 1997;155:165-74
11． Carosella ED, Rouas-Freiss N, Paul P, et al. HLA-G: a tolerance molecule from the major histocompatibility complex. Immunol Today, 1999;20:60-62
12． Gosselin P, Mason LH, Willette-Brown J, et al. Induction of DAP12 phosphorylation, calcium mobilization, and cytokine secretion by Ly49H. J Leukoc Biol, 1999;66:165-171
13． 李蓁，李树浓，徐鸿绪，等．H-2Kb基因转移诱导小鼠心肌移植免疫耐受．免疫学杂志，2000：16：4-7
14． Sentman CL, Olsson-Alheim MY, Lendahl U, et al. H-2Dp transgene alters natural killer cell specificity at the target and effector cell levels. Comparison with an H-2Dd transgene. J Immunol, 1996; 156:2423-2429
15． Held W, Cado D, Raulet DH. Transgenic expression of the Ly49A natural killer cell receptor confers class I major histocompatibility complex (MHC)-specific inhibition and prevents bone marrow allograft rejection. J Exp Med, 1996; 184:2037-2041
16． Nicholas M, Peter Parbam. Nature killer cells, HLA class I molecules, and marrow transplantation. Biology of Blood and Marrow Transplantation.l993,3:229-235
17． Chan PY, Takei F. Expression of a T cell receptor-like molecule on normal and malignant murine T cells detected by rat monoclonal antibodies to nonclonotypic determinants. J Immunol, 1986; 136: 1346-1353．
18． Chan PY, Takei F. Characterization of a murine T cell surface disulfide-linked dimer of 45-kDa glycopeptides (YE1/48 antigen). Comparison with T cell receptor, purification, and partial amino acid sequences. J Immunol, 1988;140: 161-169．
19． Chan PY, Takei F. Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48． J Immunol. 1989; 142:1727-1736
20． Margulies DH, Evens GA, Ozato K, et al. Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer. J of Immunol, 1983, 130:463-470
21． Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning-a Laboratory Manual. 2nd Ed. New York: CSH, 1989．
22． Young NT. KIR genes, Killer cells and clinical transplantation. Transplantation, 1999;68) : 1626-1628
23． Tanaka J, Tutumi Y, Mori A, et al. sequential analysis of HLA-C-specific killer cell inhibitory receptor (CD158b) expressing peripheral blood mononuclear cells during chronic graft-versus-host disease. Bone Marrow Transplant, 2000;26:287-290
24． Germann UA, Gottesman MM, Pastan I. Expression of multidrug resistance-adenosine deaminase fusion gene. J Biol Chem, 1989;264:7418-7424
25． Bowtell DD, Cory S, Johnson GR, et al. Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes. J Virol, 1988; 62:2464-2473
26． Emerman M, Temin HH. Genes with promoter in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell, 1984;39:459-467
27． Ghattas IR, Sanes JR, Majors JE. The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in the culture cells and in embros. Mol Cell Biol, 1991; 11:5548-5859
28． Karger BD, Feigner PL, Gadek TR, et al. Evaluation of procedures for transfection using lipofectin regeagent. Focus, 1990; 12: 25-29
29． Lu L, Ge Y, Li ZH, et al. CD34 stem/progenitor cells purified from cryopreserved normal cord blood can be transduced with high efficiency by a retroviral vector and expanded ex vivo with stable integration and expression of Fanconi anemia complementation C gene. Cell Transplant, 1995; 4:493-503
30． 彭秀玲，袁汉英．基因工程实验技术．第一版．湖南科学技术山版社，1987，57
31． Hawley RG, Hawley TS, Fong AZ, et al. Thrombopoietic potential and serial repopulating
ability of murine hematopoietic stem cells constitutively expressing intedeukin 11. Proc Natl Acad Sci USA, 1996; 93:10297-10302
32. Keller G, Wall C, Fong AZ, et al. Overexpression of HOX11 leads to the immortalization of embryonic precursors with both primitive and definitive hematopoietic potential. Blood, 1998; 92:877-897
33. Nienhuis AW, McDomagh KT, Bodine DM, et al. Gene transfer into hematopoietic stem cell. Cancer, 1991; 67(10 Suppl):2700-2704
34. Robbins PD, Ghivizzani SC. Viral vectors for gene therapy. Pharmacol Ther, 1998;80:35-47
35． 李家增，王鸿利，韩忠朝主编．血液实验学．上海科学技术出版社，1997，1st Ed： 126-128
36. Niks M, Otto M, Busova B, Stefanovic J. Quantification of proliferative and suppressive responses of human T lymphocytes following ConA stimulation.J Immunol Methods, 1990; 126: 263-271
37. Van Buskirk AM, Adams PW, Orosz GC. Nonradioactive alternative to clinical mixed lymphocyte reaotion. Hum Immumol, 1995; 43:38
38． 刘海鹰，周玲，曾毅．检测Epstein-Barr病毒特异性细胞毒性T淋巴细胞方法的建立及其初步研究．中华实验和临床病毒学杂志，1998；12：357-360
39． 何金生，宗庭益．LDH释放法检测NK细胞活性的方法研究．中国实验临床免疫学杂志，1996；8：10-12
40. Bellone G, Valiante NM, Viale O, et al. Regulation of hematopoiesis in vitro by alloreactive natural killer cell clones. J Exp Med, 1993; 177:1117-1120
41. Moretta A. A major histocompatibility complex class 1 specific receptors on human natural killer and T lymphocytes. Immunol Rev, 1997; 155: 105-106
42. Loung NT, Uhrberg M. KIR expression shapes cytotoxic repertoires: a developmental program of survival. Trends Immumol, 2002;23:71-75
43. Colonna M. Natural killer cell receptors specific for MHC class 1 molecules. Curt Opin Immunol, 1996; 8:101
44. Warner NL, Moore MA, Metcalf D. A transplantable myeiomonocytic leukemia in BALB-c mice: cytology, karyotype, and muramidase content. J Natl Cancer Inst. 1969; 43(4): 963-982
45. Tsukada N, Kobata T, Aizawa Y, et al. Graft versus leukemia effected graft versus host
disease can be differentiated by cytotoxic mechanisms in a murine model of allogeneic bone marrow transplantation. Blood, 1999;93: 2738-2747
46． 李家增，王鸿利，韩忠朝主编．血液实验学．上海科学技术出版社，1997，1st Ed：440-441
47． Daniel HF, Kazuhiro K, Rhett S, et al. Donor CD4 enriched cells of Th2 cytokine phenotype regulate graft versus host disease without impaired allo-genetic graft in sublethally irradiated mice. Blood, 1994; 84:3540-3543
48． Daniel HF, Bernard W, Michael L, et al. Non-host-reactive donor CD8+ T cells of Tc2 phenotype potently inhibit marrow graft rejection. Blood, 1998;91:4045-4050
49． Schmidt H, Ehninger G, Dopfer R, et al. Correlation between low CSA plasma concentration and severity of acute GvHD in bone marrow transplantation. Blut, 1988;57: 139-142
50． Norin AJ, Brewer RJ, Lui Q, et al. Donor antigen-independent effector mechanisms of graft rejection: a novel cell surface target of IL-2-activated natural killer cells. Transplant Proc, 1999;31:787-788