FasL逆转录病毒体系的构建及诱导大鼠肝移植免疫耐受的实验研究
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摘要
目的：（1）构建大鼠FasL基因逆转录病毒体系，并研究FasL在
包装细胞PA317中的表达；（2）用PA317/pLXSN-FasL+病毒上清体外
转染肿瘤细胞，以观察其对肿瘤细胞的诱导凋亡作用；（3）将FasL基
因导入供体源的树突状细胞，注入受体体内后，观察其对肝移植大鼠
生存期的影响，并探索转FasL基因治疗诱导免疫耐受的可能机理。
方法：（1）将含FasL基因的质粒pBL-KA15用XhoⅠ酶切，获得
大鼠全长FasL cDNA片段，将该片段与同样经XhoⅠ酶切的逆转录
病毒载体pLXSN在T4嗜菌体DNA连接酶作用下进行相同突出端的
连接反应，并鉴别其正反向，将正向单拷贝插入的重组子pLXSN-FasL
用脂质体转染试剂盒导入双嗜性逆转录病毒包装细胞PA317中，用
G418筛选，获得病毒滴度高的抗性克隆，命名为PA317/pXSN-
FasL+；用PCR及RT-PCR方法检测该包装细胞是否有FasL基因整
合及mRNA的表达，并以流式细胞仪检测包装细胞表面FasL的表达。
（2）将PA317/pLXSN-FasL+体外转染淋巴瘤细胞株Jurkat、Raji、Daudi，
肝癌细胞株HepG-2及骨髓瘤细胞株SP2/0，用流式细胞仪分别检测
这些细胞株表面Fas及FasL的表达，并用Annexin V-PI法检测这些
细胞的凋亡，通过细胞增殖实验观察PA317/pLXSN-FasL+病毒上清
对Raji、Daudi、SP2/0及HepG-2细胞株的生长抑制。(3）首先建立“二
袖套法”大鼠肝移植模型，并在袖套管制作，受体麻醉，肝上下腔
静脉、门静脉及肝下下腔静脉吻合等方面进行技术改进，行供体为SD
大鼠、受体为Wistar大鼠的肝移植48例，并分为4组：①对照组12
例，不作任何治疗；②mdrl组12例，移植前腹腔注射转mdrl基因
的DC细胞；③CsA治疗组，术后给予环孢霉素（CsA）治疗；④转FasL
南京医科大学悍十学位论文
基因治疗组，移植前腹腔注射转FasL基因的DC细胞。肝移植术后
3d及 7d分别杀死各组4只大鼠，取出外周血及肝脏，检测肝功能，
半定量 RT－P皿检测移植肝 FasI虬、IL4 的 InRNA表达，TU’N’E‘
法检测肝细胞及外周血淋巴细胞凋亡；光镜及透射电镜观察移植肝的
病理及超微结构，同时观察各组大鼠肝移植后生存期。
结果：O 重组子经酶切鉴定后获得正向单拷贝插入
子，脂质体介导法导入N3细胞后，m18 筛选出抗性克隆，命名
为 PA3 7MSNlasL＋，测得病毒滴度为 4．7 xlo’CFUth。PCR及
RTPCR证实 PA317细胞有 FasL基因整合及 wA表达，流式细
胞仪检测出PA3 17包装细胞表面有高强度的F虬分子表达。仅）流式
细胞仪检测 Jim’ai、oaudi、Rat、SPz／0及 HyG上细胞株 eas阳性率
分别为 99．8％、33．9％、68＊％、72％、sl．7％Z用 PA317／pLXSN－FasL＋
病毒上滑转染 oaucti’ Rat’ sn：vo及 Hpcrz细胞株后，east阳性率
分别为 99．l％、99．4％、86．l％、72．3％，AnneAn VFI法捡测细胞凋亡
率分别为 30．50、31．30、12．l％、19．40；细胞增殖实验提示转 FasL
后肿瘤细胞株生长受到明显抑制。o）对“二袖套法”大鼠肝移植进
行技术改进后，无肝期明显缩短，肝移植成功率明显提高；供体SD
大鼠的骨髓经体外扩增后，获得表型特异的DC，将之转染FasL基
因后移植前受体腹腔注射，结果发现对照组及mdrl组移植后945d
死亡，血清胆红素及转氨酶明显高于 CsA组及 FasL组，病埋学及透
射电镜提示有肝细胞变性、坏k，6 CsA fAA F吼组肝细胞免疫排
斥较轻，肝移植大鼠生存期已超过4个月；半定量RTPCR结果示
对照组及mdrl组术后7d F嘛、IL《2表达增高，而CsA组及F札
组F虬及几12则呈不表达或低表达；刀l田L法检测发现FIL组
有明显外周血淋巴细胞凋亡，透射电镜结果也证实肝组织内有凋亡的
淋巴细胞。
结论：＊）建立的 PA317／pLXSNFasL＋逆转录病毒体系能有效地
表达 F虬八 PA3 7／pLX狲F虬＋能有效地诱导 Fas高表达肿瘤细
胞凋亡，井对这些肿瘤细胞的生长有抑制作用。o准D大鼠为供体，
4
南京医科大学博士学位论文
Wistar大鼠为受体的肝移植是一个高排异组合，是用于研究移植耐受
埋想的模型；经转染FasL基因的DC治疗后可有效地诱导肝移植免
疫耐受，其机理是诱导活化T细胞凋亡，并一定程度地减少ILJ 的
分泌。
The experimental study on inducement of rat liver transplantation tolerance by constructing retroviral vector contained FasL gene
Sun Bei-cheng Wang Xue-hao
Abstract
Objectives The purposes of this study are (1) to construct the retroviral vector containing rat FasL gene and study FasL-expression in the packaging cell system PA3 17; (2) to transfect the tumor cell with supematant of PA3 1 7/pLXSN-FasL+ in vitro and observe the apoptosis in these tumor cell; (3)to introduce FasL gene into the dendritic cells from the donor, then inject the cells intraperitoneally into the recipient ,observe its survival time of the liver transplantation rat, and search the possible mechanism of immune tolerance induced by transfecting FasL gene therapy.
Methods (1)The plasmid pBL-KA15 containing FasL gene was cut with Xho I , and the rat full-length FasL cDNA was obtained at the same time, then subcloned it into the retroviral vector (pLXSN) ligated by T4 DNA ligase,and acquired pLXSN-FasL+ recombinants. Subsequently, pLXSN-FasL+ which in direct inserting were transfected into the amphotropic PA3 17 packaging cells using Lipofectamine, then we acquired G4 18 resistence clones ,and named it PA3 1 7/pLXSN-FasL+ .The polymerase chain reaction (PCR) and RT-PCR were used to detennine whether there were integration of FasL gene and expression of mRNA in the packing cell. The FasL expression on the surface of the packing cell was determined with flow cytometry (FCM). (2)The lymphoma cell strain Jurkat~ Raji~ Daudi, hepatocellular carcinoma cell strain HepG-2 and myeloma cell strain SP2IO were transfected with PA3 I 7/pLXSN-FasL+ in
6
vbo. The Fas and FasL eXPression on the sdse of these cells was
dMed with fiow Cy'tOmny (FCM), and the aPoPtosis of these cel1s
was observed with Annexin V-PI methd, the growth allbihon treated
with suPeman of PA317/PLXSN-FasL+ tOWed the. Rai. Daudi.
HePG2 and SP2/0 ceIls was evaIuated by the cell Proliboon test.(3)
Wc liver twlWon in 48 Wister rats Which receiVed SD 1iver
wer established by using "the tWoChff tecboque" with some
modifications in cuff tUbe, recipients anaeSthesia and the anastOmoses of
suPrahepatic vena, pond vein, in~atc vena cava, then, the rats wer
randoInly divided into 4 grouPs:@the contrl grou (n=12); @mdr
grou(n==l2) Which treated with Mc cells transfected with mdrl gene;
@CsA groUPbe12) haed with cyclOsPorine;@FasL grouP(n=12)
treated wdri the deWc cells tafected with FasL gene by tweritoneaI
injection. 4 rats in each gouPs wer killed on the POD3 and POD 7
resPectively The liver and periPheral blood were obtaind to detendne
liver functiOty W Wssion of Fas. FasL. K-l2 in the gh with
sendopantificational RTPCR, the aPoPtosis in hepatocytes and periPherl
lymPhcyte wer eValUated by W method, the pathological bocter
and ultrasboCtUr of the gh were observed through microscoPe and
electron ndcroscoPe.MeanWhile, the post operaon sUrVval time of each
grou was recorded.
ReSulis (lW were FasL direct inserting with single coPy in the
combinants of PLXSMasL+, and it was namd pLXSN/FasL+. The titer
of vha in its suPematan reaChe 4.7 X 107CFUffi. It was PrOVed by PCR
and RT-PCR tha there wer the integraion of FasL gene and exPression of
InRNA in the paCkin cell. The FasL eXPression with stron intenSity on
the lere of the Packin cell was determined with FCM.(2)The Fas
posthe the of the cell sha all. Daudi. Rai. SP2/0 and HePG2
detennind wh FCM was 99.8%. 33.9%. 68.6%. 7.2%. 51.7%,
7
respectively; and the FasL wtssion in the cells ase. Rai. SP2/0 and
HePG2 tranSfected wtth FasL gene Was 99.l%. 99.4%. 86.l%. 72.3%,
resPectivelyand the aPOPtsis rate was 30.5%' 3l.3%' l2.l%'
19.4O/,resPectively;the cell PrOliedn eXPerimen suggested that the
grOwth of tUInr cell sha was reshad Signiflcantiy (3)Afor some
modificationS in the lha forlantation with ..the tWO-cufftedrique",
the anhePatc period was reduced sighflcanly the survival rate of liver
wtlantation in rats was wtd reMly; the marrw from SD rat
was amPlifled i
包装细胞PA317中的表达；（2）用PA317/pLXSN-FasL+病毒上清体外
转染肿瘤细胞，以观察其对肿瘤细胞的诱导凋亡作用；（3）将FasL基
因导入供体源的树突状细胞，注入受体体内后，观察其对肝移植大鼠
生存期的影响，并探索转FasL基因治疗诱导免疫耐受的可能机理。
方法：（1）将含FasL基因的质粒pBL-KA15用XhoⅠ酶切，获得
大鼠全长FasL cDNA片段，将该片段与同样经XhoⅠ酶切的逆转录
病毒载体pLXSN在T4嗜菌体DNA连接酶作用下进行相同突出端的
连接反应，并鉴别其正反向，将正向单拷贝插入的重组子pLXSN-FasL
用脂质体转染试剂盒导入双嗜性逆转录病毒包装细胞PA317中，用
G418筛选，获得病毒滴度高的抗性克隆，命名为PA317/pXSN-
FasL+；用PCR及RT-PCR方法检测该包装细胞是否有FasL基因整
合及mRNA的表达，并以流式细胞仪检测包装细胞表面FasL的表达。
（2）将PA317/pLXSN-FasL+体外转染淋巴瘤细胞株Jurkat、Raji、Daudi，
肝癌细胞株HepG-2及骨髓瘤细胞株SP2/0，用流式细胞仪分别检测
这些细胞株表面Fas及FasL的表达，并用Annexin V-PI法检测这些
细胞的凋亡，通过细胞增殖实验观察PA317/pLXSN-FasL+病毒上清
对Raji、Daudi、SP2/0及HepG-2细胞株的生长抑制。(3）首先建立“二
袖套法”大鼠肝移植模型，并在袖套管制作，受体麻醉，肝上下腔
静脉、门静脉及肝下下腔静脉吻合等方面进行技术改进，行供体为SD
大鼠、受体为Wistar大鼠的肝移植48例，并分为4组：①对照组12
例，不作任何治疗；②mdrl组12例，移植前腹腔注射转mdrl基因
的DC细胞；③CsA治疗组，术后给予环孢霉素（CsA）治疗；④转FasL
南京医科大学悍十学位论文
基因治疗组，移植前腹腔注射转FasL基因的DC细胞。肝移植术后
3d及 7d分别杀死各组4只大鼠，取出外周血及肝脏，检测肝功能，
半定量 RT－P皿检测移植肝 FasI虬、IL4 的 InRNA表达，TU’N’E‘
法检测肝细胞及外周血淋巴细胞凋亡；光镜及透射电镜观察移植肝的
病理及超微结构，同时观察各组大鼠肝移植后生存期。
结果：O 重组子经酶切鉴定后获得正向单拷贝插入
子，脂质体介导法导入N3细胞后，m18 筛选出抗性克隆，命名
为 PA3 7MSNlasL＋，测得病毒滴度为 4．7 xlo’CFUth。PCR及
RTPCR证实 PA317细胞有 FasL基因整合及 wA表达，流式细
胞仪检测出PA3 17包装细胞表面有高强度的F虬分子表达。仅）流式
细胞仪检测 Jim’ai、oaudi、Rat、SPz／0及 HyG上细胞株 eas阳性率
分别为 99．8％、33．9％、68＊％、72％、sl．7％Z用 PA317／pLXSN－FasL＋
病毒上滑转染 oaucti’ Rat’ sn：vo及 Hpcrz细胞株后，east阳性率
分别为 99．l％、99．4％、86．l％、72．3％，AnneAn VFI法捡测细胞凋亡
率分别为 30．50、31．30、12．l％、19．40；细胞增殖实验提示转 FasL
后肿瘤细胞株生长受到明显抑制。o）对“二袖套法”大鼠肝移植进
行技术改进后，无肝期明显缩短，肝移植成功率明显提高；供体SD
大鼠的骨髓经体外扩增后，获得表型特异的DC，将之转染FasL基
因后移植前受体腹腔注射，结果发现对照组及mdrl组移植后945d
死亡，血清胆红素及转氨酶明显高于 CsA组及 FasL组，病埋学及透
射电镜提示有肝细胞变性、坏k，6 CsA fAA F吼组肝细胞免疫排
斥较轻，肝移植大鼠生存期已超过4个月；半定量RTPCR结果示
对照组及mdrl组术后7d F嘛、IL《2表达增高，而CsA组及F札
组F虬及几12则呈不表达或低表达；刀l田L法检测发现FIL组
有明显外周血淋巴细胞凋亡，透射电镜结果也证实肝组织内有凋亡的
淋巴细胞。
结论：＊）建立的 PA317／pLXSNFasL＋逆转录病毒体系能有效地
表达 F虬八 PA3 7／pLX狲F虬＋能有效地诱导 Fas高表达肿瘤细
胞凋亡，井对这些肿瘤细胞的生长有抑制作用。o准D大鼠为供体，
4
南京医科大学博士学位论文
Wistar大鼠为受体的肝移植是一个高排异组合，是用于研究移植耐受
埋想的模型；经转染FasL基因的DC治疗后可有效地诱导肝移植免
疫耐受，其机理是诱导活化T细胞凋亡，并一定程度地减少ILJ 的
分泌。
The experimental study on inducement of rat liver transplantation tolerance by constructing retroviral vector contained FasL gene
Sun Bei-cheng Wang Xue-hao
Abstract
Objectives The purposes of this study are (1) to construct the retroviral vector containing rat FasL gene and study FasL-expression in the packaging cell system PA3 17; (2) to transfect the tumor cell with supematant of PA3 1 7/pLXSN-FasL+ in vitro and observe the apoptosis in these tumor cell; (3)to introduce FasL gene into the dendritic cells from the donor, then inject the cells intraperitoneally into the recipient ,observe its survival time of the liver transplantation rat, and search the possible mechanism of immune tolerance induced by transfecting FasL gene therapy.
Methods (1)The plasmid pBL-KA15 containing FasL gene was cut with Xho I , and the rat full-length FasL cDNA was obtained at the same time, then subcloned it into the retroviral vector (pLXSN) ligated by T4 DNA ligase,and acquired pLXSN-FasL+ recombinants. Subsequently, pLXSN-FasL+ which in direct inserting were transfected into the amphotropic PA3 17 packaging cells using Lipofectamine, then we acquired G4 18 resistence clones ,and named it PA3 1 7/pLXSN-FasL+ .The polymerase chain reaction (PCR) and RT-PCR were used to detennine whether there were integration of FasL gene and expression of mRNA in the packing cell. The FasL expression on the surface of the packing cell was determined with flow cytometry (FCM). (2)The lymphoma cell strain Jurkat~ Raji~ Daudi, hepatocellular carcinoma cell strain HepG-2 and myeloma cell strain SP2IO were transfected with PA3 I 7/pLXSN-FasL+ in
6
vbo. The Fas and FasL eXPression on the sdse of these cells was
dMed with fiow Cy'tOmny (FCM), and the aPoPtosis of these cel1s
was observed with Annexin V-PI methd, the growth allbihon treated
with suPeman of PA317/PLXSN-FasL+ tOWed the. Rai. Daudi.
HePG2 and SP2/0 ceIls was evaIuated by the cell Proliboon test.(3)
Wc liver twlWon in 48 Wister rats Which receiVed SD 1iver
wer established by using "the tWoChff tecboque" with some
modifications in cuff tUbe, recipients anaeSthesia and the anastOmoses of
suPrahepatic vena, pond vein, in~atc vena cava, then, the rats wer
randoInly divided into 4 grouPs:@the contrl grou (n=12); @mdr
grou(n==l2) Which treated with Mc cells transfected with mdrl gene;
@CsA groUPbe12) haed with cyclOsPorine;@FasL grouP(n=12)
treated wdri the deWc cells tafected with FasL gene by tweritoneaI
injection. 4 rats in each gouPs wer killed on the POD3 and POD 7
resPectively The liver and periPheral blood were obtaind to detendne
liver functiOty W Wssion of Fas. FasL. K-l2 in the gh with
sendopantificational RTPCR, the aPoPtosis in hepatocytes and periPherl
lymPhcyte wer eValUated by W method, the pathological bocter
and ultrasboCtUr of the gh were observed through microscoPe and
electron ndcroscoPe.MeanWhile, the post operaon sUrVval time of each
grou was recorded.
ReSulis (lW were FasL direct inserting with single coPy in the
combinants of PLXSMasL+, and it was namd pLXSN/FasL+. The titer
of vha in its suPematan reaChe 4.7 X 107CFUffi. It was PrOVed by PCR
and RT-PCR tha there wer the integraion of FasL gene and exPression of
InRNA in the paCkin cell. The FasL eXPression with stron intenSity on
the lere of the Packin cell was determined with FCM.(2)The Fas
posthe the of the cell sha all. Daudi. Rai. SP2/0 and HePG2
detennind wh FCM was 99.8%. 33.9%. 68.6%. 7.2%. 51.7%,
7
respectively; and the FasL wtssion in the cells ase. Rai. SP2/0 and
HePG2 tranSfected wtth FasL gene Was 99.l%. 99.4%. 86.l%. 72.3%,
resPectivelyand the aPOPtsis rate was 30.5%' 3l.3%' l2.l%'
19.4O/,resPectively;the cell PrOliedn eXPerimen suggested that the
grOwth of tUInr cell sha was reshad Signiflcantiy (3)Afor some
modificationS in the lha forlantation with ..the tWO-cufftedrique",
the anhePatc period was reduced sighflcanly the survival rate of liver
wtlantation in rats was wtd reMly; the marrw from SD rat
was amPlifled i
引文
1． Wood KJ. New concepts in tolerance.Clin Transplant. 1996; 10(1) :93-99
2． Field EH,Gao Q,Chen NX,et al.Balancing the immune system for tolerance,Transplantation, 1997;64(1) : 1-7
3． Pennisi E. Teetering on the brink of danger.Science. 1996; 271(5256) :1665-1667．
4． Ridge JR,Fuchs EJ,Matziger P.Neonatal tolerance revisited:Turning on newborn T cells with dendritic cells.Science,1996;271(5256) :1723-1726
5． Sarzotti M,et al. Induction of protective CTL responses in newborn mice by a murine retrovirus.Science. 1996;271(5256) :1726-1728．
6． Benichou G, Tam RC, Soares LR, et al. Indirect T-cell allorecognition: perspectives for peptide-based therapy in transplantation.Immunol Today. 1997;18(2) :67-71．
7． Freeman GJ,Borriello F,Hodes RJ,et al.Murine B7-2,an alternative CTLA4 counter-receptor that costimulation T cell proliferation and interleukin-2 production.J Exp Med, 1993; 178:2185-2192
8． Lin H,Rathmell JC,Gray GS,et al.Cytoxic T lymphocyte antigen 4(CTLA4) blockade accelerates the acute rejection of cardiac allograft in CD28-deficient mice.CTLA4 can function independently to CD28． J Exp Med,1998;188:199-204
9． Larsen CP,Alexander DZ,Hollenbaugh D,et al.CD40-gp39 interaction play a critical role during allograft rejection.Suppression of allograft rejection by blockade of CD40-gp39 pathway.Transplantation,1996;61:4-9
10． Judge TA,Wu ZH,Zheng XG,et al.The role of CD80,CD8,and CTLA4 in alloimmune responses and induction of long-term allograft survival.J Immunol,1999;162:1947-1951
11． Carlquist JF,et al. Cytokines and rejection of mouse cardiac allografts. Transplantation,1996;62(8) :1160-1166
12． Griffith TS, Yu X, Herndon JM, et al. CD95-induced apoptosis of lymphocytes in an immune privileged site induces immunological tolerance. Immuniry,1996;5(1) :7-16
13． Kalechman Y, Strassmann G, Albeck M, et al. Up-regulation by ammonium trichloro(dioxoethylene-0,0') tellurate (AS101) of Fas/Apo-1 expression on B16 melanoma cells: implications for the antitumor effects of AS101 J Immunol, 1998; 161(7) : 3536-3542
14． Lau HT,Yu M,Fontana A,et al.Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice.Scince,1996;273:109-112
15． George JF, et al. An essential role for Fas ligand in transplantation tolerance induced by donor bone marrow. Nat Med, 1998; 4(3) : 333-335
16． Li XK,Okuyama A,Tamura M,et al.Prolonged survival of recipient rats with Fas Ligand transfected liver allografts by using HVJ-liposome.Transpiantation Proc, 1998;30:943
17． Min WP,Gorczynski R,Huang XY,et al.Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival.J Immunol,2000; 164:161-167
18． Starzl TE, Demetris AJ, Murase N, et al. Cell migration, chimerism, and graft acceptance.Lancet. 1992;339(8809) : 1579-1582
19． Wessman M, Popp S, Ruutu T, et al. Detection of residual host cells after bone marrow transplantation using non-isotopic in situ hybridization and karyotype analysis.Bone Marrow Transplant, 1993; 11 (4) :279-284
20． Starzl TE, Demetris AJ, Murase N ,et al. Donor cell chimerism permitted by immunosuppressive drugs: a new view of organ transplantation.Immunol Today,1993; 14(6) :326-332
21． Starzl TE, Demetris AJ, Trucco M, et al. Cell migration and chimerism after whole-organ transplantation: the basis of graft acceptance.Hepatology,1993;17(6) :1127-1152
22． Ardavin C, Wu L, Li CL, et al. Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature,1993;362(6422) :761-763
23． Kyewski BA, Fathman CG, Rouse RV. Intrathymic presentation of circulating non-MHC antigens by medullary dendritic cells. An antigen-dependent microenvironment for T cell differentiation.J Exp Med,1986;163(2) :231-246
24． Suss G, Shortman K. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J Exp Med, 1996; 183(4) : 1789-1796
25． Lu L, McCaslin D, Starzl TE, et al. Bone marrow-derived dendritic cell progenitors (NLDC 145+, MHC class Ⅱ+, B7-1dim, B7-2-) induce alloantigen-specific hyporesponsiveness in murine T lymphocytes. Transplantation,1995;60(12) :1539-1545
26． Mcrae BL, Semnani RT, Hayes MP, et al. Type I IFNs inhibit human dendritic cell IL-12 production and Th1 cell development. J Immunol,1998;160(9) :4298-4304
27． Finkelman FD, Lees A, Birnbaum R, et al. Dendritic cells can present antigen in vivo in a tolerogenic or immunogenic fashion. J Immunol,1996;157(4) :14061414
28． Steptoe RJ, Thomson AW. Dendritic cells and tolerance induction Clin Exp Immunol,1996;105(3) :397-402
29． 龚非力.器官移植基础研究的现状和展望.中华器官移植杂志,2000;21(4) :195
2． Field EH,Gao Q,Chen NX,et al.Balancing the immune system for tolerance,Transplantation, 1997;64(1) : 1-7
3． Pennisi E. Teetering on the brink of danger.Science. 1996; 271(5256) :1665-1667．
4． Ridge JR,Fuchs EJ,Matziger P.Neonatal tolerance revisited:Turning on newborn T cells with dendritic cells.Science,1996;271(5256) :1723-1726
5． Sarzotti M,et al. Induction of protective CTL responses in newborn mice by a murine retrovirus.Science. 1996;271(5256) :1726-1728．
6． Benichou G, Tam RC, Soares LR, et al. Indirect T-cell allorecognition: perspectives for peptide-based therapy in transplantation.Immunol Today. 1997;18(2) :67-71．
7． Freeman GJ,Borriello F,Hodes RJ,et al.Murine B7-2,an alternative CTLA4 counter-receptor that costimulation T cell proliferation and interleukin-2 production.J Exp Med, 1993; 178:2185-2192
8． Lin H,Rathmell JC,Gray GS,et al.Cytoxic T lymphocyte antigen 4(CTLA4) blockade accelerates the acute rejection of cardiac allograft in CD28-deficient mice.CTLA4 can function independently to CD28． J Exp Med,1998;188:199-204
9． Larsen CP,Alexander DZ,Hollenbaugh D,et al.CD40-gp39 interaction play a critical role during allograft rejection.Suppression of allograft rejection by blockade of CD40-gp39 pathway.Transplantation,1996;61:4-9
10． Judge TA,Wu ZH,Zheng XG,et al.The role of CD80,CD8,and CTLA4 in alloimmune responses and induction of long-term allograft survival.J Immunol,1999;162:1947-1951
11． Carlquist JF,et al. Cytokines and rejection of mouse cardiac allografts. Transplantation,1996;62(8) :1160-1166
12． Griffith TS, Yu X, Herndon JM, et al. CD95-induced apoptosis of lymphocytes in an immune privileged site induces immunological tolerance. Immuniry,1996;5(1) :7-16
13． Kalechman Y, Strassmann G, Albeck M, et al. Up-regulation by ammonium trichloro(dioxoethylene-0,0') tellurate (AS101) of Fas/Apo-1 expression on B16 melanoma cells: implications for the antitumor effects of AS101 J Immunol, 1998; 161(7) : 3536-3542
14． Lau HT,Yu M,Fontana A,et al.Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice.Scince,1996;273:109-112
15． George JF, et al. An essential role for Fas ligand in transplantation tolerance induced by donor bone marrow. Nat Med, 1998; 4(3) : 333-335
16． Li XK,Okuyama A,Tamura M,et al.Prolonged survival of recipient rats with Fas Ligand transfected liver allografts by using HVJ-liposome.Transpiantation Proc, 1998;30:943
17． Min WP,Gorczynski R,Huang XY,et al.Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival.J Immunol,2000; 164:161-167
18． Starzl TE, Demetris AJ, Murase N, et al. Cell migration, chimerism, and graft acceptance.Lancet. 1992;339(8809) : 1579-1582
19． Wessman M, Popp S, Ruutu T, et al. Detection of residual host cells after bone marrow transplantation using non-isotopic in situ hybridization and karyotype analysis.Bone Marrow Transplant, 1993; 11 (4) :279-284
20． Starzl TE, Demetris AJ, Murase N ,et al. Donor cell chimerism permitted by immunosuppressive drugs: a new view of organ transplantation.Immunol Today,1993; 14(6) :326-332
21． Starzl TE, Demetris AJ, Trucco M, et al. Cell migration and chimerism after whole-organ transplantation: the basis of graft acceptance.Hepatology,1993;17(6) :1127-1152
22． Ardavin C, Wu L, Li CL, et al. Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature,1993;362(6422) :761-763
23． Kyewski BA, Fathman CG, Rouse RV. Intrathymic presentation of circulating non-MHC antigens by medullary dendritic cells. An antigen-dependent microenvironment for T cell differentiation.J Exp Med,1986;163(2) :231-246
24． Suss G, Shortman K. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J Exp Med, 1996; 183(4) : 1789-1796
25． Lu L, McCaslin D, Starzl TE, et al. Bone marrow-derived dendritic cell progenitors (NLDC 145+, MHC class Ⅱ+, B7-1dim, B7-2-) induce alloantigen-specific hyporesponsiveness in murine T lymphocytes. Transplantation,1995;60(12) :1539-1545
26． Mcrae BL, Semnani RT, Hayes MP, et al. Type I IFNs inhibit human dendritic cell IL-12 production and Th1 cell development. J Immunol,1998;160(9) :4298-4304
27． Finkelman FD, Lees A, Birnbaum R, et al. Dendritic cells can present antigen in vivo in a tolerogenic or immunogenic fashion. J Immunol,1996;157(4) :14061414
28． Steptoe RJ, Thomson AW. Dendritic cells and tolerance induction Clin Exp Immunol,1996;105(3) :397-402
29． 龚非力.器官移植基础研究的现状和展望.中华器官移植杂志,2000;21(4) :195