hDAF转基因猪器官移植实验研究
摘要
移植用供体器官在全世界范围的严重缺乏，已经促使在此领域的学者，对异种移植产生
了极大的兴趣。目前，转基因猪由于可以无限量繁殖，以及在解剖，生理很多方面和
人的相似性原因，已经被认为是人供体器官的可能来源。在完全不同种系之间的异种移
植，如，猪-狒狒，因为受体体内预先存在的天然抗体与供体器官血管内皮细胞上的α
-galactosyl(α-Gal)糖表位结合，随后发生补体系统的激活，而最终将导致超急性排斥
（HAR）。
补体的激活可以被一组有种间特异性的蛋白-补体激活调解因子（RCAs），在可溶性状态和
细胞表面状态加以抑制。在体外表达人补体激活调解因子如，人衰变加速因子（hDAF），在
猪细胞表面表现了保护细胞免于人补体的溶解。出于此方面的考虑，剑桥大学通过显微
注射的方法，注射一段长6.5kb的DAF微小基因于猪的受精卵内，已经成功的培植出带有
hDAF的转基因猪。既往的实验已经表明，当hDAF转基因的猪心脏异位移植入cynomolgus
猴腹内后，心脏没有显示超急性排斥的表现，而且通过同时应用其他的免疫抑制剂如
：cyclosporine,cyclophosphamide和methylpridnisolone可以获得生存期的延长。
在人类和高等灵长类动物体内的异种天然抗体的受体主要是Gal(Galα1-3Gal)。这种抗体
和受体的结合被认为是对猪异种移植器官超急性排斥的起点。很多发现也证明anti-Galα
1-3Gal抗体是异种天然抗体的主要部份而且对超急性排斥的病理起决定性的重要作用。
目前已经有很多实验证明，去除预先存在的天然抗体能够克服异种移植的超急性排斥，
尽管此时补体活性仍然保持在正常水平的90％，超急性排斥仍可以被抑制。去除天然抗
体的方法包括，体外连接异种器官吸附抗体；含Galα1-3Galbl-4GlcNAc吸附柱预先吸附
抗体等。
NOVARTIS目前已经生产出多价可注射的QGAL残基pAS）可以直接注射吸附抗体。
以往的研究之所以用Cyllolllolgt1S猴作为11DAF转基因猜的心脏异位移植的受体主要是由于
数量多的原因。然而，这种猴浪少有体重能够超过5k8的个体。因此，由于器官体积和
技术上的原因，该种灵长类动物不适合作为hDAF转基因猪器官的移植实验受体。所以
必须寻找一种比较大的灵长类幼物作为受体。恨多大的灵长类动物已经濒于灭绝，道义
上不允济用于实验研究。体积较大，而且数量众多的另外一种灵长类动物－拂狒被认为
是最适台的受体幼物。然而，已经有实验证明hDAF下凋非人灵长类动物补体活性的能
力不同。11DAF下调人血浆总补体活性的84％，CyllOllolgUS猴72％，拂拂朴体活性69％，和
恒河猴28％。明显恒河猴不适合作为受体，但是在CyllO。mIgtlS猴和拂狭之闯的3％的差别
是否会导致超急性排斥还不清笼，所以用狒狒模型作为hDAF转基因猜的实验受体成为
其识。本研究的主要目的有：（1）离体肝脏，肾脏灌注实验研究设计目的在干检验
IIDAF转基因保护肝脏，肾脏免于超急性抹斥的理论。（2）活体外肝脏，肾脏支持肝衰竭
及无’署模型的研究的目的是为临床应用提供技术经验，及检验11DAF转基因的保护作用。
（3）’肾脏移植研究的实施在于确定一个涅想的免疫抑制剂方案以达到异种移植器官的长期
存活并且进一步证实11DAFty基因能够保护’肾脏免于超急性抹斥反应。O）检测最新免疫
抑制剂，多价可注射Q名al残基闪AS）与其他免疫抑制剂同时应用能否显著降低预存的天
然抗体并且改善川4XR。
第一部分11DAF转基因猜肝脏离体滔注的实验研究
l，目的
使用新鲜人血灌注普通猪肝和转基因猜肝；以及用猪血滔注猜肝脏以比较、检验
1。DAF转基因保护异种移植肝脏免干超急性排斥反应的效果。
2，方法
全麻下取供体肝并用UW液经肝动脉和门静脉灌注。行脏忌。管内插管。冷缺血时间少于
］小时。灌往循环采用一个生物PUMP，氧会器，和一个热交换器，用一个热水浴箱保存
肝脏。血液由门静脉开始滔注，经肝脏至下腔静脉插管，储血池，膜氧台器，热交换器，
6
然后经门静脉回肝脏。ACT水平用肝素保持高于 1000。
3，结果
我们做了 16例灌注：1组：猪血－猎肝则叫h 2组：人血0AFffi肝州＜八 3组：人血－普通
猎肝…一人第一组病理显禾正常。第3组表现明显的出血，血小板沉积，纤维蛋白沉积。
第2组表现相对正常的组织结构，一例DAF肝脏表规轻度的血管和内皮变化。在第H组
和第三组均见弥撒的内皮细胞的IgG，lgM沉积。叫。时电镜显示：2组和3组均有内皮损伤。
叫。时，第3组显示内皮细胞完全丢失，伴随明显的纤维蛋白沉积和血小板聚集。相反，
DAF肝，在叫。时多数的肝脏表现正常的组织结构，偶见血小板和红细胞聚集和内皮细
胞孪缩。
4；结论
该实验肯定了DAF转基因对灌注肝脏超急。往排斥反应的保护作用。为使用DAF转基
因猪干将来临床的试验提供了有力的理论基础。使用此异种灌注系统，人血滔注转基因
猜肝脏与灌注普通猜肝比较；在脉管炎；血栓，出血等方面均有显著的改善。转基因猪
肝脏同时表现了更好的肝脏功
The world-wide shortage of human organs available for transplantation has led to an interest in
xenotransptantation.The pig has been suggested as a possible source of organs because of its plentiful
supply and many anatomic and phsiological similarities with human hcings.Xenotransplantation between
widely disparate species such as pig to primate results in hypercute rejection because of the binding of
preformed natural antibodies to 0. -galactosyl carbohydrate cpitopcs on vascular endothclial
cells,followed by activation of the complement cascade.Complement activation is inhibited both in the
soluble phase and at the cell surface by a species-specific group of proteins called regulators of
complement activation.
In vitro expression of human regulators of complement activation,such as human decay-accelerating
factor(hDAF) on the surface of pig cells has been show to protect them from lysis by human
comoplemcnt.ln light of this observation pigs transgenic for hDAF have been developed by Cambridge
group by microinjecting a 6.5 kb DAFmini gene in to porcine fertilized ova.
It has previously been shown that hearts from hDAF transgenic pigs are not hyperaeutely rejected when
transplanted heterotopically into the abdomen of cynornolgus monkeys and that prolonged survival can
be achieved by use of immunosuppression with cyclosporine,cyclophosphamide,and methylpredn isolone.
Xenoreactive natural antibodies in humans and higher primates are directed predominantly at Gal
al-3Gal.These antibodies are thought to initiate hyperacute rejection of porcine organ xenografts.Some
studies have confirmed that anti-Gal a-l-3GAI antibodies is the main part of xeno preformed natural
antibodies and play a important role to the pathology of HAR.Many assay show that depiction of
antibodies from reeil)icnt animals can pretect xenograft from hypcracutc rcjcction,even though
complement activity was--900/oof baseline at the time of transplantation.The methods of depletion of
antibodes includes: extracorporeal absorption of antibodies from blood using a xenografts conrmected to
the animals.extracorporeal absorption of antibodies from plasma using columans with a matrix bearing
Gala I -3Gal hi -4G lcNAc and etc.Novartis has developed a new immusupresssion(polyvalent injectable
Gal ----GAS) which can be used through IV and absorpt antibodies.
Jnprevious studies the cynomolgus were chosen as the recipient of hctcrotopic hDAF transgenic pig
hearts largely because of the plentiful supply.However~such captive-bred animals are rarely availal)le
with a body weight greater than Skg.Hence,the cynomolgus monkey is an unsuitable recipient for
xenotransplantation of hDAF pig because of the technical difficulties Therefore it was necessary to
choose a larger primate for such studies.Many of larger primates are endangered species,and it was
deemed morally unacceptable to use such aiiimals for this purpose.of the larger primates readily
available in sufficient numbers,the baboon was considered to be the most suitable recipient.llowever,it
has been shown that hDAF has a variable ability to down-regulate complement of nonhuman
primates.lndeed,hDAF has been shown to down-regulate human complement activity by 84%of total
plasma complement activity,cynomolgus complement by 72%,baboon complement by 69%,and rhesus
complement by 28%.Clearly the rhesus monkey would not be a good choice of experimental recipient
for hDAF transgenic organs.i-Jowever,it was not clear whether the 3% difference between the
了极大的兴趣。目前，转基因猪由于可以无限量繁殖，以及在解剖，生理很多方面和
人的相似性原因，已经被认为是人供体器官的可能来源。在完全不同种系之间的异种移
植，如，猪-狒狒，因为受体体内预先存在的天然抗体与供体器官血管内皮细胞上的α
-galactosyl(α-Gal)糖表位结合，随后发生补体系统的激活，而最终将导致超急性排斥
（HAR）。
补体的激活可以被一组有种间特异性的蛋白-补体激活调解因子（RCAs），在可溶性状态和
细胞表面状态加以抑制。在体外表达人补体激活调解因子如，人衰变加速因子（hDAF），在
猪细胞表面表现了保护细胞免于人补体的溶解。出于此方面的考虑，剑桥大学通过显微
注射的方法，注射一段长6.5kb的DAF微小基因于猪的受精卵内，已经成功的培植出带有
hDAF的转基因猪。既往的实验已经表明，当hDAF转基因的猪心脏异位移植入cynomolgus
猴腹内后，心脏没有显示超急性排斥的表现，而且通过同时应用其他的免疫抑制剂如
：cyclosporine,cyclophosphamide和methylpridnisolone可以获得生存期的延长。
在人类和高等灵长类动物体内的异种天然抗体的受体主要是Gal(Galα1-3Gal)。这种抗体
和受体的结合被认为是对猪异种移植器官超急性排斥的起点。很多发现也证明anti-Galα
1-3Gal抗体是异种天然抗体的主要部份而且对超急性排斥的病理起决定性的重要作用。
目前已经有很多实验证明，去除预先存在的天然抗体能够克服异种移植的超急性排斥，
尽管此时补体活性仍然保持在正常水平的90％，超急性排斥仍可以被抑制。去除天然抗
体的方法包括，体外连接异种器官吸附抗体；含Galα1-3Galbl-4GlcNAc吸附柱预先吸附
抗体等。
NOVARTIS目前已经生产出多价可注射的QGAL残基pAS）可以直接注射吸附抗体。
以往的研究之所以用Cyllolllolgt1S猴作为11DAF转基因猜的心脏异位移植的受体主要是由于
数量多的原因。然而，这种猴浪少有体重能够超过5k8的个体。因此，由于器官体积和
技术上的原因，该种灵长类动物不适合作为hDAF转基因猪器官的移植实验受体。所以
必须寻找一种比较大的灵长类幼物作为受体。恨多大的灵长类动物已经濒于灭绝，道义
上不允济用于实验研究。体积较大，而且数量众多的另外一种灵长类动物－拂狒被认为
是最适台的受体幼物。然而，已经有实验证明hDAF下凋非人灵长类动物补体活性的能
力不同。11DAF下调人血浆总补体活性的84％，CyllOllolgUS猴72％，拂拂朴体活性69％，和
恒河猴28％。明显恒河猴不适合作为受体，但是在CyllO。mIgtlS猴和拂狭之闯的3％的差别
是否会导致超急性排斥还不清笼，所以用狒狒模型作为hDAF转基因猜的实验受体成为
其识。本研究的主要目的有：（1）离体肝脏，肾脏灌注实验研究设计目的在干检验
IIDAF转基因保护肝脏，肾脏免于超急性抹斥的理论。（2）活体外肝脏，肾脏支持肝衰竭
及无’署模型的研究的目的是为临床应用提供技术经验，及检验11DAF转基因的保护作用。
（3）’肾脏移植研究的实施在于确定一个涅想的免疫抑制剂方案以达到异种移植器官的长期
存活并且进一步证实11DAFty基因能够保护’肾脏免于超急性抹斥反应。O）检测最新免疫
抑制剂，多价可注射Q名al残基闪AS）与其他免疫抑制剂同时应用能否显著降低预存的天
然抗体并且改善川4XR。
第一部分11DAF转基因猜肝脏离体滔注的实验研究
l，目的
使用新鲜人血灌注普通猪肝和转基因猜肝；以及用猪血滔注猜肝脏以比较、检验
1。DAF转基因保护异种移植肝脏免干超急性排斥反应的效果。
2，方法
全麻下取供体肝并用UW液经肝动脉和门静脉灌注。行脏忌。管内插管。冷缺血时间少于
］小时。灌往循环采用一个生物PUMP，氧会器，和一个热交换器，用一个热水浴箱保存
肝脏。血液由门静脉开始滔注，经肝脏至下腔静脉插管，储血池，膜氧台器，热交换器，
6
然后经门静脉回肝脏。ACT水平用肝素保持高于 1000。
3，结果
我们做了 16例灌注：1组：猪血－猎肝则叫h 2组：人血0AFffi肝州＜八 3组：人血－普通
猎肝…一人第一组病理显禾正常。第3组表现明显的出血，血小板沉积，纤维蛋白沉积。
第2组表现相对正常的组织结构，一例DAF肝脏表规轻度的血管和内皮变化。在第H组
和第三组均见弥撒的内皮细胞的IgG，lgM沉积。叫。时电镜显示：2组和3组均有内皮损伤。
叫。时，第3组显示内皮细胞完全丢失，伴随明显的纤维蛋白沉积和血小板聚集。相反，
DAF肝，在叫。时多数的肝脏表现正常的组织结构，偶见血小板和红细胞聚集和内皮细
胞孪缩。
4；结论
该实验肯定了DAF转基因对灌注肝脏超急。往排斥反应的保护作用。为使用DAF转基
因猪干将来临床的试验提供了有力的理论基础。使用此异种灌注系统，人血滔注转基因
猜肝脏与灌注普通猜肝比较；在脉管炎；血栓，出血等方面均有显著的改善。转基因猪
肝脏同时表现了更好的肝脏功
The world-wide shortage of human organs available for transplantation has led to an interest in
xenotransptantation.The pig has been suggested as a possible source of organs because of its plentiful
supply and many anatomic and phsiological similarities with human hcings.Xenotransplantation between
widely disparate species such as pig to primate results in hypercute rejection because of the binding of
preformed natural antibodies to 0. -galactosyl carbohydrate cpitopcs on vascular endothclial
cells,followed by activation of the complement cascade.Complement activation is inhibited both in the
soluble phase and at the cell surface by a species-specific group of proteins called regulators of
complement activation.
In vitro expression of human regulators of complement activation,such as human decay-accelerating
factor(hDAF) on the surface of pig cells has been show to protect them from lysis by human
comoplemcnt.ln light of this observation pigs transgenic for hDAF have been developed by Cambridge
group by microinjecting a 6.5 kb DAFmini gene in to porcine fertilized ova.
It has previously been shown that hearts from hDAF transgenic pigs are not hyperaeutely rejected when
transplanted heterotopically into the abdomen of cynornolgus monkeys and that prolonged survival can
be achieved by use of immunosuppression with cyclosporine,cyclophosphamide,and methylpredn isolone.
Xenoreactive natural antibodies in humans and higher primates are directed predominantly at Gal
al-3Gal.These antibodies are thought to initiate hyperacute rejection of porcine organ xenografts.Some
studies have confirmed that anti-Gal a-l-3GAI antibodies is the main part of xeno preformed natural
antibodies and play a important role to the pathology of HAR.Many assay show that depiction of
antibodies from reeil)icnt animals can pretect xenograft from hypcracutc rcjcction,even though
complement activity was--900/oof baseline at the time of transplantation.The methods of depletion of
antibodes includes: extracorporeal absorption of antibodies from blood using a xenografts conrmected to
the animals.extracorporeal absorption of antibodies from plasma using columans with a matrix bearing
Gala I -3Gal hi -4G lcNAc and etc.Novartis has developed a new immusupresssion(polyvalent injectable
Gal ----GAS) which can be used through IV and absorpt antibodies.
Jnprevious studies the cynomolgus were chosen as the recipient of hctcrotopic hDAF transgenic pig
hearts largely because of the plentiful supply.However~such captive-bred animals are rarely availal)le
with a body weight greater than Skg.Hence,the cynomolgus monkey is an unsuitable recipient for
xenotransplantation of hDAF pig because of the technical difficulties Therefore it was necessary to
choose a larger primate for such studies.Many of larger primates are endangered species,and it was
deemed morally unacceptable to use such aiiimals for this purpose.of the larger primates readily
available in sufficient numbers,the baboon was considered to be the most suitable recipient.llowever,it
has been shown that hDAF has a variable ability to down-regulate complement of nonhuman
primates.lndeed,hDAF has been shown to down-regulate human complement activity by 84%of total
plasma complement activity,cynomolgus complement by 72%,baboon complement by 69%,and rhesus
complement by 28%.Clearly the rhesus monkey would not be a good choice of experimental recipient
for hDAF transgenic organs.i-Jowever,it was not clear whether the 3% difference between the
引文
1． William Porker，Soheyla Saadi，Shu S．Lin．TranspIantation of discordant xenograft：a challange revisited．Immunology Today，1996，17：373-378．
2． UNOS 1996 Annual Report．
3． Calne RY．Organ transplantation between widely disparate species．Transplantation Proc，1970，2：550．
4． Murase N．T ranspIantatiOn Proc，1993，25：425
5． Shu S Lin．David L Kooyman．The role of natural anti-Gal antibodies in hyperacute rejection of pig-to-baboon cordiac xenotransplants．Transplant Immunology，1997，5：212-218
6． D．K．C Cooper，E Kemp，J．L．Platt，J．G．White．Xenotmnsplantation．Springer，Second Edition，49．
7． Peter Braidley，David White．Concordant Organ xenOtranspIantation．Xeno，1994，2：25-30．
8． 陆佩华.异种移植研究.中华器官移植杂志，1998，1：61．
9 文军.猪异种抗原与异种移植排斥反应.国外医学外科学分册，1998，25：338-339．
10． John Furg，Abdul Rao，T Starzl．Clinical Trials and Projected Future of liver xenOtranspIalltatiOn．World J.Surg，1997，21：956-961．
11． Shu S．Lin．Bryun C．The role ofantibodies in Acute Vascular Rejection ofpig to baboon cardiac transplants．J．Clin．hwest，1998，101：1745-1756．
12． Denis Lambrigts，David H．Discordant organ xenotransplantation in primates．Transplalltation．1998，66：547-561．
13． 陆忠华.第十七届国际器官移植会议纪要.中华器官移植杂志，1999，20：59-60．
14． DJG White，GA Langford，E Cozzi，VJ Young．Protective effects of human DAF in transgenic pigs．Xeno，1995，3：48-51．
15． A Pascher，Ch Poehlein，M，Storck．Expression of human decay accelerating factor(hDAF)in
transgenic pigs regulates complement activation during ex vivo liver perfusion-immunopathological findings. Xenotransplantation,1996,[suppl]:s385-s387．
16． Schmoeckel M,Cozzi E,chavez G. Xenotransplantation of hDAF-transgenic swine hearts. Zentralbl Chir,1999,124(7) :604-8．
17． David JG White. hDAF transgenic pig organs: are they concordant for human transpIantation? Xeno, 1996,4(3) :50-54．
18． Vial CM,OstIie DJ. Life supporting function for over on month of a transgenic porcine heart in a baboon. J Heart Lung Transplant,2000,19(2) :224-229．
19． Veatman M, Daggett.cm. Human complement regulatory proteins protect swine lungs from xenogenic injury. Ann Thorac Suy, 1999 Mar, 67(3) :769-75．
20． Storek M, Abendroth D. white D J. Transgenic Swine as potential organ donors Results of the ex-vivo hemopefusion hDAF transgenic kifney with human blood. Zentnalbl Chin ,1998． ,123(70) :785-92．
21． Liver allotransplantation after extracorporeal hepatic support with transgenic (hCD55/ hCD59) porcine livers:clinical results and lack of pig-to human transmission of the porcine endgenous retrovirus.Transplantation,2000,Jan,69(2) :272-80．
2． UNOS 1996 Annual Report．
3． Calne RY．Organ transplantation between widely disparate species．Transplantation Proc，1970，2：550．
4． Murase N．T ranspIantatiOn Proc，1993，25：425
5． Shu S Lin．David L Kooyman．The role of natural anti-Gal antibodies in hyperacute rejection of pig-to-baboon cordiac xenotransplants．Transplant Immunology，1997，5：212-218
6． D．K．C Cooper，E Kemp，J．L．Platt，J．G．White．Xenotmnsplantation．Springer，Second Edition，49．
7． Peter Braidley，David White．Concordant Organ xenOtranspIantation．Xeno，1994，2：25-30．
8． 陆佩华.异种移植研究.中华器官移植杂志，1998，1：61．
9 文军.猪异种抗原与异种移植排斥反应.国外医学外科学分册，1998，25：338-339．
10． John Furg，Abdul Rao，T Starzl．Clinical Trials and Projected Future of liver xenOtranspIalltatiOn．World J.Surg，1997，21：956-961．
11． Shu S．Lin．Bryun C．The role ofantibodies in Acute Vascular Rejection ofpig to baboon cardiac transplants．J．Clin．hwest，1998，101：1745-1756．
12． Denis Lambrigts，David H．Discordant organ xenotransplantation in primates．Transplalltation．1998，66：547-561．
13． 陆忠华.第十七届国际器官移植会议纪要.中华器官移植杂志，1999，20：59-60．
14． DJG White，GA Langford，E Cozzi，VJ Young．Protective effects of human DAF in transgenic pigs．Xeno，1995，3：48-51．
15． A Pascher，Ch Poehlein，M，Storck．Expression of human decay accelerating factor(hDAF)in
transgenic pigs regulates complement activation during ex vivo liver perfusion-immunopathological findings. Xenotransplantation,1996,[suppl]:s385-s387．
16． Schmoeckel M,Cozzi E,chavez G. Xenotransplantation of hDAF-transgenic swine hearts. Zentralbl Chir,1999,124(7) :604-8．
17． David JG White. hDAF transgenic pig organs: are they concordant for human transpIantation? Xeno, 1996,4(3) :50-54．
18． Vial CM,OstIie DJ. Life supporting function for over on month of a transgenic porcine heart in a baboon. J Heart Lung Transplant,2000,19(2) :224-229．
19． Veatman M, Daggett.cm. Human complement regulatory proteins protect swine lungs from xenogenic injury. Ann Thorac Suy, 1999 Mar, 67(3) :769-75．
20． Storek M, Abendroth D. white D J. Transgenic Swine as potential organ donors Results of the ex-vivo hemopefusion hDAF transgenic kifney with human blood. Zentnalbl Chin ,1998． ,123(70) :785-92．
21． Liver allotransplantation after extracorporeal hepatic support with transgenic (hCD55/ hCD59) porcine livers:clinical results and lack of pig-to human transmission of the porcine endgenous retrovirus.Transplantation,2000,Jan,69(2) :272-80．