异种移植血管内皮细胞组织特异性表达人CD59转基因小鼠的建立

目的建立用于异种移植研究血管内皮细胞组织特异性表达人CD59转基因小鼠。方法采用受精卵显微注射技术，将含有人ICAM-2启动子、人CD59基因第1个内含子、人CD59 cDNA、BGH polyA终止信号的外源基因导入小鼠受精卵的原核中。选取注射后仍健康的受精卵移植入假孕母鼠的输卵管中待分娩。聚合酶链反应（PCR）及Southern blot确定外源基因整合阳性转基因小鼠。流式细胞术用于外源基因蛋白质水平表达检测。免疫组织化学方法观察人CD59在转基因小鼠心脏、肝脏、肾脏等器官表达分布情况。结果产仔130只，9只外源基因整合阳性。整合率6％（9／130）。6只获得蛋白质水平表达。蛋白质水平表达强度为人CD59在人白细胞表达强度的80％至95％，转基因效率5％（6／130）。免疫组织化学检测显示人CD59在转基因小鼠心脏、肝脏、肾脏组织有较强表达，且表达限于血管内皮细胞。结论成功地建立了用于异种移植研究血管内皮细胞组织特异性表达人CD59转基因小鼠。     

包含UI的人衰变加速因子重组基因在转基因鼠高效表达的研究

目的建立血管内皮细胞高效表达人衰变加速因子（hDAF）基因的转基因小鼠。方法采用受精卵显微注射法，将包含杂合内含子（UI）的人hDAF导入昆明白小鼠受精卵的雄原核内，然后将注射后仍健康的受精卵植入假孕母鼠输卵管内，待其自然分娩。应用聚合酶链反应（PCR）和Southern杂交检测G0代小鼠hDAF基因的整合情况，应用流式细胞计数（FCM）检测小鼠外周血单核细胞（PBMCs）表面hDAF的表达，应用逆转录（RT）-PCR检测转基因小鼠心脏、肝脏、肾脏和肺脏人hDAF基因mRNA的表达。结果注射后卵的存活率和幼鼠出生率分别为83．8％（941／1123）和9．5％（89／941），外源基因的整合率为22．5％（20／89），FCM检测发现其中有7只转基因小鼠PBMCs有人hDAF表达，表达强度为人PBMCs的152％～243％，并且相应小鼠的心、肝、肾及肺组织均有人hDAFmRNA表达。结论UI可以使hDAF在转基因鼠各主要脏器高效表达。     

用于人顶体蛋白酶原基因表达调控和蛋白分选研究的转基因小鼠模型的建立

分别将构建的人顶体蛋白酶原基因（ｈｕｍａｎ ｐｒｏａｃｒｏｓｉｎ,ｈＡＣＲ）５’侧冀序列,第１外显子和编码β－半乳糖苷酶的基因（ｌａｃＺ）的融合基因（载体１）,以及ｈＡＣＲ基因５’侧冀序列与报告基因ｌａｃＺ的融合基因（载体２）,通过显微注射受精卵法制备转基因小鼠,结果如下：载体１：注射８９只卵,移植入９只母鼠单侧卵管中,３只怀孕,产仔９只,存活７只,基因组Ｓｏｕｔｈｅｒｎ杂交检出整合有外源基因的阳     

人纤溶酶原激活物抑制物1转基因小鼠的建立

目的 建立人纤溶酶原激活物抑制物1（PAI-1）转基因小鼠。方法 利用构建的高效真核表达载体pCAGGS-PAI-1,制备注射用DNA。以显微注射法将目的DNA注入小鼠受精卵,再将受精卵移植到受体母鼠,制备转基因小鼠。经鼠尾组织DNA提取、PCR筛选、Southern杂交、DNA序列测定对转基因小鼠进行鉴定。结果 对获得的29只小鼠经PCR筛选、Southern杂交分析、DNA序列测定,得到了2只整合了人PAI-1 cDNA的转基因小鼠。在刚出生的转基因小鼠尾部及后肢发现出血性改变,免疫组织化学观察到肾脏局部PAI-1表达稍有增高。结论 本研究中,我们建立了人PAI-1基因传基因小鼠,为进一步研究PAI-1在肾脏疾病中调控细胞外基质降解的机制提供了动物模型,为研究利用抗凝、促纤溶疗法慢性肾炎奠定了基础。     

肾小管间质纤维化中转化生长因子β1的表达特征

目的 研究大鼠肾小管间质纤维化中转化生长因子β１（ＴＧＦ－β１）的表达特征。方法 以腺嘌呤灌胃法建立大鼠肾小管间质纤维化实验模型,应用免疫组织化学（组化）、逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）、原位杂交及Ｎｏｒｔｈｅｒｎ ｂｌｏｔ检测６只正常及３０只模型大鼠肾组织ＴＧＦ－β１蛋白及基因表达。结果 ６只正常大鼠肾组织ＴＧＦ－β１主要位于皮髓交界处肾小管上皮细胞,ＴＧＦ－β１免疫组化平均阳性表达率为（     

转移生长因子—β,碱性成纤维细胞生长因子诱导人成骨细胞表达血小板衍生生

目的 探讨转移生长因子（ＴＧＦ）－β、碱性成纤维细胞生长因子（ｂＦＧＦ）对人成骨细胞血小板衍生生长因子（ＰＤＧＦ）－ＢｍＲＮＡ表达的影响及其意义。方法 体外分离培养人成骨细胞。在体外培养的成骨细胞中分别加入１０、２０、４０、８０、１６０ｕｇ／Ｌ梯度浓度的ＰＤＧＦ－ＢＢ培养细胞２４ｈ,以摄取＾３Ｈ－ＴｄＲ为细胞增殖指标检测细胞增殖状况。以４ｕｇ／Ｌ的ＴＧＦ－β和１０ｕｇ／Ｌ的ｂＦＧＦ培养细胞２４ｈ,寡核苷酸探针检测细胞ＰＤＧＦ－ＢｍＲＮＡ的表达。结果 １０～１６０ｕｇ／Ｌ的ＰＤＧＦ－ＢＢ可促进成骨细胞增殖（Ｐ〈０．０５）。在普通培养条件下,细胞下表达ＰＤＧＦ－ＢｍＲＮＡ;当培养体系中加入ＴＧＦ－β和ｂＦＧＦ,可见ＰＤＧＦ－ＢｍＲＮＡ的表达。结论 ＰＤＧＦ－Ｂ基因的表达可能是骨组织生长的储备因素,ＴＧＦ－β和ｂＲＮ     

腹膜透析对转化生长因子β1在残存肾中表达的影响

探讨清除循环中毒素后生长因子和残存肾小球的改变。方法采用分子杂交技术和组织病理分析方法在肾次全切除的残存肾大鼠模型上研究腹膜透析（ＰＤ）对转化生长因子β１（ＴＧＦ－β１）ｍＲＮＡ在残存肾中表达的影响和肾小球硬化的变化。分模型组和ＰＤ组，第８周ＰＤ组开始作腹膜透析至第１２周。分别在第７周和１２周各组杀检６只作肾病理检查（ＨＥ．ＰＡＳ染色）和ＴＧＦ－β１ｃＤＮＡ缝隙点样杂交。结果在实验的第７周两组间肾小球硬化指数（ＧＳＩ）及ＴＧＦ－β１ｍＲＮＡ表达均无显著性差异。第十二周，ＰＤ组的ＧＳＩ及ＴＧＦ－β１ｍＲＮＡ表达均显著低于模型组。ＧＳＩ改变程度与ＴＧＦ－β１ｍＲＮＡ表达丰度变化呈明显正相关（ｒ＝０．６５Ｐ＜０．０５）。结论腹膜透析可明显减少ＴＧＦ－β１ｍＲＮＡ的表达并延缓残存肾小球的硬化     

胞嘧啶脱氨酶基因体外特异性前药转换抗大肠癌作用研究

目的：为使胞嘧啶脱氨酶（ＣＤ）基因在大肠癌细胞中特异表达。方法：构建了以ＣＥＡ基因顺式转录调控序列驱动ＣＤ基因的组织特异性重组逆转录病毒载体Ｇ１ＣＥＳＡＣＤＮａ，以脂质体法将重组载体及转录受ＬＴＲ驱动的ＣＤ基因逆转录病毒载体ｐＣＤ２分别转导入高分泌ＣＥＡ的大肠癌细胞ＬｏＶｏ中，经Ｇ４１８完全选择后进行前５－ＦＣ敏感试验。结果：转导普通型及组织特异型ＣＤ基因的ＬｏＶｏ细胞较亲代细胞对５－ＦＣ的前药转     

疼痛刺激神经生长因子基因在大脑皮层的过量表达

本文用福尔马林 (Ｆｏｒｍａｌｉｎ)制作了小鼠疼痛及炎症模型 ,以甘油醛 3 磷酸脱氢酶 (ＧＡＰＤＨ)基因为内参照 ,采用半定量反转录聚合酶链反应 (ＲＴ ＰＣＲ)技术 ,观察了疼痛刺激神经生长因子基因(ＮＧＦｍＲＮＡ)在大脑皮层的变化以及硫酸镁 (ＭｇＳＯ4 )对ＮＧＦｍＲＮＡ表达的影响。材料和方法选用二级成年Ｂａｌｂ/ｃ雌鼠 32只 ,随机分为Ａ、Ｂ二组 ,每组 16只。Ｂ组每只腹部皮下注射 10 %ＭｇＳＯ45 0 0ｍｇ ｋｇ-1[1] ,Ａ组注射等体积生理盐水。 10ｍｉｎ后Ａ、Ｂ二组每只小鼠左后肢掌面皮下注射 5 %Ｆｏｒｍａｌｉｎ 40 μｌ。…     

建立血管内皮细胞组织特异性表达人衰变加速因子的转基因小鼠

目的为了研究异种移植，建立血管内皮细胞组织特异性表达人衰变加速因子(DAF)的转基因小鼠。方法采用受精卵显微注射技术，将含有人内皮细胞粘附分子-2(ICAM-2)基因启动子、人DAF cDNA(插有人DAF基因第一个内含子)、SV40splice／polyA的外源基因导人小鼠受精卵的原核中；选取注射后仍健康的受精卵移植入假孕母鼠的输卵管中待分娩。聚合酶链(PCR)技术及Southern印迹杂交法确定外源基因整合阳性转基因小鼠。RT-PCR方法和流式细胞术分别用于外源基因mRNA及蛋白质水平表达的检测。免疫组织化学方法观察人DAF在转基因小鼠心脏、肝脏、肾脏等器官的表达分布。采用Langendorff心脏灌流装置，用200g／L的稀释人血清灌注转基因小鼠离体心脏，检测其抗超急性排斥反应能力。结果共产仔鼠133只，21只整合有外源基因，整合率16％(21／133)。8只实现mRNA及蛋白质水平表达，蛋白质水平表达强度为人DAF基因在人白细胞表达强度的70％至95％，转基因效率60A(8／133)。免疫组织化学法显示人DAF在转基因小鼠器官组织切片上有较强表达，且表达限于血管内皮细胞。与普通鼠对比，转基因小鼠离体心脏存活时间明显延长，且60min灌注期间内做功仍维持在最大值20％以上。结论成功地建立了血管内皮细胞组织特异性表达人DAF的转基因小鼠。这种小鼠有一定的抗超急性排斥反应能力。     

转人CRP基因在异种移植中的研究

目的：研究转入补体调节蛋白（CRP）DAF、MCP和CD59基因对抑制人补体激活从而克服超急性排斥反应的作用。方法：利用显微注射建立转人衰变加速因子（hDAF)小鼠和猪的模型和转梁hMCP及hCD59真核表达质粒的猪内皮细胞（EC）,研究小鼠和猪EC表达抑制人补体激活的人补体调节蛋白（CRP）对异种移植超急性排斥反应的抑制作用。结果（1）转人DAF基因小鼠心脏用新鲜人血连续丛外灌注,转基因组心脏搏动时间（174．6min)比对照组（106．5min)明显延延长。（2）转hDAF基因猪心脏异位移植给猕猴、移植心最长存活90h,受者死亡前移植心仍有功能,移植心病理检查未见超急性排斥反应病理改变。（3）转DAF基因基因猪EC死亡率在不同浓度血清时均明显低于对照组,在转hDAF基因猪EC上再分别转染hMCP及hCD59真核表达质粒,转hDAF hMCP或hDAF hCD59在不同血清浓度时EC死亡率较单纯hDAF组明显下降（P＜0．05）。结论,转人DAF及MCP、CD59补体调节蛋白基因能克服人对异种器官或组织的超急性排斥反应。     

A line of transgenic pigs in which the expression of human decay-accelerating factor by endothelial cells is increased in the presence of inflammatory stimuli

Abstract: Aortic endothelial cell cultures (PAE) from four lines of pigs transgenic for human decay-accelerating factor (hDAF) have been used to study the response to the inflammatory stimuli bacterial lipopolysaccharide (LPS) and recombinant human TNF-α. Human umbilical vein endothelial cells (HUVEC) and PAE from normal, non-transgenic pigs were used as controls. The expression of hDAF and E-selectin on the cell surface was determined by flow cytometry. After overnight incubation, HUVEC, normal and transgenic PAE increased the relative expression of E-selectin 2–5-fold in response to LPS (25 μg/ml), and 5–40-fold in response to TNF-α(10 ng/ml). In both normal and transgenic PAE the increase in expression of E-selectin in response to TNF-α was maximal at 4 hr and significantly decreased after 20 hr. There was no significant increase in DAF expression by HUVECs in response to LPS or TNF-α, and three of the four lines of transgenic pigs studied did not increase expression of hDAF in response to either stimulus. However, endothelial cells from the transgenic line A74 exhibited a dose-dependent increase in expression of hDAF in response to LPS and TNF-α. A study of the time course of up-regulation triggered by incubation with TNF-α showed that, in contrast to the up-regulation of E-selectin, hDAF expression continued to increase for at least 3 days. This response may afford additional protection to organs from this line of transgenic pigs.     

转人CD46基因抑制人补体在转基因小鼠心脏组织中的沉积

目的 观察转人源性膜辅助蛋白(CD46)基因对转基因小鼠心脏中人补体沉积的抑制作用.方法 以近交系昆明小鼠为对象,采用显微注射法制备转人CD46基因小鼠,用逆转录聚合酶链法(RT-PCR法)检测外源基因的整合情况.以Fo代转基因小鼠11只为实验组,同窝非转基因小鼠10只为对照,快速切取小鼠心脏,用改良的Langendorff法经小鼠主动脉逆行灌注预先制备的含补体的B型血人血浆.观察并记录小鼠心脏搏动时间;采用免疫荧光和免疫组织化学法检测小鼠心脏组织中补体C3c及C9的沉积情况.结果 Fo代转基因小鼠外源基因的整合率为33.3%.实验组小鼠心脏搏动时间为(42.6±20.6)min(15～77 min),长于对照组的(20.2±12.5)min(7～40 min),差异有统计学意义(P＜0.01).实验组小鼠心脏组织中补体C3c及C9的沉积均少于对照组.结论 通过显微注射法制备的转人CD46基因小鼠,其外源基因可稳定表达;转人CD46基因可以抑制人补体C3c及C9在转基因小鼠心脏组织中的沉积.     

Production and characterization of a pig line transgenic for human membrane cofactor protein

A pig line transgenic for human membrane cofactor protein (hMCP) has been established. Offspring from the founder were produced by crossing the founder with pigs heterozygous for the human decay accelerating factor (hDAF) transgene. As a result, pigs transgenic for both hMCP and hDAF have been produced. Ribonuclease protection assay (RPA) indicated that hMCP was expressed in all the tissues analysed. In addition, immunohistochemical results indicated a high level of expression of hMCP on neural tissues and islets where hDAF was absent or weakly expressed. C3 fragment deposition and cytotoxicity assays indicated that hMCP expression alone on pig endothelial cells and peripheral blood lymphocytes (PBLs) provided protection against human complement mediated damage. However, we did not find that porcine endothelial cells expressing both hDAF and hMCP were better protected than those expressing hDAF alone. The expression of hMCP on tissues where hDAF is not expressed could provide these tissues with protection against human complement mediated lysis.     

Skeletal abnormalities and ultrastructural changes of cartilage in transgenic mice expressing a collagen II gene (COL2A1) with a Cys for Arg-alpha1-519 substitution

OBJECTIVE: To examine the mechanism by which the Arg-->Cys 519 mutation causes the clinical phenotype employing transgenic mice that express the mutated human COL2A1. METHODS: A DNA construct under the control of a COL2A1 specific promoter was prepared from genomic DNA isolated from fibroblasts from the proband with primary generalized osteoarthritis (OA) associated with a mild chondrodysplasia. Transgenic mice were obtained by injection of the constructs into pro-nuclei of fertilized eggs from the FVB/N inbred mouse strain. Transgenic mice harboring two alleles of the mutated human COL2A1 were examined for morphological abnormalities and for alterations of their skeletal development. Ultrastructural examination was performed to identify changes in the organization and density of collagen II fibrils in articular cartilage of the transgenic mice. RESULTS: Transgenic mice harboring two alleles of the mutated human collagen gene were smaller than their normal littermates, had a cleft palate, and disorganized growth plate. Electron microscopy of articular cartilage showed a decreased density of collagen II fibrils and revealed chondrocytes with dilated Golgi cysternae. CONCLUSIONS: Expression of a COL2A1 with an Arg-->Cys 519 substitution in transgenic mice causes retardation of skeletal development and ultrastructural alterations in articular cartilage with a profound reduction of the density of the collagen II fibrils in the tissue. These alterations may be responsible for the phenotype of precocious generalized OA and chondrodysplasia displayed by patients harboring this COL2A1 mutation.     

Immunopathology of an hDAF transgenic pig model liver xenotransplant into a primate

BACKGROUND: hDAF transgenic pigs do not display the inherent hyperacute rejection reactions of pig-to-primate xenotransplants. The purpose of this study was to determine the immunopathologic phenomena following an hDAF transgenic pig hepatic orthotopic xenotransplant into a baboon. METHODS: Donor animals were unmodified pigs (n=4) and hDAF transgenic pigs (n=2). Recipient animals were baboons (Papio anubis). Liver biopsies were immunostained using monoclonal antibodies to C3, C5b-9, IgG, IgM, CD2, CD4, CD8, CD68, CD20, Bric 216, CD31, and fibrin, and polyclonal antibody to C4. RESULTS: hDAF transgenic grafts showed IgG, IgM, and C4 endothelial deposits. However, no fibrin, C3, or C5b9 deposits were observed after reperfusion. hDAF xenografts displayed CD31 staining in the portal spaces, perilobular areas, and at hepatic sinuisoidal levels. The baboon that lived for 4 days displayed either CD4 or CD8 T-cells periportal infiltrate. CONCLUSIONS: Future studies will seek to determine the physiologic role of CD31 hepatic sinusoidal expression in transgenic xenotransplants, and will also study the role of T-cell infiltrates in xenograft rejection.     

Life-supporting human complement regulator decay accelerating factor transgenic pig liver xenograft maintains the metabolic function and coagulation in the nonhuman primate for up to 8 days

BACKGROUND: It is not known whether the pig liver is capable of functioning efficiently when transplanted into a primate, neither is there experience in transplanting a liver from a transgenic pigs expressing the human complement regulator human complement regulator decay accelerating factor (h-DAF) into a baboon. The objective of this study was to determine whether the porcine liver would support the metabolic functions of non-human primates and to establish the effect of hDAF expression in the prevention of hyperacute rejection of porcine livers transplanted into primates. METHODS: Five orthotopic liver xenotransplants from pig to baboon were carried out: three from unmodified pigs and two using livers from h-DAF transgenic pigs. FINDINGS: The three control animals transplanted with livers from unmodified pigs survived for less than 12 hr. Baboons transplanted with livers from h-DAF transgenic pigs survived for 4 and 8 days. Hyperacute rejection was not detected in the baboons transplanted with hDAF transgenic pig livers; however, it was demonstrated in the three transplants from unmodified pigs. Baboons transplanted with livers from h-DAF transgenic pigs were extubated at postoperative day 1 and were awake and able to eat and drink. In the recipients of hDAF transgenic pig livers the clotting parameters reached nearly normal levels at day 2 after transplantation and remained normal up to the end of the experiments. In these hDAF liver recipients, porcine fibrinogen was first detected in the baboon plasma 2 hr postreperfusion, and was present up to the end of the experiments. One animal was euthanized at day 8 after development of sepsis and coagulopathy, the other animal arrested at day 4, after an episode of vomiting and aspiration. The postmortem examination of the hDAF transgenic liver xenografts did not demonstrate rejection. INTERPRETATION: The livers from h-DAF transgenic pigs did not undergo hyperacute rejection after orthotopic xenotransplantation in baboons. When HAR is abrogated, the porcine liver maintains sufficient coagulation and protein levels in the baboon up to 8 days after OLT.     

KIDNEYS DERIVED FROM MICE TRANSGENIC FOR HUMAN COMPLEMENT BLOCKERS ARE PROTECTED IN AN IN VIVO MODEL OF HYPERACUTE REJECTION

Purpose

The major obstacle to successful discordant kidney xenotransplantation is hyperacute rejection (HAR). Complement plays a key role in the induction of HRA, defined by endothelial cell activation, loss of vascular integrity, hemorrhage and thrombosis. The activation of complement is tightly controlled by a number of species-specific regulatory proteins which inhibit, at different points, the cascade of events leading to the formation of the membrane attack complex (MAC). We have tested the hypothesis that kidneys derived from transgenic mice expressing two human complement inhibitors, Decay Accelerating Factor (hDAF) and Membrane Cofactor Protein (MCP), could be protected from human complement-mediated damage.

Materials and Methods

Control and transgenic mice were perfused with human plasma by cannulation of the right jugular vein, at a perfusion rate of 10 micro L./min. for two hours. Complement C3 deposition was detected on kidney sections by immunohistochemistry using specific FITC antibody. Complement-induced tissue damage was evaluated by histopathological examination.

Results

Heavy deposition of complement C3 was observed on kidneys derived from perfused control mice. This was associated with a characteristic HAR pathology of severe interstitial hemorrhage, inflammatory reaction, loss of glomerula and tubuli structure. Kidneys derived from mice transgenic for hDAF or hMCP were partially protected from both complement C3 deposition and tissue damage. The expression of both dDAF and hMCP in double transgenic mice significantly increases the protection from human complement-mediated damage.

Conclusions

A novel model of in vivo perfusion with human plasma has been adopted to recreate the initial event of HAR. Our data show that this murine model could be very valuable to determine the effect of transgenic human molecules in protecting vascularized organs from human complement attack.