二例成人间双活体供肝移植的手术重建技巧

目的探讨成人间双活体供肝移植的手术重建技巧。方法2例成人患者,1例原发病为乙型肝炎后肝硬化、巨块型肝癌,接受其2位姐姐的左半肝移植,移植物与受者体重比(GR/WR)为1.5%,移植于原肝位置左侧的左半肝,其肝左静脉、门静脉分别于受者的肝左静脉、门静脉左支端端吻合,胆肠吻合方式重建供肝胆道;移植至右侧的左半肝,沿矢状位旋转180°,其肝左静脉、门静脉分别与受者的肝右静脉、门静脉右支端端吻合,左肝管与受者的肝总管端端吻合。另1例原发病为乙型肝炎后肝硬化,接受其母亲的右半肝移植,但由于供肝存在大泡状空泡变性,且供、受者的体重差较大,为能满足患者的生理需要,故该例同时行尸体左半肝移植,GR/WR为1.2%,右半肝的肝右动脉、肝右静脉分别于受者的同名血管端端吻合,其门静脉与受者的门静脉右支端端吻合,右肝管与受者的肝总管端端吻合;左半肝的肝左静脉、门静脉及肝动脉分别与受者的肝左静脉、门静脉左支及脾动脉端端吻合,胆肠Roux-en-Y吻合方式重建供肝胆道。结果除母亲供者术后3d出现短暂乳糜漏,经对症治疗11d后痊愈外,供者无其它并发症发生。2例患者术后恢复良好,未发生排斥反应及全身感染,现已随访10个月以上,肝功能正常,均恢复正常工作和生活。结论成人间双活体供肝移植可为受者提供更大的肝脏,又可减少供者的风险,但手术操作复杂,需要对供、受者的条件进行充分评估后施行。     

Adenovirus-mediated gene transfer using in-situ perfusion of the liver graft

To establish an efficient technique for adenovirus-mediated gene transfer in liver transplantation, we evaluated the in situ perfusion of liver grafts. The grafts were perfused in situ with 1 × 10¹⁰ of E1-deleted, replication-defective adenoviral vectors encoding the LacZ gene driven by the human CMV promoter, either through the hepatic artery (group 1) or the portal vein (group 2). Group 3 animals served as negative controls; their liver grafts were perfused with lactated Ringer's solution through the portal vein. PCR confirmed the presence of viral DNA in every graft perfused with viral vectors. In X-gal staining, positive staining was observed almost exclusively at the portal triad in group 1, whereas in group 2 minimal staining was observed, predominantly in the parenchymal area. Protein production from the transfected gene was confirmed by a functional protein assay; the values were 0.16 % ± 0.07 % liver protein in group 1, 0.13 % ± 0.02 % in group 2, and 0.007 % ± 0.0003 % in group 3 on postoperative day 2. In conclusion, in situ perfusion of the viral vectors through the hepatic artery resulted in an effective expression of the transfected gene, predominantly at the portal triad. Received: 15 July 1996 Received after revision: 1 November 1996 Accepted: 12 November 1996     

Adenovirus-mediated gene transfer to glomerular cells in newborn mice

The systemic delivery of recombinant adenoviral (rAd) vectors to renal glomeruli has been problematic due to the rapid clearance of the circulating virus by the liver. We have previously shown that prolonged retention of rAd vectors in the circulation by liver bypass improves the transduction of renal glomerular cells in adult mice and rats. This study was done to determine whether newborn mice have a delayed clearance of rAd vectors from the circulation and a more efficient transduction of glomerular cells after a systemic injection of rAd vectors. Newborn (1 day old) and adult (6 months old) C57Bl6/J mice ( n =20 in each group) were injected with rAd vectors carrying the lacZ gene (rAd.lacZ) through the retro-orbital venous plexus (2×10⁹ particles/g body weight). The renal expression of Coxsackie and Adenoviral Receptors (CAR) and lacZ gene were evaluated at different time points by Western blots, immunohistochemistry, -galactosidase staining, enzyme assay activity, and RT-PCR studies in newborn and adult mice. The clearance rate of rAd.lacZ was significantly delayed in newborn mice, and the concentration of circulating virus in these mice was almost ten times higher than that in adult mice. Newborn kidneys showed increased expression of CAR, predominately localized in glomerular cells. These findings were associated with an efficient gene transfer of the lacZ gene into glomeruli and tubules of newborn mice. This study demonstrates for the first time the feasibility of using systemic intravenous injections of rAd vectors to express foreign genes in developing glomeruli of young mice.     

Impaired metabolism in donor kidney grafts after steroid pretreatment

We recently showed in a randomized control trial that steroid pretreatment of the deceased organ donor suppressed inflammation in the transplant organ but did not reduce the rate or duration of delayed graft function (DGF). This study sought to elucidate such of those factors that caused DGF in the steroid‐treated subjects. Genome‐wide gene expression profiles were used from 20 steroid‐pretreated donor‐organs and were analyzed on the level of regulatory protein–protein interaction networks. Significance analysis of microarrays (SAM) yielded 63 significantly down‐regulated sequences associated with DGF that could be functionally categorized according to Protein ANalysis THrough Evolutionary Relationships ontologies into two main biologic processes: transport (P < 0.001) and metabolism (P < 0.001). The identified genes suggest hypoxia as the cause of DGF, which cannot be counterbalanced by steroid treatment. Our data showed that molecular pathways affected by ischemia such as transport and metabolism are associated with DGF. Potential interventional targeted therapy based on these findings includes peroxisome proliferator‐activated receptor agonists or caspase inhibitors.     

Adenovirus-mediated gene transfer into selected liver segments using a vascular exclusion technique.

Adenovirus-mediated gene therapy is hampered by severe virus-related toxicity, especially to the liver. The aim of the present study was to test the ability of a vascular exclusion technique to achieve transgene expression within selected liver segments, thus minimizing both viral and transgene product toxicity to the liver. An E1-E3-deleted replication-deficient adenovirus expressing a green fluorescent protein (GFP) reporter gene was injected into the portal vein of BDIX rats, with simultaneous clamping of the portal vein tributaries to liver segments II, III, IV, V, and VIII. GFP expression and inflammatory infiltrate were measured in the different segments of the liver and compared with those of the livers of animals receiving the viral vector in the portal vein without clamping. The GFP expression was significantly higher in the selectively perfused segments of the liver as compared with the non-perfused segments (p < 0.0001) and with the livers of animals that received the vector in the portal vein without clamping (p < 0.0001). Accordingly, the inflammatory infiltrate was more intense in the selectively perfused liver segments as compared with all other groups (p < 0.0001). Fluorescence was absent in lungs and kidneys and minimal in spleen. The clinical usefulness of adenovirus-mediated gene transfer to the liver largely depends on the reduction of its liver toxicity. Clamping of selected portal vein branches during injection allows for delivery of genes of interest to targeted liver segments. Transgene expression confined to selected liver segments may be useful in the treatment of focal liver diseases, including metastases.     

Adenovirus-mediated p53 gene transfer to rat testis impairs spermatogenesis

The tumor suppressor protein p53 participates in normal cell differentiation as well as induction of programmed cell death. The authors investigated the effect of p53 overexpression on spermatogenesis by transferring p53 gene into the rat testes. Replication-deficient recombinant adenovirus vectors were constructed to include cytomegalovirus (CMV) promoter driving wild-type p53 (Ad-CMV-p53) or beta-galactosidase (Ad-CMV-beta-gal). Virus was delivered to cells of the tubules by slow retrograde injection through the rete testis. At 0, 4, 7, and 14 days, testes were removed, weighed, and analyzed histopathologically, including immunohistochemistry for p53, Bcl-2, Bax, and interleukin-1beta converting enzyme (ICE). Testicular weight was decreased in Ad-CMV-p53 group at 14 days after injection, while no change occurred in phosphate-buffered saline-injected controls or Ad-CMV-beta-gal-infected testes. Beyond 4 days, cell degradation in tubules interfered with immunohistochemical observation in the Ad-CMV-p53 group. At 4 days, p53 was expressed mostly in spermatocytes. Bax showed greater expression in the p53 group than in the control or Ad-CMV-beta-gal group. ICE, expressed mostly in spermatids, was more abundant in the p53 group than in controls. Overall, p53 overexpression in the testis impaired spermatogenesis.     

Protection of the ischemic liver by donor pretreatment before transplantation

Canine livers ischemically damaged for thirty minutes prior to auxiliary transplantation did not survive for long periods of time unless a combination of isoproterenol, allopurinol, and heparin was administered intravenously to the donor animal before the ischemic damage. These drugs had no protective effect when given individually. Ischemic livers treated with adenosine prior to transplantation also showed no improved recovery of function.     

Adenovirus-mediated gene transfer of human inducible nitric oxide synthase in porcine vein grafts inhibits intimal hyperplasia.

OBJECTIVE: The aim of this study is to determine whether adenoviral inducible nitric oxide synthase (iNOS) gene transfer could inhibit intimal hyperplasia (IH) in porcine internal jugular veins interposed into the carotid artery circulation. METHODS: Porcine internal jugular veins were transduced passively with 1 x 10(11) particles of an adenoviral vector carrying either the human iNOS (AdiNOS) or beta-galactosidase (AdlacZ) cDNA for 30 minutes and then interposed into the carotid artery circulation. Segments of each vein graft were maintained in an ex vivo organ culture to measure nitrite accumulation, a marker of nitric oxide synthesis. The grafts were analyzed immunohistochemically for the presence of neutrophils, macrophages, and leukocytes by staining for myeloperoxidase, ED1, and CD45, respectively, at 3 (n = 4) and 7 (n = 4) days. Morphometric analyses and cellular proliferation (Ki67 staining) were assessed at 3 (n = 4), 7 (n = 4), and 21 days (n = 8). RESULTS: AdlacZ-treated vein grafts demonstrated high levels of beta-galactosidase expression at 3 days with a gradual decline thereafter. Nitrite production from AdiNOS-treated vein grafts was approximately fivefold greater than AdlacZ-treated grafts (P =.00001). AdiNOS or AdlacZ treatment was associated with minimal graft inflammation. Cellular proliferation rates were significantly reduced in AdiNOS-treated grafts as compared with controls at both 3 (41%, P =.000004) and 7 days (32%, P =.0001) after bypass. This early antiproliferative effect was most pronounced at the distal anastomosis (65%, P =.0005). The iNOS gene transfer reduced the intimal/medial area ratio in vein grafts at 7 (36%, P =.009) and 21 days (30%, P =.007) versus controls. This inhibition of IH was again more prominent in the distal segments of the grafts (P =.01). CONCLUSION: Adenovirus-mediated iNOS gene transfer to porcine internal jugular vein grafts effectively reduced cellular proliferation and IH. Although iNOS gene transfer reduced IH throughout the entire vein graft, the most pronounced effect was measured at the distal anastomosis. These results suggest potential for iNOS-based genetic modification of vein grafts to prolong graft patency.     

静脉桥基因转录及表达的实验研究

目的：研究静脉桥基因的转移及表达。方法：将转染Ａｄｖ ５－ＣＭＶ／ＬａｃＺ质粒的颈静脉桥移植于同一大鼠的颈总动脉，行β－半乳糖苷酶活性测定和组织化学染色。结果腺病毒载体能使外源性基因有效地转入静脉桥并获得高效的基因表达。结果：用腺病毒载体将治疗性基因导入静脉桥，通过基因高效表达来防止血栓形成、内膜增生和粥样硬化，可能是提高静脉桥近期和远期通畅率的一个有效方法。     

Adenovirus-mediated gene transfer into tendon and tendon sheath

In this study, we successfully transferred the Escherichia coli β-galactosidase gene, LacZ, into the chicken tendon and tendon sheath by a recombinant adenovirus. The recombinant adenovirus Adv-βgal that carried the E. coli LacZ gene was constructed by homologous recombination in 293 cells (human transformed embryonic kidney) between the expressing vector and the ClaI large fragment of adenovirus 5 genome. Each chicken received a 10 μl injection, containing 10⁵ plaque-forming units of recombinant virus Adv-βgal, into the tendon sheath of the long toe. Samples of tendon and tendon sheath were harvested at 3, 30, and 75 days after the injection. The LacZ gene transfer was detected for its coding product β-galactosidase by staining with X-gal solution. The results showed that all tendon and tendon sheath samples from the three harvest times stained positive (blue). The tendon sheath samples were more extensively stained; staining of the tendon was limited to the epitenon layer. These data suggest that a functional exogenous gene can potentially be transferred into the tendon and tendon sheath by similar techniques; such techniques may be used to improve healing and reduce adhesion formation.     

腺病毒载体介导反义Src基因治疗胶质瘤

目的 探讨腺病毒载体介导的反义Src基因对胶质瘤生长的作用及其机制.方法 应用携带反义Src基因的重组腺病毒体外感染C6胶质瘤细胞,观察其对细胞形态、生长曲线和克隆形成率的影响.建立大鼠胶质瘤动物模型,原位注射重组腺病毒.观察其治疗作用,Western blot检测肿瘤组织Src、Ras、MAPK蛋白的表达,实时逆转录-聚合酶链反应(Real-time RT-PCR)检测肿瘤组织Caspase-3、Caspase-8 mRNA的表达.结果 体外研究表明感染反义Src基因的C6胶质瘤细胞生长受到显著抑制.体内研究表明应用携带反义Src基因的腺病毒原位注射治疗大鼠胶质瘤能够抑制肿瘤生长,减少Src、Ras、MAPK蛋白的表达,增加Caspase-3、Caspase-8 mRNA的表达.结论 腺病毒载体介导的反义Src基因能够抑制大鼠胶质瘤细胞的生长.Src-Ras-MAPK信号通路参与胶质瘤的生长,抑制该信号通路可以增加凋亡通路关键蛋白Caspase-3、Caspase-8的表达.     

Adenovirus-mediated OX40Ig gene transfer induces long-term survival of orthotopic liver allograft in rats

BackgroundTo discuss the effect and mechanism of adenovirus-mediated OX40Ig gene transfer in inducing long-term survival of liver allografts in rats.MethodsOrthotopic liver transplantation was performed from Lewis to Brown Norway (BN) rats through the modified two-cuffed technique, and all rats were randomly divided equally into four groups: control, AdEGFP, AdOX40Ig, and FK506. The survival times of the rats were recorded. The rats' liver function, serum cytokines, hepatocyte pathology, OX40Ig protein level, and mixed lymphocyte reaction (MLR) with or without recombinant interleukin-2 (rIL-2) were evaluated.ResultsCompared with the control and AdEGFP groups, the rats in the AdOX40Ig and FK506 groups survived longer (P < 0.05), experienced less damage to hepatic function (P < 0.05), and showed milder hepatic cellular rejection and less hepatic cellular apoptosis. Interferon (IFN)-γ and IL-2 content in the serum were lower after operation (P < .05) in the AdOX40Ig and FK506 groups. On the contrary, IL-4 and IL-10 content in the serum was higher after operation (P < 0.05) in the AdOX40Ig and FK506 groups. OX40Ig protein was significantly expressed in the AdOX40Ig group and reached the highest level on the 7th day after operation. With respect to the MLR between BN and Lewis rats, the AdOX40Ig group showed a lighter reaction for the same strain than the control and AdEGFP groups (P < 0.05), which is different from the MLR between BN and F344 rats. After adding rIL-2 to the MLR system between BN rats in the AdOX40Ig group and Lewis rats, MLR was aggravated.ConclusionThrough OX40/OX4OL pathways, OX40Ig created an immunosuppressive effect after liver transplantation in rats. This immunosuppressive effect is associated with reduced IL-2 and can be reversed by adding IL-2 with antigen specificity.     

边缘性供肝的研究进展

供肝短缺已成为限制肝移植广泛应用的主要因素.如何扩大供体来源成为目前研究焦点之一.边缘性供肝用于移植有助于改善这一临床困境.然而,边缘性供肝存在术后原发性移植肝无功能、功能低下、迟发型移植物失活及疾病传染等风险.研究边缘性供肝危险因素及预防策略有助于推动肝脏移植的发展.本文总结了近年来边缘性供肝的研究进展.结合本中心的经验,提出边缘性供肝应用于临床有助于改善供体短缺的现状,并强调缺血再灌注损伤是边缘性供肝的核心问题及未来主要研究方向.     

Adenovirus-mediated gene transfer to healing tendon--enhanced efficiency using a gelatin sponge.

Adenovirus-mediated gene transfer is a potential method for enhancing tendon healing. We investigated the transfection of Ad5CMVntLacZ, an adenovirus containing the reporter gene LacZ, in primary cultured human rotator cuff tendon cells and in a rat Achilles tendon healing model in vivo. Ad5CMVempty, the adenoviral vector containing no inserted gene, was used as a control for adenoviral transfection alone. Activity of beta-galactosidase,the protein expressed by LacZ gene, was measured using a beta-galactosidase assay and detected visually by X-gal staining. Cultured cells were successfully transfected without impairing cell viability. Maximal beta-galactosidase activity was detected when cells were transfected at the dose of 1000 PFU/cell. The duration of LacZ expression was six days with a peak value at 24 h post-transfection. A transfection rate of 100% was obtained at 5000 PFU/cell. Successful in vivo transfection by Ad5CMVntLacZ was obtained in healing rat Achilles tendon as confirmed by X-gal staining. 0.4% of tendon cells were transfected when Ad5CMVntLacZ was injected into the tendon at a dose of 10(6) PFU. The rate rose to 2% with 10(8) PFU and 3% with 10(9) PFU. The duration of LacZ expression in vivo was 17 days. Transfection efficiency was enhanced threefold and localization improved when a gelatin sponge was used to deliver the adenovirus. The results demonstrate that adenovirus can be used to deliver a gene of interest to cultured human rotator cuff tendon cells and healing tendon, with gelatin sponge implantation enhancing adenoviral transfection efficiency in vivo.     

Adenovirus-mediated gene transfer in the midgestation fetal mouse.

BACKGROUND: The development of strategies for gene transfer in utero will make possible the amelioration, and eventually the cure, of genetic diseases associated with pre- and postnatal morbidity and mortality. We have developed a murine model for in utero, intrahepatic, adenovirus-mediated gene transfer in Day 15 fetuses and compared the level and distribution of luciferase reporter gene expression in newborns with those observed in adult animals injected intravenously. MATERIAL AND METHODS: CD-1 fetuses underwent intrahepatic injection on Day 15 of gestation with 1 x 10(7) particle-forming units (PFU) of an E1- and E3-deleted recombinant adenovirus containing the luciferase reporter gene or with normal saline. At birth, pups were euthanized, and the brain, heart, intestine, liver, lungs, and spleen harvested and analyzed for luciferase activity. RESULTS: Two adenovirus-injected litters proceeded to term and one female aborted. Tissues from 10 newborn mice in the experimental group and 5 newborns in the control group were analyzed; tissues from the remaining newborns were reserved for other studies. High-level luciferase expression was detected in all adenovirus-injected newborn livers. Lower levels of luciferase activity were detected in distant organs. Hepatic toxicity as determined by serum transaminase elevations was observed in adult, but not in newborn mice previously injected with the adeno-luciferase virus. CONCLUSIONS: In utero intrahepatic gene delivery with adenoviral vectors in the developing murine fetus is feasible and produces high-level gene expression. These studies suggest that viral and nonviral gene delivery vectors may be useful in the development of future approaches to prenatal treatment of genetic disorders.