Drazan K E, Olthoff K M, Wu L, Shen X D, Gelman A, Shaked A
Department of Surgery, University of California at Los Angeles, USA.
Transplantation. 1996 Oct 27;62(8):1080-4. doi: 10.1097/00007890-199610270-00010.
We hypothesized that adenovirus mediated gene transfer of TGF-beta1 into liver grafts would enhanced local expression of this recombinant protein and down-regulate inflammatory and alloreactive immune response. A full length DNA encoding the murine TGF-beta1 was used to replaced the E1 region of adenovirus type 5 (AdmTGF-beta1). Expression and protein production of biologically active murine TGF-beta1 was tested in AdmTGF-beta1-transduced Hep G2 cells and TGF-beta-sensitive MV1 cells. In the transplant setting, the replication-defective vector was used to perfused cold preserved ACI liver allograft prior to transplantation into Lewis recipients. Control livers were similarly perfused with cold lactated Ringer's solution and were followed without immunosuppression. Animals were sacrificed at 1, 3, and 5 days after transplantation. Intragraft cytokine levels of TNFalpha, and IFNgamma were determined using ELISA and quantitative PCR. TGF-beta1 ELISA of culture supernatants from AdmTGF-beta1 transduced hepatocyte cell line Hep G2 excreted TGF-beta1 in quantities directly correlated with multiplicity of infection (MOI, vector:hepatic cell ratio). The biological activity of the excreted recombinant protein was confirmed by growth inhibition of MV1 TGF-beta-sensitive cells. Enhanced production of TGF-beta1 in transduced allografts was associated with decreased levels of TNFalpha and IFNgamma when compared with nonimmunosuppressed controls. Adenovirus-mediated gene transfer of murine TGF-beta1 into hepatic cells results in the expression of biologically active protein. Transduction of allografts with TGF-beta1 down-regulates TNFalpha and IFNgamma production early after orthotopic transplantation. Graft transduction with TGF-beta1 offers a novel approach to study the effects of single immune modulator on alloreactive immune response, T cell function, and cytokine cascade.
我们推测，腺病毒介导的转化生长因子β1（TGF-β1）基因转移至肝移植物中，会增强该重组蛋白的局部表达，并下调炎症和同种异体反应性免疫应答。使用编码小鼠TGF-β1的全长DNA替换5型腺病毒（AdmTGF-β1）的E1区。在AdmTGF-β1转导的Hep G2细胞和对TGF-β敏感的MV1细胞中检测生物活性小鼠TGF-β1的表达和蛋白质产生。在移植实验中，使用复制缺陷型载体在移植入Lewis受体之前灌注冷保存的ACI肝同种异体移植物。对照肝脏同样用冷乳酸林格液灌注，且不进行免疫抑制处理。在移植后1天、3天和5天处死动物。使用酶联免疫吸附测定（ELISA）和定量聚合酶链反应（PCR）测定移植物内肿瘤坏死因子α（TNFα）和干扰素γ（IFNγ）的细胞因子水平。来自AdmTGF-β1转导的肝细胞系Hep G2的培养上清液的TGF-β1 ELISA显示，分泌的TGF-β1量与感染复数（MOI，载体：肝细胞比率）直接相关。分泌的重组蛋白的生物活性通过MV1 TGF-β敏感细胞的生长抑制得到证实。与未免疫抑制的对照相比，转导的同种异体移植物中TGF-β1的产生增加与TNFα和IFNγ水平降低相关。腺病毒介导的小鼠TGF-β1基因转移至肝细胞中导致生物活性蛋白的表达。TGF-β1转导同种异体移植物可在原位移植后早期下调TNFα和IFNγ的产生。用TGF-β1进行移植物转导为研究单一免疫调节剂对同种异体反应性免疫应答、T细胞功能和细胞因子级联反应的影响提供了一种新方法。
