Qin L, Chavin K D, Ding Y, Woodward J E, Favaro J P, Lin J, Bromberg J S
Department of Surgery, Medical University of South Carolina, Charleston.
Ann Surg. 1994 Oct;220(4):508-18; discussion 518-9. doi: 10.1097/00000658-199410000-00009.
The authors tested the ability of plasmid gene transfer to express transforming growth factor-beta 1 (TGF-beta 1), prolong allograft survival, and evaluate promoter effects on gene expression.
Delivery of immunosuppressants directly to allografts using gene transfer and gene therapy approaches may inhibit immune activation while avoiding the systemic toxicity of conventional immunosuppression. Candidate genes include soluble cytokines, which could be expressed at low levels throughout the graft while inducing a local immunosuppressive effect. Transforming growth factor-beta 1 is a soluble cytokine that has pleiotropic immunosuppressive effects.
Cardiac grafts from syngeneic (CBA/J, H-2k) or allogenic (C57BL/6, H-2b) donors were placed into CBA/J recipients. Purified plasmid DNA-encoding murine TGF-beta 1 or beta-galactosidase (Lac Z) under the control of RSV, SV40, MMTV, or pancreatic elastase promoters was injected into grafts at surgery. The Lac Z expression was determined by histologic examination and TGF-beta 1 expression by graft survival. Cytotoxic T lymphocyte and flow cytometric analyses were performed to evaluate the immunosuppressive effects of TGF-beta 1 in vitro.
Plasmid DNA-encoding TGF-beta 1 prolonged survival from 12.6 +/- 1.1 days to 26.3 +/- 2.5 days (p < 0.02, Student's t test). The SV40 promoter was superior to the MMTV promoter in its ability to prolong survival. The effects of the plasmids were specific because Lac Z, antisense TGF-beta 1 inserts, or pancreatic elastase promoter did not prolong allograft survival. Histologic examination demonstrated Lac Z expression at least 14 days post-transplant in myocardial cells. Both RSV and SV40 promoters were effective in this respect, while a control null promoter was not. Toxicity testing showed that gene transfer of TGF-beta 1 did not alter survival or histology of syngeneic grafts. In addition, plasmids and purified TGF-beta 1 protein were not toxic to myoblasts in vitro. Recombinant TGF-beta 1 inhibited cytotoxic T lymphocyte generation and altered T cell surface receptor expression and subset expansion in vitro.
Gene transfer/therapy with plasmid DNA encoding TGF-beta 1 in vivo achieves immunologic effects that prolong allograft survival. Multiple promoters effectively induce plasmid expression, which is achieved in cardiac myocytes for at least 2 weeks without toxicity or adverse systemic effects. Transforming growth factor-beta 1 inhibits immune responses by different mechanisms, revealed by in vitro analysis of T cell cytolytic function, subset distribution, and receptor display.
作者测试了质粒基因转移表达转化生长因子-β1（TGF-β1）、延长同种异体移植物存活时间的能力，并评估启动子对基因表达的影响。
使用基因转移和基因治疗方法将免疫抑制剂直接递送至同种异体移植物，可能会抑制免疫激活，同时避免传统免疫抑制的全身毒性。候选基因包括可溶性细胞因子，其可在整个移植物中低水平表达，同时诱导局部免疫抑制作用。转化生长因子-β1是一种具有多效性免疫抑制作用的可溶性细胞因子。
将来自同基因（CBA/J，H-2k）或异基因（C57BL/6，H-2b）供体的心脏移植物植入CBA/J受体。在手术时将在RSV、SV40、MMTV或胰腺弹性蛋白酶启动子控制下编码小鼠TGF-β1或β-半乳糖苷酶（Lac Z）的纯化质粒DNA注入移植物中。通过组织学检查确定Lac Z表达，通过移植物存活情况确定TGF-β1表达。进行细胞毒性T淋巴细胞和流式细胞术分析以评估TGF-β1在体外的免疫抑制作用。
编码TGF-β1的质粒DNA将存活时间从12.6±1.1天延长至26.3±2.5天（p<0.02，学生t检验）。SV40启动子在延长存活时间的能力上优于MMTV启动子。质粒的作用具有特异性，因为Lac Z、反义TGF-β1插入片段或胰腺弹性蛋白酶启动子并未延长同种异体移植物的存活时间。组织学检查显示，移植后至少14天心肌细胞中有Lac Z表达。在这方面，RSV和SV40启动子均有效，而对照无效启动子则无效。毒性测试表明，TGF-β1的基因转移未改变同基因移植物的存活或组织学。此外，质粒和纯化的TGF-β1蛋白在体外对成肌细胞无毒。重组TGF-β1在体外抑制细胞毒性T淋巴细胞的生成，并改变T细胞表面受体表达和亚群扩增。
体内用编码TGF-β1的质粒DNA进行基因转移/治疗可产生延长同种异体移植物存活时间的免疫效应。多种启动子可有效诱导质粒表达，在心肌细胞中至少可持续2周，且无毒性或不良全身影响。通过对T细胞溶细胞功能、亚群分布和受体展示的体外分析揭示，转化生长因子-β1通过不同机制抑制免疫反应。
