血红素加氧酶-1诱导大鼠肝移植免疫耐受的研究

Heme oxygenase-1 gene transfer mediated by adeno-associated virus induces immunological toler- ance in liver allograft in rats

目的探讨腺相关病毒介导血红素加氧酶-1（AAV—HO-1）诱导大鼠肝移植免疫耐受的有效性及其分子机制。方法按Kamada二套管法建立大鼠原位肝移植模型。在供肝冷保存阶段分别经门脉灌注磷酸盐缓冲液（PBS）、EmptyAAV或AAV—HO-1，孵育2h后行DA—Lewis大鼠原位肝移植，检测移植术后大鼠的中位存活时间（MST）；血清中白细胞介素（IL）-2、肿瘤坏死因子（TNF）-d的表达；移植肝中CD4＋、CD8＋，调节性T细胞（Treg细胞、CD＋CD25＋Foxp3＋）的表达；耐受组脾脏中Treg细胞百分率并对耐受组脾脏行混合淋巴细胞培养（MLC）观察免疫耐受。结果AAV—HO-1组MST为30d，显著长于PBS组（11d）或EmptyAAV（12d）（P〈0．01），其中20％存活超过90d；血清中IL-2、TNF-0l的表达明显降低，CIM＋和CD8＋T细胞的浸润减少，Treg细胞在移植肝中的浸润增加。耐受组脾脏中Treg细胞为6．7％，显著提高，行MLC后几乎无淋巴细胞反应。结论AAV—HO-1可通过增加Treg细胞，延长大鼠的存活期，证实AAV．HO-1可诱导肝移植免疫耐受。

Objective To investigate the efficacy and molecular mechanisms of AAV-HO-1 indu- cing immuno-telorance in rats. Methods An orthotopic liver transplantation model by DA to Lewis was set up using Kamada＇ s two cuff technique. PBS, empty AAV or purified AAV-HO-1 was injected into the portal vein and incubated for 2 h at the donor liver cold preserved stage, and then OLT was done. The media survival time （MST） , serum levels of interleukin （IL）-2 and tumor necrosis factor （TNF）-α, infiltration of CD4 ＋ , CD8＋ and Treg （ CD4＋ CD25 ＋ Foxp3 ＋ ） cells into donor livers and percent of Treg in the spleen were examined. A mixed lymphocyte reaction （MLR） was performed. Results Recipients transplanted with AAV-HO-l-perfused liver allografts had a median survival time of 30 days, which was significantly longer than that in the PBS group （11 days） and empty AAV group （12 days） （both P 〈 0. 01 ）. Overex- pression of HO-1 reduced the production of IL-2 and TNF-α, inhibited infiltration of CD4＋ and CD8＋ cells, and increased infiltration of Treg cells into donor livers. Splenocytes from the tolerant recipients had higher percentages of Treg cells, and responded poorly to the allogeneic donor splenocytes. Conclusion Persistent expression of HO-1 in the donor livers by intraportal delivery of AAV-HO-1 can prolong the sur- vival by expanding Treg cells, suggesting that AAV-HO-1 can induce immuno-tolerance in liver allograft in rats.