大鼠心脏移植后心肌组织内支链氨基酸代谢对移植物血管病变的影响

Dysfunction of branded-chain amino acids catabolism in rat cardiac allograft

目的探讨大鼠心脏移植后心肌组织内支链氨基酸（BACC）代谢对移植物血管病变发生、发展的影响。方法实验分为同系移植组和同种移植组，采用改良的Ono模型建立大鼠腹部异位心脏移植模型。移植后2周和8周，取两组受鼠，分别获取其血液和移植心脏标本。观察两组受鼠的血管狭窄程度，使用比较蛋白质组学技术观察移植心肌内蛋白质表达的变化，获得一些影响BACC代谢的差异蛋白质，并采用免疫组织化学法进行验证。用氨基酸自动分析仪测定心脏组织和血浆中BACC浓度。结果移植后2周和8周，同系移植组冠脉血管未出现狭窄，而同种移植组冠脉血管出现明显狭窄（P〈0．01），并且随时间的延长，其血管狭窄程度逐渐加重（P〈0．01）。经搜索得到37个差异蛋白质（肽）点，其中3个为支链酮酸脱氢酶（BCKDH）亚单位E1α、E1β和E3BCKDH E1α、E1β在同种移植心脏组织和血管组织中的表达均显著下调（P〈0．05）。同时，移植心脏心肌组织内BACC浓度升高，而血浆内BACC浓度并未明显升高。结论同种移植心脏组织内支链氨基酸氧化代谢的限速酶受到抑制，使心脏组织内支链氨基酸（缬氨酸、亮氨酸和异亮氨酸）的浓度升高，其中亮氨酸作为蛋白质翻译的启动因子，通过哺乳动物雷帕霉素靶蛋白（mTOR）途径和非mTOR途径促进了蛋白质合成，可能在移植心脏心肌肥厚和移植物血管病变的发生、发展中发挥了一定的作用。

Objective Allograft vasculopathy （AV）, feature of chronic rejection, is a major serious long-term post-operation complication in organ transplantation. The accurate mechanisms for AV have not been definitively established, but extensive basic and clinical studies demonstrate AV is triggered by immune reaction and nonimmunologic factors, and also possibly attributed to the metabolism of branched-chain amino acids （BCAA）. Methods The transplanted hearts from Lewis to Sprague-Dawely rats served as allografts and those from Lewis to Lewis rats as isografts based on Ono＇s model. The differential proteins in transplanted hearts were separated by comparative proteomic technique, and some enzymes which regulated the metabolism of BCAA were identified and validated. Results All transplanted hearts at second week postoperation were characterized by lumen loss （total area-luminal area/total area） in coronary artery, but more predominant at 8th week. All samples from the left ventricles were analyzed by proteomic techniques and the subunits E1α, E1β and E3 of branched-chain α-ketoacid dehydrogenase （BCKDH） complex were decreased in the heart allografts. Immunohistological detection also showed the expression of BCKDH was reduced not only in the cardiac muscle but also more significantly in blood vessels with cardiac allograft vasculopathy （CAV）. BCAA concentrations were increased in the cardiac allografts, but there was no difference in the serum. Conclusion These findings suggest that the catabolic pathways of the BCAA may be inhibited owing to the reduced expression of BCKDH complex, and elevated intracellular concentrations of leucine. The vascular smooth muscle cell and cardiac muscle cell proliferation is stimulated via roTOR-dependent and mTOR-independent pathways, hypertrophy and AV in the heart allografts.