一种供氧并表达鸢尾素的新型生物系统的构建及其对术中血流阻断期间器官保护作用的初探

Hepatic blood occlusion, the essential step in hepatectomy often gives rise to liver injury. A recent study has reported a novel strategy that alleviated the cardiac ischemic injury caused by blood occlusion. It is the photosynthesis through intramyocardial delivery of the cyanobacterium Synechococcus elongatus. By using light as a source of energy, photosynthetic therapy increases tissue oxygenation and yields durable improvements in cardiac function during and after induction of ischemia. However, as prokaryote, Synechococcus elongatus is very difficult to purify and is not a proper vector for expression of active heterogenous molecular, which limits its therapeutic utilization. Chlorella, a type of eukaryotic microalgae, is easy to purify and a readily used vector for expression of many advanced heterogenous proteins. Moreover, our previous studies showed that Chlorella has a good biocompatibility and high photosynthesis efficiency. Irisin, a recently identified hormone, plays a pivotal role in energy expenditure and oxidative metabolism. Our previous study also showed that irisin has protective effects on hepatic I/R injury. Therefore, we proposed a novel biologic system by combining the photosynthesis and irisin to protect ischemic liver. We will initiate our study by constructing the irisin-expression Chlorella using genetic engineering approach. Then we will investigate the effect of light intensity on the photosynthetic efficiency and the irisin expression efficiency of the modified Chlorella in the ischemic liver in vivo. At last we will investigate the protect effect of the genetic modified Chlorella on the ischemic liver. This novel biologic system offers a new strategy for protecting the ischemic organ caused by blood occlusion.

血流阻断是肝切除术的关键环节也是导致的术后肝功能不全的重要原因，临床尚无有效治疗方法。最近有研究报道将聚球藻注射入血流阻断的小鼠心脏并给予光照，通过藻的光合作用供氧来减轻心脏损伤，这为解决术中血流阻断期的缺氧问题带来了希望。但聚球藻无杂菌藻株难以获得，且属于原核生物难以表达复杂、有生物活性的外源蛋白，临床转化前景不佳。属于真核生物的小球藻，无菌藻种易得且有完善外源蛋白表达修饰系统。我们前期研究证实小球藻具有高生物相融性和高光合产氧率，并证实鸢尾素这种肌肉分泌蛋白对肝脏缺血再灌注损伤有显著治疗效果。因此我们提出利用小球藻构建具有光合供氧功能并表达鸢尾素的新型生物系统，用以保护血流阻断期的肝脏。我们将首先构建表达分泌型鸢尾素的小球藻，之后研究光照强度对该小球藻注射入肝内后产氧及鸢尾素分泌的影响，最后验证其对血流阻断肝脏的保护作用。这一新型生物系统将为解决血流阻断期器官缺氧问题提供新思路。

术中因血流阻断和血流复通导致的器官缺血再灌注损伤是外科手术中难以避免的并发症且目前临床没有有效治疗手段。缺血再灌注损伤的一个重要诱因是血流阻断过程中氧供不足。因此在术中血流阻断期间为缺血组织器官提供充足氧供，缩短缺氧时间，是解决缺血再灌注损伤的有效途径。由于术中器官处于血流阻断状态，通过自然管腔进行供氧不易实现，而在术中建立人工腔道供氧也会对器官造成损伤。基于以上问题，本研究设计出利用小球藻光合作用为术中血流阻断器官供氧从而减轻器官缺血再灌注损伤新方法。. 本项目建立了细胞缺氧复氧模型、小鼠肾缺血再灌注模型和大鼠肝缺血再灌注模型，通过细胞实验和动物实验探究了小球藻光合供氧对器官缺血再灌注损伤的影响。研 究结果表明小球藻具有良好的生物相容性，在缺血缺氧阶段可显著增加细胞和器官中的氧分压，在复氧或再灌注阶段可显著提高肝功和肾功水平，减轻炎症反应，降低氧化应激水平，改善线粒体形态等，从而减缓缺血再管损伤。通过比较组间线粒体钙离子单向转运体（MCU）的表达水平发现，缺血再灌注vehicle 组中MCU表达较sham组显著升高，而小球藻治疗组中MCU表达与sham组无统计学差异。上述结果说明利用小球藻为术中缺血缺氧器官供氧光合供氧可减轻缺血再灌注损伤，其机制与有关改善线粒体钙动态失衡密切相关。