硝酸盐预防腮腺放射损伤及经典wnt通路对其调控的机理研究

A significant complication following IR is its serious damage to the parotid gland, resulting in dry mouth and series of other sufferings, and finally seriously influences patient’s quality of life. However, no effective conventional prevention exists for dysfunction of parotid gland. Our pilot study demonstrated that dietary nitrate (NO3-) significantly protected parotid glands against IR and improved its secretory function in swine. However, the definite mechanism remains unknown. Wnt/β-catenin signaling pathway has been proven to enhance proliferation and angiogenesis of endothelial progenitor during ischemia. Here, to explore whether the radioprotective function of nitrate is regulated by Wnt/β-catenin signaling pathway via NO3-_ NO2-_NO, miniature pigs are subjected to a single irradiation at dose of 20 Gy targeting at one side of parotid gland, and before IR, these animals are treated with sodium nitrate or sodium nitrate (1mmol/kg body weight) + mouth water (to interrupt NO3- generating NO ) for 14 days. Parotid saliva flow, apoptosis of parotid cells, parotid blood flow, capillary density and expression of β-catenin, GSK-3β and p-GSK-3β will be determined. Further, the preventive effects of nitrate will be evaluated when Wnt/β-catenin signal pathway is activated or inhibited. This aspect of nitrate biology provides a novel strategy for effective and safe nutrition-based prevention of IR induced damage, which also shows a new sight into the function of nitrate, much different from the conventional conception..

放疗引起腮腺损伤，常导致唾液分泌低下，引起口干及系列并发症，严重影响了患者生活质量；目前无满意疗法。课题组前期研究发现：硝酸盐可维持腮腺血流、预防腮腺放射损伤，效果显著且安全，但具体机制尚不明确。经典Wnt通路已被证明可促进缺血组织血管新生。我们假设硝酸盐是通过转化为NO后，受经典Wnt通路调节，促进血管新生、维持血管功能来预防腮腺放射损伤。为验证此假设，本项目拟采用20 Gy单次放疗建立小型猪腮腺放射损伤模型，单独加载硝酸盐（1mmol/kg 体重）或联合抗生素（阻断硝酸盐转变为NO）14天，检测唾液分泌功能、组织结构、腮腺血流、血管状态及Wnt通路主要信号分子的表达情况等，并观察抑制或激活Wnt通路对硝酸盐预防效果的影响；进一步证实硝酸盐对腮腺放射损伤的预防效果，并探讨其机理。这一研究不仅打破人们对硝酸盐安全性的传统偏见，而且有望为临床上腮腺放射损伤患者，提供一种新的、有效的预防模式。

头颈部恶性肿瘤放疗引起腮腺损伤、唾液分泌低下，引起口干及系列并发症，严重降低了患者生活质量；目前无满意疗法。本项目模拟临床放疗条件，采用20 Gy单次放疗建立小型猪腮腺放射损伤模型，给予1mmol/kg硝酸钠（预防组）或1mmol/kg氯化钠（对照组）2周，检测血液和唾液中硝酸盐浓度，腮腺唾液分泌功能、组织结构，及经典Wnt通路主要信号分子的表达情况等，观察硝酸盐对腮腺放射损伤的预防效果，及经典Wnt通路对其调控作用；结果发现：（1）给予硝酸盐后，预防组唾液和血液的硝酸盐浓度显著增加，停止给予后，其浓度又恢复到基础水平；.（2）放疗后，预防组腮腺唾液流率为下降不明显或基本处于稳定高分泌水平的状态，而对照组腮腺唾液流率显著下降，且不能恢复；.（3）放疗后，预防组腮腺唾液成分无明显变化，而对照组腮腺钠离子、氯离子分泌显著减少，唾液淀粉酶显著下降，钾离子浓度显著上升；.（4）大体观示：对照组腮腺体积和重量明显萎缩，腺小叶被间质组织替代，而预防组变化不明显；.（5）HE染色示：预防组腮腺结构相对完整，与正常腮腺组织近似；而对照组腮腺腺泡结构混乱，间质内可见大量淋巴细胞浸润；.（6）电镜下观察示：预防组腺泡细胞膜相对完整，分泌小泡基本充盈，线粒体丰富，总体其超微结构近似正常腮腺组织；对照组腺泡细胞内细胞核固缩，染色质皱缩成团；残存的分泌小泡欠充盈；.（7）TUNEL染色示：预防组凋亡细胞较对照组明显减少；而内皮细胞明显增多；.（8）Westblot示：经典Wnt通路的主要信号分子如Wnt3a，β-Catenin，Gsk-3β，磷酸化Gsk-3β在预防组的放疗腮腺中的表达较未预防的放疗腮腺中的表达明显下调；.（9）血常规、血生化、重要组织器官组织结构等方面，未发现硝酸盐对小型猪产生明显的安全性问题。. 本研究利用与人类腮腺体积、结构等相似的小型猪作为研究对象，模拟临床放疗条件建模，从功能、结构、机理等方面均证实了硝酸盐能预防小型猪腮腺放射损伤，并初步探讨了安全性问题；为硝酸盐预防腮腺放射损伤应用于临床奠定了一定的研究基础。