人羊膜间充质干细胞促进放射性损伤小鼠模型唾液腺功能修复及机制研究

Treatments for head and neck cancer such as radiotherapy can lead to salivary gland hypofunction and yet there is no effective treatment. Stem cell therapy has provided new ideas for the disease. In the past, the stem cell treatment mechanism was usually explained as stem cells homing, differentiation, and replacement of damaged cells, which has been found closely related to the paracrine effects of stem cells in recent years, and hypoxia treatment can increase the paracrine effects of stem cells. Human amniotic mesenchymal stem cells have many advantages, such as wide sources and noninvasive sampling. Our recent studies show that the ability of proliferation, secretion and anti-apoptosis of hAMSCs is promoted after hypoxic preconditioning. Based on this, we propose to co-culture hAMSCs or its' lysates after hypoxic preconditioning with radiation-damaged salivary gland cells, then detect the morphology, anti-apoptosis, anti-oxidation, revascularization-promoting and expression of functional proteins of salivary gland cells, and the repair system of radiation injury was also optimized. Finally, the repair effect and signal transduction mechanism of hypoxia preconditioning hAMSCs were verified in the salivary gland radiation injury model. This study provides a new idea for the treatment of radiation-damaged salivary gland from the new perspective of promoted paracrine effects of hAMSCs after hypoxia preconditioning.

头颈部恶性肿瘤放射治疗等可导致涎腺功能损伤，目前尚无有效治疗方法。干细胞治疗为该疾病提供了新思路。以往认为干细胞治疗机制是干细胞归巢、分化并替代损伤细胞，近年发现其治疗机制与干细胞的旁分泌作用密切相关，且低氧处理可增加干细胞的旁分泌作用。人羊膜间充质干细胞（Human amniotic mesenchymal stem cells ，hAMSCs）具有来源广泛、取材无创等优点。我们新近研究显示：hAMSCs经低氧预处理后，可促进其增殖、分泌及抗凋亡能力。在此基础上，本研究拟将低氧预处理hAMSCs或其裂解物与放射损伤涎腺细胞共培养，检测涎腺细胞形态学、抗凋亡、抗氧化、促进血管再生及功能蛋白表达情况，优化修复放射损伤的体系。最终，在涎腺放射损伤模型中，验证低氧预处理hAMSCs的修复效果和信号转导机制。本研究从低氧预处理促进hAMSCs旁分泌这个新视点，为放射性涎腺损伤的治疗提供新思路。

头颈部恶性肿瘤的放射治疗、舍格伦综合征、发育异常均可导致涎腺分泌功能降低或丧失，引起口干、继发性龋、吞咽困难，严重影响患者的生活质量和身体健康，目前尚无有效的治疗方法。显微镜下观察不同体积分数氧气预处理组人羊膜间充质干细胞的形态无明显差异，低氧预处理能提高人羊膜间充质干细胞的增殖、抗凋亡和旁分泌细胞因子的能力，且体积分数1%氧气预处理效果优于体积分数3%，5%，10%，21%氧气预处理。体外hAMSCs及hAMSCs-Exos对放射性损伤涎腺上皮细胞的修复有促进修复效果。体内常氧hAMSCs-Exos组、低氧hAMSCs-Exos组大鼠体重、唾液量均高于PBS组。低氧hAMSCs-Exos组唾液量高于常氧hAMSCs-Exos组，在第7、28天 低氧hAMSCs-Exos组大鼠体质量高于常氧hAMSCs-Exos组且差异有统计学意义（P＜0.05）。颌下腺组织HE染色显示：低氧hAMSCs-Exos组的腺体修复能力最强。颌下腺组织AQP5表达的定量分析结果表明：低氧hAMSCs-Exos组及常氧hAMSCs-Exos组较PBS组在第14、28天光密度值增加（P＜0.05），且低氧hAMSCs-Exos组高于常氧hAMSCs-Exos组，差异有统计学意义（P＜0.05）。将人尿源干细胞外泌体注射入放射损伤涎腺大鼠体内，检测发现hUSC-Exos可能通过激活Wnt3a/GSK3β途径修复放射损伤的唾液腺。