IRF-6定向诱导分化iPS细胞三维导向法再生腮腺组织研究

Samples of human parotid tissue were isolated, extracted, then cultured human parotid mesenchymal stem cells(hPMSCs). The cells were then reprogrammed to induced pluripotent stem cells(hPMSC-iPSCS) by episomal vector-mediated transduction of reprogramming factors with plasmid mixture on a feeder-free cells system. The iPSCs clones were authenticated by genome identification to ensure its safety and reliability. 24 SCID mice were randomly divided into three groups, each group of 8 mice. The first group was the control group; the second group was the animal model group of parotid radiation injury; the third group was the animal model group of lesion after partial parotidectomy. The iPSCs transfected with irf-6 plasmid were co-cultured with the mouse parotid gland tissue. Parotid gland stem cells, which were selected to differentiate into parotid tissue cells from hPMSC-iPSCs, were observed. After cell proliferation and passaging, the mice were injected with parotid gland stem cells into one side of parotid gland by Matrigel three dimensional guide technique, another side of paritod gland injected normal saline. Mice were sacrificed after four weeks, then remove bilateral parotid glands. The weight of the parotid gland tissue was compared between the transplanted side and the control group. HE staining was used to compare the regeneration of parotid gland tissue. The ultrastructural changes were observed by electron microscopy. Immunofluorescence technique was used to observe the changes of microvessel and the expression of AQP5, AMY1B,CK14 and CK15 in the regenerated parotid gland tissue.

取人腮腺组织样本，分离、提取、培养腮腺间充质干细胞。使用细胞核转染系统，用游离质粒电转染细胞，在无饲养层体系下重编程培养，诱导生成hPMSC-iPSCs克隆，行基因组鉴定以确定安全可靠。取联合免疫缺陷（SCID）小鼠24只，随机分为三组，每组8只。第一组为对照组；第二组为腮腺放射损伤组；第三组为腮腺部分切除术后损伤组。将用IRF-6质粒转染的iPS细胞与小鼠腮腺组织块共培养，筛选向腮腺组织细胞定向分化的hPMSC-iPSCs，传代增殖。通过Matrigel导管注射三维导向法将诱导成功干细胞注射移植到各组小鼠单侧腮腺。移植四周后处死小鼠，摘除双侧腮腺。比较小鼠移植侧与对照侧腮腺；HE染色比较再生情况；电镜检测细胞超微结构改变；免疫荧光染色观察再生腮腺组织的微血管改变与水通道蛋白、人腺泡细胞标记物、人导管结构角蛋白标记物以及人腺泡结构角蛋白标记物的表达。

目前临床上各种病因（头颈部肿瘤接受放射治疗、舍格伦综合征等）可导致的涎腺组织不可逆性破坏，引发唾液分泌功能低下导致严重的口腔干燥症。诱导性多潜能干细胞(iPSCs)是在基因表达、多潜能分化和表观遗传上类似于 ESCs 的全能干细胞，是组织修复再生研究重要的干细胞资源。IRF-6 是涎腺发育过程中起重要作用的基因通路。本研究利用IRF-6过表达在体外诱导形成涎腺干细胞并进行组织学培养；通过建立小鼠涎腺损伤模型，用IRF-6+IPSC进行动物研究再生腮腺组织。体外研究结果显示，iPS细胞的NANOG、SSEA4、OCT4呈阳性，具有类胚胎干细胞特性；经定向腮腺诱导分化后镜下观察呈上皮样改变，形成类导管及腺泡样结构，细胞上清液淀粉酶检测呈阳性；转录组测序结果显示IRF-6+诱导组与空白对照组相比，差异表达基因共643个，上调基因365个，下调基因278个，IRF-6+诱导组中唾液腺相关基因HAPLN1、CCL2、MSX2、ANXA1、CYP11A1、HES1和LUM均为高表达。动物实验研究结果表明，小鼠腮腺损伤模型建立后，小鼠唾液分泌的质（唾液淀粉酶活性）与量（唾液流率）均有不同程度下降，后缓慢上升，于模型建立后4周达到稳定；进行IRF-6+IPSC尾静脉注射治疗后发现，损伤模型治疗组小鼠唾液流率及唾液中淀粉酶活性明显高于损伤模型对照组、模型组血清及腮腺中的IL-6、IL-17a和TNF-α含量明显高于正常组，但损伤模型治疗组血清及腮腺中IL-6和TNF-α含量明显低于损伤模型对照组，IL-17含量低于模型组但差异不明显；模型组腮腺中的CD105和AQP-5的表达明显低于正常组，而损伤模型治疗组腮腺中的CD105和AQP-5的表达明显高于模型损伤对照组。本研究结果表明IRF-6过表达对IPSCs成涎腺分化有促进作用，其促进分化的机制可能是IRF-6上调了HAPLN1、CCL2、MSX2、ANXA1、CYP11A1、HES1和LUM的表达以促进上皮分化；同时动物研究结果表明进行iPS细胞定向诱导腮腺细胞全身注射治疗后改善了模型小鼠的口干症状、降低炎症反应、一定程度上加快涎腺血管组织及涎腺组织再生并加快恢复涎腺分泌功能，为涎腺损伤的组织工程学治疗提供了新的方法。