分子影像学方法示踪胚胎干细胞移植治疗急性肝损伤

摘要

BACKGROUND:Embryonic stem cel s have the capacity of multi-differentiation potential, and have been utilized for the therapy of acute liver injury. However, the migration and proliferation of embryonic stem cel s after transplantation remains not wel characterized. OBJECTIVE:To track the transplanted embryonic stem cel s in repairing acute liver injury by bioluminescence imaging technology. METHODS:Murine embryonic stem cel s (D3) were transducted with a construct composed of firefly luciferase, monomeric red fluorescence protein and herpes simplex virus truncated thymidine kinase triple fusion reporter genes by lentivirus system. Stable D3 embryonic stem cel s integrating three report genes were screened. The undifferentiated embryonic stem cel s or differentiated embryonic stem cel s from the 6-day-old embryoid body were transplanted into acute liver injury model of SV129 mouse through spleen, and the transplanted cel s were monitored by bioluminescence imaging technology. RESULTS AND CONCLUSION:Reverse transcription PCR results showed that the expression level of Oct-4 and Nanog was not affected in embryonic stem cel s transducted with triple fusion reporter gene compared with wild-type embryonic stem cel s. The migration process of transplanted cel s was visualized by bioluminescence imaging technology. Teratomas were found in both triple fusion-embryonic stem cel s treatment group and triple fusion-embryoid body cel s treatment group at liver, and the teratoma formation could be suppressed by ganciclovir administration because ganciclovir can react with herpes simplex virus truncated thymidine kinase and trigger cel necrosis process. Histological analysis showed that teratomas comprised tissues from al three germ layers. These results demonstrate that triple gene fusion does not affect differentiation potential of embryonic stem cel s and it is risky to utilize embryonic stem cel s for cel therapy, because it affects repair of liver injury. The therapy strategy requires further improvement and real-time visualizing of embryonic stem cel s in vivo is absolutely necessary.%背景：胚胎干细胞具有多向分化潜能，已被用于急性肝损伤的治疗，但其在体内治疗过程中的增殖迁移情况尚不清楚。　　目的：利用分子影像技术监测移植胚胎干细胞在急性肝损伤修复过程中的行为。　　方法：利用慢病毒载体，将萤火虫荧光素酶、红色荧光蛋白以及单纯疱疹病毒胸苷激酶三融合基因转入小鼠胚胎干细胞(D3)中，筛选得到稳定整合3个报告基因的D3胚胎干细胞系。将上述胚胎干细胞或分化了6 d的拟胚体细胞通过脾脏注射到急性肝损伤SV129模型小鼠体内，利用生物发光成像技术监测移植的细胞。　　结果与结论：RT-PCR结果显示，三融合基因的转入并未影响胚胎干细胞Oct-4和Nanog的表达。利用小动物活体成像系统可以观察到移植细胞从脾脏迁移到肝脏的过程。移植的胚胎干细胞和拟胚体细胞都在肝脏处形成畸胎瘤，由于单纯疱疹病毒胸苷激酶同时也是自杀基因，可以与更昔洛韦作用诱导转基因细胞死亡，通过注射更昔洛韦来抑制畸胎瘤的生长并逐步将其杀死。组织学分析显示，畸胎瘤里包含来自于3个胚层的组织。提示三融合基因的转入并未影响胚胎干细胞的多向分化潜能，且胚胎干细胞用于细胞治疗有潜在的成瘤风险，影响了对肝损伤的修复，治疗策略有待进一步改进，并需要实时监控其在体内的行为。