在体角膜上皮细胞去分化追踪及相关生物学特性的研究

Dedifferentiation is a cellular process in which a partially or terminally differentiated cells reverts to there earlier adult stem cells. Clinically, we found that, in some total limbal stem cell deficiency cases, the central island corneal epithelial somatic cells (CECs) could keep self-stable and even self-healing after pieces of detachment. Further, we transplanted the EGFP+ mice's CECs to the corneal stroma of wild type mice after the whole corneal epithelium were debrided, and found that the LSCs related genes: ADCG2 and integrinα9 could over expressed in the transplanted CECs. Based on this previous work, we put forward a new hypothesis: The inhibition signal of LSCs will decrease or vanish after impairment or debridement, followed by the activation of CECs' dedifferentiation. In this project, we will first establish 8 transgenic mice models, using cellular linage tracing technique, intended to precisely testify, such as the respiratory tract epithelium, the phenomenon of dedifferentiation is also exist in the CECs. Further, Some biological characteristics of this phenomenon related to in vivo LSCs and CECs micro-environment changes will be studied. The ability of dedifferentiation in different location and different layers of CECs will also be evaluated. Hopefully, the results of this study will explain some uncertainty questions appeared in recent published papers, and should provide a theoretical foundation for the finding of new CECs sources.

去分化是指已分化的细胞，逆转成其早期干细胞的过程。临床中我们发现有些病例在角膜缘干细胞（LSCs）完全缺失后，中央的孤岛状角膜上皮体细胞（CECs）仍可长期维持稳定，甚至可在小片状损伤后再生修复。进而我们把小鼠EGFP+的中央CECs移植给完全去除角膜上皮组织的野生型小鼠，发现LSCs相关基因ADCG2和integrin α9可在该移植CECs高表达。据此，我们提出LSCs损伤时其抑制去分化的信号会减弱或消失，CECs的去分化进程会启动的新假设。本课题首先制备多种转基因小鼠模型，采用细胞谱系追踪等技术，意在精确验证与呼吸道粘膜上皮类似，CECs亦存在去分化现象，并对LSCs及CECs相关的“在体”微环境改变导致的这种去分化的生物学特性进行研究。研究结果有望解释新近多篇报道提出的LSCs地位的不明确性问题，并为发现新的CECs再生源奠定理论基础。

角膜缘干细胞缺失(limbal stem cells deficiency，LSCD)是一种可致盲的眼表性疾病，角膜缘干细胞（limbal stem cells，LSCs）移植是当今条件下治疗此类眼表疾病的重要手段。然而，角膜缘干细胞缺失患者其本身可用于移植的角膜缘干细胞残存量很少，临床上的治疗颇受限制。本研究依据新近人体内不同组织内发现的成体细胞去分化现象，意在探讨中央角膜上皮细胞在体“去分化”的可能性，有望为临床治疗角膜缘干细胞缺失提供新的思路。研究内容：通过20%乙醇联合机械刮除法建立小鼠全角膜上皮细胞缺失模型，后观察角膜缘是否缺失状态下角膜上皮移植后各模型的荧光素钠染色、眼表参数评分，免疫荧光检测LSCs标志物表达情况等各项指标，后取下各组的植片，对其LSCs标志物的表达情况采用Western-blot及RT-PCR方法进行检测。另外，通过慢病毒载体(Lentivirus vectors, LVs)介导的增强型绿色荧光蛋白(Enhanced green fluorescence protein, eGFP)基因转染离体小鼠中央角膜上皮细胞实验进行细胞示踪，并制作转染后角膜上皮细胞的细胞植片，对比移植到不同小鼠模型后经转染的角膜上皮细胞的不同变化，并观察和检测植片细胞的各项指标。结果：成功建立了小鼠全角膜上皮细胞缺失模型，角膜缘干细胞缺失组小鼠的角膜中央上皮体细胞LSCs标志物ABCG2等表达水平较正常对照组升高，表明角膜上皮体细胞存在向细胞上一阶段去分化的现象；通过细胞示踪，证实了小鼠中央角膜上皮中的非干细胞可以去分化为与LSCs类似的上皮干细胞，且去分化的角膜上皮细胞具有向角膜缘移形的趋势。全角膜上皮剥除联合同种异体角膜中央上皮移植组的LSCs标志物表达水平较角膜中央上皮剥除联合同种异体角膜中央上皮移植组更高，角膜缘组织对小鼠角膜中央上皮体细胞的去分化具有抑制作用。