p75NTR在细胞核和细胞浆不同部位表达与食管癌细胞分化程度的关系及其作用研究

Cancer stem cells are the leading cause of high cancer mortality for advanced cancer. Therefore, there is an increasing need to understand the biology of cancer stem cells so that new therapeutic developments could tackle this cell population. However, comparatively little is known about the biology of esophageal cancer stem cells (ECSC) compared to other solid tumor stem cells because the special marker of ECSC has not been found. We suppose a hypothesis that the cell expressing p75NTR in nucleus is likely ECSC or the more primitive ECSC in the hierarchy of esophageal cancer stem cell, on account of the results we have done. So in this grant,we will separate the cell clones of p75NTR+ expression in nucleus and in cytoplasm by single cell clone and cell spheres formation from the human esophageal cancer cell line and the primary culture esophageal cancer cells of patient, respectively, to detect its expression of esophageal epithelial stem cells marker with immunofluorescent cell technology ,Western-blot and PcR,respectively; to analysis their proliferation, self-renewal, invasion and tumorigenesis by MTT, Flat clone, Transwell and xenografted into immunodeficient mice, respectively, to prove the hypothesis we supposed.

肿瘤干细胞是晚期癌症死亡率高的主要原因，认识肿瘤干细胞有助于进一步了解肿瘤细胞的生物学特点，发展直接靶向肿瘤干细胞的治疗技术。但是有关食管癌干细胞生物学知之甚少。基于前期研究结果，我们提出食管癌细胞中p75NTR核阳性表达的细胞可能就是食管肿瘤干细胞或分化程度比较低的干细胞的假设。本项目将通过采用单细胞克隆和细胞球培养技术分别从食管癌细胞株和原代培养的细胞中分离p75NTR核阳性表达细胞克隆和p75NTR浆阳性表达细胞克隆，采用荧光免疫细胞技术、Western-blot技术和PCR技术，分析两种细胞克隆的食管上皮干细胞标签蛋白表达情况，采用MTT、平板克隆、Transwell和体内种植致瘤检测其增殖和自我更新能力、侵袭性和致瘤能力，证明p75NTR核阳性表达细胞克隆是食管癌干细胞或分化程度比较低的干细胞，并在此基础上探讨p75NTR对维持食管癌干细胞的生存及自我更新的作用及机制。

肿瘤干细胞是晚期癌症死亡率高的主要原因，认识肿瘤干细胞有助于进一步了解肿瘤细胞的生物学特点，发展直接靶向肿瘤干细胞的治疗技术。但是有关食管癌干细胞生物学知之甚少。基于前期研究结果，我们提出食管癌细胞中p75NTR核阳性表达的细胞可能就是食管肿瘤干细胞或分化程度比较低的干细胞的假设。.本项目将通过采用单细胞克隆和细胞球培养技术从食管癌细胞株中分离p75NTR核阳性表达细胞克隆和p75NTR浆阳性表达细胞克隆，通过检测食管上皮干细胞标签蛋白p63、外皮蛋白、CK-13、整合素-β1、整合素-β4、与多药耐药有关蛋白CTR1和ABCG2表达，分析p75NTR核阳性癌细胞的性质和其在癌细胞中所处地位。通过三种途径：分别诱导p75NTR核、质阳性表达细胞分化，观察细胞p75表达的变化、细胞增殖能力等；通过体外增殖实验和体内致瘤实验，比较两种细胞的自我更新能力和致瘤性；分析p75NTR核、质阳性表达细胞自分泌神经营养因子情况以及对p75NTR核、质阳性表达的细胞自我更新和分化的影响。以及共受体，主要是TrA受体表达情况以及对p75NTR核、质阳性表达的细胞自我更新和分化的影响及可能的信号通路。.结果无血清连续培养5代以上，细胞球均由p75NTR核阳性癌细胞组成。采用荧光免疫细胞化学技术检测5代及以上细胞球细胞的p75NTR和Ki67的表达，发现 p75NTR核阳性细胞球由5种细胞组成：①仅表达p75NTR；②p75NTR和Ki67共表达；③p75NTR++和Ki67+；④p75NTR+和Ki67++；⑤p75NTR+和Ki67+DAPI++。 初步筛选出分离p75NTR核阳性细胞的方案：p75NTR核阳性+Nanog细胞浆（膜）阳性。初步证实p75NTR核阳性的食管癌细胞是食管癌干细胞，p75NTR核阳性细胞仍然是一群异质性细胞群，处于不同的增殖和分化时期，大概能分成5群细胞①仅表达p75NTR；②p75NTR和Ki67共表达；③p75NTR++和Ki67+；④p75NTR+和Ki67++；⑤p75NTR+和Ki67+DAPI++。初步证明p75NTR核阳性的食管癌细胞分泌NGF，通过自分泌作用，激活p75NTR受体，提高γ-secretase活性，产生ICD，进入细胞核，激活p38/MAPK 信号通路，促进p75NTR核阳性的食管癌细胞增殖和自我更新。