Her2 信号通路在胃癌肿瘤干细胞自我更新性及侵袭致瘤性中的作用研究

Therapies targeting cancer stem cells (CSC) have the potential to become an effective treatment for cancer in the future. However, related studies are very rare. Researches have found Her2 signal pathway in which AKT is an important member of downstream molecules involved closely in carcinogenesis and expansion of gastric cancer. Our previous studies have identified gastric CSCs from cancer tissues and the peripheral blood of gastric cancer patients. It has been observed that Her2 was expressed widely in gastric cancer stem cell. However, the expression of Her2 and lung metastatic ability of circulating cancer stem cell were higher than those of tissue cancer stem cell, with the higher expression of AKT in circulating cancer stem cell. Based on these, whether Her2 signal pathway facilitates to maitain the self-renewal, invasion and carcinogenicity of gastric cancer stem cell? Whether the role of Her2 signal pathway could be targeted as therapy strategy need to be resolved in this research. The research aims to investigate the impact of Her2 signal pathway on self-renewal, invasion and carcinogenicity of gastric cancer stem cell, as well as the feasibility and validity of Her2 signal pathway as biotherapy target in gastric cancer stem cell through RT-PCR, Western blot, immunohistochemistry and RNA interference, which might shed the lights on elucidating the mechanism further how gastric cancer stem cell maitain the self-renewal, invasion and carcinogenicity and show the theoretic basis of anti-tumor comprehensive therapies targeting Her2 signal pathway of gastic cancer stem cell.

靶向肿瘤干细胞的治疗成为目前研究热点，但相关研究寥寥。研究表明Her2信号通路与胃癌的发生、发展关系密切，AKT为该轴重要下游分子。我们前期已同时从原代胃癌组织及外周血中捕获了胃癌肿瘤干细胞，发现胃癌肿瘤干细胞普遍表达Her2，组织肿瘤干细胞Her2的表达及肺转移能力均低于循环肿瘤干细胞，AKT在循环肿瘤干细胞中高表达。基于此，Her2信号通路是否有助于维持肿瘤干细胞自我更新性及侵袭致瘤性？是否能以此作为治疗靶点？均为本课题拟解决的关键问题。本项目拟通过RT-PCR、免疫印迹、免疫组化、RNA干扰等研究Her2信号通路在维持胃癌肿瘤干细胞自我更新性及侵袭致瘤性方面所起作用，并初步探索胃癌肿瘤干细胞Her2信号通路作为生物治疗靶点的可行性及有效性。本项目或能为进一步揭示胃癌肿瘤干细胞维持其自我更新、侵袭致瘤性的机制，及为Her2信号通路用于胃癌肿瘤干细胞的靶向治疗提供理论基础。

本研究成功从胃癌细胞系及胃腺癌组织中培养出了细胞球，并证实了胃癌细胞球中含共表达CD54和CD44的胃癌肿瘤干细胞。研究发现Her2在胃癌组织肿瘤干细胞和胃癌细胞系中均有表达，但胃癌肿瘤干细胞中Her2表达远高于胃癌细胞系；对 Her2转录异构体Herstatin、Δ16HER2进行检测发现在胃癌组织肿瘤干细胞中及胃癌细胞系中均有较高表达。通过慢病毒转染，成功构建了Her2表达抑制的胃癌肿瘤干细胞。通过CCK8体外细胞增殖实验，发现Her2表达下调能降低胃癌肿瘤干细胞增殖能力。Her2表达下调也能降低胃癌肿瘤干细胞单细胞自我更新能力。利用Transwell模型发现，Her2表达下调使胃癌肿瘤干细胞的迁移和侵袭能力降低。通过细胞增殖-毒性实验，结果发现Her2表达下调后,胃癌肿瘤干细胞对化疗药物奥沙利铂及5-Fu敏感性增强。但对胃癌肿瘤干细胞体内外成瘤能力的检测发现，Her2表达下调后，胃癌肿瘤干细胞非锚定依赖性生长能力增加，裸鼠成瘤能力较对照组有所上升。此外，本项目收集了219例患者的胃癌组织标本并通过免疫组化检测Her2的表达，发现121人呈阴性表达，1+ 63人，2+ 31人，3+ 3人。进一步分析胃癌组织中Her2的表达情况、血清Her2的浓度及肿瘤分期三者之间均无相关性。生存分析发现胃癌组织不同程度Her2表达之间生存并无统计学的差异，血清Her2浓度高组生存曲线优于血清Her2浓度低组，但无统计学的差异。该结果与肿瘤干细胞体内外致瘤能力实验的结果显示出了一致性。为进一步解释上述结果，对两组细胞凋亡系统分子进行了RT-PCR及基因芯片检测。研究结果发现干扰组中的Bcl-2、mTOR表达上调，PI-3K和P53表达变化不大，而AKT和Caspase-9表达下调。基因芯片检测的结果来看，提示在胃癌肿瘤干细胞中目的基因Her2或许对Jak-STAT, mTOR等信号传导通路有调控作用，并提示该基因在生长调控、凋亡、蛋白质磷酸、和药物跨膜转运等功能和疾病中发挥作用，但具体作用尚需进一步验证。本项目研究了Her2分子在维持肿瘤干细胞自我更新性及侵袭致瘤性方面所起的初步作用，并初步探索了胃癌肿瘤干细胞Her2分子作为生物治疗靶点的可行性及有效性，为胃癌的靶向治疗奠定了理论基础。