Notch信号通路调控骨肉瘤干细胞的实验研究

Cancer stem cells (CSCs) are involved in the recurrence, metastasis and drug resistance of osteosarcoma. It is reported that Notch signaling pathway promotes the proliferation and metastasis of osteosarcoma. Osteosarcoma is thought to derived from abnormal bone marrow stem/progenitor cells, and Notch pathway participates in the self-renewal of bone marrow stem/progenitor cell, and inhibits its differentiation, we thus assume that Notch pathway plays a role in the regulation of osteosarcoma stem cells. Preliminary data has also shown that Notch pathway is activated in osteosarcoma stem cells and Inhibition of Notch pathway can prevent the tumor formation both in vitro and in vivo. In this project, we will transduce the osteosarcoma cells with pGreenFire Notch pathway reporter lentivirus, and select for stably transfected osteosarcoma cell lines, and the cells are then divided into different groups according to their Notch pathway activity. To evaluate the stem cell-like properties, the in vitro and in vivo capacity to self-renew, differentiation, chemotherapy sensitivity, and invasion/metastasis are examined. The project aims to disscuss the relationship between Notch signaling and osteosarcoma stem cells, and provide the basis for cancer stem cell-targeted therapy in osteosarcoma.

肿瘤干细胞同骨肉瘤复发、转移及耐药密切相关。Notch促进骨肉瘤增殖转移，骨肉瘤起源于异常骨髓间质干/祖细胞，而Notch参与其调节，故推测异常Notch调控骨肉瘤干细胞。课题组前期预实验发现骨肉瘤干细胞中Notch通路活性增高，而抑制Notch通路可抑制骨肉瘤成瘤。本项目在此基础上，构建Notch荧光报道慢病毒载体，感染并筛选稳转的骨肉瘤细胞，根据Notch活性分组，离体及在体水平比较不同Notch活性组细胞的自我更新、多向分化、药物敏感性和侵袭转移能力。项目旨在探讨Notch信号通路与骨肉瘤干细胞的关系，为骨肉瘤靶向治疗提供理论基础。

肿瘤干细胞同骨肉瘤复发、转移及耐药密切相关，我们推测异常Notch调控骨肉瘤干细胞。利用悬浮培养富集的骨肉瘤干细胞中Notch通路活性增高，但Notch报道的骨肉瘤细胞荧光表达呈正态分布，无法将细胞分为两群，需更改实后续验方案。. 由于耐药是肿瘤干细胞导致复发、转移的核心，本研究建立了骨肉瘤耐药模型，耐药细胞成间质样变，细胞长/短径比值较对照细胞显著增大，免疫荧光染色发现N-cadherin处理组较对照组荧光明显增高，流式细胞术发现处理组细胞Stro-1/CD117表达率显著增高，实时荧光定量PCR结果显示Sox2、Oct4及hTERT与对照组相比均有不同程度升高，以上结果表明顺铂处理后骨肉瘤细胞具有干细胞特性。. 为证实骨肉瘤干细胞同Notch通路活性的关系，我们检测了耐药细胞的Notch靶靶基因表达情况，实时荧光定量PCR结果显示发现Hes1、Hes5、Hey1及HeyL均有不同程度增高，Western Blot结果也证实Hes1蛋白表达量较对照组增加。构建裸鼠皮下移植瘤模型，分为顺铂及对照组，顺铂处理后体内肿瘤明显缩小，但仍有组织残留，取出肿瘤组织行免疫组化，发现顺铂处理后Hes1表达量增加，也支持Hes1参与骨肉瘤化疗耐药。. 研究过程中，拟行悬浮培养检测干细胞比例，结果发现对照组及处理组成球数（10细胞以上）无明显差异，但处理组有大量3-5个细胞的小球，推测细胞处于静止状态，进一步检测发现处理组较对照组G0/G1期增加，而G2/M期减少，与假设相符。. 综上所述，本研究证实耐药的骨肉瘤细胞具有较强干细胞样特性，Notch信号通路可能参与其中。