中文摘要：

目的：体外分离培养人视网膜母细胞瘤肿瘤干细胞（RTSC），建立RTSC的鉴定方法。方法：新鲜Rb组织经酶消化法获取单个瘤细胞，接种于无血清化学限定培养基培养。对培养7-15d的神经样球体，分别进行克隆形成、增殖能力、分化能力等干细胞特性鉴定；采用RT-PCR及免疫荧光等方法对RISC及其分化细胞的特性进行鉴定。结果：Rb肿瘤在无血清培养基中可以形成神经球体样肿瘤球体，后者可以多次传代，具有自我再续能力。RTSC来源的神经球体表达神经干细胞或视网膜早期发育的相关基因Nestin、CD133、Bmi-1、Pax6、RX及Chx10。在分化培养基上，肿瘤球体内细胞可以向神经元和胶质细胞分化，形成神经网络样结构，该分化细胞部分表达GFAP、MAF2、GAP43、PKC-a、Syntaxin及Recoverin等。RTSC分化后的神经样细胞为谷氨酸能神经元细胞。结论：本结果揭示Rb中存在具有自我增殖及分化能力的视网膜前体细胞样的肿瘤干细胞。此类细胞可能是Rb恶性增殖的根源，也将成为Rb治疗的新靶点。

英文摘要：

AIM： To isolate and culture tumor stem cells in human retinoblastomas （RTSC）. METHODS： Retinoblastoma （RB） single cells acquired from fresh tumors of RB patients by enzyme digestion were seeded in serum - free medium at a density of 1×10^8 cells/L. Clonal cultures were plated at a density of 1×10^6 eells/L. Secondary tumor spheres were triturated again and passaged in fresh medium. The sphere - forming, proliferation and differentiation assays were performed. RT - PCR and immunocytochemistry were performed to identify the RTSC and differentiated cells. RESULTS： All RB tumors studied produced proliferating neurosphere- like tumor spheres, which were also passaged multiple times. These tumor spheres had the capability to self- renew, proliferate in SFM medium, expressed retinal progenitor cell related genes, and differentiated into neurons and glia when they were transferred to differentiation conditions. CONCLUSION： Our findings demonstrated that there were subsets of tumor stem cells resembling retinal progenitor cells in human RB, which can be isolated, cultured in SFM. The RTSC may be original cells of RB tumor, and also become the new target of tumor therapy.