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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RTSC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、MAP2、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。
OBJECTIVE: To isolate and culture human retinoblastoma tumor stem cells (RTSC) in vitro and establish an RTSC identification method. Methods: Fresh Rb tissue was obtained by enzymatic digestion of single tumor cells, seeded in serum-free chemically defined medium. The neurospheres were cultured for 7-15 days, respectively. The characteristics of stem cells such as clonality, proliferation and differentiation were identified. The characteristics of RTSC and its differentiated cells were identified by RT-PCR and immunofluorescence. Results: Rb tumor can form neurosphere-like tumor spheroids in serum-free medium, which can pass through many times with self renewal ability. RTSC-derived neurospheres express Nestin, CD133, Bmi-1, Pax6, RX, and Chx10 on neural stem cells or early retinal development. On the differentiation medium, the cells in the tumor sphere can differentiate into neurons and glial cells to form neural network-like structures, and the differentiated cells partially express GFAP, MAP2, GAP43, PKC-a, Syntaxin and Recoverin and the like. RTSC-differentiated neuronal-like cells are glutamatergic neurons. CONCLUSIONS: This result revealed that retinal precursor cell-like tumor stem cells with self-proliferating and differentiating abilities exist in Rb. Such cells may be the source of Rb malignant proliferation, will also become a new target for Rb treatment.