SK-N-SH神经母细胞瘤干细胞培养及鉴定

Culture and identification of SK-N-SH neuroblastoma stem cells

背景:神经母细胞瘤是儿童较为常见的实体瘤,儿童肿瘤受环境因素影响小,其与发育的关系紧密。而悬浮法是一种有效并可靠的获得肿瘤干细胞的方法。目的:采用悬浮法培养人神经母细胞瘤SK-N-SH,获得具有干细胞特性的克隆球,并对其生物学特性进行鉴定。方法:采用无血清悬浮培养的方法,获得具有克隆性增生的肿瘤球,免疫荧光鉴定克隆球细胞是否具有干细胞特性;通过荧光素酶标记SK-N-SH神经母细胞瘤,将所得克隆球与普通培养肿瘤细胞种植在裸鼠背部,活体成像监测细胞在裸鼠体内的增殖特性。结果与结论:悬浮培养法可将SK-N-SH神经母细胞瘤培养成克隆球,将无血清培养的克隆球免疫荧光检测分子生物学标记,显示强阳性表达,将其种植于裸鼠背部皮下显示其具有较强增殖与转移的生物学特性。提示悬浮法培养的SK-N-SH神经母细胞瘤克隆球,分子生物学标记巢蛋白Nestin强表达,胶质原纤维酸性蛋白、特异性神经元管蛋白(Tuj-1)弱表达,具有神经干细胞的特性,在裸鼠体内具有较强的增殖性与转移侵袭性。

BACKGROUND：Neuroblastoma is a common solid tumor in children. Pediatric tumors are little affected by environmental factors, but closely related to child development. The suspension method is an effective and reliable method to harvest neoplastic stem cel s. OBJECTIVE：To culture the cel clones of human neuroblastoma cel line SK-N-SH and to assess the biological properties of the cel clones. METHODS：Using the suspension method with no serum media, tumor cel clones were obtained. Immunofluorescence method was used to identify whether tumor cel clones exhibit stem cel properties. SK-N-SH neuroblastoma was labeled by luciferase, and tumor cel clones and tumor cel s were seeded onto the back of nude mice to monitor cel proliferative properties in nude mice using in vivo imaging. RESULTS AND CONCLUSION：Using the suspension culture method, SK-N-SH neuroblastoma cel s could successful y develop into cloning bal s. Under serum-free culture, cloning bal s were immunofluorescently used to detect molecular markers that showed strong positive expression. Cloning bal s subcutaneously implanted into 〈br〉 nude mice showed the strong ability of self-renewal and differentiation as stem cel s. The cel clones cultured by the suspension method strongly expressed Nestin, but weakly expressed glial fibril ary acidic protein, neuron-specific tubulin, possessing stem cel characteristics and strong proliferation and metastasis in nude mice.