人类神经上皮肿瘤中Nestin^+细胞的分离培养和表达

Isolation,culture of Nestin positive cells and Nestin expression in human neuroepithelial tumors

目的:检测巢蛋白(Nestin)在神经上皮肿瘤组织、细胞中的表达,评价Nestin+细胞体外生物学特性并探讨其意义.方法:采用免疫组化法检测神经上皮肿瘤组织中Nestin的表达情况,免疫磁珠法分选肿瘤组织中的Nestin+细胞,悬浮细胞培养法进行Nestin+细胞的增殖、分化与鉴定,RT-PCR法检测Nestin蛋白在其中的活性表达情况.结果:Nestin在肿瘤内皮细胞中均有阳性染色,而在神经上皮肿瘤细胞中阳性染色比例为9.5%～96.2%,且Nestin+细胞比例及染色强度与肿瘤的恶性程度正相关.Nestin+细胞能在无血清培养基中形成典型的脑肿瘤球,在含血清培养基中分化为成熟的神经细胞系.Nestin蛋白在Nestin+细胞中活性表达强度约为阴性细胞的18.5倍,其表达强度取决于细胞的来源.结论:Nestin表达检测有助于正确评价神经上皮肿瘤的生物学行为和恶性程度,并可作为判断肿瘤病理级别和患者预后的重要依据.

AIM： To investigate the expression of Nestin in human neuroepithelial tumors and to evaluate the biological characteristics of Nestin positive tumor stem cells in vitro and the significance. METHODS： Nestin expression was detected in 33 cases of neuroepithelial tumors using the immunocytoehemical staining. The Nestin positive and negative cells were isolated by magnetic activated cell sorting （MACS）. Nestin positive cells were culutered, identified, and induced to differentiate. The expression of Nestin protein was detected in Nestin^＋ cells by RTPCR. RESULTS： All the vascular endothelial cells of tumors were positive for Nestin. The percentage of Nestin positive cells was 9.5% - 96.2% in the ncuroepithelial tumors. The positive expression rate and staining intensity of Nestin^＋ cells were positively correlated with the tumor pathological grades. Nestin protein activity in Nestin^＋ tumor stem cells was 18.5 times more than that of Nestin^- cells, and expression intensity was related to cell origin. Nestin positive cells proliferated into form typical tumor neurospheres in serum-free medium and further differentiated into neuron-like, glia-like and oligodendroglia-like mature cells. CONCLUSION：The detection of Nestin expression contributes to correctly evaluate the malignant degree and biological behaviors and is an important clue to ascertain the pathological grades and prognosis of neuroepithelial tumors.