人原代胶质瘤起始细胞的生物学行为初步研究

目的研究人原代胶质瘤起始细胞（GICs）的表型、自我更新能力、成瘤能力和耐药性等生物行为学特性。方法运用常规培养法和条件培养法对来自手术切除的同一胶质母细胞瘤组织分别进行培养,同时获得贴壁生长的人原代胶质瘤细胞（AGCs）和GICs;通过免疫荧光染色法测定其神经胶质酸性蛋白（GFAP）、CD133和巢蛋白的表达,通过单克隆形成实验和梯度密度成瘤实验检测其成球和成瘤能力,利用细胞计数药盒-8检测两种细胞在嘧啶亚硝脲（ACNU）和长春新碱（VCR）作用下的增殖曲线。结果 GICs呈神经球样生长且表达CD133和巢蛋白;47.8%±5.6%的GICs能够自我更新,而可表达GFAP的AGCs中仅有4.3%±1.6%能形成单克隆;ACNU对GICs的IC₅0=1×10^-3.1mol/L,对AGCs的IC₅0=1×10^-3.9mol/L,两者相比较,相差显著（P<0.05）;VCR对GICs的IC₅0=1×10^-4.3mol/L,对AGCs的IC₅0=1×10^-5.5mol/L,两者相比较,亦相差显著（P<0.05）。结论利用条件培养法可以获得具有明显干性的GICs,与分化的AGCs相比具有更强的自我更新能力和成瘤能力,且GICs对ACNU和VCR较AGCs更为耐药。

Preliminary study of biological behavior of primary human glioma-initiating cells

Objective To investigate the biological behavior properties of human primary glioma-initiating cells（GICs） including the phenotype,self-renewal,tumorigenicity and drug resistance.Methods The cells derived from the same human glioblastoma sample were cultured in the routine culture medium and conditioned medium shorting system respectively to obtain primary adhesive glioma cells（AGCs） and GICs.The expressions of the stem cell markers including CD133 and nestin in GICs were detected by immunofluorescence staining.The self-renewal ability and tumorigenic capacity of GICs and AGCs were determined by monoclonal formation assay and intracranial transplant glioma model.The sensitivities of both the cells to nimustine（ACNU） and vincristine（VCR） were detected by cell counting kit-8 assay.Results The neurosphere of GICS where CD133 and nestin were expressed could be obtained by conditioned medium sorting system.AGCs expressed differentiated marker GAFP.The monoclonal formation assay showed that 47.8%±5.6% GICs could self-renew,and only 4.3%±1.6% AGCs did.The small amount（1×10³） of GICs could form tumors while the tumor formation needed at least 5×10³ AGCs.The IC₅0 of ACNU to GICs and AGCs were 1×10^-3.1 mol/L and 1×10^-3.9mol/L respectively and The IC₅0 of VCR to GICs and AGCs were 1×10^-4.3mol/L and 1×10^-5.5 mol/L respectively.Conclusions GICs obtained by conditioned medium sorting method have significant stem cell-like properties,stronger self-renewal ability and tumorigenic capacity.GICs are more resistant to ACNU and VCR than AGCs（P <0.05）.