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目的:建立一种新的可动态观察肿瘤细胞生长的大鼠神经胶质瘤原位移植瘤模型。方法:利用脂质体将含有强化绿色荧光蛋白(enhanced green fluorescence protein,EGFP)基因的真核表达质粒pEGFP-N1转染大鼠神经胶质瘤C6细胞,经G418筛选及亚克隆,获取稳定表达EGFP的细胞系C6-gfp。采用CCK-8试剂盒检测C6及C6-gfp细胞增殖情况;将C6-gfp细胞接种于Wistar大鼠颅内,通过B超、大体标本、荧光体视镜、H-E染色和免疫组化观察颅内成瘤情况及瘤组织中绿色荧光蛋白的表达。结果:流式细胞术分析体外培养的C6-gfp细胞97.7%产生绿色荧光;C6-gfp细胞与亲代C6细胞相比,生长曲线无明显不同(P>0.05)。C6-gfp接种于颅内3周后成瘤率达70%;利用B超可动态观察肿瘤生长,利用荧光体视镜可见肿瘤组织发出绿色荧光,可与周围正常组织区别。H-E染色可见肿瘤细胞核大,染色深,核分裂明显,瘤内有很多新生血管。免疫组化可见GFP阳性细胞染色深浅不一。结论:成功构建了能稳定高水平表达EGFP的大鼠神经胶质瘤C6-gfp细胞株,其生物学行为未发生改变,能在同系大鼠颅内成瘤。该模型为神经胶质瘤防治研究提供了良好的基础。
OBJECTIVE: To establish a new rat glioma orthotopic xenograft tumor model which can dynamically observe the growth of tumor cells. Methods: Glioma C6 cells were transfected with eukaryotic expression plasmid pEGFP-N1 containing enhanced green fluorescence protein (EGFP) gene by lipofectamine. The cells were screened and subcloned by G418 to obtain stable expression EGFP cell line C6-gfp. The proliferation of C6 and C6-gfp cells was detected by CCK-8 kit. C6-gfp cells were intracranially inoculated into Wistar rats, and the intracranial sites were observed by B ultrasound, gross specimen, fluorescence microscope, HE staining and immunohistochemistry Tumorigenesis and expression of green fluorescent protein in tumor tissue. Results: Flow cytometry analysis showed that 97.7% of C6-gfp cells produced green fluorescence in vitro. There was no significant difference in growth curve between C6-gfp cells and parental C6 cells (P> 0.05). C6-gfp inoculated intracranial 3 weeks after the tumor formation rate of 70%; the use of B-dynamic observation of tumor growth, the use of fluoroscopic mirror visible tumor tissue emitting green fluorescence, with the surrounding normal tissue difference. H-E staining showed large tumor nuclei, deep staining, mitotic obvious tumor has a lot of neovascularization. Immunohistochemistry shows that GFP-positive cells have different shades. CONCLUSION: C6 glioma cell line C6 glioma stably expressing high level of EGFP has been successfully constructed and its biological behavior has not changed. This model provides a good foundation for the prevention and treatment of glioma.