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目的研究猿肾病毒40大T抗原基因永生化大鼠星形胶质细胞株(IAST)的生物学特性,探讨其作为转基因细胞镇痛载体的可行性。方法取大鼠大脑皮层星形胶质细胞(AST)和IAST 进行体外培养,观察细胞的形态学、超微结构以及传代复苏的情况;绘制细胞生长曲线,免疫细胞化学法检测细胞胶质纤维酸性蛋白的表达;5’溴-2-脱氧尿苷掺入法和流式细胞仪检测细胞增殖周期;双层软琼脂克隆形成实验和裸鼠接种实验检测细胞的致瘤性。结果AST体外传代至10代左右即出现复制衰老现象,而IAST可连续传代,未出现衰老迹象;细胞生长呈单层,锚着依赖和接触抑制;传代、冻存和复苏对IAST的存活率无影响,而第6代和第10代AST的存活率降低(P<0.01);与第6代和第10代AST相比,IAST增殖较为旺盛,增殖指数较高,群体倍增时间较短、增殖率较高(P<0.01); IAST胶质纤维酸性蛋白免疫染色阳性,软琼脂培养无集落形成,裸鼠接种无致瘤性。结论大鼠IAST是增殖活跃的非恶性转化细胞,可安全地用于转基因细胞移植镇痛研究。
Objective To study the biological characteristics of the immortalized rat astrocyte cell line 40 of the simian nephrotic virus (TSP) gene and investigate its feasibility as a carrier for the transfection of transgenic cells. Methods The rat cerebral cortical astrocytes (AST) and IAST were cultured in vitro to observe the morphological and ultrastructural changes of the cells and the passage of resuscitation. The cell growth curve was drawn, and the immunocytochemistry was used to detect the glial fibrillary acidic Protein expression; 5 ’bromo-2-deoxyuridine incorporation and flow cytometry cell proliferation cycle; double soft agar colony formation assay and nude mice inoculation experiments to detect cell tumorigenicity. Results Adenosine triphosphate (AAST) was found to replicate and senescently in passages 10 to 10, but IAST was passaged continuously with no sign of aging. Cell growth was monolayer, anchorage dependent and contact inhibition. Survival rate of IAST with passage, cryopreservation and resuscitation was no (P <0.01). Compared with the sixth and the tenth generation AST, IAST had stronger proliferation, higher proliferation index, shorter population doubling time and more proliferation (P <0.01). IAST glial fibrillary acidic protein immunostaining positive, soft agar culture without colony formation, nude mice vaccination non-tumorigenic. Conclusion Rat IAST is an actively proliferating non-malignant transformed cell that can safely be used in the study of analgesic effect of transgenic cell transplantation.