摘要
目的对新型永生化人肝细胞系Hep ZJ进行安全性研究。方法获取Hep ZJ培养上清液,通过PCR联合琼脂糖凝胶电泳法进行支原体检测,通过显色基质法进行内毒素检测;将Hep ZJ细胞悬液经尾iv进入SD大鼠后观察其生存情况并于不同时间点进行剖检,通实时荧光定量PCR(q RT-PCR)法分析该细胞系的生物分布;将Hep ZJ细胞悬液sc进入BALB/c-nu裸鼠后分析其致瘤性。结果 Hep ZJ支原体检测电泳图未见阳性条带;内毒素检测浓度<2 EU/m L;SD大鼠尾iv Hep ZJ后生存情况良好,血液、肺脏、脾脏在不同时间点出现不同程度的GAPDH-Human和TERT-Human基因表达,血液中12 h后、肺脏和脾脏中1周后基本被清除,剖检过程中未见肿块形成;裸鼠sc Hep ZJ后无硬性肿物形成。结论新型永生化人肝细胞系Hep ZJ无支原体、细菌污染,进入体内后无明显副反应及致瘤性,具备应用安全性。
Objective To complete the safety research for the new immortalized hepatocyte cell line Hep ZJ to confirm that it has applied security. Methods Obtaining the supernatant fluid of Hep ZJ for mycoplasma detection by PCR and bacterial endotoxin detection by Chromogenic End-point Tachypleus Amebocyte Lysate. Through tail vein Hep ZJ suspension was injected into SD rats to observe its survival and have autopsies at different time points for purpose of analyzing biodistribution of Hep ZJ by RT-PCR. The Hep ZJ suspension was injected subcutaneously into BALB/c nude mice to analyze its tumorigenicity. Results Hep ZJ mycoplasma detection electrophoregram did not show up positive stripe and endotoxin detection concentration was lower than 2 EU/m L; SD rats after injecting Hep ZJ intravenously survived well and its blood, lung, and spleen had different Hep ZJ gene expressions at different time but they were basically cleared away after one week. No lumps was discoverd in the process of autopsy. Nude mice injecting Hep ZJ subcutaneously hadn't found any tumor. Conclusion The new immortalized hepatocyte cell line Hep ZJ had no mycoplasma, bacterial contamination, and obvious secondary reaction nor tumorigenicity. Therefore, Hep ZJ is secure to be a new cell line for clinical and basic research.
引文
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