[关键词]
[摘要]
目的 对新型永生化人肝细胞系HepZJ进行安全性研究。方法 获取HepZJ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HepZJ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(qRT-PCR)法分析该细胞系的生物分布;将HepZJ细胞悬液sc进入BALB/c-nu裸鼠后分析其致瘤性。结果 HepZJ支原体检测电泳图未见阳性条带;内毒素检测浓度<2 EU/mL;SD大鼠尾iv HepZJ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 HepZJ后无硬性肿物形成。结论 新型永生化人肝细胞系HepZJ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。
[Key word]
[Abstract]
Objective To complete the safety research for the new immortalized hepatocyte cell line HepZJ to confirm that it has applied security. Methods Obtaining the supernatant fluid of HepZJ for mycoplasma detection by PCR and bacterial endotoxin detection by Chromogenic End-point Tachypleus Amebocyte Lysate. Through tail vein HepZJ suspension was injected into SD rats to observe its survival and have autopsies at different time points for purpose of analyzing biodistribution of HepZJ by RT-PCR. The HepZJ suspension was injected subcutaneously into BALB/c nude mice to analyze its tumorigenicity. Results HepZJ mycoplasma detection electrophoregram did not show up positive stripe and endotoxin detection concentration was lower than 2 EU/mL; SD rats after injecting HepZJ intravenously survived well and its blood, lung, and spleen had different HepZJ 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 HepZJ subcutaneously hadn't found any tumor. Conclusion The new immortalized hepatocyte cell line HepZJ had no mycoplasma, bacterial contamination, and obvious secondary reaction nor tumorigenicity. Therefore, HepZJ is secure to be a new cell line for clinical and basic research.
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[基金项目]
国家高技术研究发展计划(863计划)资助(2012AA020505);国家自然科学基金资助项目(81470875);国家重点研发计划课题(2016YFA0101503);广东省级科技计划项目(2014B020227002、2015B090903069、2015B020229002、2013B091100001);广东省自然科学基金资助项目(2014A030312013);中国食品药品检定研究院学科带头人课题(2015X1)