逆转录病毒作为基因治疗载体的生物安全检测

Bio safety testing for retroviral vector as gene therapy delivery system

目的在以逆转录病毒为载体的抗乙型肝炎病毒（ＨＢＶ）细胞内免疫基因治疗研究过程中，建立较完整的生物安全检测系统，为临床应用奠定基础。方法包括无菌，支原体和可复制性逆转录病毒的检测。支原体检测采用聚合酶链反应（ＰＣＲ）方法，可复制性逆转录病毒的检测应用Ｓ＋／Ｌ－试验，ＮＩＨ３Ｔ３细胞扩增试验和ｎｅｏ基因补救分析。结果在所建立的靶基因包装细胞系中，无菌试验均为阴性，一株包装细胞支原体呈阳性，全部包装细胞系未测到可复制性逆转录病毒。结论本研究采用的生物安全检测系统具有稳定性好，敏感染性高的优点，对基因治疗临床试验的生物安全检测具有重要的应用价值。

Objective To generate a bio safety testing system including the testing of sterility, mycoplasma and replication competent retroviruses(RCR) in the study of gene therapy anti hepatitis B virus by intracellular immunization.Methods Mycoplasma was detected by polymerase chain reaction. S +/L - assay, NIH3T3 amplification and rescue assay of neo gene were performed to determine RCR. Results The results showed that all of the packaging cell lines were negative for aerobic, anaerobic bacteria and fungi. One of the packaging cell lines was positive for mycoplasma. No RCR was detected in all of the packaging cell lines.Conclusions The results suggested that the methods using in present report are stable and sensitive, it is very useful for bio safety testing of gene therapy in clinical trials.