携带反义c-myc的重组腺病毒安全性研究

Safety evaluation of Ad ASmyc in vitro and in vivo

目的 研究携带反义c myc的重组腺病毒 (Ad ASmyc)潜在的副作用 ,评价其安全性 ,为其进一步进入临床试验提供依据。方法 用Ad ASmyc感染HeLa细胞 ,制备细胞提取液后反复两次感染HeLa细胞 ,观察Ad ASmyc的繁殖、扩增 ,以及对HeLa细胞的作用 ,RT PCR检测其DNA的转录 ;确定Ad ASmyc对作为正常细胞的人胚肺二倍体细胞 2BS的感染能力 ,绘制细胞生长曲线 ,观察Ad ASmyc对正常细胞的毒性 ;给Balb/c小鼠腹腔注射重组腺病毒后 ,每日观察一般状况 ,化验肝、肾功能 ,PCR检测Ad ASmyc在重要脏器中的分布 ,显微镜观察重要脏器的病理学变化。结果 Ad ASmyc是一复制缺陷型腺病毒 ,它能高效感染 2BS细胞 ,但并不影响其生长 ,小鼠体内应用后 ,无急性毒性症状发生 ,在肝、肾、胃、脾内可检测到腺病毒的分布。但在注射 10 9pfu第 6天、第 12天的小鼠肝组织内可观察到轻微炎症。结论 Ad ASmyc应用是安全的 ,可以进入临床试验。

Objective To assess the safety of adenovirus mediated transfer of anti sense c myc(Ad ASmyc).Methods RT PCR was used to detect the transcription of Ad ASmyc in HeLa cells infected with extracts from HeLa cells previously infected with Ad ASmyc. Cell count was used to observe the effects of Ad ASmyc on the growth of normal human fetal lung diploid cell line 2BS. Ad ASmyc was given to 2 groups of Balb/c mice by intraperitoneal injection at a dosage of 10 7 pfu and 10 9 pfu respectively. Blood samples were obtained from the mice for liver and renal function tests. PCR was used to screen the vital organs for the presence of adenovirus DNA. Microscopic examination of the vital organs was performed to observe the pathogenicity of Ad ASmyc.Results As a replication defective virus, Ad ASmyc could infect 2BS cells effectively, but did not inhibit 2BS cell growth. No mouse died and no signs of general toxicity following intraperitoneal injection of Ad ASmyc were observed. In mice injected with 10 9 pfu Ad ASmyc, the adenoviral vector was found in the liver, spleen, kidney, and stomach. On day 6 and day 12 after injection, mild inflammatory infiltration of mononuclear cells was observed.Conclusion The use of adenoviral vector for antisense c myc gene transfer in vitro and in vivo is considered to be safe for clinical trial. Subject