中文摘要
孤独症严重影响青少年情感和智力正常发育。既往在孤独症人群中发现MeCP2基因拷贝数增加，MeCP2基因突变小鼠具有孤独症表型，证实MeCP2参与孤独症的发生，但机制未明。我们通过慢病毒介导的转基因方法构建了携带人类MECP2基因的转基因食蟹猴。我们通过免疫组化与生化试验验证转基因在食蟹猴脑内成功表达，并通过多种行为学实验发现MECP2转基因食蟹猴有重复刻板行为，焦虑水平上升，与社交行为障碍等类孤独症表型。我们并得到了稳定遗传人类MECP2基因的转基因第二代食蟹猴，并发现第二代转基因食蟹猴仍然具有社交障碍等孤独症核心症状。通过此项研究，对阐明MeCP2在孤独症发生发展中的作用，揭示MeCP2在孤独症发病中的机制，为干预孤独症提供新的思路。
英文摘要
Methyl-CpG binding protein 2 (MeCP2) plays critical roles in transcriptional regulation and microRNA processing. Mutations in MECP2 gene are found in 90% of patients with Rett syndrome, a severe form of developmental disorders with autistic phenotypes. Duplications of MECP2–containing genomic segments cause the MECP2 duplication syndrome, which shares core symptoms with autism spectrum disorders (ASD). Although the Mecp2- mice recapitulate most developmental and behavioral defects found in Rett syndrome patients, it has been difficult to identify autism-like behaviors in the mouse model with MeCP2 overexpression. Here we report that lentivirus-based transgenic cynomolgus monkey (Macaca fascicularis) expressing human MeCP2 in the brain exhibited autism-like behaviors and showed germline transmission of the transgene. Expression of the MECP2 transgene was confirmed by Western blotting and immunostaining of brain tissues of transgenic monkeys. Genomic integration sites of the transgenes were characterized by deep-sequencing. As compared to wild-type (WT) monkeys, MECP2 transgenic (TG) monkeys exhibited higher frequency of repetitive circular locomotion and elevated stress responses, as measured by the threat-related anxiety and defensive (TAD) test. The TG monkeys showed less interaction with WT monkeys within the same group and also reduced interaction time when paired with another TG monkey in social interaction tests. The cognitive functions of TG monkeys were largely normal in Wisconsin General Test Apparatus, although some showed signs of stereotypic cognitive behaviors. Notably, we succeeded in generating 5 F1 offspring of MECP2 TG monkeys by intracytoplasmic sperm injection with sperms from one F0 TG monkey, showing germline transmission and Mendelian segregation of multiple MECP2 transgenes in the F1 progeny. Moreover, F1 TG monkeys also showed reduced social interactions when tested in pairs, as compared to WT monkeys of similar age. Together, these results indicate the feasibility and reliability of using genetically engineered non-human primates for studying brain disorders.
结题摘要
孤独症严重影响青少年情感和智力正常发育。既往在孤独症人群中发现MeCP2基因拷贝数增加，MeCP2基因突变小鼠具有孤独症表型，证实MeCP2参与孤独症的发生，但机制未明。我们通过慢病毒介导的转基因方法构建了携带人类MECP2基因的转基因食蟹猴。我们通过免疫组化与生化试验验证转基因在食蟹猴脑内成功表达，并通过多种行为学实验发现MECP2转基因食蟹猴有重复刻板行为，焦虑水平上升，与社交行为障碍等类孤独症表型。我们并得到了稳定遗传人类MECP2基因的转基因第二代食蟹猴，并发现第二代转基因食蟹猴仍然具有社交障碍等孤独症核心症状。通过此项研究，对阐明MeCP2在孤独症发生发展中的作用，揭示MeCP2在孤独症发病中的机制，为干预孤独症提供新的思路。