恒河猴甲基苯丙胺依赖肠屏障损伤动物模型的建立

Establishment of an intestinal injury non-human primate model of methamphetamine-dependent in Rhesus monkeys

目的以剂量递增方法形成甲基苯丙胺依赖，建立恒河猴甲基苯丙胺成瘾的肠道损伤动物模型。方法选用体重8～10 kg的成年恒河猴5只，正常对照组A组（A1组：正常对照12周；A2组：正常对照24周）和甲基苯丙胺处理组B组（B1组：甲基苯丙胺浓度递增处理12周；B2组：甲基苯丙胺浓度递增处理12周后稳定给药至24周组；B3组：甲基苯丙胺浓度递增处理24周），每组各1只。静脉给药，起始浓度为每次0.1 mg/kg，2次/天，每周浓度递增每次0.05 mg/kg，1周加药5次，每2周称体重；抽血进行肠屏障损伤标志物测定；所有组取肠道组织进行苏木素-伊红（HE）染色。结果正常对照组实验期间体重无明显变化，甲基苯丙胺处理的各组于8周开始出现体重下降2.5%～4.8%，12周开始显著下降7.6%～10.8%，最终B1～B3组体重分别减轻7.6%、25.6%、33.3%；处理组血液二胺氧化酶（DAO）于第10周开始出现轻微上升17.5%～29.2%，第12周开始明显上升152.4%～188.4%，24周实验结束B2、B3组DAO值分别为27.32、49.69 U/ml。血液D-乳酸（D-LA）于12周出现明显上升200.0%～220.7%，后持续上升至24周（B2、B3组分别为28.47、38.36 μg/ml）；血液内毒素（LPS）变化趋势与D-LA一致，24周时B2、B3组分别为33.70、46.12 pg/ml。组织学结果显示正常对照组肠道除了轻微细胞浸润，无明显病理改变；而处理组各肠段出现不同程度的黏膜上皮糜烂、黏膜固有层腺体破坏及消失、肠绒毛变性坏死、肠壁变薄等情况。结论采用甲基苯丙胺浓度递增处理，恒河猴血液肠屏障损伤标志物及肠道组织学异常改变，符合肠屏障损伤表型；数据表明成功建立甲基苯丙胺依赖肠道损伤非人类灵长类动物模型。

Objective To establish an intestinal injury animal model of methamphetamine-dependent in Rhesus monkeys by weekly increasing the dose of methamphetamine.Methods 5 adult Rhesus monkeys which weighed 8-10 kg, 1 for each group. All treated monkeys were injected through vain with methamphetamine twice per day at the beginning concentration of 0.1 mg/kg every time, with 0.05 mg/kg increasing every week. Group A1 was control which was killed at 12 weeks; group A2 was another control which was killed at 24 weeks; group B1 was treated for 12 weeks; group B2 was treated for 12 weeks, and then maintained the last concentration without increasing to 24 weeks; group B3 was treated for 24 weeks. Animals were treated 5 days per week, weighed and phlebotomize every two weeks. Intestinal injury markers was tested by enzyme linked immunosorbent assay （ELISA）, hematoxylin and eosin （HE） stain was used to analyze intestinal tissue pathological changes.Results The control groups had little significant change in weight, while the treated groups were off 2.5%-4.8% at 8 weeks, and markedly decreasing of 7.6%-10.8% from 12 weeks. B1-B3 had respectively 7.6%, 25.6%, 33.3% weight off. In treated groups, the blood levels of DAO ascended of 17.5%-29.2% at 10 weeks, of 152.4%-188.4% at 12 weeks, and 27.32, 49.69 U/ml of B2 and B3 separately at the end of 24 weeks. The levels of D-LA rose of 200.0%-220.7% at 12 weeks, constantly rose to 28.47, 38.36 μg/ml of B2 and B3 separately to 24 weeks, which showed the same trends of LPS in group B2 and B3 to 33.70, 46.12 pg/ml separately. Histological results showed inflammatory infiltrate in control groups, while the treated groups, had varying degrees of epithelia erosion, glandular destroyed or disappeared in mucosa lamina propria, degeneration and necrosis of intestinal villus. The thickness of intestinal wall changed in accordance with epithelia erosion.Conclusion The intestinal injury markers, intestinal histologic features and other phenotype of the methamphetamine-dependent animal model were in accordance with intestinal injury, which showed the animal model was successfully established.