异丙酚干预下人羊膜间充质干细胞移植对大鼠脑损伤的影响

Propofol intervention on mesenchymal stem cell transplantation on brain injury in rats between Human amniotic

目的人羊膜间充质干细胞（AM-MSCs）移植同时予以异丙酚治疗，观察两者对脑损伤大鼠恢复的影响。方法健康SD鼠80只，雌雄各半，体质量300～350g。体外复苏并培养人羊膜间充质干细胞（CM-Dil标记）移植前备用。采用液压颅脑损伤仪，给予液压冲击力，制成重型液压颅脑损伤模型，伤后6h给予相应治疗随机分成4组：损伤组（培养液移植组）、AM-MSCs移植组、异丙酚组、AMMSCs+异丙酚组。利用CM-Dil标记在荧光显微镜下观察各组的阳性细胞数。移植后4周各组随机处死6只，并用RT-PCR、WesternBlot检测脑组织中GAP-43、AQP4基因表达和蛋白合成的变化。移植后24h，3天及1、2、3、4周行动物神经学缺损评分，在移植后4周进行Morris水迷宫（Morris Water Maze,MWM）试验。四周后处死行免疫组化、HE染色。结果CM-Dil的阳性细胞数AM-MSCs+异丙酚组最多，异丙酚组、AM-MSCs移植组次之，损伤组最少，且各组之间差异有显著性意义(P＜0.05)。移植后4周脑损伤周围组织AQP4及其mRNA的表达损伤组高于AM-MSCs移植组、异丙酚组；AM-MSCs移植组、异丙酚组高于AM-MSCs+异丙酚组（P＜0.05）；GAP-43及其mRNA的表达损伤组低于AM-MSCs移植组、异丙酚组，AM-MSCs移植组、异丙酚组低于AM-MSCs+异丙酚组（P＜0.05）。移植后1周，大鼠神经功能障碍评分AM-MSCs+异丙酚组低于异丙酚组、AM-MSCs移植组；异丙酚组、AM-MSCs移植组低于对照组（P＜0.05）。AM-MSCs+异丙酚组穿越平台次数高于异丙酚组、AM-MSCs移植组（P＜0.05），明显高于损伤组（P＜0.01）。观察伤后4周病理切片，损伤组未见神经轴索通过，异丙酚组、AM-MSCs移植组可见少量神经轴索样结构，AM-MSCs+异丙酚组可见较多神经轴索样结构。结论异丙酚联合人羊膜间充质干细胞移植治疗大鼠脑损伤可明显改大鼠的神经学功能。

Objective Human amniotic mesenchymal stem cells(AM-MSCs)transplantation and to investigate the effect of propofoltreatment,recovery of brain injury in rats.Methods80SD rats,male and female,weight300-350g.Human amniotic mesenchymal stem cellscultured in vitro and recovery(CM-Dil)before transplantation standby.The fluid percussion brain injury device,give2.5-3.0ATM hydraulic impact,severe fluid percussion brain injury model was made.After injury6h give corresponding treatment which were randomly divided into4groups:injurygroup(medium injection group),AM-MSCs transplantation group and propofol group,AM-MSCs+propofol group.The number of positive cells ineach group was observed under the fluorescence microscope using CM-Dil markers.4weeks after transplantation,6rats were randomly executed,and RTPCRand Blot Western were used to detect the changes of GAP-43,AQP4gene expression and protein synthesis in brain tissues.After transplantation,24h,3days and1,2,3,4weeks for animal neurological deficit score,in4weeks after transplantation of Morris water maze(Morris Water Maze,MWM)test.Theywere killed after four weeks by immunohistochemistry,HE staining.Results Number of CM-DIL positive cell was the most in AM-MSCs+propofol group,whichwas in propofol group and AM-MSCs transplantation group were lower than that in the AM-MSCs+propofol group,which in injury group was the least,and the differences among the groups had statistically significant(P<0.05).4weeks after transplantation,AQP4and its mRNA expression of brain damagesurrounding tissue in injury group were higher than those in AM-MSCs transplantation group and propofol group;which in AM-MSCs transplantation groupand propofol group was higher than those in AM-MSCs+propofol group(P<0.05);GAP-43protein and mRNA expression in injury group were lower thanthose in AM-MSCs transplantation group and propofol group,which in AM-MSCs transplantation group and propofol group were lower than those in AMMSCs+propofol group(P<0.05).1weeks after transplantation,rat nerve dysfunction score in AM-MSCs+propofol group was lower than that in propofolgroup and AM-MSCs transplantation group;which in propofol group and AM-MSCs transplantation group was lower than that in the control group(P<0.05).The times of crossing platform in AM-MSCs+propofol group was higher than that in propofol group and AM-MSCs transplantation group,significantlyhigher than that in the injury group(P<0.01).The pathological section was observed4weeks after injury,there was no axonal passage in the injurygroup.In propofol group and AM-MSCs transplantation group,a few axonal like structures were observed,more axonal like structures were observedin AM-MSCs+propofol group.Conclusion Propofol combined with human amniotic mesenchymal stem cells transplantation can significantlyimprove the neurological function of rats with brain injury.