丹参联合骨髓间充质干细胞移植治疗新生鼠缺氧缺血性脑损伤的实验研究

Study on Cotransplantation of Bone Mesenchymal Stem Cells and Salvia Miltiorrhiza into Neonatal Rats with Hypoxic-Ischemic Brain Damage

目的研究骨髓间充质干细胞(MSCs)植入缺氧缺血性脑损伤(HIBD)新生大鼠脑后,植入MSCs在脑组织中的迁移、神经细胞抗原分化率的变化,探讨丹参联合MSCs移植治疗新生儿HIE的可行性。方法将36只7日龄新生SD大鼠随机分为正常对照组(8只)、HIE组(8只)、MSCs移植组(10只)、MSCs移植+丹参组(10只)。在MSCs移植后18d取脑组织后做石蜡切片,免疫组织化学法检测并计数进入脑组织的MSCs,观察植入细胞在脑组织中的分布、迁移;免疫荧光双标记法检测植入细胞神经元巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)的表达,观察其向神经细胞的分化,并进行组间比较。结果植入MSCs主要分布于HIBD组左侧大脑皮质区,MSCs移植+丹参组标本中MSCs更多向右侧大脑半球迁移,且分布范围更广,与MSCs移植组比较,不同脑组织层面、两侧大脑半球MSCs计数均有统计学差异(Pa<0.05);免疫荧光双标记法观察MSCs主要在左侧大脑皮质、海马等部位分化为神经细胞,Nestin、NSE、GFAP均可表达。MSCs移植+丹参组植入MSCs的Nestin、NSE分化率更高,且有统计学意义;MSCs移植组和MSCs移植+丹参组GFAP分化率比较差异无统计学意义。结论 MSCs植入HIBD新生大鼠脑组织后能存活,并在脑组织中移行,植入MSCs主要在HIBD新生鼠左侧脑皮质、海马等部位分化为神经细胞,表达Nestin、NSE及GFAP,加用丹参后可促进植入MSCs表达Nestin及NSE,对GFAP表达无影响。

Objective To explore the migration and differentiation of bone mesenchymal stem cells（MSCs）implanted in the brain of neonatal rat with hypoxic-ischemic brain damage（HIBD）,and observe the results of salvia miltiorrhiza injection inducing MSCs to differentiate into neuron-like cells and the influence on neural differentiation ratio of MSCs implanted in the brain of neonatal rat with HIBD.Methods Seven-day-old SD neonatal rats（n=36）were divided randomly into normal control group（n=8）,HIBD group（n=8）,cellular transplant of MSCs group（n=10）,and cotransplantation of MSCs and salvia miltiorrhiza group（n=10）.Animal models of HIBD were established in neonatal rats of HIBD group,cellular transplantation of MSCs group,cotransplantation of MSCs and salvia miltiorrhiza group as described by Rice.Eighteen days after cellular transplantation,all rats were sacrificed and their cerebra were made in paraffin sections.Immunohistochemical staining with chromogen diaminobenzidine（DAB）was used to detect and count the cells derived from MSCs,so as to explore the distribution and migration of MSCs implanted in the brain of neonatal rats with HIBD.The neural differentiation ratio of the BrdU positive cells entering the brains,immunofluorescence double labeling for BrdU and neural cells specific antigens——Nestin,neuron specific enolase（NSE）,glial fibrillary acidic protein（GFAP）were performed,then the ratios between 2 groups were compared.Results More BrdU positive cells were observed in the left cerebral cortex.Compared with cellular transplant of MSCs group,MSCs distributed more extensively,and more MSCs migrated to the right cerebral hemisphere in cotransplantation of MSCs and salvia miltiorrhiza group.There was a significant difference（P0.05）of the MSCs count between 2 groups in each different brain section and both cerebral hemisphere.Majority of MSCs located at the cerebral cortex,hippocampus of left cerebral hemisphere should be differentiated into neuronal-like cells,expressed Nestin,NSE and GFAP.There was a significant difference in the neural differentiation ratio（Nestin,NSE）of MSCs between 2 groups（P0.05）,but no difference in GFAP.Conclusions After implanted in the brain of neonatal rats with HIBD,MSCs could survive and migrate throughout the cerebra in both groups,and distribute more extensively by using salvia miltiorrhiza at the same time.Majority of MSCs located at the cerebral cortex,hippocampus of left cerebral hemisphere should be differentiated into neuron-like cells,expressed Nestin,NSE and GFAP.Using salvia miltiorrhiza in addition should promote MSCs to express Nestin and NSE,but had no significant influence on GFAP.