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目的探讨混合培养的人脐血间充质干细胞(MSCs)对缺氧缺血性脑损伤(HIBD)新生大鼠的疗效。方法无菌条件下收集20份健康足月新生儿脐血,采用密度梯度离心法分离出脐血单个核细胞,再通过贴壁细胞培养法培养出人脐血MSCs。当细胞传至第3代时,随机取培养的8份脐血MSCs(半量)为A组,8份脐血MSCs(半量)为B组,将A、B组剩余的半量脐血MSCs混合为C组,3组细胞密度均为1×106L-1。混合培养的第3代脐血MSCs用流式细胞仪检测细胞表面抗原CD29、CD34、CD44、CD105的表达。选择P4代人脐血MSCs作为移植细胞,并经5-溴-2-尿嘧啶脱氧核苷(BrdU)体外标记。7日龄SD大鼠45只制备HIBD模型,造模中死亡3只,余42只分为混合移植组(n=16)、单份移植组(n=16)和对照组(n=10)。造模第3天,在立体定位条件下,移植组大鼠经左侧大脑皮质注入人脐血MSCs,对照组在相同部位注入相同体积的PBS。在细胞移植第7天,随机选择6只移植组大鼠处死取脑,采用免疫组织化学方法观察移植细胞在脑内的存活、迁移和分化情况。移植第1、7、14、21、28天采用改良神经功能损害评分(mNSS)对3组大鼠的神经功能进行评价。结果活细胞计数显示,C组细胞增殖较A组、B组快(Pa<0.05)。移植组大鼠脑组织免疫组织化学检测发现移植细胞在脑内能够存活,并以移植点为中心向周围迁移;混合移植组BrdU阳性细胞数多于单份移植组(P<0.05)。移植第1天,移植组大鼠mNSS低于对照组,但差异无统计学意义(P>0.05);第7、14、21、28天,移植组大鼠mNSS低于对照组,差异有统计学意义(P<0.05);第7、14、21、28天,混合移植组大鼠mNSS低于单份移植组,差异有统计学意义(P<0.05)。结论混合培养的人脐血MSCs脑移植对HIBD新生大鼠具有较好的治疗作用。
Objective To investigate the effects of mixed culture of human umbilical cord blood mesenchymal stem cells (MSCs) on neonatal rats with hypoxic-ischemic brain damage (HIBD). Methods Umbilical cord blood was collected from 20 healthy full-term neonates under aseptic conditions. Cord blood mononuclear cells were isolated by density gradient centrifugation. MSCs were cultured by adherent cell culture. When the cells reached the third generation, eight cord blood MSCs (half amount) were randomly divided into group A, eight cord blood MSCs (group B), and the remaining half amount of cord blood MSCs from group A and group B The cell density in group C and group 3 was 1 × 106L-1. The 3rd generation of cord blood MSCs mixed culture were used to detect the expression of cell surface antigen CD29, CD34, CD44 and CD105 by flow cytometry. P4 generation of cord blood MSCs were selected as transplanted cells and labeled with 5-bromo-2-uracil deoxynucleoside (BrdU) in vitro. Forty-five SD rats of 7 days old were used to prepare HIBD model. Three of them died in the modeling. The other 42 mice were divided into mixed transplantation group (n = 16), single transplantation group (n = 16) and control group (n = 10) . On the third day of modeling, human umbilical cord blood MSCs were implanted into the left cerebral cortex under stereotaxic conditions, and the control group was injected with the same volume of PBS in the same place. On the 7th day of cell transplantation, 6 rats in the transplantation group were randomly selected to sacrifice their brains. Immunohistochemistry was used to observe the survival, migration and differentiation of the transplanted cells in the brain. The neurological function of the three groups of rats was evaluated on the 1st, 7th, 14th, 21st and 28th day after transplantation by using modified neurological impairment score (mNSS). Results The viable cell count showed that the cell proliferation in group C was faster than that in group A and group B (Pa <0.05). The results of immunohistochemistry showed that the transplanted cells survived in the brain and migrated around the transplantion site. The number of BrdU positive cells in the mixed transplantation group was more than that in the single transplanted group (P <0.05). On the first day after transplantation, the mNSS in the transplantation group was lower than that in the control group, but the difference was not statistically significant (P> 0.05). On the 7th, 14th, 21st and 28th days, the mNSS in the transplantation group was lower than that in the control group (P <0.05). On the 7th, 14th, 21st and 28th days, the mNSS in the mixed transplantation group was lower than that in the single transplantation group (P <0.05). Conclusion Mixed culture of human cord blood MSCs transplantation has a good therapeutic effect on HIBD neonatal rats.