【作者】 周先虎； 冯世庆； 陈有； 王颖； 郝岩； 宁广智； 刘洋； 班德翔；

【机构】 天津医科大学总医院；

【摘要】 【目的】观察激活态雪旺细胞(Activated Schwann cells,ASCs)分泌神经营养因子对诱导人脐血间充质干细胞(Human Umbilical Cord Blood Mesenchymal Stem Cells,HUCBMSCs)神经方向分化的作用。同时评价脊髓损伤局部微环境对HUCBMSCs分化影响。探讨ASCs与HUCBMSCs联合移植在促进大鼠脊髓损伤后轴突再生和功能恢复方面所起的作用。【方法】结扎成年Wistar大鼠双侧隐神经近端,一周后取下结扎处远端的隐神经,采用双酶消化组织块法结合机械分离法分离培养ASCs;Ficoll法分离得到的人脐血单个核细胞(Human Cord BloodMononuclear Cells,HCMNCs)并经贴壁培养、分离后获得HUCBMSCs。采用全反式微甲酸+bFGF+EGF(含50ng/ml人碱性成纤维细胞生长因子(bFGF)、50ng/m1人表皮生长因子(EGF)、0.5μmol/L全反式维甲酸和10%FBS的DMEM/F12培养液,对照组)、体外ASCs培养基半量换液法(实验组)及体内共移植诱导HUCBMSCs向神经方向分化(空白对照组、ASCs移植组、HUCBMSCs移植组、ASCs+HUCBMSCs移植组),应用免疫组化、Western-Bolt半定量、Real-time PCR(NF200、GFAP、MBP)定量分析等方法在诱导培养后1、2、3、4周进行综合评价HUCBMSCs诱导分化情况,分析其神经标志性蛋白的表达情况。同时,用NYU Impactor-Ⅱ打击器制作成年雌性Wistar大鼠胸10脊髓损伤模型(打击重量和高度为:10g×50mm)。将80只大鼠随机分成4组:空白对照组(注射DMEM);ASCs移植组;HUCBMSCs移植组;ASCs与HUCBMSCs联合移植组。分别将DMEM,消化后的ASCs,HUCBMSCs,以及ASC+BMSC移植入对应组的脊髓损伤部位。术后采用BBB评分对大鼠的后肢功能恢复情况进行评价。12周后,从每组随机选取8只大鼠进行10%BDA顺行示踪标记。标记两周后处死动物,将损伤段脊髓取出作5μm快速冰冻切片后,进行BDA显影、HE染色,NF200、GFAP、MBP免疫组化染色。综合以上数据并进行统计学分析。【结果】ASCs和HUCBMSCs经分离,纯化后可以在体外分别稳定的传4代和6代以上;NF200、GFAP、MBP免疫组化染色结果可以证实HUCBMSCs在ASCs和全反式微甲酸及bFGF、EGF的作用下向神经源性细胞分化。Western-Bolt半定量、Real-time PCR(NF200、GFAP、MBP)定量分析结果显示两种诱导方法比较无统计学差异。损伤后4周开始,共移植组动物的BBB评分显著高于其余3组(p<0.05)。BDA顺行示踪标记显示共移植组较ASCs移植组和HUCBMSCs移植组有较多的再生神经纤维通过损伤区,而空白对照组几乎未见再生神经纤维穿越。HE染色显示细胞共移植组损伤空洞明显小于其余三组(p<0.05)。【结论】ASCa可以分泌多种神经营养因子并促进HUCBMSCs在体外及Wistar大鼠体内的增值和向神经源性细胞分化,与传统全反式微甲酸诱导方法相比更简单。动物实验结果显示HUCBMSCs联合ASCs共移植后HUCBMSCs在脊髓损伤区域可以向神经元和少突胶质细胞分化,填充组织缺损,弥补脊髓空洞,有利于神经再生轴突的延伸和突触之间的连接。ASCs联合HUCBMSCs移植治疗脊髓损伤可促进更多比例的HUCBMSCs向神经元方向分化,促进轴突再生,改善脊髓损伤后功能的恢复。更多还原

【Abstract】 objective To observe the capability of Human Umbilical Cord Blood mesenchymal Stem Cells(HUCBMSCs) to differentiate into neurogenic cells in vitro and vivo under the impact of Activated Schwann cells’(ASCs) secreting sorts of neuronotrophins.The influence of microenvironment after spinal cord injury(SCI) on differentiation of HUCBMSCs were also evaluated.Finally,we discuss whether these two types of cells grafted into the injured site of spinal cord,have the abilyty oi promoting axonal regeneration and functional recovery after SCI. Method Firstly,bilateral saphenous nerves of four weeks’ old Wister rats were ligated.One week later, far end of saphenous nerves were isolated and cultured by way of dual-enzyme digestion method combined with mechanical separation.HUCBMSCs were cultured through isolating HCMNCs by Ficoll’s way and then Plastic-adherent culture.Firstly,two methods were used for HUCBMSCs culture:all-trans retinoic acid+bFGF+EGF(control group),1/2 DMEM+ASCs culture(medium after ASCs cultured 2 days,experimental group).Immunohistochemistry,Westem-Bolt semi-quantitative and Real-time PCR(NF200、GFAP、MBP ) quantitative analysis were used to evaluate neurogenic differentiation of HUCBMSCs.Then,SCI model in Wistar rat(Female,8weeks’ old) was created at T10 with NYU Impactor-Ⅱmachine with 10g x 50mm weight drop.80 SCI rats were classified into 4 groups randomly and evenly:blank control group(DMEM),ASCs transplantation group,HUCBMSCs transplantation group and co-transplantation group of ASCs and HUCBMSCs.1,2,3,4 weeks after operation,2 animals of each group were sacrificed,and injured spinal cord tissue were used for Westem-Bolt and Real-time PCR analysis(NF200、GFAP、MBP).BBB score was carried out at the 3rd day and at weekly after intervals injury.12 weeks later,8 rats in each group were chosen out to carry out 10%BDA anterograde tracing mark.Then,2 weeks later,the animals were sacrificed,the spinal cord tissue with injured lesion was taken out and fast cryostat sections(5μm) was made followed by BDA developing,HE and immunochemistry(NF200,MBP,GFAP) staining.The pictures were processed by microimage analysis software(Image pro plus 5.0),the IOD value of positive response area was counted to compare among four groups. Result ASCs and HUCBMSCs can be steadily cultured at least 4 and 6 passages separately after isolation and purification in vitro.The results of immunohistochemistry staining,such as NF200、GFAP and MBP, confirm that HUCBMSCs can differentiate into neurogenic cells under the effect of ASCs and RA+bFGF+EGF. The statistical analysis of Westem-Bolt and RT-PCR(NF2(XK GFAP> MBP) indicate that there exists differences between ASCs and RA+bFGF+EGF group.From the 4th week after injury,BBB score in co-graft group was significandy higher than that in other 3 groups(p<0.05).The rats of co-graft group recoverd to the largest extent BDA anterograde tracing mark showed that there were more regenerated nerve fibers pass through the injured site in co-graft group than that in other 3 groups,while little in control group(p<0.05).HE staining showed that the injured cavity was the smallest one in cell co-graft group when compared with other 3 groups. Conclusion HUCBMSCs can proliferate and differentiate into neurogenic cells under the effect of numerous neurotrophic factors in vivo and in vitro.The animal experiment results indicate that the co-transplantation of HUCBMSCs and ASCs is more effective dian single cell in promoting axonal regeneration and functional recovery from rat’s spinal cord injury.HUCBMSCs differentiates into neuron and oligodendrocytein,which are beneficial to regeneration axonal extension and the link between synapses by filling the tissue gap and making up the spinal cavity,in lesion area.The co-transplantation fully takes advantage of ASCs,which have a higher activity dian Schwann cells,to promote HUCBMSCs differentiate into neurons,enhance axonal regeneration and functional recovery.更多还原