【作者】 樊旭辉；

【导师】 王如密； 郑兆聪；

【作者基本信息】 福建医科大学 ， 神经外科学， 2010， 硕士

【摘要】 第一部分嗅鞘细胞对原代培养神经干细胞增殖、分化的影响【目的】观察在体外培养条件下嗅鞘细胞(OECs)对神经干细胞(NSCs)增殖、分化的影响规律,为两者共移植于体内促进神经功能恢复提供可借鉴的理论依据。【方法】取胎鼠脑OECs和NSCs分别进行原代培养,采用免疫荧光及免疫细胞化学方法鉴定相关细胞,每天在倒置显微镜下观察细胞生长情况。设立实验组和对照组观察OECs对原代培养NSCs增殖、分化的影响。其中实验组:取原代OECs和NSCs浓度均为1×105个/ml各2ml分别置入培养板的两个相邻培养孔中并去除间隔,使OECs和NSCs共用一培养体系,加入维持培养基观察1~7d悬浮的NSCs增殖情况并进行计数;另一培养板中加入分化培养基,分别于4d、7d用免疫细胞化学法行NF200和GFAP染色,显微镜下观察NSCs分化情况。对照组:单独培养NSCs,其余同实验组。数据以均数士标准差显示,分别采用SPSS13.0两样本t检验和方差分析进行统计分析,以P<0.05为有显著差异。【结果】原代培养的OECs和NSCs经鉴定均符合实验要求,OECs表达神经生长因子受体(P75NGFR),神经球表达巢蛋白(Nestin),神经球分化的细胞表达神经丝200(NF200)和胶质纤维酸性蛋白(GFAP)。在增殖实验中,前3天两组细胞数量无统计学意义,自第4天起实验组NSCs数量较对照组明显增多,差异有统计学意义(P<0.05)。实验组细胞数量在第5天达到高峰,以后悬浮细胞逐渐贴壁,并开始分化,部分细胞变黑皱缩;对照组在第3天细胞数达到高峰,以后逐渐贴壁分化或皱缩。在诱导分化实验中,实验组4d、7d时NF200阳性细胞率较对照组明显增加,差异有统计学意义(P<0.05),说明两种细胞共培养4d、7d时,OECs提高了NSCs向NF200阳性细胞分化率;实验组7d时GFAP阳性细胞数较对照组明显减少,差异有统计学意义(P<0.05),提示NSCs分化培养7d时,OECs降低了NSCs向GFAP阳性细胞分化率。【结论】体外培养条件下OECs可促进NSCs增殖,并提高了NSCs向神经元分化效率。第二部分神经干细胞与嗅鞘细胞共移植治疗大鼠创伤性脑损伤的实验研究【目的】观察NSCs和OECs联合移植对大鼠颅脑损伤的修复作用。【方法】选用清洁级SD大鼠(上海斯莱科公司提供)80只,参考王占祥等介绍的动物模型制作方法,直接以牙科钻头损伤运动区制作动物模型,剔除死亡或失败模型后,获得成功创伤性脑损伤模型大鼠64只采用随机数字表法分成A、B、C、D4组,每组16只,分别在立体定向下行脑损伤灶边缘注射移植。A组为对照组,单纯注射生理盐水10ul,不行细胞移植;B组为NSCs组,单独注射NSCs悬液浓度4×104/ul,共10ul;C组为OECs组,单独注射OECs悬液浓度4×104/ul,共10ul;D组为共移植组,注射OECs+NSCs混合悬液10ul,其中含OECs数量为40×104,NSCs数量为40×104。NSCs以Brdu标记。分别于移植后3d、7d、14d随机处死3只大鼠,采用免疫荧光染色法观察NSCs存活、迁移及分化的情况,其余大鼠于30d时处死,采用免疫荧光染色法观察B、D组的Brdu+/NF200+细胞数和Brdu+细胞数;采用Bederson神经缺损体征评分法检测各组大鼠的运动功能。采用SPSS13.0统计软件分别行两样本t检验和重复测量方差分析,以P<0.05为差异有统计学意义。【结果】1. 80只大鼠中因麻醉过深死亡3只,因运动区定位不准确致评分过高13只被剔除,其余64只大鼠均于术后2h左右清醒,神经功能评分均>2分,提示模型制作成功。各组大鼠神经功能缺失评分随时间延长呈总体下降趋势。其中对照组只是术后30d的评分较第3d减低(P<0.05),而NSCs组、OECs组及共移植组在术后14d、30d时的评分即较术后3d时明显减低(P<0.05)。NSCs组及OECs组仅在术后30d时的评分明显低于对照组(P<0.05),而共移植组在术后14d时的评分即较对照组明显减低(P<0.05),其术后30d的评分也明显低于NSCs组和OECs组(P<0.05)。NSCs组与OECs组各时间的评分相比,差异无统计学意义(P>0.05)。2. HE染色光镜下见损伤区脑组织破坏明显,正常结构不清,周围形成明显的水肿带,血管受压变形,细胞呈不规则状,细胞核偏移,移植的NSCs以针道为中心,分布在针道两侧,中心细胞密度较高,随着向两边的延伸,细胞密度逐渐降低。移植7d后,移植细胞进一步向两侧迁移,可观察到少量坏死的细胞,损伤区组织有一定程度修复。3.荧光显微镜下Brdu免疫荧光染色阳性的NSCs呈较强的绿色,细胞呈圆形,部分呈梭形,少数形态不规则,个别细胞呈碎片状。单独NSCs组和共移植组可见Brdu+细胞大量出现在损伤中心区域,沿针道分布,损伤区远隔处较少。7d时可见Brdu+细胞明显向周围迁移,14d时Brdu+细胞出现在远隔部位,最远距离可达2mm。对照组及OECs移植组免疫荧光染色均未发现Brdu+细胞。4.对照组14d后在损伤区出现零星的NF200+细胞;OECs移植组在7d出现NF200+细胞,数量很少,14d后数量有所增加,30d时数量变化不大;单独NSCs组和共移植组在7d时针道附近就可见大量绿色荧光Brdu+细胞,呈椭圆形,突起较少,两组Brdu+/NF200+细胞较少,主要集中在移植区域内。14d时两组Brdu+细胞明显增多,Brdu+/NF200+细胞也逐渐增多。30d时NSCs组的Brdu+细胞数量变化不大,共移植组Brdu+细胞数量明显较NSCs组多,差异有统计学意义(349.846士1.068vs312士2.000,p<0.05);Brdu+/NF200+细胞数量NSCs+OECs组也较NSCs组多,差异有统计学意义,(40.231士1.166vs23.231士6.572,p<0.05)。以上结果提示在受损脑组织中,OECs有利于NSCs存活、迁移并可促进向神经元分化。【结论】NSCs和OECs体内共移植可促进NSCs存活、迁移、分化,及大鼠脑损伤的恢复。更多还原

【Abstract】 PART One Effect of olfactory ensheathing cells on proliferation and differentiation of neural stem cells in primary culture.【Objective】To explore the effect of olfactory ensheathing cells(OECs) on the proliferation and differentiation of neural stem cells(NSCs).【Methods】The cells from the embryonic rat brains were primarily cultured and identified by Immunofluorescence and immunocyochemistry method. The cells will be abserved everyday.The effect of olfactory ensheathing cells on proliferation and differentiation of neural stem cells in primary culture were abserved by comparing the experimental group and the control group.NSCs in the two-chambe shared medium with OECs were cultured in two adjacent holes in the experimental group.The proliferation of NSCs were abserved in maintenance medium at 1-7day.Simultaneously,NSCs and OECs were co-cultured in differential medium in the other corning costar . The differentiation of NSCs were abserved by Immunofluorescence and immunocyochemistry method at 4day and 7day.NSCs were cultured alone in the control group and the factors were the same as the experimental group.The datas were shown by means士standard deviation and using SPSS 13. 0 statistic software to deal with data and make 0.05 the boundary of statistical significance.【Results】Nerve growth factor receptor(P75NGFR) was observed in the primarily cultured OECs;nestin was expressed in the primarily cultured neurosphere and the cells differentiated from neurosphere expressed neurofilament 200(NF200) and glial fibrillary acidic protein(GFAP) .In the proliferation experiment,the datas of two groups were not statistical significance in first three days ,but from the 4th day the number of NSCs was increased significantly in the experimental group compared with that in the control group (P<0.05).The number of NSCs in the experimental group was on the top,then decreased and the cells were adherence and polarization.On the 4th and 7th day of differentiation,the percentage of NF200-positive cell was higher in the experimental group than that in the control group (P<0.05),indicating that the appearance of OECs increased the differentiation of NSCs into NF200-positive cells.【Conclusion】OECs can promote the proliferation of NSCs and induce the differentiation of NSCs into neurons.PART Two Effect of co-graft of Olfactory ensheathing cells with Neural stem cells on rats with traumatic brain injury.【Objective】To investigate the functional recovery of traumatic brain injury when co-transplanting NSCs and OECs in rat brain injury.【Methods】Eighty male SD rats in a SPF grade were selected.TBI model of rats in the motor cortex by electric micro-drilling was established referencing to Wang Zhanxiang’method.Sixty-four of rats were randomly divided into 4 groups(n=16 in each group),group A(NS transplantation group),group B(NSCs marked with Brdu transplation group,4×104/ul),group C(OECs transplation group,4×104/ul),group D(NSCs 40×104 and OECs 40×104 co- transplation group) .Three rats were killed in each group at 3rd,7th,14th and 30th day after transplantation and the rest was killed at 30th day.The migration,survival,differentiation of NSCs were investigated with immunofluorescene staining.On the 30th day after the operation,the number of Brdu/NF200-positive cell and Brdu-positive cell was compared in the NSCs group and co-graft group by immunofluorescene staining.Functional recovery of injury brain was assessed by Bederson scores.Using SPSS 13. 0 statistic software to deal with data and make 0.05 the boundary of statistical significance.【Results】1. In 80 SD rats,16 rats were removed and 64 rat models with TBI were successful.The scores of nervous functional def iciency in all groups were beyond 2 after operation,and all tended to decrease after the transplantatio,including the control group.In the control group,the score at the 30th day after operation was significantly decreased as compared with that at the 3rd day(P<0.05);The scores in NSCs,OECs and co-graft groups at the 14th and 30th were all significantly decreased as compared with that at the 3rd day(P<0.05);The score in the co-graft group was significantly decreased compared with that in NSCs and OECs group at the 30th day(P<0.05) .2. The cellular figure was damaged seriously and exhibited irregular cell morphology.The damage area was edema with obscured structural outlines.NSCs were located on the side of the injection.The number of cells decreased gradually away from transplanted area.After 7 days,transplanted cells migrated around the injury site and the tissue was gradully repaired.3. The result of immunofluorescene staining show the green ectogenesis BrdU positive cells were round and oval monocytes.Brdu-positive cells were seen aroud the injury site,but at 14th day the cells obviously migrated into other area away from the transplanted site.In contral group and OECs group,Brdu -positive cells were not found at the injury site.4. Few NF200-positive cells were seen around the injury site in contral and OECs group.Compare with the contral and OECs group,more NF200-positive cells and Brdu-positive cells were found on the side of the injection in NSCs and co-graft group.At 30th day,the number of Brdu-positive cell and Brdu/NF200-positive cell was increased significantly in the co-graft group (P<0.05) compared with that in the NSCs group,indicating that the appearance of OECs promoted migration,survial and differentiation of NSCs.【Conclusion】In vivo co-transplantation of NSCs and OECs can promote migration,survival and differentiation of NSCs as well as functional recovery of injury brain.更多还原