POSTN、OPC及亚低温治疗新生大鼠缺氧缺血性脑损伤的实验研究
发布时间：2019-06-13 07:42
【摘要】：[目的]研究POSTN对新生大鼠缺氧缺血(hypoxic-ischemic, HI)后神经干细胞增殖分化的影响,为临床新生儿脑损伤的治疗提供新的思路。 [方法]通过7日龄新生大鼠左侧颈总动脉结扎离断后8%氧气缺氧2h制备动物模型,随机分为HI组和假手术组。干预组进行侧脑室注射POSTN蛋白干预治疗,非干预组注射PBS。采用BrdU标记结合免疫荧光化学方法观察各组大鼠不同时间点脑室下区(subventricular zone, SVZ)和海马齿状回(dentate gyrus,DG)神经干细胞的增殖及其向神经元、星形胶质细胞分化情况。采用Morris水迷宫实验方法观察各组大鼠学习记忆功能变化。 [结果]POSTN干预治疗能够显著增加SVZ和海马DG新生细胞(BrdU+)数量,其中新生神经干细胞(BrdU+/Nestin+)在术后7d达高峰,而新生神经元(BrdU+/Map-2+)和星形胶质细胞(BrdU+/GFAP+)在14d达高峰,差异有统计学意义。Morris水迷宫实验结果显示,POSTN干预治疗能够降低大鼠的逃避潜伏期和游泳距离,增加站台穿越次数和目标象限停留时间比例,差异有统计学意义。 [结论]POSTN干预可促进新生大鼠缺氧缺血性脑损伤后的神经干细胞增殖和分化,改善其学习记忆功能,有助于脑损伤后的神经修复。 [目的]通过体外培养获得的Olig2+-GFP+-OPC移植入新生大鼠缺氧缺血性(hypoxia-ischemia, HI)脑损伤模型,观察Olig2+-GFP+-OPC定向分化成熟为少突胶质细胞的能力和植入细胞的存活情况,探讨外源性植入神经前体细胞OPC治疗早产儿脑室周白质软化(periventricular leukomalacia, PVL)的可行性。 [方法]Olig2+-GFP+-mES细胞通过拟胚体(embryonic body, EB)介导的神经诱导法,即综合全反式维甲酸(retinoic acid, RA)诱导法和五步法使Olig2+-GFP+-mES定向分化为OPC。采用免疫细胞化学染色,利用倒置相差显微镜和荧光显微镜来观察OPC的分化成熟能力。3日龄新生大鼠左侧颈总动脉结扎离断后37℃条件下6%氧气缺氧2.5h制备动物模型,通过HE(haematoxylin-eosin)染色方法评估模型情况。通过免疫荧光染色方法观察侧脑室注射植入的Olig2+-GFP+-OPC存活情况。 [结果]Olig2+-GFP+-mES在胚胎干细胞培养基中形成EB,在含有RA(0.5μM)和SHH(100ng/ml)神经分化培养基中,逐渐发育为神经前体细胞球。在EB4d开始出现Olig2+-GFP+-阳性细胞,在EB12d时表达出现高峰。Olig2+-GFP+-阳性细胞具有体外条件下分化为OPC,再定向分化为成熟少突胶质细胞并具备成髓鞘的能力。HI组脑室下区神经细胞出现变性、坏死,细胞层次紊乱、排列松散,部分细胞出现核固缩、核溶解,胞浆深染,结构不清。对照组则无明显病理变化。在侧脑室周边及附近脑区有GFP的表达,证实植入OPC成功,并向周围迁移。 [结论]体外EB介导的神经诱导法能够高效诱导产生Olig2+-GFP+-OPC,并具有分化为成熟少突胶质细胞且有成髓鞘能力。在HI脑损伤模型中侧脑室植入Olig2+-GFP+-OPC细胞能够存活。 [目的]研究缺氧缺血后亚低温对新生大鼠海马部位细胞的存活及星形胶质细胞活化增殖的影响,探讨亚低温对缺氧缺血脑损伤保护作用的机制。 [方法]3日龄新生大鼠海马脑片,培养至第4天进行氧糖剥夺(oxygen-glucose deprivation, OGD)制备模型,分为亚低温组(33℃,24h)和常温组(37℃)；对照组不进行OGD处理,37℃培养7d。采用碘化丙啶(propidium iodide, PI)和免疫荧光染色观察海马脑片的细胞存活和星形胶质细胞的活化增殖。动物实验：7日龄新生大鼠采用左侧颈总动脉结扎离断并在8%02+92%N2中缺氧2h,制备缺氧缺血模型,分为手术常温组(肛温36-37℃,24h)和手术亚低温组(肛温32-33℃,24h)。假手术组仅分离左侧颈总动脉,不结扎,也不缺氧,分为假手术常温组和假手术亚低温组。各组于术后3d和7d处死取材,采用DAPI (4'-6-diamidino-2-phenylindole)和免疫组织化学方法检测海马组织细胞存活和星形胶质细胞的活化增殖。 [结果]体外和体内实验结果都表明缺氧缺血后3d新生大鼠海马组织中存活的细胞常温组较亚低温组少。海马脑片培养GFAP (glial fibrillary acidic protein, GFAP)染色显示,亚低温组GFAP阳性细胞数较常温组明显减少,两组比较具有统计学差异(P0.05)。动物实验海马组织GFAP染色结果也显示,缺氧缺血后3d和7d手术亚低温组GFAP阳性细胞较手术常温组显著减少,同一时间点两组比较具有统计学意义(P0.05)。 [结论]亚低温能减轻新生大鼠缺氧缺血后海马星形胶质细胞的活化增殖。
[Abstract]:[Objective] To study the effects of POSTN on the proliferation and differentiation of neural stem cells after hypoxic-ischemic (HI) in neonatal rats. [Methods] The animal model was prepared by ligation of the left common carotid artery of 7-day-old neonatal rats and 8% oxygen hypoxia for 2 h. The model was divided into HI group and sham operation. Group. The intervention group was treated by intraventricular injection of POSTN protein, and the non-intervention group was injected with PB. S. The proliferation of the subventricular zone (SVZ) and the dentate gyrus (DG) neural stem cells in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampal dentate gyrus (DG) and the differentiation of the neurons and astrocytes were observed by means of the BrdU label and the immunofluorescence chemical method. The learning and memory function of each group was observed by the Morris water maze test method. [Results] POSTN intervention could significantly increase the number of the newly-born cells (BrdU +) in the SVZ and the hippocampus, in which the new neural stem cells (BrdU +/ Nestin +) reached the peak at 7 d after the operation, while the new neurons (BrdU +/ Map-2 +) and the astrocytes (BrdU +/ GFAP +) peaked at 14 d and the difference was statistically significant. The results of the Morris water maze show that the POSTN intervention can reduce the escape latency and the swimming distance of the rat, increase the number of crossing times of the platform and the proportion of the residence time of the target quadrant, and the difference is statistics. [Conclusion] POSTN intervention can promote the proliferation and differentiation of neural stem cells after hypoxic-ischemic brain injury in neonatal rats, improve their learning and memory function, and contribute to brain injury Nerve repair.[Objective] To observe the ability and implantation of the Olig2 +-GFP +-OPC (Olig2 +-GFP +-OPC) transplanted into the hypoxic-ischemic (HI) brain injury model of the neonatal rats by in-vitro culture, and observe the ability and the implantation of the oligodendrocytes in the Olig2 +-GFP +-OPC directional differentiation. The survival of the cells was investigated, and the periventricular leukomalacia of the premature infants treated with the exogenous pre-implantation of the neural precursor cells (OPC) was discussed. The feasibility of VL is that the OLlig2 +-GFP +-mES cell is mediated by an embryonic body (EB), i.e., an integrated all-trans-retinoic acid (RA) method and a five-step method to make the Oli2 +-GFP +-mE S-directional differentiation was OPC. The differentiation and maturation of the OPC were observed by using an inverted phase-contrast microscope and a fluorescence microscope. The animal model was prepared by using an inverted phase-contrast microscope and a fluorescence microscope at 37 鈩，
本文编号：2498370
[Abstract]:[Objective] To study the effects of POSTN on the proliferation and differentiation of neural stem cells after hypoxic-ischemic (HI) in neonatal rats. [Methods] The animal model was prepared by ligation of the left common carotid artery of 7-day-old neonatal rats and 8% oxygen hypoxia for 2 h. The model was divided into HI group and sham operation. Group. The intervention group was treated by intraventricular injection of POSTN protein, and the non-intervention group was injected with PB. S. The proliferation of the subventricular zone (SVZ) and the dentate gyrus (DG) neural stem cells in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampal dentate gyrus (DG) and the differentiation of the neurons and astrocytes were observed by means of the BrdU label and the immunofluorescence chemical method. The learning and memory function of each group was observed by the Morris water maze test method. [Results] POSTN intervention could significantly increase the number of the newly-born cells (BrdU +) in the SVZ and the hippocampus, in which the new neural stem cells (BrdU +/ Nestin +) reached the peak at 7 d after the operation, while the new neurons (BrdU +/ Map-2 +) and the astrocytes (BrdU +/ GFAP +) peaked at 14 d and the difference was statistically significant. The results of the Morris water maze show that the POSTN intervention can reduce the escape latency and the swimming distance of the rat, increase the number of crossing times of the platform and the proportion of the residence time of the target quadrant, and the difference is statistics. [Conclusion] POSTN intervention can promote the proliferation and differentiation of neural stem cells after hypoxic-ischemic brain injury in neonatal rats, improve their learning and memory function, and contribute to brain injury Nerve repair.[Objective] To observe the ability and implantation of the Olig2 +-GFP +-OPC (Olig2 +-GFP +-OPC) transplanted into the hypoxic-ischemic (HI) brain injury model of the neonatal rats by in-vitro culture, and observe the ability and the implantation of the oligodendrocytes in the Olig2 +-GFP +-OPC directional differentiation. The survival of the cells was investigated, and the periventricular leukomalacia of the premature infants treated with the exogenous pre-implantation of the neural precursor cells (OPC) was discussed. The feasibility of VL is that the OLlig2 +-GFP +-mES cell is mediated by an embryonic body (EB), i.e., an integrated all-trans-retinoic acid (RA) method and a five-step method to make the Oli2 +-GFP +-mE S-directional differentiation was OPC. The differentiation and maturation of the OPC were observed by using an inverted phase-contrast microscope and a fluorescence microscope. The animal model was prepared by using an inverted phase-contrast microscope and a fluorescence microscope at 37 鈩，
本文编号：2498370
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