去小胶质细胞协同神经干细胞移植治疗缺血性脑卒中

发布时间：2018-06-03 11:36

本文选题：神经干细胞 + 分化　；参考：《兰州大学》2017年硕士论文

【摘要】：新生小鼠神经干细胞的体外分离培养及诱导分化鉴定目的:建立小鼠神经干细胞分离培养体系,并对其分化产物进行鉴定分析。方法:无血清悬浮培养技术对新生C57BL/6小鼠海马神经干细胞进行分离培养及传代,第三代神经干细胞分别行Brd U、Ed U标记,Brd U/Nestin/Hochest33258染色鉴定所培养细胞“干性”,血清促分化培养后,NSE、GFAP荧光染色鉴定神经干细胞分化产物。Brdu/Hochest33342及Edu/Hochest33342双标阳性细胞计数,比较Brd U和Ed U增殖标记效率。结果:新生小鼠海马源性神经干细胞在体外可悬浮生长传代,巢蛋白染色阳性,血清诱导分化后可以产生特异性烯醇化酶、胶质纤维酸性蛋白阳性细胞。Brd U标记组与Ed U标记组阳性细胞数相比较,有统计学差异(p0.05)。结论:证实本实验采用的体外分离培养技术所获得的神经干细胞具有自我更新复制增殖及多潜能分化能力。Ed U标记效率更高,更为灵敏方便。新生小鼠海马神经干细胞的冰冻保存与复苏目的:观察不同冰冻保存液冷冻保存不同时间的神经干细胞复苏后,其存活率、增殖能力。方法:采用两种冰冻保存液进行神经干细胞冻存,分别在冻存后1、3、6个月复苏细胞,台盼蓝染色检测复苏后活细胞率,CCK8法检测复苏后1-7d神经干细胞的增殖能力。结果:同一种冰冻保存液冻存不同时间的细胞复苏后其活细胞率没有明显统计学差异(p0.05),相同冻存时间两种冰冻保存液冻存的细胞复苏后其活细胞率均具有统计学差异(p0.05)。CCK-8检测不同冰冻保存液及不同冻存时间保存的细胞复苏后其增殖能力,第6天各组之间均具有统计学差异(p0.05)。结论:两种配方冰冻保存液均可用于神经干细胞冻存,但冻存时间越久,细胞增殖能力可能会有所下降。神经干细胞移植治疗缺血性脑卒中的研究目的:观察移植NSCs对急性缺血性脑卒中小鼠损伤后的修复作用,并探讨其作用机制。方法:光化学诱导方法构建小鼠急性脑缺血模型后进行神经干细胞移植。造模前1d,移植后1d、3d、7d、14d、21d、28d、35d进行加速转棒及前肢抓力测试;免疫荧光染色观察NSCs分化为神经元、星形胶质细胞情况。结果:加速转棒测试:B、C两组相比较无统计学差异(p0.05),B、D两组在移植后1d无明显统计学差异(p0.05),移植后第3d(▲p0.05),其余各时间点(☆p0.01)。前肢抓力测试发现各时间点B、C两组无统计学差异(p0.05),B、D两组在移植后1d无明显统计学差异(p0.05),其余各时间点均存在明显统计学差异(☆p0.01)。免疫荧光染色结果:移植后5d可见Edu/GFAP、Edu/NG-2双阳性细胞。移植后14d可见Edu/GFAP、Edu/MAP-2双阳性细胞。移植后28d、35d可见Edu/GFAP、Edu/MAP-2、Edu/Neu N双阳性细胞。结论:外源性的NSCs移植可在体内存活分化为神经元及星形胶质细胞,发挥神经再生及修复作用促进缺血性脑卒中后行为功能恢复。去小胶质细胞协同神经干细胞移植治疗缺血性脑卒中目的:观察比较单纯移植NSCs和去小胶质细胞协同移植神经干细胞在缺血性脑卒中的治疗效果,并探讨其作用机制。方法:A、B两组都进行外源性神经干细胞移植,同时B组在建模后当天开始口服PLX3397,连续7d,两组分别于建模前后进行加速转棒、前肢抓力、自发活动测试;移植后3d进行Ed U、Nestin,14d进行Ed U、GFAP、MAP-2染色观察NSCs分化情况。结果:加速转棒测试:A、B两组在移植后7d、14d、21d、28d、35d相比较有统计学差异(▲p0.05),前肢抓力测试发现A、B两组在移植后14d、21d、28d、35d相比较有统计学差异(▲p0.05;☆p0.01),A、B两组自发活动总路程无统计学差异(p0.05)。免疫荧光染色结果显示在移植后第3天A、B两组外源性神经干细胞均有存活,同时B组存活数量观察上优于A组。结论:去小胶质协同神经干细胞移植对于外源性的神经干细胞存活分化更为有利,其在行为恢复方面也有着更好的效果。
[Abstract]:In vitro isolation, culture and differentiation of neural stem cells from newborn mice and identification of differentiation and differentiation of neural stem cells in mice and identification and analysis of their differentiation products. Methods: serum-free suspension culture was used to isolate and subculture hippocampal neural stem cells of newborn C57BL/6 mice and the third generation of neural stem cells were separated. Brd U, Ed U markers and Brd U/Nestin/Hochest33258 staining were used to identify the "dry" cells of the cultured cells. After serum differentiation and culture, NSE, GFAP fluorescence staining was used to identify the number of.Brdu/Hochest33342 and Edu/Hochest33342 double standard positive cells in the differentiation products of neural stem cells. The stem cells could be suspended in vitro, the nestin staining was positive, and the specific enolase could be produced after the differentiation of the serum. The.Brd U marker group of glial fibrillary acidic protein positive cells was compared with the number of positive cells in the Ed U marker group (P0.05). Conclusion: it is proved that the isolation and culture techniques used in this experiment have been obtained. Neural stem cells have the ability of self renewal, replication, replication, proliferation and pluripotency, the.Ed U marker is more efficient and more sensitive and convenient. The cryopreservation and resuscitation of neural stem cells in the hippocampus of newborn mice: the survival rate and proliferation ability of different frozen preservation solution for different time cryopreservation of neural stem cells. Two frozen preservation solutions were stored for neural stem cells in 1,3,6 months after cryopreservation, trypan blue staining was used to detect the living cell rate after resuscitation, and the proliferation ability of 1-7d neural stem cells after resuscitation was detected by CCK8 method. Results: the living cell rate of the same cryopreservation solution after the cryopreservation was not significantly worse after the cryopreservation. Different (P0.05), the same frozen storage time two frozen storage cells resuscitated the living cells after the resuscitation, the living cell rates were statistically different (P0.05).CCK-8 detection of different frozen preservation solution and the storage time preserved cells after the recovery of their proliferation ability, sixth days of statistical difference between groups (P0.05). Conclusion: two kinds of frozen preservation solution It can be used for cryopreservation of neural stem cells, but the longer the cryopreservation time, the cell proliferation ability may decrease. The purpose of neural stem cell transplantation in the treatment of ischemic stroke: To observe the repair effect of transplanted NSCs on acute ischemic stroke in mice and to explore its mechanism. 1D, 1D, 3D, 7d, 14d, 7d, 14d, 21d, 28d, 35d were used to carry out the acceleration stick and forelimb grasping force test, and the immunofluorescence staining was used to observe the differentiation of NSCs into neurons and astrocytes. Results: there was no statistical difference between the B, C two groups (P0.05). No significant statistical difference (P0.05), post transplant 3D (P0.05), and the rest of the time point (P0.01). The forelimb grasping force test found no statistical difference (P0.05) at all time points B, C two groups, B, D two groups after transplantation, there was no significant statistical difference (P0.05), the rest of the time points were significant statistical differences (P0.01). Immunofluorescence staining results: Migration After transplantation, Edu/GFAP, Edu/NG-2 double positive cells were found. Edu/GFAP, Edu/MAP-2 double positive cells were found in 14d after transplantation. Edu/GFAP, Edu/MAP-2, Edu/Neu N double positive cells were seen in 28d after transplantation. Conclusion: exogenous transplantation can survive in vivo and differentiate into neurons and astrocytes, and play the role of nerve regeneration and repair to promote ischemia in the body. Behavioral function recovery after cerebral apoplexy. The treatment of ischemic stroke with microglia and neural stem cell transplantation for the treatment of ischemic stroke: compare the therapeutic effects of simple transplantation of NSCs and microglia transplantation of neural stem cells in ischemic stroke, and explore its mechanism of action. Methods: A, B two groups are performed exogenous neural stem cells. At the same time, group B began to take orally PLX3397 and continuous 7d on the same day after modeling. The two groups carried out acceleration rods, forelimb grasping force, spontaneous activity test before and after modeling, and 3D carried out Ed U, Nestin, 14d for Ed U, GFAP, MAP-2 staining to observe the differentiation. There were statistical differences (P0.05). The test of forelimb grasping force found that A, B two were compared with 14d, 21d, 28d, and 35d, and there was no statistical difference (P0.05) in the total spontaneous activity of the group of A and B two (P0.05). The results of immunofluorescence staining showed that the two groups of exogenous neural stem cells were alive at third days after transplantation. The survival number is superior to that of the A group. Conclusion: microglia synergistic neural stem cell transplantation is more beneficial to the survival and differentiation of exogenous neural stem cells, and it also has a better effect on the recovery of behavior.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.3

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