骨髓基质细胞移植治疗大鼠实验性脑梗死的疗效分析及组织病理学观察

目的 观察大脑中动脉闭塞(MCAO)模型大鼠脑内移植骨髓基质细胞fBMSCs)的治疗作用,分析植入梗死灶不同区域的BMSCs的存活、迁移情况以及植入细胞的行为与脑内微环境中GFAP阳性细胞的形态关系.方法 75只成年SD大鼠采用随机数字表法分为MCAO组(n=50)和BMSCs移植组(n=25),所有动物均采用线栓法制作MCAO 1 h模型,24 h后BMSCs移植组脑内注射BrdU标记的同种异体BMSCs(2x106个),MCAO组注射等量PBS.MCAO前及MCAO后第1(移植前)、3、5、7、10、14天应用加速转轮试验和贴纸去除试验检测神经功能缺损情况;第14天处死动物,取脑组织切片应用HE染色观察两组的缺血病灶范围,行BrdU和GFAP免疫组化染色观察BMSCs在不同区域和不同胶质细胞环境下的存活和迁移情况.结果 BMSCs移植组MCAO后7 d加速转轮试验结果优于MCAO组,差异有统计学意义(P＜0.05);组织学观察发现植入缺血半暗带区的细胞存活数量最多,且向病变方向放射状迁移,植入缺血病灶核心的细胞甚少存活,且无迁移现象.结论 BMSCs脑内移植可改善MCAO后大鼠神经运动功能;活化的星形胶质细胞构成适合植入细胞存活、迁移的环境,而胶质瘢痕阻碍了细胞的迁移.     

针刺任脉和督脉对脑缺血大鼠海马星形胶质细胞的影响

目的 研究针刺任脉和督脉经穴对局灶性脑缺血大鼠缺血海马星形胶质细胞的调节作用。方法 线栓法制作大鼠大脑中动脉栓塞(MCAO)模型。通过免疫荧光双标方法研究各组GFAP阳性细胞和GFAP/NSE双标细胞的表达。结果 针刺督脉治疗可以下调脑缺血后海马齿状回的GFAP阳性细胞数,同时使GFAP/NSE双标细胞增多;针刺任脉和督脉治疗的GFAP阳性细胞增长幅度小于针刺督脉组,而GFAP/NSE双标细胞的增长幅度则大于针刺督脉组。结论 针刺任脉和督脉可抑制脑缺血后海马星形胶质细胞的过度增殖并可促进细胞分化     

rTMS促进大鼠局灶性脑缺血海马内源性神经干细胞分化的研究

目的探讨重复经颅磁刺激（rTMS）对局灶性脑缺血大鼠海马内源性神经干细胞分化的影响。方法线栓法制备大鼠大脑中动脉闭塞（MCAO）模型,随机分为脑缺血自然恢复组和rTMS治疗组,用荧光显微镜和共聚焦显微镜观察缺血14d、28d后各组大鼠海马中5-溴脱氧尿核苷（BrdU）与神经元特异核蛋白（NeuN）、神经胶质酸性蛋白（GFAP）共同标记的阳性细胞,并在高倍荧光显微镜下对双标阳性细胞计数。结果脑缺血后14d、28d,rTMS治疗组大鼠海马BrdU／NeuN双标阳性细胞数量分别为17．12±2．91、23．20±5．97,较相应自然恢复组12．96±2．79、15．92±2．52明显增加,两组同一时间点组间比较有统计学差异（P〈0．01）。而脑缺血后14d、28d,rTMS治疗组大鼠海马BrdU／GFAP双标阳性细胞数量分别为30．48±4．58、36．48±4．90,较相应自然恢复组37．44±3．58、43．60±5．96减少,两者相比有统计学差异（P〈0．05）。结论局灶性脑缺血大鼠海马增殖的内源性神经干细胞,可分化为神经元或神经胶质细胞,而rTMS可促进海马内源性神经干细胞向神经元的分化。     

移植Noggin基因修饰的神经干细胞对于大鼠MCAO模型梗死区域生长相关蛋白GAP-43MAP-2表达的影响

目的探讨移植携带Noggin基因的神经干细胞对MCAO动物模型神经功能修复及相关蛋白表达的作用。方法应用RT-PCR技术扩增大鼠Noggin基因,构建载体pEGFP-N1-Noggin,转染入原代培养的大鼠神经干细胞(NSCs)。线栓法制作大鼠MCAO模型,造模后3 d,移植入Noggin基因修饰的NSCs。分别于移植后2 d、7 d、14 d,应用免疫组化方法检测移植后缺血脑组织中GAP-43、MAP-2的表达情况。结果转染神经干细胞后可以稳定表达Noggin,神经干细胞移植后2 d、7 d、14 d,NOG组MAP-2表达显著高于生理盐水移植组(NS组)和空质粒组(P<0.05),神经干细胞移植后2 d、7 d、14 d,NOG组GFAP表达显著低于生理盐水移植组(NS组)和空质粒组(P<0.05)。结论 Noggin可以促进脑梗死区域生长相关蛋白GAP-43及微管结合蛋白MAP-2的表达。     

老年大鼠脑出血后海马齿状回神经干细胞的增殖与分化

目的 观察老年大鼠脑出血后海马齿状回神经干细胞(NSCs)的增殖与分化,探讨脑出血后NSCs的变化规律.方法 制作老年大鼠脑出血模型,5-溴脱氧尿核苷(BrdU)腹腔注射标记增殖细胞,用免疫组化法检测大鼠海马齿状回BrdU、神经元核抗原(NeuN)、胶质纤维酸性蛋白(GFAP)阳性细胞数的变化.结果 正常组和假手术组老年大鼠海马齿状回均有少量BrdU阳性细胞,脑出血后大鼠各时间段的BrdU阳性细胞数目均较正常组和假手术组明显增加,7d组达到峰值后逐渐下降,28d组仍高于正常组和假手术组.正常老年大鼠海马齿状回可见少量BrdU/NeuN和BrdU/GFAP双标阳性细胞,脑出血后双标阳性细胞数较正常组明显增加.结论 脑出血后老年大鼠海马齿状回NSCs增殖明显,且可以向神经元和神经胶质细胞分化.     

东菱迪夫对脑缺血大鼠侧脑室下区神经干细胞分化及迁移的影响

目的 观察东菱迪夫对脑缺血损伤后大鼠神经干细胞分化、迁移的影响,并探讨东菱迪夫治疗急性脑缺血损伤的可能机制.方法 采用大脑中动脉阻塞法(MCAO)建立局灶性脑缺血再灌注模型;运用神经功能缺陷评分和TTC染色评定神经功能与脑梗死体积;免疫组化检测缺血侧脑室下区(SVZ)5-溴脱氧尿嘧啶核苷(BrdU)以及胶质纤维酸性蛋白GFAP(作为检测脑缺血后神经干细胞的分化、迁移的指标),从而评价东菱迪夫对缺血脑损伤后神经干细胞分化迁移的作用.结果 东菱迪夫组(DLDF)再灌后第7天脑梗死灶体积明显小于模型组(P<0.01),DLDF组大鼠在第7、8、9、16天的神经功能缺陷评分明显优于模型组(P<0.05);和模型组相比,缺血损伤后第7天,东菱迪夫组大鼠SVZ的GFAP阳性细胞数明显增多(P<0.05);缺血损伤后第14天,东菱迪夫组大鼠SVZ的BrdU阳性细胞数明显增多(P<0.05),并且观察到从SVZ至其背外侧带的BrdU阳性细胞的迁移带.结论 东菱迪夫能减少脑缺血大鼠的脑梗死灶体积和改善神经功能评分;缺血脑损伤后,脑内神经干细胞发生分化、迁移;东菱迪夫可促进SVZ神经干细胞的分化、迁移.     

大鼠脑损伤后脑内移植骨髓基质细胞的研究

目的研究大鼠脑损伤后骨髓基质细胞(BMSCs)颅内移植对内源性神经干细胞(NSCs)的作用。方法 48只雄性Wistar大鼠随机分为3组:正常对照组、移植组、非移植组。大鼠脑损伤后24 h立体定向下局部注射BMSCs,然后每天腹腔注射5-溴脱氧尿嘧啶核苷(BrdU)2次。损伤后14 d和28 d随机处死取脑,行BrdU免疫组化染色以及BrdU和神经元特异性烯醇化酶(NSE)、胶质原纤维酸性蛋白(GFAP)免疫组化双染色。结果局部注射BMSCs的移植组大鼠表达BrdU/NSE阳性的细胞较非移植组为多。结论大鼠脑损伤后BMSCs对内源性NSCs的分化有促进作用。     

神经干细胞移植治疗大鼠创伤性脑外伤的实验研究

目的 将神经干细胞(NSCs)移植于大鼠创伤性脑损伤部位,观察NSCs对创伤性脑损伤的治疗作用.方法 首先进行NSCs的体外培养,同时采用5-2-溴脱氧尿嘧啶(BrdU)标记,脑外伤模型采用自由落体撞击大鼠左侧大脑皮质运动感觉区来制作,脑外伤后24h内移植NSCs,分别于脑外伤前、脑外伤后1、2、3周时行神经运动行为学评分(NMFE)和免疫组织化学染色检测BrdU、巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)和半乳糖脑苷酯(GalC)蛋白表达和TUNEL原位杂交染色,经计算机图像分析系统处理.结果 大鼠自由落体撞击伤后,右下肢神经运动功能障碍明显.神经运动行为学评分在第1周时,实验组与对照组无明显差异,而在第2、3周时,实验组的评分明显低于对照组,有统计学意义(P＜0.05).第2、3周时实验组NSE、GFAP和GalC染色阳性细胞数要多于对照组,nestin染色在第1周时表达最高,而后逐渐下降.BrdU阳性细胞在损伤灶中心区最多,在损伤灶的远隔部位也有少许发现,而TUNEL染色则对照组明显比NSCs组高,有统计学意义(P＜0.05).结论 脑外伤移植NSCs可以在损伤区存活、增殖及分化,并在损伤的远隔部位有少许NSCs迁移、分化.NSCs移植有利于大鼠脑自由落体撞击伤后期的功能恢复.     

动员自体骨髓干细胞对大鼠缺血/再灌注损伤后细胞增殖的影响

目的 探讨应用粒细胞集落刺激因子(G-CSF)动员自体骨髓干细胞对大鼠脑缺血/再灌注损伤后细胞增殖的影响.方法 大鼠随机分为G-CSF治疗组及对照组,根据取样时间点的不同每组又分为7、14、21d共3组,采用线栓法制备大鼠大脑中动脉缺血/再灌注模型(MCAO/R),治疗组于MCAO/R后24h予G-CSF 50μg/(kg·d),连续5d,对照组给予皮下注射等量生理盐水.观察不同时间点大鼠的神经功能评分.采用5-溴-2′-脱氧尿嘧啶核苷(BrdU)标记处于增殖状态的细胞,应用免疫组化法检测BrdU阳性细胞.结果 大鼠脑缺血/再灌注后7、14、21d G-CSF治疗组神经功能评分明显优于对照组,有显著性差异(P＜0.05),G-CSF治疗组大鼠在脑缺血/再灌注后7、14、21d均可检测到BrdU阳性细胞,可见接近梗死的同侧皮质、室管膜下区域及血管腔周围,BrdU阳性细胞计数显示各个时间点G-CSF治疗组阳性细胞数明显多于对照组(P＜0.05).结论 应用G-CSF动员自体骨髓干细胞治疗MCAO/R大鼠,能够改善脑缺血动物的神经功能,促进骨髓干细胞在病损脑组织周围的增殖.     

尾静脉移植骨髓间充质干细胞治疗脑缺血损伤大鼠的研究

目的通过尾静脉途径移植大鼠骨髓间充质干细胞(MSCs)到大脑中动脉闭塞模型(MCAO)的大鼠体内,观察MSCs移植对大鼠缺血性脑损伤后神经功能恢复的作用并探讨其作用机制。方法分离和培养大鼠的MSCs,线栓法制作脑缺血再灌注模型。将48只SD大鼠随机分为对照组和尾静脉移植组2组,移植组在造模7d后尾静脉途径将MSCs植入MCAO大鼠体内,对照组仅造模。比较两组大鼠在不同时间的神经功能评分的差异,免疫组化染色和脑源性神经生长因子(BDNF)分泌的情况。结果移植后1d两组之间无统计学差异;在2w、1m、2m、3m时移植组NSS评分均低于对照组;免疫组化结果发现尾静脉组见到双侧半球均可见较多的Brdu阳性细胞。移植后1m即可见到MSCs分化为Brdu+NSE、Brdu+GFAP免疫组化双阳性细胞。不同时间点移植组的BDNF分泌较对照组明显增高。结论 MSCs可以在体外分离、培养和传代,尾静脉移植的MSCs可在宿主脑内存活和分化并改善神经功能。MSCs移植后可促进脑内BDNF的分泌。     

Electroacupuncture improves neurological deficits and enhances proliferation and differentiation of endogenous nerve stem cells in rats with focal cerebral ischemia

Abstract

Objectives: This study was carried out to observe the effect of electroacupuncture (EA) on neurological deficits, proliferation and differentiation of nerve stem cells (NSCs) in adult rats with middle cerebral artery occlusion (MCAO) and to study its possible role in the treatment of cerebral ischemic injury.

Methods: A rat model of MCAO was established and interfered with EA. On days 4, 7, 14 and 21 after ischemic injury, neurological deficits were scored. On days 4, 7, 14 and 21 after injury, effect of EA interference on the proliferation and differentiation of rat NSCs was observed with BrdU/NeuN and BrdU/GFAP immunofluorescence double labeling.

Results: A significant difference was found in the scores of rat neurological deficits between the EA and model groups 7, 14 and 21 days after cerebral ischemic injury (p<0·05). BrdU positive cells were found in the subventricular zone (SVZ) 4, 7, 14 and 21 days after ischemic injury. The number of positive BrdU cells in the SVZ reached its peak 7 days after injury and was greater in the EA group than in the model group 7 and 14 days after injury (p<0·05). The number of BrdU/GFAP doubly labeled positive cells in the SVZ was greater in the EA group than in the model group 7 and 14 days after ischemic injury (p = 0·012 and p = 0·025, respectively). There was no difference in the number of BrdU/NeuN doubly labeled positive cells 4, 7 and 14 days in the striatum, but a significant difference 21 days (p = 0·033) after ischemic injury between the two groups.

Discussion: Cerebral ischemic injury induces proliferation of NSCs, some of which will differentiate into both astroglia and neurons. EA may promote cells proliferation, stimulate the proliferating cells to differentiate into astroglia and mature into neurons, which may be one of the important reasons why EA can alleviate neurological deficits.     

缺氧预处理后骨髓源神经干细胞联合脑源性神经生长因子移植治疗大鼠脑缺血再灌注损伤的研究

目的 研究缺氧预处理后骨髓源性神经干细胞（source n eural s tem c ells o f b one m arrow,BMSCs- NSCs）联合脑源性神经生长因子（brain-derived neurotrophic factor,BDNF）立体定向移植治疗大鼠脑缺 血再灌注损伤的疗效,为高原地区脑梗死的细胞移植治疗提供动物实验基础。 方法 72只SD大鼠随机分为缺氧预处理组和常氧组,每组各36只,缺氧预处理组造模前3 d进行低 氧预处理（hypoxic preconditioning,HPC）。两组均制作大脑中动脉阻塞再灌注（middle cerebral artery occlusion/reperfusion,MCAO/R）模型。每组分为3个亚组（BMSCs-NSCs+BDNF组、BMSCs-NSCs组和对 照组,每组各12只）,分别梗死灶同侧尾状核内立体定向移植BMSCs-NSCs+BDNF、BMSCs-NSCs和 DMEM/F12培养基。移植后3 d、7 d、14 d、21 d、28 d、35 d进行神经功能缺损评分,每个时间点每组 取2只大鼠,断头取脑后行免疫组织化学染色,观察5-溴脱氧尿嘧啶（5-Bromo-deoxyuridine,Brdu）阳 性细胞的迁移路径,行CD133、Nestin、微管相关蛋白2（microtubule-associated protein 2,MAP-2）、兔 抗微管蛋白（β-tubulin）、胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）、半乳糖神经酰胺 （Galactosylceramidase,Galc）免疫荧光染色,了解骨髓源性神经球分化情况。 结果 常氧BMSCs-NSCs+BDNF组、常氧BMSCs-NSCs组、缺氧预处理BMSCs-NSCs+BDNF组、缺氧预处 理BMSCs-NSCs组7 d、14 d、21 d、28 d和35 d的神经功能评分均显著低于同组3 d时的神经功能评分。移 植3 d时缺氧预处理对照组神经功能学评分显著低于常氧对照组（P =0.040）;移植7 d时缺氧预处理 BMSCs-NSCs+BDNF组神经功能评分显著低于常氧BMSCs-NSCs+BDNF组（P =0.031）。无论缺氧预处 理组还是常氧组,BMSCs-NSCs+BDNF组CD133、Nestin、MAP-2、β-tubulli n、GFAP、Gal c免疫荧光染色光 密度值（integral optical density,IOD）均显著高于BMSCs-NSCs组（均P＜0.001）;BMSCs-NSCs+BDNF组、 BMSCs-NSCs移植组各检测指标IOD均高于对照组（均P＜0.001）。 结论 大鼠缺氧预处理后BMSCs-NSCs联合BDNF立体定向移植可显著提高BMSCs-NSCs的效果。缺氧 预处理并不能促进外源性BMSCs-NSCs分化,但却能明显改善大鼠神经功能。     

Behavioral and histological evaluation of a focal cerebral infarction rat model transplanted with neurons induced from bone marrow stromal cells

Neurons can be specifically induced from bone marrow stromal cells (MSCs) with extremely high efficiency using gene transfection of the Notch intracellular domain and subsequent treatment with basic-fibroblast growth factor, forskolin, and ciliary neurotrophic factor. We investigated the behavioral and histologic efficacy of such bone marrow stromal cell-derived neuronal cell (MSDNC) transplantation into a focal cerebral infarction model in rats. A left middle cerebral artery occlusion (MCAO) was performed on adult Wistar rats. MSDNC transplantation into the ipsilateral hemisphere was performed on day 7 after MCAO. The behavioral analyses were conducted on days 14, 21, 28, 35, and 36-40, and a histologic evaluation was performed on day 41. MSDNC-transplanted rats showed significant recovery compared with controls (MCAO without cell transplantation) in beam balance, limb placing, and Morris water maze tests. Histologically, transplanted cells migrated from the injection site into the ischemic boundary area, including the cortex, corpus callosum, striatum, and hippocampus. Transplanted MSDNCs were positive for MAP-2 (84% +/- 8.11%), whereas only a small number of cells were positive for GFAP (1.0% +/- 0.23%). The survival rates of MSDNCs and MSCs 1 month after transplantation were approximately 45% and 10%, respectively. These results suggest that use of MSDNCs may be a promising therapeutic strategy for cerebral infarction.     

经鼻给予酸性成纤维细胞生长因子对脑梗死大鼠的血管和神经再生作用

目的研究经鼻给予酸性成纤维细胞生长因子（acidic fibroblast growth factor, aFGF）对脑梗死后神经和血管再生及神经功能恢复的影响。方法健康雄性SD大鼠30只,随机分为aFGF组（n=12）、对照组（n=12）和假手术组（n=6）;aFGF组和对照组大鼠建立大脑中动脉闭塞模型,脑缺血再灌注24h后经鼻分别给予10μg aFGF（200μl）和等容积生理盐水,1次／d,连续7d;同时腹腔注射5-溴脱氧尿核苷（Bromodeoxyuridine,BrdU）50mg／kg,1次/d,连续13d;假手术组操作过程和给药与对照组相同,但不用线栓阻塞大脑中动脉;分别在术前和术后第1、7、14d采用改良神经功能评分评价大鼠神经功能改变情况,并于术后第14d经尾静脉注入异硫氰酸荧光素右旋糖酐（FITC—dextran）,采用免疫组化及激光共聚焦方法分别检测梗死灶周、室管膜下区和纹状体BrdU阳性细胞及微血管的数量。结果术后第7及14d aFGF组神经功能评分显著低于对照组;术后第14d aFGF组缺血侧室管膜下区和纹状体BrdU阳性细胞数与对照组相比均显著增高（P〈0.01）,假手术组仅见少量BrdU阳性细胞;激光共聚焦显示aFGF组梗死灶周微血管数较对照组显著增加（P〈0.05）;同时,与对照组相比经鼻给予aFGF能增加Brdu阳性细胞在血管内皮细胞中的百分比,且有统计学意义（P〈0.05）。结论经鼻给予aFGF能有效促进神经和血管再生,改善脑缺血后神经功能评分,对于治疗脑梗死具有潜在的应用前景。     

Therapeutic effects of lipo-prostaglandin E1 on angiogenesis and neurogenesis after ischemic stroke in rats

Previous studies have demonstrated that prostaglandin E1 (PGE1) has a neuroprotective effect on cerebral ischemia. However, it remains unknown whether PGE1 promotes angiogenesis and neurogenesis after ischemic stroke. In this study, adult male Sprague-Dawley rats were subjected to permanently distal middle cerebral artery occlusion (MCAO). Rats were treated with lipo-prostaglandin E1(lipo-PGE1, 10 μg/kg/d) or the same volume of 0.9% saline starting 24 hours after MCAO daily for 6 consecutive days. All rats were injected 5'-bromo-2'-deoxyuridine (BrdU, 50 mg/kg) intraperitoneally every 12 hours for 3 consecutive days before being sacrificed. At 7 and 14 days after MCAO or sham-operation, rats were sacrificed. Post-stroke neurological outcome, infarction volume, angiogenesis and neurogenesis were evaluated. Treatment with lipo-PGE1 significantly increased the vascular density in the peri-infarct areas at 7 and 14 days after MCAO. The lipo-PGE1 treatment significantly enhanced the proliferation and migration of endogenous neural stem cells in the ipsilateral subventricular zone. The neural stem cells associated with blood vessels closely within a neurovascular niche in lipo-PGE1-treated rats after stroke. The lipo-PGE1 treatment also significantly improved the neurological recovery after MCAO. These results indicate that treatment with lipo-PGE1 promotes post-stroke angiogenesis, neurogenesis and their interaction, which would contribute to neurological recovery after cerebral infarction. Our study provides novel experimental evidences for the neuroprotective roles of PGE1 in ischemic stroke.     

自体骨髓内皮祖细胞移植治疗动脉粥样硬化大鼠急性脑缺血的实验研究

目的 探讨自体骨髓内皮祖细胞移植治疗动脉粥样硬化大鼠急性局灶性脑缺血的有效性.方法 高脂膳食制备20只动脉粥样硬化大鼠模型,采集骨髓,分离血管内皮祖细胞(EPCs)并扩增培养,检测其表面标记物的表达;第7天采用线栓法制作急性局灶性脑缺血模型,造模后3 h进行移植,其中实验组经颈静脉自体移植BrdU标记的EPCs,对照组给予等量的PBS.急性脑缺血术后6 h和第1、3、7、10、14天通过神经功能缺损评分(mNSS)量表行行为学评价,免疫组化染色观察BrdU标记的EPCs在缺血脑组织的分布和血管密度.结果体外培养大鼠骨髓来源的EPCs,细胞数目可达到5×106;CD34免疫荧光和FLK-1免疫组化鉴定呈阳性,并能特异性吸附FITC-UEA和内吞DIL-Ac-LDL.第14天实验组mNSS得分为6.13±0.30.对照组为8.50±0.46,比较差异具有统计学意义(P<0.05),实验组神经功能的恢复优于对照组.第28天实验组脑缺血区和血管壁可见BrdU标记的EPCs,而对照组则为阴性.免疫组化染色显示实验组大鼠缺血脑组织血管数为16.87±5.52,对照组大鼠缺血脑组织血管数为12.76±4.94,比较差异具有统计学意义(P<0.05).结论 从活体大鼠骨髓中分离的EPCs通过自体移植后可以进入脑缺血区并长期存活,其能够促进神经功能恢复,这可能与血管再生有关.     

脑栓通抑制凋亡并减轻脑梗死后丘脑继发性损害的研究

目的 研究脑栓通对脑梗死后同侧丘脑继发性损害的神经保护作用及其可能机制。方法 采用高血压大鼠（RHRSP）建立一侧大脑中动脉皮层支闭塞（MCAO）模型,随机分为：假手术组、溶剂对照组和脑栓通组,每组8只。脑栓通组大鼠MCAO术后24h经灌胃给予2ml/kg（10mg/ml）脑栓通,溶剂对照组给予等剂量溶剂,连续6天。MCAO术后1周感觉功能评估后行尼氏染色评价脑梗死灶体积和丘脑损害,并行免疫组化检测丘脑TUNEL、MAP-2和GFAP表达。结果 脑栓通组大鼠患肢感觉功能较溶剂对照组出现明显改善（P〈0.05）。两组间脑梗死灶体积无显著差异（P〉0.05）。脑栓通治疗后同侧丘脑神经细胞数量、MAP-2表达较溶剂对照组显著增多（P均〈0.05）,但GFAP表达显著下降（P〈0.05）。脑梗死后同侧丘脑TUNEL＋细胞数量明显高于假手组,脑栓通可显著减少同侧丘脑TUNEL＋细胞数量（P均〈0.05）。结论 脑栓通能够改善脑梗死后同侧丘脑继发性神经损害和神经功能,其机制可能与抑制同侧丘脑细胞凋亡有关。     

Endogenous neurogenesis and neovascularization in the neocortex of the rat after focal cerebral ischemia

The present study was designed to examine whether endogenous neurogenesis and neovascularization occur in the neocortex of the ischemic rat brain after unilateral middle cerebral artery occlusion (MCAO). Sprague-Dawley rats were divided into six groups (n = 29): one control group (n = 4) and five groups composed of animals sacrificed at increasing times post-MCAO (2 days and 1, 2, 4, and 8 weeks; n = 5 per group). To determine the presence of neurogenesis and neovascularization in the ischemic brain, nestin, Tuj1, NeuN, GFAP, Tie2, RECA, and 5-bromo-2'-deoxyuridine (BrdU) were analyzed immunohistochemically. In addition, nestin, GFAP, and Tie2 expression was determined by Western blotting. Triple-labeling of nestin, BrdU, and laminin was performed to visualize the interaction between endogenous neurogenesis and neovascularization. The number of BrdU- and nestin-colabeled cells increased markedly in the neocortex and border zone of the ischemic area up to 1 week after MCAO and decreased thereafter. Western blot analysis revealed that the expression of nestin, Tie-2, and GFAP was amplified in the ipsilateral hemisphere 2 days after MCAO and peaked 1 week after MCAO, compared with that in the normal brain. After ischemic injury, nestin- and BrdU-colabeled cells were observed in the vicinity of the endothelial cells lining cerebral vessels in the ipsilateral neocortex of the ischemic brain. Endogenous neurogenesis and neovascularization were substantially activated and occurred in close proximity to one other in the ipsilateral neocortex of the ischemic rat brain.     

神经干细胞移植治疗大鼠脑缺血再灌注损伤实验研究

目的探讨大鼠胚胎神经干细胞移植治疗局灶性脑缺血再灌注损伤的可行性。方法孕龄8~10d的大鼠神经干细胞在体外扩增后,用免疫组织化学方法分别检测神经干细胞及其分化后代的特异性标志蛋白nestin、胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)的表达。分别于缺血后不同时间窗将神经干细胞移植到局灶性脑缺血大鼠模型的缺血半暗带和梗塞中心,移植4w后比较不同移植部位神经干细胞存活、增殖和迁移的差异。结果从胎鼠中成功培养出悬浮生长的可表达nestin的神经球,其在含血清条件下可分化为表达GFAP的胶质细胞和表达NSE的神经元。神经干细胞移植4w后可见所有移植动物的细胞都存活,梗塞中心移植的细胞存活、增殖水平明显低于半暗带移植的细胞。结论大鼠胚胎神经干细胞移植到局灶性脑缺血再灌注损伤大鼠梗塞中心和半暗带均可长期存活,其增殖能力与移植部位密切相关。