脂肪来源干细胞移植对脑缺血大鼠运动功能的影响

目的 观察脂肪来源干细胞(ADAS)移植入脑缺血大鼠后存活、迁移、分化以及大鼠的运动功能障碍恢复情况,探讨ADAS移植治疗大鼠局灶性脑缺血的有效性和可能机制. 方法 将雄性SD大鼠饲养至250～300 g时,制作左侧大脑中动脉阻塞模型(MCAO),按照随机数字表法分为未处理组、对照组和移植组,每组6只.未处理组造模后不作特殊处理,对照组在造模后3h通过尾静脉注射杜氏改良培养基(DMEM),移植组造模后3 h通过尾静脉注射ADAS.造模后14 d处死大鼠,通过免疫荧光染色观察5-溴脱氧尿嘧啶核苷(BrdU)、神经元特异性烯醇化酶(NSE)、人微管相关蛋白2(MAP-2)和神经胶质纤维酸性蛋白(GFAP)的表达.造模后1、7及14 d时神经功能缺损评分评价大鼠运动功能改善情况. 结果 (1)移植后,标记了BrdU的ADAS大量出现在缺血灶周围;(2)MCAO后14d,缺血灶周围出现了少量BrdU/GFAP双染阳性细胞;同时出现少量BrdU/NSE和BrdU/MAP-2双染阳性细胞;(3)14d时移植组大鼠神经功能缺损评分与对照组相比明显降低,差异有统计学意义(P＜0.05). 结论 (1)成年大鼠ADAS在MCAO大鼠体内存活并少量分化为神经元样细胞和星形胶质细胞样细胞;(2)移植ADAS可使大鼠脑缺血所致的运动功能缺损得到改善.     

骨髓间充质干细胞静脉注射移植对脑缺血大鼠脑内神经细胞增殖和分化的影响

目的观察骨髓间充质干细胞（bone marrow stromal cells,BMSCs）经静脉注射移植对缺血/再灌注脑内神经细胞增殖和分化的影响。方法体外培养和扩增成年雄性大鼠BMSCs;以“四管阻断法”制作大鼠前脑缺血/再灌注模型;造模后3、5、7d通过尾静脉注射Hoechst33342标记的BMSCs（2×10^6/1ml/只）,另设对照组于相同时间点通过尾静脉注射载体溶液。于造模后4周处死大鼠,取脑切片,HE染色观察大鼠海马缺血性损伤情况;BrdU/GFAP、BrdU/MAP2和BrdU/Hoechst33342免疫荧光双标染色,观察大鼠脑内神经细胞的增殖及分化情况。结果BMSCs移植组海马CA1区存活锥体细胞数显著多于载体溶液对照组（P〈0.05）。BMSCs移植大鼠脑内海马结构BrdU阳性细胞数显著多于载体溶液对照组（P〈0.05）,BrdU/MAP-2双标阳性细胞占BrdU阳性细胞的比例显著高于载体溶液对照组（P〈0.05）。结论BMSCs经静脉注射移植能够减轻缺血性脑损伤,促进宿主脑内自体神经细胞增殖,并提高其分化为神经元样细胞的比例。     

经枕大池神经干细胞移植治疗大鼠创伤性脑损伤的研究

目的 将神经干细胞经枕大池移植到创伤性脑损伤模型大鼠蛛网膜下腔中并观察其存活、迁移和分化,从而为神经干细胞的体内存活、迁移和分化机理研究和临床应用提供实验依据.方法 体外培养BrdU标记的胚胎神经干细胞并应用免疫荧光细胞化学染色对BrdU、神经干细胞标记物nestin的表达进行鉴定:采用Feeney自由落体撞击法制做大鼠脑损伤模型,伤后24 h将BrdU标记的胚胎神经十细胞经立体定向注射移植到蛛网膜下腔;制作大鼠脑绢织石蜡切片,应用免疫组织化学染色检测BrdU、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)表达;伤前24h、伤后24 h及1、2周行动物运动神经功能评分.结果 免疫荧光检测显示神经球的表面细胞表达nestin及BrdU:免疫组织化学染色检测到脑内损伤灶存在BrdU阳性神经干细胞、MAP2阳性神经元和GFAP阳性胶质细胞;接受神经十细胞移植的大鼠神经运动功能评分的恢复较对照组有明显提高,差异有统计学意义(P<0.05).结论 经枕大池移植到脑损伤大鼠蛛网膜下腔中的神经干细胞能存活且具有远距离迁移能力,并明显有助于脑损伤大鼠神经运动功能的恢复.     

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脑缺血再灌注损伤的实验研究

目的探讨胎鼠皮质培养的神经干细胞移植治疗大鼠脑缺血再灌注损伤的可行性及疗效。方法取孕龄15d Sprague-Dawley(SD)胎鼠皮质细胞培养为神经干细胞;用线栓法制备大鼠脑缺血再灌注损伤模型;将24只健康SD大鼠分为假手术组、缺血对照组、缺血移植组,在移植后2周、4周依据Garcia的18分评分法对各组大鼠的神经功能进行评分;行脑灌注固定取材,免疫组化染色观察移植后神经干细胞的分化、迁移和整合情况。结果用胎鼠皮质培养的细胞nestin表达阳性;缺血移植组大鼠的神经功能评分明显高于缺血对照组(P<0.05);缺血移植组免疫组织化学染色能够检测到存活的BrdU阳性细胞,移植后4周时可见移植细胞向周围迁移、分化、参与血管形成,并可见梗死区边缘微血管明显增生;缺血移植组大鼠脑GFAP阳性细胞数较缺血对照组明显增多(P<0.05)。结论分离、培养胎鼠皮质细胞Nestin表达阳性,即是大鼠的神经干细胞;移植体外培养的神经干细胞能在脑缺血大鼠脑内存活、迁移、分化,并且对脑梗死大鼠的神经功能修复起到了积极作用。     

神经生长因子基因修饰骨髓间充质干细胞移植治疗脑梗死大鼠的实验研究

目的观察神经生长因子基因(NGF)修饰的骨髓间充质干细胞(BMSCs)移植治疗脑梗死的作用及可能机制。方法采用线栓法制备大鼠脑梗死模型,将符合条件的30只脑梗死模型大鼠按随机数字表法分为模型组(n=10),BMSCs移植组(n=10)和NGF-BMSCs移植组(n=10),分别经尾静脉注射PBS、BrdU标记的BMSCs和NGF-BMSCs各1mL。分别于术后1d、7d和14d采用改良神经功能损害评分(mNSS)对各组大鼠进行神经功能评估,于术后14d应用HE染色观察脑组织病理情况,应用免疫荧光组织化学检测BrdU标记的移植细胞存活状况和TUNEL法检测脑组织中细胞凋亡情况。结果NGFBMSCs组和BMSCs组mNSS评分和TUNEL阳性细胞数较Model组减低(P0.05),且NGF-BMSCs组较BMSCs组更低(P0.05);HE染色显示NGF-BMSCs组和BMSCs组较Model组脑组织损伤及细胞丢失较轻,NGF-BMSCs组更明显;并且NGF-BMSCs组中的BrdU阳性细胞数较BMSCs组增多(P0.05)。结论 NGF基因修饰的BMSCs移植较单纯BMSCs移植能进一步改善脑梗死大鼠的神经功能,其机制为能够促进植入的BMSCs在脑内存活和减轻神经细胞凋亡。     

大鼠骨髓基质细胞经枕大池移植后在损伤脑组织中的存活和分化

目的:探讨MSCs经枕大池移植到创伤性脑损伤模型大鼠的蛛网膜下腔后迁移到损伤脑组织存活并分化为神经细胞的能力。方法:采用Feeney’s自由落体脑创伤模型,建立23个大鼠脑创伤模型。从SD大鼠的后肢骨髓分离培养得MSCs,将第三代的MSCs以BrdU在体外标记。随机取15个脑创伤大鼠模型接受MSCa枕大池注射(脑创伤细胞移植组);随机取6个脑创伤大鼠模型接受枕大池注射生理盐水(脑创伤生理盐水组);取6个正常大鼠接受枕大池注射MSCs(正常大鼠细胞移植组)。定期杀死大鼠制作脑组织石蜡切片,行BrdU、BrdU-GFAP、BrdU-MAP2的免疫组织细胞化学染色。另取两个脑创伤大鼠在创伤1周后脑石蜡切片行HE染色。结果:观察到脑创伤细胞移植组石蜡切片BrdU染色可见BrdU阳性细胞,细胞进入皮质下的最大距离为3 mm。双标记染色可见部分BrdU阳性细胞胞质呈现GFAP或MAP_2染色阳性。而另外两组均未见阳性细胞。结论:大鼠MSCs具有从蛛网膜下腔迁移入损伤脑组织存活一定时期并分化为神经细胞的能力。     

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.     

大鼠骨髓间充质干细胞静脉移植治疗大鼠脑损伤

目的了解大鼠骨髓间充质干细胞（BMSCs）经鼠尾静脉移植后在脑内的表达情况及对大鼠脑创伤后神经运动功能的影响。方法将溴脱氧尿苷（BrdU）标记培养的BMSCs分别在大鼠脑创伤后第1天（实验组1，n=6）、第3天（实验组2，n=10）、第7天（实验组3，，F7）经大鼠尾静脉注射移植，另取6只脑创伤大鼠（实验组4）在伤后第3天经尾静脉移植BrdU标记的全骨髓，尾静脉注射仅．MEM脑创伤大鼠作为对照组（n=6）。应用免疫组化染色，了解移植细胞在脑内的表达情况。脑创伤后第1、3、7、14天评价移植对大鼠神经运动功能的影响。结果脑创伤后14d时神经运动功能评分分别为实验组1：31．83±1．60，实验组2：31．10±1．79，实验组3：28，43±1．72，实验组4：28．67±1．37，对照组：26．00±1．00．各组间差异有显著性（P=0．000）。在移植各组的创伤侧脑组织，可见BrdU染色阳性细胞，并以脉络丛、脑室周围、血管周围分布较多。对实验组2进行免疫组化双染色，未发现BrdU＋神经元特异性烯醇化酶（NSE1、BrdU＋胶质纤维酸蛋白（GFAP）双染色细胞。结论大鼠BMSCs经鼠尾静脉移植．在一定时期内能改善大鼠脑创伤后神经运动功能；脑内可以发现移植细胞存活．但移植细胞是否转化为神经元仍然需要进一步的实验证实。     

人脐血单个核细胞移植在缺氧缺血性脑损伤新生大鼠脑内的迁移及分化

背景：研究发现脐血单个核细胞脑内移植后可以存活，并减轻缺氧缺血性脑损伤程度，显著改善脑损伤大鼠的远期行为学，但作用机制仍未阐明。 目的：将人脐血单个核细胞经侧脑室移植入缺氧缺血性脑损伤新生大鼠脑内，观察植入细胞在脑内的迁移与分化。 设计、时间及地点：细胞学体内观察，于2007-12/2008-08在潍坊医学院分子影像学研究中心完成。 材料：脐血来源于健康、足月妊娠产妇，由潍坊医学院附属医院产科提供。清洁级健康7 d龄SD新生大鼠50只，由山东省中医药大学动物中心提供。 方法：Ficoll法体外分离培养人脐血单个核细胞，移植前3 d行BrdU标记。50只大鼠均采用Rice-Vannucci法复制缺氧缺血性脑损伤模型，造模后24 h，在左侧侧脑室(AP：-0.5 mm，ML：-2 mm，DV：-2 mm)注入脐血单个核细胞悬液，2 μL/点，共1×105个活细胞。分别于细胞移植后24 h，3 d，7 d，14 d，28 d取材制备脑组织切片，10只/时间点。 主要观察指标：采用免疫荧光双标法检测移植脐血单个核细胞的脑内迁移、神经干细胞的表达、分化情况。 结果：50只大鼠均进入结果分析。①移植后24 h侧脑室内可见BrdU+细胞；移植后3，7 d移植细胞迁移到室管膜下区；移植后14，28 d移植细胞迁移到大脑皮质及海马。②移植后24 h侧脑室内可见BrdU+nestin+细胞；移植后3 d室管膜下区出现BrdU+nestin+细胞；移植后7 d BrdU+nestin+细胞数增加；移植后14 d BrdU+nestin+细胞数下降。③移植后14 d大脑皮质、海马开始出现BrdU+NSE+细胞与BrdU+GFAP+细胞，移植后28 d双阳性细胞数进一步增加，BrdU+NSE+ 细胞及BrdU+ GFAP+细胞占BrdU+的比例也增加。 结论：脐血单个核细胞侧脑室移植入脑内后可以继续存活，并迁移到大脑皮质与海马部位，分化为成熟的神经元与神经胶质细胞。     

Transplantation of Flk-1+ human bone marrow-derived mesenchymal stem cells promotes angiogenesis and neurogenesis after cerebral ischemia in rats

Transplantation of bone marrow‐derived mesenchymal stem cells (BMSCs) is a potential therapy for cerebral ischemia. Although BMSCs‐induced angiogenesis is considered important for neurological functional recovery, the neurorestorative mechanisms are not fully understood. We examined whether BMSCs‐induced angiogenesis enhances cerebral tissue perfusion and creates a suitable microenvironment within the ischemic brain, which in turn accelerates endogenous neurogenesis and leads to improved functional recovery. Adult female rats subjected to 2 h middle cerebral artery occlusion (MCAO) were transplanted with a subpopulation of human BMSCs from male donors (Flk‐1+ hBMSCs) or saline into the ipsilateral brain parenchymal at 3 days after MCAO. Flk‐1+ hBMSCs‐treated rats exhibited significant behavioral recovery, beginning at 2 weeks after cerebral ischemia compared with controls. Moreover, rats treated with Flk‐1+ hBMSCs showed increased glucose metabolic activity and reduced infarct volume. Flk‐1+ hBMSCs treatment significantly increased the expression of vascular endothelial growth factor and brain‐derived neurotrophic factor, promoted angiogenesis, and facilitated cerebral blood flow in the ischemic boundary zone. Further, Flk‐1+ hBMSCs treatment enhanced proliferation of neural stem/progenitor cells (NSPCs) in the subventricular zone and subgranular zone of the hippocampus. Finally, more NSPCs migrated toward the ischemic lesion and differentiated to mature neurons or glial cells with less apoptosis in Flk‐1+ hBMSCs‐treated rats. These data indicate that angiogenesis induced by Flk‐1+ hBMSCs promotes endogenous neurogenesis, which may cause functional recovery after cerebral ischemia.     

Distribution and differentiation of bone marrow-derived mesenchymal stem cells in vivo after intraperitoneal and tail vein injection into rats in the recovery phase of stroke Which path is better?

BACKGROUND:Stereotactic injection(striatum or lateral ventricle)and vascular injection(tail vein or carotid artery)are now often used in cellular therapy for cerebral infarction.Stereotactic injection can accurately deliver cells to the infarct area,but requires a stereotactic device and causes secondary trauma;vascular injection is easy and better for host neurological deficit recovery,but can cause thrombosis.OBJECTIVE: To compare the therapeutic potential of adult bone marrow-derived mesenchymal stem cells(BMSCs)transplantation by intraperitoneal versus intravenous administration to cerebral ischemic rats.DESIGN,TIME AND SETTING: A randomized controlled animal experiment was performed at the Cell Room and Pathology Laboratory,Brain Hospital Affiliated to Nanjing Medical University from November 2007 to September 2008.MATERIALS: BMSCs were derived from 20 healthy Sprague-Dawley rats aged 4-6 weeks.METHODS: Forty-five adult middle cerebral artery occlusion(MCAO)rats were randomly divided into control,intravenous and intraperitoneal injection groups,with 15 rats in each group.At 21 bromodeoxyuridine(BrdU)via intravenous or intraperitoneal injection.MAIN OUTCOME MEASURES: Angiogenin expression and survival of transplanted cells were measured by immunohistochemical staining of brain tissue in infarction hemisphere at 7,14 or 21 days after BMSC transplantation.Co-expression of BrdU/microtubule-associated protein 2 or BrdU/glial fibrillary acidic protein was observed by double-labeled immunofluorescence of cerebral cortex.Evaluation of nerve function using the neurological injury severity score and the adhesion-removal test was performed on the 1st and 21st day before and after MCAO,and at 3,7,14 or 21 days after BMSCs treatment.RESULTS: Angiogenin-positive new vessels were distributed in the bilateral striatum,hippocampus and cerebral cortex of each group of rats at each time point,most markedly in the intravenous injection group.There were significantly more BrdU-positive cells in the intravenous injection group than in the intraperitoneal injection group(P < 0.01).Co-expression of BrdU/ microtubule-associated protein 2 or BrdU/glial fibrillary acidic protein were almost only seen in theintravenous group by fluorescence microscopy.After transplantation,BMSCs significantly restored nerve function in rats,particularly in the intravenous injection group.CONCLUSION: BMSCs were able to enter brain tissue via the tail vein or peritoneal injection and improve neurological function by promoting the regeneration of nerves and blood vessels in vivo,more effectively after intravenous than intraperitoneal injection.     

骨髓基质细胞及血管内皮祖细胞移植对局灶性脑缺血大鼠神经功能恢复的影响

目的 观察经静脉移植骨髓基质细胞(BMSCs)及血管内皮祖细胞(EPCs)后,缺血性脑损伤大鼠神经系统功能恢复情况.方法 40只健康成年Wistar大鼠按随机数字表法分为模型组、移植BMSCs组、移植EPCS组、移植BMSCs/EPCs组,每组10只.线栓法建立大鼠大脑中动脉闭塞(MCAO)模型.造模后24h取1 mLBMSCs、EPCs、BMSCs/EPCs细胞悬液(3×10~6个/mL)分别经尾静脉注射移植入后3组大鼠.模型组大鼠注射等量生理盐水.移植前、移植后1、7、14、28 d,采用旋转试验、躯体感觉试验、神经系统功能评分(NSS)评估各组大鼠神经功能的恢复情况.移植后28d,免疫荧光染色检测各组大鼠缺血脑组织的微血管密度(MVD).结果 移植后第7天,旋转试验显示移植BMSCs/EPCs组大鼠旋转时间长于其他3组,躯体感觉试验和NSS评分分别显示移植BMSCs/EPCs组大鼠移物时间和NSS评分低于其他3组,差异有统计学意义(P<0.05);移植后28 d,免疫荧光染色检测结果显示缺血脑组织MVD明显高于其他3组,差异有统计学意义(P<0.05).结论 静脉BMSCs/EPCS联合移植可增强缺血性脑损伤功能的恢复.     

Transplantation of human mesenchymal stem cells promotes functional improvement and increased expression of neurotrophic factors in a rat focal cerebral ischemia model

Previous studies have suggested that intravenous transplantation of mesenchymal stem cells (MSCs) in rat ischemia models reduces ischemia‐induced brain damage. Here, we analyzed the expression of neurotrophic factors in transplanted human MSCs and host brain tissue in rat middle cerebral artery occlusion (MCAO) ischemia model. At 1 day after transient MCAO, 3 × 10⁶ immortalized human MSC line (B10) cells or PBS was intravenously transplanted. Behavioral tests, infarction volume, and B10 cell migration were investigated at 1, 3, 7, and 14 days after MCAO. The expression of endogenous (rat origin) and exogenous (human origin) neurotorphic factors and cytokines was evaluated by quantitative real‐time RT‐PCR and Western blot analysis. Compared with PBS controls, rats receiving MSC transplantation showed improved functional recovery and reduced brain infarction volume at 7 and 14 days after MCAO. In MSC‐transplanted brain, among many neurotrofic factors, only human insulin‐like growth factor 1 (IGF‐1) was detected in the core and ischemic border zone at 3 days after MCAO, whereas host cells expressed markedly higher neurotrophic factors (rat origin) than control rats, especially vascular endothelial growth factor (VEGF) at 3 days and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) at 7 days after MCAO. Intravenously transplanted human MSCs induced functional improvement, reduced infarct volume, and neuroprotection in ischemic rats, possibly by providing IGF‐1 and inducing VEGF, EGF, and bFGF neurotrophic factors in host brain. © 2009 Wiley‐Liss, Inc.     

慢病毒介导bFGF基因修饰的骨髓基质细胞对脑梗死后血管新生的影响

目的探讨慢病毒介导碱性成纤维细胞生长因子（basic fibrobla stgrowth factor,bFGF）基因修饰的骨髓基质细胞（bone marrow stromal cells,BMSCs）对脑梗死后血管新生的影响。方法利用慢病毒载体介导bFGF基因修饰BMSCs,获得稳定转染bFGF的BMSCs。脑梗死后1d立体定向移植至梗死灶周围。在MCAO术前及术后1、3、7、14d进行神经功能评分。术后14d股静脉注射异硫氰酸荧光素右旋糖酐（FITC-dextran）标记微血管,结合共聚焦显微镜和3DDoctor3．5版软件分析梗死灶周围区微血管的直径、面积及血管分支数目。结果bFGF-BMSCs组术后3d神经功能恢复明显优于对照组与BMSCs组（P〈0．05）,BM—SCs组和bFGF-BMSCs组术后7d及14dmNSS评分显著低于对照组（P〈0．05）,而且bFGF-BMSCs组神经功能恢复好于BMSCs组（P〈O．05）。bFGF-BMSCs组微血管直径、分支数目及面积显著性高于对照组（P〈0．01）和BMSCs组（P〈0．05）。结论bFGF修饰的B^假瞄能更好地促进脑梗死后神经功能恢复及血管新生。     

骨髓基质细胞静脉移植治疗大鼠短暂性局灶性脑缺血

目的探讨骨髓基质细胞静脉移植治疗大鼠短暂性局灶性脑缺血的可行性及其机制。方法将大鼠骨髓基质细胞在体外纯化、扩增并经BrdU标记后,经尾静脉移植到局灶性脑缺血大鼠体内,通过神经缺损评分观察移植后大鼠神经行为学改善情况,通过组织学方法观察移植到脑内的骨髓基质细胞表达脑源性神经营养因子、缺血灶周围细胞凋亡及脑微血管密度的变化。结果骨髓基质细胞移植组大鼠的神经缺损评分显著低于对照组(P<0.05);移植到脑内的骨髓基质细胞主要选择性分布于缺血灶周围区域并表达BDNF;骨髓基质细胞移植组大鼠梗死灶周围的凋亡细胞明显少于对照组(P<0.01)、微血管密度显著高于对照组(P<0.001)。结论经静脉注射移植骨髓基质细胞能够明显促进局灶性脑缺血大鼠的神经行为功能恢复;抗凋亡及促微血管增生可能是骨髓基质细胞移植治疗局灶性脑缺血的机制之一。     

Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and neurogenesis after cerebral ischemia in rats

Hypoxic preconditioning of stem cells and neural progenitor cells has been tested for promoting cell survival after transplantation. The present investigation examined the hypothesis that hypoxic preconditioning of bone marrow mesenchymal stem cells (BMSCs) could not only enhance their survival but also reinforce regenerative properties of these cells. BMSCs from eGFP engineered rats or pre-labeled with BrdU were pre-treated with normoxia (20% O(2), N-BMSCs) or sub-lethal hypoxia (0.5% O(2). H-BMSCs). The hypoxia exposure up-regulated HIF-1α and trophic/growth factors in BMSCs, including brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF) and its receptor FIK-1, erythropoietin (EPO) and its receptor EPOR, stromal derived factor-1 (SDF-1) and its CXC chemokine receptor 4 (CXCR4). Meanwhile, many pro-inflammatory cytokines/chemokines were down-regulated in H-BMSCs. N-BMSCs or H-BMSCs were intravenously injected into adult rats 24h after 90-min middle cerebral artery occlusion. Comparing to N-BMSCs, transplantation of H-BMSCs showed greater effect of suppressing microglia activity in the brain. Significantly more NeuN-positive and Glut1-positive cells were seen in the ischemic core and peri-infarct regions of the animals received H-BMSC transplantation than that received N-BMSCs. Some NeuN-positive and Glut-1-positive cells showed eGFP or BrdU immunoflourescent reactivity, suggesting differentiation from exogenous BMSCs into neuronal and vascular endothelial cells. In Rotarod test performed 15days after stroke, animals received H-BMSCs showed better locomotion recovery compared with stroke control and N-BMSC groups. We suggest that hypoxic preconditioning of transplanted cells is an effective means of promoting their regenerative capability and therapeutic potential for the treatment of ischemic stroke.     

骨髓基质细胞立体定向移植治疗大鼠脑缺血损伤的实验研究

目的:观察骨髓基质细胞立体定向移植对大鼠脑缺血损伤后神经功能恢复的作用并探讨其作用机制。方法:制作SD大鼠大脑中动脉缺血模型(MCAO);体外培养骨髓基质细胞,观察其生物学特性以及立体定向移植后对脑缺血损伤后神经功能改善情况。结果:骨髓基质细胞体外可以长期传代、扩增,分泌NGF、VEGF等多种神经保护性因子;立体定向移植后,骨髓基质细胞在脑内存活、迁徙,小部分分化成具有神经元表面标志的细胞,与对照组相比,骨髓基质细胞移植组神经功能改善情况好于对照组。结论:骨髓基质细胞具有多向分化潜能,表达并分泌多种神经保护性营养因子。立体定向移植MSCs,对改善脑缺血损伤后的神经功能状况具有积极作用。     

Comparison of ischemia-directed migration of neural precursor cells after intrastriatal, intraventricular, or intravenous transplantation in the rat

Cell replacement therapy may have the potential to promote brain repair and recovery after stroke. To compare how focal cerebral ischemia affects the entry, migration, and phenotypic features of neural precursor cells transplanted by different routes, we administered neuronal precursors from embryonic cerebral cortex of green fluorescent protein (GFP)-expressing transgenic mice to rats that had undergone middle cerebral artery occlusion (MCAO) by the intrastriatal, intraventricular, and intravenous routes. MCAO increased the entry of GFP-immunoreactive cells, most of which expressed neuroepithelial (nestin) or neuronal (doublecortin) markers, from the ventricles and bloodstream into the brain, and enhanced their migration when delivered by any of these routes. Transplanted neural precursors migrated into the ischemic striatum and cerebral cortex. Thus, transplantation of neural precursors by a variety of routes can deliver cells with the potential to replace injured neurons to ischemic brain regions.