DAPI标记骨髓间充质干细胞在胶质瘤模型大鼠脑内的迁徙和定位

背景：骨髓间充质干细胞作为载体细胞行脑内移植治疗胶质瘤,如何证实其为最好的药物载体？ 目的：将DAPI标记的大鼠骨髓间充质干细胞移植入模型鼠脑内,观察骨髓间充质干细胞在肿瘤内的迁徙与定位。 方法: 体外培养骨髓间充质干细胞。利用立体定向仪将培养好的C6细胞注入大鼠脑内,建立大鼠脑内胶质瘤模型。将DAPI标记于培养好的骨髓间充质干细胞上,利用微量注射器注入模型鼠脑内;移植后第3,7天处死大鼠,利用共聚焦显微镜观察骨髓间充质干细胞在肿瘤内的分布及与肿瘤细胞的关系。 结果与结论:实验成功建立了大鼠脑内胶质瘤模型。以DAPI标记的骨髓间充质干细胞。在模型鼠脑内主动聚集于肿瘤血管周围,并能与肿瘤细胞发生融合。结果证实骨髓间充质干细胞可以作为肿瘤基因治疗的良好载体。     

骨髓基质干细胞向大鼠脑胶质瘤的趋向迁移作用

目的 研究骨髓基质干细胞(BMSCs)向大鼠脑胶质瘤的趋向性.方法 在体外分离培养Fisher344大鼠BMSCs.流式细胞仪检测细胞免疫表型;Transwell法分别将BMSCs、NIH3T3细胞与9L细胞进行共培养.24 h后计算细胞迁移率:立体定向法建立Fisher344大鼠/9L细胞脑胶质瘤模型,2周后经神经行为学、MRI、HE染色证实模型成功后.将5-溴脱氧核苷尿嘧啶(BrdU)标记的BMSCs、N1H3T3细胞由颈内动脉进行移植,2周后,Fisher344大鼠灌注固定取脑,免疫组织化学检测BMSCs的趋向迁移规律.结果 体外分离培养的3～6代BMSCs表面标志CD34、CD45阴性而CD29、CD44阳性;BMSCs在体外能够向9L细胞迁移;立体定向法建立Fisher344大鼠/9L细胞脑胶质瘤模型符合胶质瘤的特征:颈内动脉移植的BMSCs也能够向9L细胞胶质瘤趋向迁移,主要分布在肿瘤组织与正常组织的边界.结论 BMSCs在体内、外均能够向脑胶质瘤迁移,颈内动脉移植是一种简单、有效的方法.     

骨髓间质干细胞移植对大鼠颅脑损伤后氧化应激的影响

目的观察骨髓间质干细胞移植对大鼠外伤性脑损伤的治疗效应和对大鼠颅脑损伤后氧化应激的影响。方法体外分离培养MSCs;建立大鼠TBI模型;经颈内动脉移植MSCs,观察大鼠神经功能的改善状况和两组大鼠脑组织内氧化应激指标的差异。结果MSCs在体外可长期培养扩增,生物学特性稳定;经颈动脉移植4周后,移植治疗的大鼠其神经功能状况改善较好。与对照组相比较,实验组大鼠脑组织内SOD的活性较高,羟自由基的含量较低。结论移植MSCs对TBI所造成的大鼠神经功能缺损有一定的治疗效应。而移植MSCs后能上调脑组织的抗氧化能力,这可能是产生治疗效应的机制之一。     

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。     

超顺磁氧化铁标记骨髓基质干细胞移植治疗帕金森病鼠的实验研究

目的:研究应用超顺磁氧化铁(SPIO)标记骨髓基质干细胞(MSCs)移植治疗帕金森病(PD)大鼠后的在体MRI观察。方法:分离、获取大鼠骨髓基质细胞,脂质体转染法将SPIO标记MSCs;制作PD模型,SPIO标记的MSCs移植到PD大鼠右侧纹状体区,应用MRI在体观察脑内移植的骨髓基质细胞的存活和迁徙情况。结果:体外SPIO标记的骨髓基质细胞普鲁蓝染色阳性;脑内移植SPIO标记MSCs的PD大鼠磁共振T2和T2GRE扫描检查显示在移植区呈低信号改变。随时间的延长,移植区信号向周围扩大。脑纹状体区的铁染色也可见SPIO标记MSCs从移植部位向四周迁移。结论:SPIO可用于体外标记MSCs,通过MRI技术可以对标记细胞脑内移植后进行初步的活体示踪,有利于MSCs移植治疗PD后的疗效观察。     

神经胶质瘤诱导骨髓基质干细胞定向迁移的初步研究

目的 探讨骨髓基质干细胞(BMSCs)对神经胶质瘤的定向迁移能力及其在脑内的分布规律. 方法 从雄性Wistar大鼠股骨和胫骨中分离出BMSCs并进行传代培养,采用免疫荧光检测BMSCs表面抗原并将其向脂肪细胞和成骨细胞方向诱导分化.证明BMSCs性质.立体定向接种C6胶质瘤细胞于Wistar大鼠脑内基底节区以建立鼠腩胶质瘤模型,7 d后将5-溴脱氧尿嘧啶(BrdU)标记的BMSCs接种入鼠脑对侧半球、对侧脑室和鼠尾静脉.BMSCS移植14d后心脏灌注取脑制成石蜡切片.以HE染色确定胶质瘤性质.SABC法免疫荧光染色显示BMSCs对神经胶质瘤组织的定向迁移能力及其在脑内的分布规律. 结果 对侧半球组和对侧脑室组均可见移植的BMSCs多沿针道直线分布并向对侧胶质瘤迁移.在胶质瘤与正常脑组织交界处可见激发出绿色荧光的阳性BMSCs:鼠尾静脉组亦可见少量BMSCs阳性细胞分布在胶质瘤组织周围. 结论 BMSCs通过脑实质、脑室及静脉注射途径均可定向迁移到胶质瘤组织,提示BMSCs可作为一种新的细胞载体用于胶质瘤的基因治疗.     

骨髓间质干细胞联合脑源性神经营养因子用于大鼠脑出血的实验研究

目的观察骨髓间质干细胞(MSCs)联合脑源性神经营养因子(BDNF)治疗大鼠脑出血(ICH)的疗效。方法建立大鼠ICH模型,体外培养标记纯化的MSCs,经侧脑室植入脑部,同时局部注入BDNF。记录对照组、BDNF组、MSCs组和MSCs+BDNF组7d、14d、21d大鼠神经功能改善程度;免疫组化法检测MSCs脑内迁移及分化;电子显微镜观察神经凋亡细胞。结果 MSCs组大鼠运动功能有明显改善,MSCs+BDNF组对ICH损伤的修复作用最明显。MSCs+BDNF组各时间点MSCs阳性细胞数及NEUN、GFAP、CNP免疫阳性细胞数均高于MSCs组(P<0.01),且MSCs+BDNF组神经细胞凋亡程度最轻。结论 MSCs脑部移植可促进大鼠ICH损伤部位结构和功能修复,BDNF对其修复具有协同作用。     

骨髓间充质干细胞自体移植治疗大鼠脑冷冻伤的实验研究

目的探索骨髓间充质干细胞（MSCs）对脑损伤的治疗作用。方法将Wistar大鼠自体骨髓MSCs在体外扩增并经Brdu标记后，通过颈内动脉注射将其植入冷冻伤脑水肿动物体内，从组织化学和神经功能评分两个方面观察骨髓MSCs自体移植的治疗作用。实验动物分为4组；A组给予常规治疗；B组给予干细胞自体移植；C组在进行干细胞自体移植的同时给予神经节苷脂和丹参注射液治疗；D组在干细胞自体移植前给予罂粟碱开放血脑屏障。结果经颈内动脉注射的Brdu标记的大鼠自体骨髓MSCs细胞可向脑损伤区域迁移。B、C、D组Brdu阳性细胞的数量和神经功能恢复的程度均高于A组（P〈0．05）。与B组相比，C组未增加Brdu阳性细胞的数量，但是神经功能恢复的程度明显升高（P〈0．05）。D组Brdu阳性细胞的数量、神经功能恢复的程度均高于B组（P〈0．05），但神经恢复的程度与C组相比无明显差异（P〉0．05）。结论骨髓间充质干细胞可以通过循环系统向脑损伤区迁移而发挥其治疗作用。     

PKH26和DAPI联合示踪骨髓干细胞在脑缺血模型小鼠脑内迁移的实验研究

目的 探讨PKH26和DAPI联合标记骨髓干细胞在脑内迁移过程中的示踪作用.方法 用PKH26和DAPI联合标记从Balb/c小鼠骨髓中分离出的骨髓单个核细胞(BMMCs),用改良电凝法制备局灶性脑缺血模型后,将标记的BMMCs经尾静脉移植入脑缺血小鼠体内,对照组经尾静脉注入PBS.移植2w后取脑组织,通过激光共聚焦荧光显微镜观察BMMCs向脑内迁移的情况.结果 实验组小鼠脑组织内可发现PKH26和DAPI双标的神经样细胞.结论 PKH26和DAPI可以联合标记向脑缺血模型小鼠脑组织迁移的骨髓干细胞,为体内示踪骨髓干细胞脑内迁移提供更加准确的实验方法.     

骨髓间质干细胞经静脉移植后在脑缺血大鼠的体内分布与迁移

目的 观察骨髓间质干细胞经静脉移植后在脑缺血大鼠体内的存活、分布及迁移情况,为细胞静脉移植奠定基础。方法密度梯度离心法分离大鼠骨髓间质干细胞(rat bone mescnchymal stem cels,rMSCs),经体外培养并扩增,线拴法制作大鼠大脑中动脉闭塞(MCAO)脑局灶缺血模型,rMSCs Hoechst33342标记后,通过静脉移植,经过1周、3周、5周后处死大鼠,取脑、心、肺、肾、肝、脾组织切片,在倒置荧光显微镜下观察细胞在体内的存活、分布及迁移情况。结果大鼠脑缺血后,经静脉移植骨髓间质干细胞在脑内能较长时间存活,静脉移植的细胞集中于脑缺血灶周围,而脑缺血对侧及心、肺、肾、肝、脾等组织少有细胞发现。结论rMSCs通过静脉途径进行移植后,能够向损伤部位迁移,与宿主脑组织整合在一起。骨髓间质干细胞经静脉移植用于治疗脑缺血具有可行性。     

脑胶质瘤经颈内动脉灌注ACNU的疗效观察

目的比较盐酸尼莫司汀（ACNU）经动脉灌注与经静脉灌注的疗效，并了解不同剂量的疗效。方法培养大鼠C6胶质瘤细胞株．建立大鼠C6胶质瘤模型。分为对照组及动、静脉灌注组，将ACNu按15、30、45mg／kg分别通过颈内动脉和股静脉注入荷瘤大鼠体内。10d后处死大鼠，取脑标本，行苏木精-伊红染色，测量肿瘤体积，比较不同给药方式和剂量对肿瘤生长的影响。结果ACNU治疗大鼠颅内肿瘤体积均小于对照组（P≤0．01）；ACNU颈内动脉灌注组肿瘤体积缩小较静脉灌注组更明显（P≤0．05）。结论单次经颈内动脉灌注ACNU较单次经静脉灌注能更有效地抑制脑胶质瘤的生长。     

No evidence that severity of stroke in internal carotid occlusion is related to collateral arteries

Background/Aim

The neurological effects of internal carotid artery (ICA) occlusion vary between patients. The authors investigated whether the severity of symptoms in a large group of patients with ipsilateral or/and contralateral ICA occlusion at presentation with ocular or cerebral ischaemic symptoms could be explained by patency of other extra or intracranial arteries to act as collateral pathways.

Methods

The authors prospectively identified all patients (n = 2881) with stroke, cerebral transient ischaemic attack (TIA), retinal artery occlusion (RAO), and amaurosis fugax (AFx) presenting to our hospital over five years, obtained detailed history and examination, and examined the intra and extracranial arteries with carotid and colour‐power transcranial Doppler ultrasound. For this analysis, all those with intracranial haemorrhage on brain imaging and cerebral events without brain imaging were excluded.

Results

Among 2228/2397 patients with brain imaging (1713 ischaemic strokes, 401 cerebral TIAs, 193 AFx, and 90 RAO) who underwent carotid Doppler, 195 (9%) had ICA occlusion. Among those patients with cortical events, disease in potential collateral arteries (contralateral ICA, external carotid, ipsilateral or contralateral vertebral or intracranial arteries) was equally distributed among patients with severe and mild ischaemic presenting symptoms.

Conclusion

The authors found no evidence that the clinical presentation associated with an ICA occlusion was related to patency of other extra or intracranial arteries to act as collateral pathways. Further work is required to investigate what determines the clinical effects of ICA occlusion.     

骨髓间充质干细胞趋脑胶质瘤性迁移机制的实验研究

目的探讨骨髓间充质干细胞（MSC）向脑胶质瘤选择性迁移的可能机制。方法从大鼠骨髓组织中分离培养MSC。选取第3-4代MSC,与胶质瘤细胞共培养或利用胶质瘤细胞条件培养基进行诱导培养,倒置相差显微镜下观察培养后MSC的细胞形态学变化：采用细胞免疫荧光染色方法检测培养后MSC表达内皮细胞标志物KDR、Ⅷ因子的情况。结果与胶质瘤细胞共培养和利用胶质瘤细胞条件培养基诱导培养后的MSC在形态上均表现出内皮细胞特征,细胞免疫荧光染色显示KDR、Ⅷ呈阳性表达。结论胶质瘤细胞能够诱导MSC分化为血管内皮细胞样细胞,这可能与MSC的趋胶质瘤性迁移的特性密切相关。     

Transcranial Doppler in cerebrovascular disease

Doppler analysis of flow in intracranial arteries is now possible using a 2 MHz probe allowing sufficient penetration of bone to obtain signals noninvasively. Thirty-two normal subjects, and 11 patients with cerebrovascular diseases including vasospasm following subarachnoid hemorrhage, middle cerebral artery stenosis, and extracranial internal carotid artery stenosis were studied by transcranial Doppler. Increased peak velocity and spectral broadening of the reflected signal corresponded to clinical and angiographic evidence of middle cerebral artery vasospasm or stenosis. Decreased peak velocity and blunted waveforms occurred in the middle cerebral artery ipsilateral to severe extracranial internal carotid stenosis with poor crossfilling from the contralateral carotid artery. Abnormalities resolved following carotid endarterectomy. Transcranial Doppler identifies vasospasm or stenosis of the middle cerebral artery and may allow noninvasive evaluation of collateral flow across the anterior circle of Willis in patients with extracranial carotid artery stenosis.     

TCD对颈内动脉中度及重度狭窄患者脑血流动力学的评估价值

目的应用经颅多普勒超声(transcranial doppler,TCD)评价颈内动脉中度及重度狭窄患者的脑血流动力学变化。方法经数字减影血管造影(digital subtraction angiography,DSA)确诊单侧颈内动脉中度及重度狭窄的患者69例,采用TCD检测其大脑中动脉血流参数,评价其侧支循环开放情况和脑血流储备能力(CRV)。结果 TCD显示侧支循环开放者患侧收缩期峰时血流速度(Vs)、搏动指数(PI)及CVR明显高于无侧支循环开放患者(P0.05),颈内动脉中度狭窄组患侧收缩期峰时血流速度(Vs)、PI值及CVR明显高于重度组(P0.05)。结论颈内动脉狭窄时侧支开放可以改善远端血流动力学指标,但改善程度有限。TCD可检测颈内动脉狭窄患者颅内血流动力学变化,评价颅内侧支循环的建立情况,为临床治疗和评估提供可靠依据。     

Usefulness of carotid artery compression test in the diagnosis of cerebral circulation disorders in patients with cerebral aneurysm. Evaluation by the rheoencephalographic method

Rheoencephalographic investigations (REG) was carried out in patients with intracranial aneurysms and in healthy subjects. The purpose of the study was to obtain approximate data on the efficiency of collateral circulation of the brain before ligation of the internal carotid artery. For this purpose REG was associated with common artery compression test. It was found that this combined examination gives good prediction of the efficiency of collateral circulation after ligation of one carotid artery. This method is more sensitive than the Matas test combined with EEG used previously.     

Differentiation and neurological benefit of the mesenchymal stem cells transplanted into the rat brain following intracerebral hemorrhage

Spontaneous intracerebral hemorrhage (ICH) is often a fatal event. In a patient who survives the initial ictus, the resulting hematoma within brain parenchyma can trigger a series of events that lead to secondary insults and severe neurological deficits. Great efforts have been focused on searching for new approaches to help patients recover neurological function after ICH. Previous studies indicate that mesenchymal stem cells (MSCs) grafted into the ischemic rat brain can improve neurological function. However, there is no report regarding whether MSCs can be used in the same way to improve the neurological function after ICH. We generated the ICH model by injecting collagenase VII into rat brain. Subsequently, 5-bromo-2-deoxyuridine (BrdU)-labeled mesenchymal stem cells were delivered into the brain through carotid artery, cervical vein or lateral ventricle. The distribution and differentiation of MSCs were investigated by methods of immunohistochemistry. We found that MSCs were able to differentiate into neural cells in vitro as well as in the rat brain after ICH. The injected MSCs were able to migrate into hippocampus, blooding foci and ipsilateral cortex. In the hippocampus, MSCs differentiated into neurons; but in surrounding bleeding foci, they differentiated into neurons and astrocytes. In the ipsilateral cortex, MSCs differentiated into neurons, astrocytes and oligodendrocytes. Notably, the motor function of the rats in the carotid artery (CA) group and the lateral ventricle (LV) group improved significantly. Collectively, our study indicates that MSCs are able to differentiate into neural cells in the rat brain after ICH and can significantly improve motor function.