小鼠气管异位移植后模拟肺移植慢性排斥反应模型的建立*☆

目的：闭塞性细支气管炎是引起肺移植后移植物功能异常的主要并发症，原位肺移植动物模型技术难度高且费时，限制了其在闭塞性细支气管炎研究中的应用。通过小鼠的气管异位移植来建立模拟肺移植慢性排斥反应模型，以期为国内的闭塞性细支气管炎研究提供一种新的模型选择。 方法：①实验材料及分组：实验于2007-07/08在上海市肺科医院动物实验室完成，动物实验方法符合动物伦理学要求。实验组采用10只C57BL/6小鼠为供体，10只BALB/c小鼠为受体；对照组10只供体、10只受体均采用BALB/c小鼠。②实验方法：取供体小鼠的气管、左右主支气管连续气道作为供体，两端结扎后异位移植到受体小鼠背部皮下。③实验评估：移植28 d后获取移植气管标本，石蜡包埋，行苏木精-伊红染色观察移植气管病理变化；比较两组小鼠的移植气管闭塞性细支气管炎发生率。 结果：20例模型动物全部存活，无感染。①模型动物手术时间（12±2）min。②苏木精-伊红染色病理切片示实验组小鼠移植气管管腔闭塞，慢性炎细胞浸润广泛，气管上皮完全脱落，纤维组织增生明显，呈现闭塞性细支气管炎表现；对照组小鼠移植气管的组织形态未见明显异常。③实验组小鼠移植气管闭塞性细支气管炎发生率高于对照组（P=0.000）。 结论：小鼠气管异位移植后模拟肺移植慢性排斥反应模型作为研究闭塞性细支气管炎的动物模型具有诸多优点。     

环孢素聚乳酸微球在同种异体气管移植中的应用

背景：前期实验已获得了最优的环孢素聚乳酸微球制备工艺，且证实微球具有明显的缓释性能，无明显毒、副作用。 目的：观察同种异体原位气管移植后，局部置入环孢素聚乳酸微球对免疫排斥的抑制作用。 设计、时间及地点：随机对照动物实验，于2007-02/04在解放军第二五二医院医院动物实验中心完成。 材料：选取SD大鼠60只，按体质量配对后行同种异体气管原位移植。移植后按随机数字表法分为3组，即空白对照组、环孢素注射组及环孢素聚乳酸微球局部应用组，每组10只。 方法：空白对照组不做特殊处理；环孢素注射组于气管原位移植后给予环孢素肌肉注射；环孢素聚乳酸微球局部应用组于气管原位移植后分别将10，30 mg环孢素聚乳酸微球埋植于气管周围肌肉层内、外。 主要观察指标：移植后4周麻醉后处死动物，对移植气管标本进行大体及病理学评估，计算上皮积分、淋巴细胞计数以及无核软骨细胞/全部软骨细胞比值等指标，并进行组间比较。 结果：①环孢素注射组、环孢素聚乳酸微球局部应用组大鼠全部存活至预杀期，空白对照组大鼠死亡1只。②移植后4周环孢素注射组、环孢素聚乳酸微球局部应用组的上皮积分均高于空白对照组（P < 0.01）；环孢素聚乳酸微球局部应用组高于环孢素注射组（P < 0.01）。③移植后4周环孢素注射组、环孢素聚乳酸微球局部应用组的淋巴细胞计数均低于空白对照组（P < 0.05，0.01）；环孢素聚乳酸微球局部应用组低于环孢素注射组（P < 0.05）。④移植后4周环孢素注射组、环孢素聚乳酸微球局部应用组无核软骨细胞与所有软骨细胞比值均低于空白对照组（P < 0.05）。 结论：同种异体气管原位移植后局部置入环孢素聚乳酸微球可以有效降低免疫排斥反应，对移植体的保护作用优于肌肉注射环孢素。     

骨髓间充质干细胞对慢性哮喘小鼠血清白细胞介素12和白细胞介素4水平的影响

背景：研究显示,骨髓间充质干细胞移植通过减轻炎症程度来改善急性肺损伤的病情,而骨髓间充质干细胞移植对哮喘疾病的研究甚少。 目的：观察慢性哮喘小鼠移植同种异体骨髓间充质干细胞后,其血清白细胞介素12和白细胞介素4水平的变化。 方法：取40只雌性BALB/c小鼠,随机分为4组,正常对照组和骨髓间充质干细胞对照组以PBS致敏及激发小鼠,于第21天激发前气管内注射PBS或骨髓间充质干细胞 30 μL。哮喘模型组和骨髓间充质干细胞治疗组用鸡卵白蛋白制备慢性哮喘模型,于第21天激发前气管内注射PBS或骨髓间充质干细胞 30 μL。采用ELISA法检测各组小鼠血清炎症因子水平。 结果与结论：病理提示哮喘模型组支气管上皮黏膜脱落,同时上皮黏膜有杯状细胞增生,部分管腔内大量黏液栓塞;气道、血管旁有大量炎性细胞浸润以及气道平滑肌细胞增生和肥大。正常对照组及骨髓间充质干细胞对照组无气道炎症及气道重塑的表现,而骨髓间充质干细胞治疗组气道炎症及气道重塑明显减轻。对比骨髓间充质干细胞对照组及正常对照组,哮喘模型组白细胞介素12明显减低,白细胞介素13 和白细胞介素4明显增高;而骨髓间充质干细胞治疗组较哮喘模型组白细胞介素12明显升高,白细胞介素4明显降低。结果表明骨髓间充质干细胞治疗哮喘可能通过降低白细胞介素4水平,提高体内白细胞介素12水平,从而改善气道炎症及气道重塑的程度。     

骨髓间充质干细胞移植修复肾损伤

背景：研究发现外源性骨髓间充质干细胞能够定居于肾脏并且分化为肾脏细胞，参与损伤肾组织的修复，但对其修复作用途径尚无公认。 目的：探讨外源性骨髓间充质干细胞移植对肾损伤的可能治疗作用。 设计、时间及地点：细胞学体内实验，于2007-03/09在南昌大学泌尿外科研究所完成。 材料：清洁级SD大鼠56只。雄鼠8只用于制备骨髓间充质干细胞，雌鼠48只随机分为细胞移植组、模型对照组、假手术组，16只/组。荧光染料DAPI为Biotium Inc产品，蓖麻血凝素为Vector Laboratories产品，BrdU为Sigma产品。 方法：以密度梯度离心法分离鼠骨髓间充质干细胞，加入DAPI进行标记，调整细胞数为1.5×1010 L-1。细胞移植组、模型对照组建立缺血再灌注肾损伤模型，假手术组开腹后不予结扎肾血管。缺血45 min后，细胞移植组经下腔静脉注入用DAPI标记的骨髓间充质干细胞悬液1 mL，模型对照组、假手术组输入1 mL无菌生理盐水。移植后各组每天腹腔注射BrdU，其后留取血标本和肾组织。 主要观察指标：荧光显微镜及免疫组织化学染色观察移植细胞在肾组织中的分布，采用能与肾小管内腔壁特异结合的蓖麻血凝素鉴定移植细胞的分化，以免疫组化方法检测Brdu反映肾组织细胞的增殖状况，检测血清肌苷、尿素氮水平。 结果：移植后第3天可见少量DAPI阳性细胞，第4天DAPI阳性细胞数明显增加（P < 0.05），且多数DAPI标记细胞能结合蓖麻血凝素，DAPI阳性细胞主要分布于肾脏外髓质肾小管中，肾皮质和内髓质阳性细胞很少，而肾小球内则未见DAPI阳性细胞。假手术组肾组织细胞增殖非常弱，移植后第4天Brdu阳性细胞数明显低于模型对照组（P < 0.05）；移植后第2，3，4天细胞移植组Brdu阳性细胞均明显多于模型对照组（P < 0.05），其分布类似于DAPI阳性细胞。与模型对照组比较，细胞移植组大鼠血清尿素氮水平移植后第1，2天明显降低（P < 0.05）；细胞移植组大鼠血清肌苷水平移植后第1，2，3天均显著降低（P < 0.05）。 结论：移植的外源性骨髓间充质干细胞能够迁移、定居于缺血再灌注损伤后的肾组织中并分化为肾小管上皮细胞；骨髓间充质干细胞移植可促进损伤肾脏本身的细胞增殖再生；对肾损伤后早期肌苷、尿素氮的升高具有一定抑制作用，这种早期对肾功能的保护与其迁移定居到肾脏局部、分化修复损伤的肾小管无关。     

精原干细胞冷冻保存后再移植的生精功能

背景：精原干细胞的冷冻保存在男性不育治疗方面具有潜在的临床应用价值,但冷冻保存过的精原干细胞移植后,其精子发生过程及生精能力目前尚不完全清楚。 目的：观测精原干细胞冷冻保存后移植的生精功能。 方法：采用复合酶消化、差速贴壁结合非连续性Percoll 密度梯度离心的方法获取雄性SD 大鼠及雄性C57BL/6 小鼠精原干细胞;以雄性BALB/c 裸小鼠为受体,出生后6 周予腹腔注射白消安破坏其内源性生精功能。将供体精原干细胞分为冷冻保存组与非冷冻保存组,分别以曲细精管微注射的方法异种移植精原干细胞,采用形态学方法观察生精过程;采集附睾精液,观察精子形态并进行体外受精实验。移植后1,2,3个月取受体睾丸组织,采用免疫组化SABC 法检测α6-Integrin 蛋白表达情况;移植后1周、1个月、3个月取受体睾丸组织,应用扫描电镜观察生精过程。 结果与结论：冷冻保存前的精原干细胞活性率高于冷冻保存后的细胞活性率(P < 0.01)。冷冻保存组与未冷冻保存组受体睾丸组织精原细胞膜和细胞质中均有α6-Integrin 的阳性表达,随时间增加而增加,两组间α6-Integrin 蛋白的表达无统计学差异;SD 大鼠精原干细胞移植后能在BALB/c 裸小鼠生精上皮中克隆增殖,并能分化形成大鼠形态特征的精子,在小鼠精子发生巢内既有大鼠来源的精子发生,又有小鼠内源性精子发生,其供体来源的精子数量较小鼠自身来源的精子数量多。通过体外受精实验提示移植后的精原干细胞在受体内增殖分化形成的精子功能正常,具有受精的能力。结果说明采用常规方法冷冻保存的精原干细胞,移植后能在受体生精上皮中克隆增殖,并能分化形成成熟的精子。 关键词：冷冻保存;精原干细胞;精子发生;移植;组织细胞移植     

气管异位移植模型大鼠气管上皮细胞对闭塞性细支气管炎的影响

背景：目前很多实验已证明气管上皮细胞是引起这种慢性炎症的关键因素,气管上皮起到免疫应答的“靶器官”作用,可激活细胞免疫和体液免疫的应答。 目的：探讨在大鼠气管异位移植中气管上皮细胞对闭塞性细支气管炎发生的影响。 方法：建立大鼠去上皮组织的气管异位模型。将大鼠随机分4组：①上皮组：Wistar大鼠→SD大鼠,气管模型建立不需要消化气管上皮,直接灌入培养液。②去上皮组：Wistar大鼠→SD大鼠,没有上皮细胞的Wistar大鼠气管,将气管模型移植于SD大鼠的背部皮下。③对照组1：Wistar大鼠→Wistar大鼠,气管灌入生理盐水植入皮下。④对照组2：Wistar大鼠→Wistar大鼠,将去上皮组织的气管植于皮下。 结果与结论：上皮组气管内发生了闭塞,闭塞率随着移植天数的增加而增加。去上皮组气管内没有堵塞。对照组管内没有闭塞。在大鼠气管异位移植模型中得到证实,气管上皮细胞在闭塞性细支气管炎的发生发展中起重要作用。     

羊膜负载表皮干细胞促进糖尿病大鼠创面的愈合

背景：表皮干细胞作为皮肤组织的特异性干细胞,具有强大增殖及多向分化潜能,与创面修复紧密相关。近期研究表明糖尿病皮肤创面愈合过程中表皮干细胞数量减少、活性降低是导致其创面难愈的重要原因。 目的：观察表皮干细胞在糖尿病大鼠创面愈合中的作用。 方法：分离培养及鉴定SD大鼠表皮干细胞,并以BrdU标记。建立糖尿病SD大鼠创面模型,抽签法随机分为3组：表皮干细胞组创面移植羊膜负载BrdU标记的表皮干细胞;羊膜组创面移植羊膜;空白对照组创面未给予干预。 观察创面愈合情况、计算创面愈合率,苏木精-伊红及免疫组织化学SP法检测创面愈合组织中BrdU及增殖细胞核抗原表达。用图像分析软件测量阳性细胞积分吸光度平均值。 结果与结论：表皮干细胞组治疗后7d创面缩小明显,治疗后14 d创面基本愈合,创面愈合率明显高于羊膜组、空白对照组 (P < 0.01)。表皮干细胞组创面及新生表皮中可见BrdU阳性细胞,而另两组皮肤创面组织中始终未见BrdU阳性细胞。各组创面组织中可见增殖细胞核抗原阳性细胞表达,但表皮干细胞组的阳性细胞积分吸光度平均值与羊膜组、空白对照组比较差异有显著性意义(P < 0.01)。结果证实糖尿病大鼠创面愈合过程中表皮干细胞与创缘表皮移行、创面的上皮化有直接关联,可有效促进其创面愈合。     

不同方法移植骨髓间充质干细胞在体内重要器官的分布及分化

背景：大量实验显示心肌损伤后,趋化或直接心肌内注射的骨髓干细胞可以在体内分化为心肌细胞、血管内皮细胞或血管平滑肌细胞。 目的：观察不同方法移植的骨髓间充质干细胞体内重要器官的分布、生长和分化情况。 方法：选用3月龄日本大耳白兔39只,均制作急性心肌梗死动物模型并随机分为4组,3个组为实验组分别以心肌内直接注射、经冠脉注射、经静脉注射3种不同移植经BrdU标记的自体骨髓间充质干细胞。对照组分为3个亚组,分别经冠脉、静脉和心肌内直接注射等量的生理盐水。4周后处死动物,取心、肺、肝、肾制作组织切片并观察BrdU阳性细胞和BrdU及α-横文肌肌动蛋白双阳性细胞。 结果与结论：①骨髓间充质干细胞移植各实验组在梗死区及其周边均可见到BrdU阳性细胞和BrdU及α-横文肌肌动蛋白双阳性细胞。BrdU阳性细胞间及与宿主心肌细胞间有细胞突起相连,并可见明显的横纹和肌小节。②各实验组新生毛细血管密度较对照组都有增加(P < 0.05),其效果顺序由好到差依次为冠脉注射组、心肌注射组和静脉注射组。③经冠脉和静脉移植组非目的器官肺、肝、肾的标本切片内均发现BrdU阳性细胞表达,且静脉移植组肺内BrdU阳性细胞数多于肝、肾,但均未见有分化趋势和恶性细胞表达。结果提示骨髓间充质干细胞经3种方法移植均能使心肌梗死区及其周边区心肌细胞和毛细血管再生,除心肌注射组外非目的器官都有BrdU阳性细胞表达,但仍以目的器官表达为主。     

Bcl-2基因修饰的骨髓间充质干细胞移植治疗脑缺血的实验研究

目的观察Bcl-2基因修饰的骨髓间充质干细胞（MSCs）移植对大鼠脑缺血性损伤的治疗作用及对移植的MSCs保护性作用。方法将114只SD大鼠随机分为假手术组、Model组、MSCs组和Bcl-2-MSCs组;线栓法制作大鼠一侧大脑中动脉缺血再灌注模型,MSCs组及Bcl-2-MSCs组在缺血24h后经尾静脉注射方式移植BrdU标记的MSCs及人Bcl-2基因修饰的MSCs;分别于术后1、7、14、28d对各组大鼠进行神经功能缺损评分（NSS）;在脑缺血14d应用TTC法观察梗死灶体积;BrdU和TUNEL免疫荧光双重标记检测移植的MSCs凋亡情况;Westernblot检测大鼠脑梗死周边区Bcl-2蛋白的表达;HE染色观察脑组织病理形态。结果MSCs-Bcl-2组和MSCs组NSS评分、脑梗死体积百分比、TUNEL阳性细胞数较Model组低（P〈0.05）,且MSCs-Bcl-2组比MSCs组更低,BrdU阳性细胞数较多（P〈0.05）;MSCs-Bcl-2组BrdU和TUNEL免疫荧光双标细胞数稍多,但差异不显著（P〉0.05）,而MSCs-Bcl-2组BrdU和TUNEL免疫荧光双标细胞占BrdU阳性细胞百分比明显较低（P〈0.05）。MSCs-Bcl-2组Bcl-2蛋白呈持续较高水平的表达,和MSCs组同时间点相比差异有统计学意义（P〈0.05）。HE染色示MSCs组和MSCs-Bcl-2组脑组织损伤及细胞丢失较轻,MSCs-Bcl-2组更明显,脑梗死周边区均未见到核大、浓染的异形细胞。结论Bcl-2基因修饰的MSCs移植较MSCs移植能进一步改善脑缺血大鼠的神经功能,减少脑梗死体积;其机制为Bcl-2基因修饰使MSCs持续、稳定表达一定量的Bcl-2蛋白,从而能保护移植的MSCs,减少其凋亡,增加其存活。     

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.     

Progesterone attenuates astro- and microgliosis and enhances oligodendrocyte differentiation following spinal cord injury

Reactive gliosis, demyelination and proliferation of NG2+ oligodendrocyte precursor cells (OPC) are common responses to spinal cord injury (SCI). We previously reported that short-term progesterone treatment stimulates OPC proliferation whereas chronic treatment enhances OPC differentiation after SCI. Presently, we further studied the proliferation/differentiation of glial cells involved in inflammation and remyelination in male rats with SCI subjected to acute (3 days) or chronic (21 days) progesterone administration. Rats received several pulses of bromodeoyuridine (BrdU) 48 and 72 h post-SCI, and sacrificed 3 or 21 days post-SCI. Double colocalization of BrdU and specific cell markers showed that 3 days of SCI induced a strong proliferation of S100β+ astrocytes, OX-42+ microglia/macrophages and NG2+ cells. At this stage, the intense GFAP+ astrogliosis was BrdU negative. Twenty one days of SCI enhanced maturation of S100β+ cells into GFAP+ astrocytes, but decreased the number of CC1+ oligodendrocytes. Progesterone treatment inhibited astrocyte and microglia /macrophage proliferation and activation in the 3-day SCI group, and inhibited activation in the 21-day SCI group. BrdU/NG2 double labeled cells were increased by progesterone at 3 days, indicating a proliferation stimulus, but decreased them at 21 days. However, progesterone-enhancement of CC1+/BrdU+ oligodendrocyte density, suggest differentiation of OPC into mature oligondendrocytes. We conclude that progesterone effects after SCI involves: a) inhibition of astrocyte proliferation and activation; b) anti-inflammatory effects by preventing microglial activation and proliferation, and c) early proliferation of NG2+ progenitors and late remyelination. Thus, progesterone behaves as a glioactive factor favoring remyelination and inhibiting reactive gliosis.     

Passive avoidance training is correlated with decreased cell proliferation in the chick hippocampus

One-trial passive avoidance learning (PAL), where the aversive stimulus is the bitter-tasting substance methylanthranilate (MeA), affects neuronal and synaptic plasticity in learning-related areas of day-old domestic chicks (Gallus domesticus). Here, cell proliferation was examined in the chick forebrain by using 5-bromo-2-deoxyuridine (BrdU) at 24 h and 9 days after PAL. At 24 h post-BrdU injection, there was a significant reduction in labelling in MeA-trained chicks in both the dorsal hippocampus and area parahippocampalis, in comparison to controls. Moreover, double-immunofluorescence labelling for BrdU and the nuclear neuronal marker (NeuN) showed a reduction of neuronal cells in the dorsal hippocampus of the MeA-trained group compared with controls (35 and 49%, respectively). There was no difference in BrdU labelling in hippocampal regions between trained and control groups of chicks at 9 days post-BrdU injection; however, the number of BrdU-labelled cells was considerably lower than at 24 h post-BrdU injection, possibly due to migration of cells within the telencephalon rather than cell loss as apoptotic analyses at 24 h and 9 days post-BrdU injection did not demonstrate differences in cell death between treatment groups. Cortisol levels increased in the chick hippocampus of MeA-trained birds 20 min after PAL, suggesting the possibility of a stress-related mechanism of cell proliferation reduction in the hippocampus. In contrast to hippocampal areas, the olfactory bulb, an area strongly stimulated by the strong-smelling MeA, showed increased cell genesis in comparison to controls at both 24 h and 9 days post-training.     

行为学训练对海马损伤梗死大鼠齿状回区BrdU和Nestin表达的影响

目的 探讨行为学训练对海马损伤梗死大鼠齿状回区神经干细胞增殖的影响。方法 采用光化学法制成单侧海马损伤梗死模型大鼠72只,随机分为训练组（n=36）和自由活动组（n=36）,每个组设1、7、14、21、28及35d 6个亚组。另设正常对照组36只,与模型组对应分为1、7、14、21、28及35d 6个亚组。训练组大鼠于造模1d后给予水迷宫训练,自由活动组大鼠自由活动,不予水迷宫训练。免疫荧光双标记法观察各不同时间点大鼠海马齿状回区溴脱氧尿嘧啶核苷（Bromodeoxyuridine,BrdU）与巢蛋白（Nestin）的双标记表达情况。结果 正常对照组大鼠海马齿状回区有少量BrdU/Nestin双标记阳性细胞,训练组及自由活动组大鼠在7、14、21及28d海马损伤梗死侧齿状回区BrdU/Nestin双标记阳性细胞数量均有显著增多（P <0.01）;训练组大鼠7、14、21、28d时海马损伤梗死侧齿状回区的BrdU/Nestin双标记阳性细胞数量显著高于自由活动组（P <0.01）;至35d时,训练组及自由活动组大鼠海马损伤梗死侧齿状回区BrdU/Nestin双标记阳性细胞数量与正常对照组无明显差异（P >0.05）。结论 行为学训练能显著增强海马损伤梗死大鼠齿状回区神经干细胞的增殖,促进神经功能恢复。     

组织工程角膜上皮移植治疗兔角膜碱烧伤：形态学观察

背景：采用健康角膜缘干细胞体外培养形成组织工程化角膜上皮,移植于碱烧伤后角膜创面可促进创面的修复愈合。 目的：观察组织工程角膜上皮移植治疗角膜碱烧伤的疗效和时机。 设计、时间及地点：对比观察动物实验,于2007-07/2008-06在解放军总医院第一附属医院实验动物科完成。 材料：新西兰雌性大白兔21只,体质量2.0~2.5 kg,随机分为对照组和移植组,其中对照组8只16眼,移植组13只再分为：早期移植组,1 d移植组2只4眼、3 d移植组3只6眼、6 d移植组3只6眼、9 d移植组2只4眼;中期移植组,14 d移植组3只6眼。 方法：取21只兔上方健康角膜缘组织体外培养制备组织工程角膜上皮。21只兔双眼采用1 mol/L氢氧化钠制作改良角膜碱烧伤模型,移植组在碱烧伤后早期1,3,6,9 d和中期14 d行自体或同种异体组织工程角膜上皮移植,对照组烧伤后持续观察4周。 主要观察指标：移植组及对照组烧伤后4周内眼表和组织病理学变化。 结果：角膜碱烧伤后1周开始出现角膜上皮的大片脱落,2周角膜上皮大片脱落或溃疡发生率达72%,持续至4周,移植组在4周时发生率仅为25%,大多获得完整的角膜上皮;烧伤后早期移植组角膜基质深层炎性细胞浸润和新生血管生长较对照组明显受到抑制,而中期移植组角膜基质层较对照组并无明显差异;4周内异体组织工程角膜上皮移植的免疫排斥反应并不大于自体移植。 结论：自体或同种异体组织工程角膜上皮移植可尽快恢复眼表完整性,且烧伤后早期移植效果明显优于中期移植。     

倒千里光碱对肝大部分切除小鼠肝脏损伤后再生修复的影响**☆

背景：目前大多数应用倒千里光碱造成肝损伤模型都是以大鼠为实验对象,有研究报道倒千里光碱并不能抑制小鼠肝细胞增殖,也有报道倒千里光碱对小鼠的肝细胞具有增殖抑制作用。 目的：观察单纯肝脏大部分切除及其联合应用倒千里光碱致小白鼠对肝损伤后再生修复的影响。 方法：40只C57BL/6J小鼠随机数字表法分为2组,每组20只。倒千里光碱/肝脏部分切除组：腹腔注射倒千里光碱溶液70 mg/kg,注射2次,每次间隔2周,4周后肝脏2/3切除。肝脏部分切除组：腹腔注射生理盐水70 mg/kg,注射2次,每次间隔2周,4周后行肝脏2/3切除。观察术后14 d肝脏大体结构恢复情况;苏木精-伊红染色观察术后第3,7天肝细胞损伤情况;BrdU染色观察术后第3天成熟肝细胞增殖情况;CK19和C-kit免疫组织化学方法观察术后第3,7,14天肝脏卵圆细胞增生情况。 结果与结论：肝脏部分切除组14 d肝大体结构基本恢复正常,而倒千里光碱/肝脏部分切除组肝脏大小没有明显的恢复。苏木精-伊红染色可见倒千里光碱/肝脏部分切除组明显的肝细胞变性改变;BrdU染色肝脏部分切除组术后第3天有明显肝细胞增殖,而倒千里光碱/肝脏部分切除组肝细胞增殖很少。CK19和C-kit免疫组织化学结果显示倒千里光碱/肝脏部分切除组术后第3天开始肝脏主要通过肝卵圆细胞增生并逐渐向肝细胞和小胆管分化,从汇管区开始向肝小叶内延伸以修复损伤。提示倒千里光碱可抑制小鼠肝脏损伤后肝细胞增殖再生。建立倒千里光碱/肝脏部分切除小鼠模型可诱导肝脏卵圆干细胞增生。     

Chronic stress and social housing differentially affect neurogenesis in male and female rats

Stress plays an important role in the development of affective disorders. Women show a higher prevalence for these disorders than men. The course of a depression is thought to be positively influenced by social support. We have used a chronic stress model in which rats received foot-shocks daily for 3 weeks. Since rats are social animals we hypothesised that 'social support' might reduce the adverse effects of chronic stress. To test this hypothesis, male and female rats were housed individually or socially in unisex groups of four rats. The proliferation marker bromodeoxyuridine (BrdU) was injected 2 weeks before the sacrifice to investigate if stress and social housing influenced the survival of proliferating cells in the dentate gyrus (DG). To investigate changes in proliferation, another group of rats was sacrificed the day after the last BrdU injection. Stress significantly decreased BrdU labelling in individually housed males and not significantly in socially housed males. In individually housed females stress increased BrdU labelling, which was prevented by social housing. The increase found in females is most likely caused by differences in survival rate, since cell proliferation was not affected by stress or housing conditions. These results indicate that social support can affect neurogenesis in both female and male rats, however in a different way.     

Prenatal phencyclidine exposure alters hippocampal cell proliferation in offspring rats

Multiple case reports have described pregnancy in phencyclidine hydrochloride (PCP) abusers. Characteristic clinical symptoms of PCP‐exposed infants have revealed neurobehavioral or physical abnormalities. We designed this study to evaluate whether chronic prenatal exposure to PCP during the last 2 weeks of gestation in rats produces alterations of hippocampal neurogenesis in offspring. Rats received repeated subcutaneous injection of PCP (5 mg/kg) once daily during the last 2 weeks of gestation. Control animals received subcutaneous injection of physiological saline during gestation. Dams receiving repeated PCP administrations showed markedly increased locomotor activities on days 1, 5, and 10 during the last 2 weeks of gestation. At 21 days after birth, 5‐bromo‐2′‐deoxyuridine (BrdU)‐positive cells of offspring were counted in the granule cell layer (GCL) and subgranular zone of the dentate gyrus. The numbers of BrdU‐positive cells in the GCL in male and female offspring of the PCP‐treated group were significantly increased by ～77% compared with those from the control group. At 56 days, the number of surviving BrdU‐positive cells also remained to be increased by 74% in the GCL in PCP‐treated group. At 21 days, locomotor activities of offspring in the PCP‐treated group were significantly decreased by ～30% compared with those in the control group. However, neuronal differentiation of newly formed cells and cell survival were not influenced at 5 weeks after BrdU injections. Some altered biochemical or physiological conditions of offspring from dams receiving repeated PCP injections during pregnancy could influence changes in cell proliferation in the GCL of offspring during early development. Changes to cell proliferation in the hippocampus may affect behavioral abnormalities during infancy in offspring. Synapse 63:729–736, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

经枕大池移植神经干细胞治疗重型颅脑损伤大鼠的实验研究

目的探讨胎脑皮质来源的神经干细胞(NSC)用于重型颅脑损伤模型大鼠的治疗作用。方法用电子颅脑损伤仪制备重型颅脑损伤大鼠模型(n=46),随即分为神经干细胞治疗组(NSC组,n=23)和对照组(DMEM/F12组,n=23)。将胎鼠皮质来源的NSC经体外培养、扩增后用BrdU标记,经枕大池移植到模型大鼠的脑脊液中,于移植后24h,3d,7d,14d,21d,28d对大鼠进行改良神经功能缺损评分(mNSS)的评估。对照组用相同体积的DMEM/F12代替NSC,mNSS评估时间和方法同NSC组。4w后NSC组取5只大鼠脑组织行免疫组化法染色检测BrdU表达。结果 NSC组大鼠在移植后第7d、14d、21d和24d,其mNSS评分与DMEM/F12组相比,均有显著性差异。对脑组织行BrdU免疫荧光染色,发现移植后第4w脑损伤灶及周围脑组织中有BrdU阳性细胞的表达。结论经枕大池移植后,NSC可在大鼠脑组织内存活、增殖,并能促进脑损伤大鼠神经功能的恢复。