人羊膜间充质干细胞移植治疗帕金森病模型大鼠的疗效评价

目的观察不同途径移植人羊膜间充质干细胞(human amniotic mesenchymal stem cells,h AMSCs)对帕金森病(Parkinson’s disease,PD)模型大鼠的生物学效应及其在体内的分化。方法采用胰蛋白酶-胶原酶消化法分离hAMSCs,流式细胞术分析表型。40只雌性Wistar大鼠随机分为假手术组、模型组、hAMSCs静脉移植组和原位移植组。采用单侧前脑内侧束(MFB)注射6-羟基多巴胺建立PD大鼠模型。通过舌下静脉或于MFB原位移植3×105个hAMSCs。腹腔注射阿朴吗啡诱导旋转观察大鼠的行为变化,免疫荧光染色法检测人细胞核抗原及神经元微管结合蛋白(microtubule-associated protein 2,MAP-2)的表达,免疫组化染色法检测酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达。结果与模型组比较,hAMSCs静脉移植组和原位移植组大鼠旋转次数均明显减少(均P<0.05),前者行为学改善可持续至移植后6 w,后者则至8 w;免疫荧光染色显示,hAMSCs在原位移植区可存活至少12 w,并表达MAP-2;免疫组化染色显示静脉和原位移植hAMSCs均可上调PD模型大鼠黑质TH表达,但后者强于前者。结论 hAMSCs能改善PD模型大鼠的运动行为,原位移植优于静脉移植,其机制可能与上调黑质TH表达有关。hAMSCs可在原位移植部位分化为多巴胺能神经元样细胞。     

人羊膜上皮细胞移植治疗帕金森病鼠的研究

目的 观察人羊膜上皮细胞在帕金森病鼠移植后的存活情况,以及它对帕金森病鼠旋转行为的改善作用.方法 采用6-羟多巴胺立体定向纹状体注射制作帕金森病鼠模型;51只大鼠随机分三组:人羊膜上皮细胞移植组、假手术PBS对照组以及空白模型对照组.制模成功后第5周用人特异性抗体Nestin和Vimentin检测人羊膜细胞的存活情况,第10周切片观察黑质部TH阳性神经元的变化情况,高效液相色谱--电化学仪测定纹状体多巴胺(DA),高香草酸(HVA),3,4-二羟基苯乙酸(DOPAC)等浓度以及脑脊液DA的含量.结果 人羊膜上皮细胞帕金森病鼠侧脑室内移植可以存活达10周;移植组大鼠旋转数较对照组明显降低(P≤0.01);黑质部TH阳性神经元数量较对照组升高(P≤0.01),纹状体区DA、HVA和DOPAC含量较PBS对照组明显升高(P＜0.01～0.05),移植组脑脊液DA含量较PBS对照组也显著增加(P＜0.01).结论 人羊膜上皮细胞侧脑室移植可以改善帕金森病鼠的旋转行为,其机制可能与其增加纹状体区多巴胺等递质水平有关.     

人羊膜上皮细胞侧脑室移植治疗脑出血大鼠实验研究

目的 探讨人羊膜上皮细胞(hAECs)侧脑室移植对脑出血(ICH)大鼠脑水肿与神经功能恢复的影响.方法 分离培养hAECs,用Hoechst33258和增强绿色荧光蛋白(EGFP)标记hAECs,将其移植入脑出血大鼠侧脑室中,测试大鼠28 d内的运动功能变化与脑水肿的动态变化,并行免疫组织化学观察.结果 移植hAECs沿大鼠侧脑室壁生长,细胞存活4 w以上.巢蛋白(nestin)与波形蛋白(vim)免疫组化检测呈阳性表达,病灶周围小胶质细胞OX-42染色阳性细胞减少.移植组ICH大鼠脑含水量减少,神经功能明显改善.结论 hAECs移植到ICH大鼠侧脑室可存活,表达神经元特异性抗原,并减轻ICH大鼠脑水肿,改善运动功能.     

人羊膜上皮细胞侧脑室移植对帕金森病大鼠模型行为、多巴胺能神经元、神经递质影响的实验研究

目的 观察人羊膜上皮细胞(HAECs)移植入帕金森病(PD)大鼠模型侧脑室后的存活及分化情况,及其对PD大鼠模型旋转行为、纹状体区多巴胺及其代谢产物的影响.方法 采用6-羟多巴立体定向脑内注射制作PD大鼠模型,将制模成功大鼠随机分成3组:人羊膜上皮细胞移植组(HAECs组)、磷酸缓冲组(PBS组)和帕金森组(PD组),1w后腹腔注射阿朴吗啡观察各组大鼠旋转行为的变化,连续观察10w,HAECs组5w后用人特异性抗体Nestin和Vimentin检测人羊膜细胞的存活情况,10w后酪氨酸羟化酶(TH)染色观察各组PD大鼠模型黑质部TH阳性神经元的变化情况及HAECs的分化情况,高效液相色谱--电化学仪测定纹状体多巴胺(DA)、高香草酸(HVA)、3,4-二羟基苯乙酸(DOPAC)等神经递质的水平.结果 HAECs在PD大鼠侧脑室内移植可以长期存活达10w,并且可以分化为DA能神经元,HAECs组大鼠旋转数较PBS组及PD组明显降低(P<0.01),黑质部TH阳性神经元数量较PD组及PBS组升高(P<0.01),HAECs组大鼠纹状体区DA及其代谢产物DOPAC、HVA含量较PBS组明显升高(P<0.05).结论 人羊膜上皮细胞移植入PD大鼠侧脑室可以改善PD大鼠的旋转行为,其机制可能与增加纹状体区DA等神经递质有关.     

人羊膜上皮细胞移植在神经系统疾病中的应用

1人羊膜上皮细胞的神经生物学特征1．1人羊膜上皮细胞的干细胞性质与分 化羊膜是由最终发育成胎儿的内细胞团在开始分化成其它组织器官之前首先分化出来的一层薄膜组织。它是包绕胎儿的两层膜的内层,来源于形成肠管之前的外胚层,人羊膜上皮细胞是克隆原细胞,原始的人羊膜上皮细胞经培养有分化成3个胚层细胞的潜能,例如：内胚层的肝细胞,胰腺细胞,中胚层的心肌细胞,外胚层的神经细胞,在一定的培养条件下,人羊膜上皮细胞可以形成球形结构,     

人羊膜上皮细胞移植治疗灵长类动物脊髓损伤的实验研究

目的探讨人羊膜上皮细胞移植治疗恒河猴脊髓半切损伤的作用。方法采用脊髓半切损伤制作恒河猴脊髓损伤模型,同时移植人羊膜上皮细胞或转染BDNF的羊膜上皮细胞,于损伤后70d行荧光金注入脊髓损伤下方4cm处,损伤后80d观察实验动物的行为学变化,并行组织学检查。结果治疗组脊髓损伤侧后肢运动功能恢复明显优于对照组。治疗组移植物内可见AchE、TH阳性神经纤维和GFAP阳性的神经胶质细胞,移植物内有荧光金染色。结论移植人羊膜上皮细胞使恒河猴脊髓半切损伤后运动功能明显改善。     

微囊化人视网膜色素上皮细胞移植治疗帕金森病的实验研究

目的 观察微囊化人视网膜色素上皮(RPE)细胞移植治疗帕金森病(PD)大鼠的疗效. 方法 采用机械分离法和酶消化法原代培养人RPE细胞,传代后用高压静电微胶囊成型装置制作海藻酸钠-多聚赖氨酸-海藻酸钠微囊化细胞,将其立体定向移植人6-羟基多巴胺(6-OHDA)所致的PD模型大鼠的右侧纹状体.实验分为模型组、裸细胞(RPE)组、空囊对照(APA)组以及微囊化细胞(APA-RPE)组.检测各组大鼠移植前后阿朴吗啡诱导的旋转行为变化和移植后8周纹状体中多巴胺(DA)的含量. 结果 APA-RPE组大鼠在移植后4周阿朴吗啡诱发的旋转次数[(6.25±1.04)r/min]开始减少,与移植前[(12.88±7.34)r/min]相比减少幅度为51.48%,至第8周[(5.87±2.03)r/min]减少更加明显,减少幅度为54.43%,差异均有统计学意义(P<0.05);与模型组[(108.14±1.89)mol/L]比较,APA-RPE组移植后8周[(342.63±28.32)mol/L]大鼠纹状体DA含量明显增加,差异有统计学意义(P<0.05),而RPE组和APA组未见明显变化. 结论 微囊化人RPE细胞对PD大鼠模型有治疗作用,可作为一种前景良好的治疗PD的方法 进一步研究.     

移植人羊膜细胞对大鼠创伤性脑损伤的实验研究

目的 探究大鼠TBI后脑内移植人羊膜细胞（HACs）对大鼠运动功能的影响。方法 HACs经分离、Hoechst33342标记后重悬调整细胞浓度为10^5/μl；采用改进的Feeney自由落体法打击大鼠脑皮层后肢运动区域，损伤后24h经微量注射器和立体定向仪将Hoechst33342标记的HACs 10μl分别移植于挫伤灶中心和挫伤灶边缘；在TBI后的28d内采用钉板平衡木行走测试大鼠运动功能变化，运动功能检测结束后取出脑组织行组织学检测。结果 治疗组滑落脚步数明显少于对照组（P〈0．05）；移植的HACs呈蓝色荧光；部分移植HACs可见MAP-2阳性表达。结论 移植HACs使大鼠TBI后运动功能明显改善。     

人羊膜细胞移植治疗大鼠脊髓损伤的实验研究

人羊膜细胞（HEACs）源自于受精卵第八天时成羊膜细胞，具有细胞来源充足，不表达HLA抗原，移植不引发免疫排斥的优点。另外羊膜细胞具有表达神经细胞和神经胶质细胞标志物的细胞以及其能分泌神经递质和营养因子的特性更利于神经移植。因此作者采用移植人羊膜细胞来治疗大鼠脊髓损伤，观察其是否对改善功能确实有益。     

人羊膜上皮细胞在体内外不同脑损伤环境中向神经元样细胞转化

移植细胞的存活及分化与局部的微环境关系密切。实验发现,RPMI 1640培养基与创伤性脑组织提取液构成的体外模拟创伤性脑损伤微环境下培养的人羊膜上皮细胞部分呈现锥形、三角形或不规则形,伸出类似神经元样的突起, 部分细胞突起相互交织相连,表现出神经元样形态,部分形态改变明显的人羊膜上皮细胞呈微管相关蛋白2阳性表达。人羊膜上皮细胞移植于创伤性脑损伤大鼠脑挫伤灶中心和边缘处后,可存活至少4周,且也呈微管相关蛋白2阳性表达,并使创伤性脑损伤大鼠后肢运动功能明显改善。     

An experimental study on intracerebroventricular transplantation of human amniotic epithelial cells in a rat model of Parkinson's disease

Abstract

Objectives: Human amniotic epithelial (HAE) cells are formed from amnioblasts, separated from the epiblast at about the eighth day after fertilization. In the present study, we attempt to investigate the effects of intracerebroventricular transplantation of HAE cells on Parkinson's disease (PD) rats.

Methods: A PD rat model was induced by 6-OHDA injections. Then the rats were transplanted intracerebroventricularly with HAE cells. Apomorphin-induced turns were used to assess the neurobehavioral deficit in rats. Immunofluorescence cytochemistry was used to track the survival of HAE cells. Tyrosinehydroxylase (TH) immunohistochemistry was used to determine the density of TH-positive cells in rat substantia nigra and the differentiation of HAE cells. High performance liquid chromatography (HPLC) was used to measure the levels of dopamine (DA) and its metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum. HVA levels in the cerebrospinal fluid of rats were also determined by HPLC.

Results: The results showed that transplanted HAE cells can survive for at least 10 weeks and differentiate into TH-positive cells in PD rats. The grafts significantly ameliorated apomorphine-induced turns in PD rats. TH immunohistochemistry showed that HAE cells attenuated the loss of TH-positive cells in rat substantia nigra. In addition, HAE cells prevented the fall of DA and its metabolites DOPAC and HVA in PD rats. Increased HVA levels in the cerebrospinal fluid of PD rats were also observed.

Conclusion: These results demonstrate that HAE cells have beneficial effect on 6-OHDA-induced PD rats, which may be due to the neurotrophic factors secrete by HAE cells.     

Monoamine alterations and rotational asymmetry in a rat model of Parkinson's disease following lateral ventricle transplantation of human amniotic epithelial cells

BACKGROUND： Human amniotic epithelial cells （HAECs） can differentiate into neurons, astrocytes and oligodendrocytes. They biologically secrete many active neurotrophins and have the capacity to metabolize dopamine enzymes. These features underlie a theoretical basis for the treatment of Parkinson＇s disease （PD）. OBJECTIVE： To investigate the survival and differentiation of transplanted HAECs in the lateral ventricle of PD model rats, and to explore its effect on circling behavior, as well as levels of dopamine （DA）, the metabolite homovanillic acid, dihydroxyphenyl acetic acid, 5-hydroxyindoleacetic acid, and 5-hydroxytryptamine in the striatum. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, and Shanghai Celstar Institute of Biotechnology from May 2007 to December 2008. MATERIALS： HAECs were derived from the placental chorion following caesarean delivery at the Shanghai International Matemal and Child Health Hospital. 6-hydroxydopamine （6-OHDA）, and mouse anti-human Vimentin monoclonal antibody were purchased from Sigma, USA; mouse anti-human nestin and tyrosine hydroxylase （TH） monoclonal antibodies were purchased from Chemicon, USA. METHODS： A total of 114 healthy, adult, Sprague Dawley rats were randomly assigned to two groups： PD model [n = 90, stereotactic microinjection of 2 μL 6-OHDA （3.5 μg/uL） into the striatum] and control （n = 24, no treatment）. The 51 successful PD model rats were randomly divided into 3 subgroups （n = 17）： HAEC, PBS, and model. The HAEC and PBS groups were respectively injected with 10 μL PBS solution containing 1 × 10^5/mL HAECs or 10 pL PBS into the lateral ventricle. The model group was not treated. MAIN OUTCOME MEASURES： TH protein expression in the striatum was evaluated by immunohistochemistry 5 weeks after HAEC transplantation. At 10 weeks, HAEC survival in the lateral ventricle was investigated by immunofluorescent staining; differentiation of HAECs in the lateral and third ventricles was examined by TH immunohistochemistry; concentrations of DA, homovanillic acid, dihydroxyphenyl acetic acid, 5-hydroxyindoleacetic acid, and 5-hydroxytryptamine in the striatum, as well as DA concentration in the cerebrospinal fluid, were measured with high-performance liquid chromatography-electrochemical detection. Circling behavior of PD model rats was consecutively observed for 10 weeks following intraperitoneal injection of amphetamine 1 week after successful model establishment. RESULTS： tn the HAEC group, the number of TH-positive cells significantly increased in the striatum, and circling behavior significantly decreased, compared with the PBS and model groups （P 〈 0.01）. In addition, monoamine concentrations in the striatum, as well as DA concentrations in the cerebrospinal fluid, significantly increased, compared with the PBS group （P 〈 0.05-0.01）. Moreover, a large number of nestin-, vimentin-, and TH-positive cells were observed in the lateral and third ventricles following HAEC injection.CONCLUSION： HAECs survived for 10 weeks with no overgrowth following transplantation into the lateral ventricle of PD model rats. Moreover, the cells differentiated into dopaminergic neurons, which increased DA secretion. HAEC transplantation improved cycling behavior in PD model rats.     

人羊膜上皮细胞分化为成骨细胞的特性

背景：研究发现人羊膜上皮细胞系WISH和FC植入裸鼠体内均可分化成软骨和成骨，作者检索关于足月分娩人胎膜的羊膜上皮细胞是否具有成骨特性国内外报道少见。 目的：以足月分娩胎膜的人羊膜上皮细胞为前体细胞，观察其在体外定向诱导条件下分化为成骨细胞的能力。 设计、时间及地点：细胞分子水平检测，于2005-09/2006-12在贵州省细胞工程重点实验室完成。 材料：经产妇知情同意，无菌采集健康足月剖宫产胎盘6份，实验经医院医学伦理委员会批准。 方法：采用机械法剥离胎盘羊膜组织，用胰蛋白酶消化法分离人羊膜上皮细胞，按3×108 L-1密度接种进行原代培养。取第1代人羊膜上皮细胞，设立两组：诱导组以1×108 L-1密度接种于培养皿内，24 h后更换为含体积分数为0.1的胎牛血清、 100 nmol/L地塞米松、50 mg/L抗坏血酸、5 mmol/Lβ-甘油磷酸的诱导培养液。对照组换液成分为仅含体积分数为0.1的胎牛血清的LG-DMEM培养液。 主要观察指标：原代细胞用流式细胞仪分析其表型，免疫细胞化学法检测细胞角蛋白19的表达。诱导后采用钙-钴法检测成骨细胞特异性碱性磷酸酶的表达，茜素红S检测钙盐沉积情况。 结果：人羊膜上皮细胞表达间充质干细胞表面标志CD29、CD44和上皮细胞标志细胞角蛋白19。向成骨细胞诱导分化21 d后，人羊膜上皮细胞由圆形变成梭形、三角形，细胞胞浆内碱性磷酸酶呈阳性表达，且可见钙盐沉积。 结论：源自足月分娩胎盘的人羊膜上皮细胞具有分化为成骨细胞的特性。     

人羊膜上皮细胞的干细胞特性***☆

背景：研究发现人羊膜上皮细胞具有类似胚胎干细胞或多能干细胞的多向分化潜能,说明其可能是未来组织工程重建的一种新型种子细胞。 目的：研究体外培养的人羊膜上皮细胞的干细胞特性。 方法：取足月剖宫产的人羊膜组织,经酶消化法和差异黏附法获得纯度高的人羊膜上皮细胞,接种于含体积分数为10%胎牛血清的DMEM/F12培养基中进行原代和传代培养,用免疫荧光法和流式细胞仪检测法检测人羊膜上皮细胞表面胚胎干细胞的表面标记蛋白OCT-4和干细胞表面分子标记CD29、CD34、CD44、CD45、CD105的表达。 结果与结论：人羊膜上皮细胞在体外培养条件下呈上皮细胞特有的铺路石样外观,其胞浆中有OCT-4免疫荧光表达,其干细胞标记分子CD29、CD34的表达是阳性,但干细胞标记分子CD44、CD45、CD105的表达为阴性。结果表明人羊膜上皮细胞具有干细胞的某些特性,其可能是未来组织工程重建的一种新型种子细胞。     

人羊膜上皮细胞有向心肌样细胞分化的特性*★

目的：通过体外诱导分化实验，评价人羊膜上皮细胞向心肌样细胞分化的能力。 方法：实验于2005-09/2006-12在贵州省细胞工程重点实验室完成。①对象：经产妇知情同意，无菌采集健康足月剖宫产胎盘6份，实验经医院医学伦理委员会批准。②实验方法：采用机械法将羊膜从胎盘组织上剥离，D-Hank’s液冲洗后剪成碎片，加入含有0.2 g/L乙二胺四乙酸的0.5 g/L胰蛋白酶溶液，离心、消化、过滤，收集滤液，加入胎牛血清终止消化，重复消化2次。合并3次消化所得的细胞悬液，离心后将细胞沉淀悬浮于L-DMEM培养基中，按1.25×108 L-1密度接种，常规培养3 d后更换培养基，待细胞达80%~90%融合后用胰蛋白酶+乙二胺四乙酸联合消化，终止后离心弃上清，细胞沉淀用培养基重新悬浮，按1×107 L-1密度传代。③实验评估：用流式细胞仪鉴定人羊膜上皮细胞表型；使用10 μmol/L 5-氮杂胞苷和1 mmol/L抗坏血酸磷酸盐诱导第2代人羊膜上皮细胞，免疫荧光染色法检测诱导后细胞中特异蛋白结蛋白和 α-辅肌动蛋白的表达，RT-PCR检测心肌特异性转录因子Nkx2.5、GATA-4和心肌特异性收缩蛋白α-肌球蛋白重链mRNA的表达。 结果：①人羊膜上皮细胞的免疫组化特征：人羊膜上皮细胞几乎不表达CD44，不表达波形蛋白，表达角蛋白19。②人羊膜上皮细胞α-辅肌动蛋白和结蛋白的表达：人羊膜上皮细胞经诱导分化后，表达肌系细胞标志α-辅肌动蛋白和结蛋白。③人羊膜上皮细胞Nkx2.5、GATA-4和α-肌球蛋白重链mRNA的表达：人羊膜上皮细胞经诱导分化后，表达心肌特异性转录因子Nkx2.5 mRNA和GATA-4 mRNA，心肌特异的可收缩蛋白α-肌球蛋白重链mRNA未见表达。 结论：通过胰蛋白酶消化法分离获得的人羊膜上皮细胞具有向心肌样细胞分化的特性，可能成为细胞心肌成形术的候选供体细胞。     

Neurotrophic function of conditioned medium from human amniotic epithelial cells

Human amniotic epithelial cells (HAEC) may have pluripotent function because they are formed from the epiblast cells at the 8th day of fertilization. Previously, we reported that HAEC have the capacity to synthesize and release acetylcholine and catecholamine associated with the binding sites of catecholamine receptors. We show the neurotrophic function of a conditioned medium from HAEC using cultured cortical neurons of E18 rats. Extensive analyses with various techniques demonstrated that HAEC and immortalized HAEC synthesize and release brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF). Other neurotrophic factors were not detected in a cultured medium of HAEC by enzyme immunoassay. Various neurotrophic factors or growth factors did not show neurotrophic effects on E18 rat neuron except for EGF. Because EGF was not detected in the conditioned medium of HAEC, these data indicate an unidentified neurotrophic factor presently that is synthesized and released from HAEC. The amniotic membrane may have a significant role in supplying neurotrophic factors to the amniotic fluid as well as neurotransmitters, suggesting an important function to the early stages of neural development in the embryo.     

FGF-2 in the MPTP model of parkinson's disease: Effects on astroglial cells

Fibroblast growth factor (FGF) is synthesized and stored by astroglial cells and regulates their proliferation and differentiation in vitro. Its implication in the transformation of quiescent astrocytes into reactive astroglia has been discussed. Using a mouse model of Parkinson's disease, in which FGF-2 has been shown to exert marked neuroprotection of nigrostriatal dopaminergic neurons, we have studied striatal levels of glial fibrillary acidic protein (GFAP), an established marker for astrocytes, and the distribution and morphologies of GFAP-immunoreactive cells following treatments with the neurotoxic drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the growth factor FGF-2, and the non-trophic control protein cytochrome C (cyt C). Systemic injections of MPTP (30 mg/kg) on 3 consecutive days, which we have previously shown to cause profound and long-lasting damage to the nigrostriatal system, induced an approximate 20% transient increase in striatal GFAP, determined by enzyme-linked immunosorbent assay (ELISA), 1 day after the final MPTP injection (= day 4), with subsequent normalization at day 7, which lasted until the end of the experiment (day 18). Morphologically, MPTP elicited a marked increase in number, size, arborization, and stainability of GFAP-immunoreactive cells at day 4 in a striatal area adjacent to the corpus callosum, which was evaluated throughout all experiments. Even on day 18, astrocytes were still apparently larger and more branched than in unlesioned controls. Administration of 4 μg of either FGF-2 or cyt C (soaked into a piece of Gelfoam unilaterally to the right striatum in either MPTP- or saline-injected controls) increased striatal GFAP levels bilaterally about 2- to 2.5-fold at 14 days, when FGF-2 showed marked protection of dopaminergic parameters. Likewise, GFAP immunocytochemistry revealed increased numbers of intensely immunoreactive astrocytes under any experimental situation. Differences in the morphologies of astrocytes in FGF-2- and cyt C-treated animals were very subtle and only noted at greater distances away from the site of application of the factors. We conclude that FGF-2, a potent neurotrophic factor for the neurotoxically lesioned nigrostriatal system, does not cause a marked astrogliotic reaction, which might be expected from previous in vitro and in vivo studies in other neural systems. This may limit concerns regarding potential applicability of FGF-2 to the parkinsonian striatum. © 1994 Wiley-Liss, Inc.