帕金森病模型大鼠纹状体肾上腺髓质移植后突触结构的观察

目的 :对移植到帕金森病模型大鼠纹状体内的嗜铬细胞与脑细胞之间形成的突触结构进行观察。方法 :实验用Wistar大鼠 4 0只 ,注射 6羟多巴胺 ( 6 -OHDA)破坏大鼠一侧黑质纹状体系统 ,制成实验性帕金森病 (PD)动物模型。将肾上腺髓质植入PD模型大鼠纹状体内 ,移植后定期检测大鼠阿朴吗啡诱发旋转行为 ,并用电镜观察嗜铬细胞与脑细胞之间形成的突触结构。结果 :移植组大鼠的阿朴吗啡诱发旋转行为明显改善 (P <0 0 5)。电镜观察发现嗜铬细胞与脑细胞之间形成突触结构 ,以嗜铬细胞为突触前膜的占 72 88% ;以嗜铬细胞为突触后膜的占 2 7 12 %。结论 :移植肾上腺髓质后 ,模型大鼠阿朴吗啡诱发旋转行为明显改善 ,嗜铬细胞与宿主脑细胞之间形成突触联系 ,并以嗜铬细胞为突触前膜的为多。     

熄风静宁冲剂抗大鼠实验性Tourette综合征

目的观察熄风静宁冲剂对大鼠Tourette综合征模型和纹状体内多巴胺(DA)和高香草酸(HVA)含量的影响.方法 Tourette综合征模型用腹腔注射阿朴吗啡(APO)和局部注射6羟基多巴胺(6-OHDA)损毁黑质的方法制造.DA和HVA用HPLC法测定.结果熄风静宁冲剂能对抗APO引起的定型活动和Tourette综合征模型大鼠的旋转行为(P＜0.01),且能提高纹状体内HVA含量(P＜0.01),作用与氟哌啶醇一致.结论熄风静宁冲剂能减轻Tourette综合征动物模型的症状,其作用机理可能与脑内DA神经有关.     

移植诱导中脑NSCs分化的多巴胺能神经元对PD大鼠治疗作用的实验研究

目的研究移植诱导中脑神经干细胞分化出的多巴胺能神经元对PD大鼠的治疗作用.方法利用纹状体提取液诱导大鼠胚胎中脑神经干细胞,使其部分分化为酪氨酸羟化酶(TH)阳性的多巴胺能神经元,继而把分化后的细胞移植到PD大鼠的纹状体内,免疫组化法证实移植细胞的存活并以诱发旋转行为的改善来观察其治疗作用.结果纹状体提取液能够诱导中脑神经干细胞分化出多巴胺能神经元而且这种经诱导后产生的细胞能够在PD大鼠体内长期存活并改善阿卟吗啡诱导的旋转行为.结论诱导中脑神经干细胞分化出的多巴胺能神经元对PD有一定的治疗作用.     

酪氨酸羟化酶基因修饰的神经干细胞脑内移植PD模型动物旋转行为及神经生化研究

目的 探讨TH基因修饰的神经干细胞脑内移植对PD模型动物旋转行为和神经生化的影响。方法 构建pN2ATH逆转录病毒载体质粒，用PA317细胞包装，G418筛选阳性克隆，病毒上清感染神经干细胞，将神经干细胞和表达TH的神经干细胞植入PD大鼠纹状体内，测定移植后不同时间PD大鼠旋转行为改善以及DA和DOPAC含量变化。结果 TH基因修饰的神经干细胞移植8周后能显著降低PD大鼠旋转行为，增加纹状体DA和DOPAC含量，疗效好于单纯神经干细胞移植组和对照组。结论 TH基因修饰的神经干细胞脑内移植能增加纹状体DA和DOPAC含量，降低PD大鼠旋转行为，从而对PD大鼠有明显的治疗作用，也为PD的治疗开辟了新途径。     

损毁帕金森病大鼠腹侧苍白球对纹状体内谷氨酸含量的影响

目的:观察腹侧苍白球(VP)损毁后帕金森病(PD)大鼠纹状体内谷氨酸(Glu)含量的变化. 方法:应用6-羟基多巴胺(6-OHDA)制备偏侧PD大鼠模型,插入电极直流电毁损VP,观察毁损后PD大鼠的旋转行为,应用高效液相色谱检测纹状体内Glu的含量. 结果:VP损毁术后PD大鼠偏侧旋转行为明显改善,纹状体内Glu的含量(μg/g) (3.0±0.4)比正常对照组(2.2±0.7) 、模型对照组(1.3±0.2)和假手术组(1.4±0.4)都要高(P<0.05),在术后第2周时达高峰,以后逐渐下降,但仍高于其他三组. 结论:VP毁损可能会减少Glu的释放或与其受体的结合,从而对神经元的毒性作用减弱,有助于PD症状的改善.     

牛磺酸抑制帕金森病模型大鼠的旋转行为及其可能机制

探讨牛磺酸对帕金森病模型大鼠由阿朴吗啡(APO)诱发的向健侧的旋转行为的影响。应用6-羟基多巴胺(6-OHDA)制备偏侧损毁的帕金森病模型大鼠，侧脑室(icv)微量注射不同浓度的牛磺酸，1w后在自动旋转测试仪中测试由APO诱发的旋转行为，并通过高效液相色谱电化学检测(HPLC-ECD)检测纹状体中多巴胺(DA)及其代谢产物的含量。     

帕金森病大鼠中脑腹侧被盖区多巴胺能神经元的改北

目的：探讨帕金森病（PD）大鼠中脑腹侧被盖区（VTA）多巴胺（DA）能神经元的改变.方法：20只3月龄Wistar雄性大鼠,随机分为模型组和对照组,每组10只.6-羟基多巴胺（6-OHDA）注射右侧黑质致密区（SNC）制作PD大鼠模型,腹腔注射阿朴吗啡后进行行为学观察;尼氏染色观察左侧中脑VTA神经元、酪氨酸羟化酶（TH）的改变;免疫组织化学ABC法观察其DA能神经元的改变并进行图像分析.结果：阿朴吗啡诱发PD大鼠模型异常旋转行为,尼氏染色见PD大鼠左侧中脑VTA有神经细胞肿胀、坏死等变化;VTA TH阳性神经元形态学改变,数量减少,与对照组相比,差异有统计学意义.结论：中脑VTA DA能神经参与PD模型大鼠的改变.     

酪氨酸羟化酶基因修饰的神经干细胞脑内移植PD模型动物旋转行为及神经生化研究

目的探讨TH基因修饰的神经干细胞脑内移植对PD模型动物旋转行为和神经生化的影响.方法构建pN2ATH逆转录病毒载体质粒,用PA317细胞包装,G418筛选阳性克隆,病毒上清感染神经干细胞,将神经干细胞和表达TH的神经干细胞植入PD大鼠纹状体内,测定移植后不同时间PD大鼠旋转行为改善以及DA和DOPAC含量变化.结果 TH基因修饰的神经干细胞移植8周后能显著降低PD大鼠旋转行为,增加纹状体DA和DOPAC含量,疗效好于单纯神经干细胞移植组和对照组.结论 TH基因修饰的神经干细胞脑内移植能增加纹状体DA和DOPAC含量,降低PD大鼠旋转行为,从而对PD大鼠有明显的治疗作用,也为PD的治疗开辟了新途径.     

电针及针药结合对PD大鼠行为学及DA含量的影响

[目的]观察电针及针药结合对帕金森病大鼠的行为学指标的影响,及其对纹状体内多巴胺含量的影响。[方法]纹状体内注入6-OHDA制作PD大鼠模型。将大鼠随机分为假手术组、手术组,将造模成功的手术组大鼠随机分为电针治疗组、美多巴治疗组、针药结合组、模型对照组。检测各组实验大鼠的旋转行为,并通过高效液相电化学法测定各组实验大鼠纹状体内多巴胺含量。[结果]对各组实验大鼠行为学检测及脑纹状体内多巴胺的含量检测结果显示,与模型对照组及假手术组比较,各治疗组差异显著(P0.05);各治疗组间比较无差异。[结论]说明电针疗法、、针药结合疗法有与口服美多巴相似的疗效,可以有效提高纹状体内DA的含量,改善帕金森病大鼠的旋转行为。     

早期帕金森病大鼠模型的建立

目的 探索建立早期帕金森病（Parkinson disease,PD）大鼠模型.方法 成年大鼠单侧纹状体内注射6-羟多巴胺（6-hydroxydopamine,6-OHDA）.注射后第7天,通过姿势不对称性、前肢始动不能、前肢使用不对称性、阿朴吗啡诱发旋转实验进行行为学评估.观察组织学变化,取鼠脑黑质节段（n=6）,固定、石蜡包埋、连续冠状切片.焦油紫（Nissl）染色显示黑质神经细胞;抗酪氨酸羟化酶（tyrosine hydroxylase,TH）抗体免疫组织化学染色显示黑质多巴胺（DA）能神经元,光镜下观察并进行细胞计数,统计学分析处理数据.而且,使另一部分模型动物（n=6）继续存活到第28天,鉴定所选动物模型的可靠性.结果 纹状体内注射6-OHDA后第7天,动物行为学出现有意义改变,黑质内神经细胞出现肿胀等变性坏死的早期形态变化,DA能神经元出现有意义减少;注射后的第28天,行为学改变更加显著,神经细胞大量减少,DA能神经元数量减少到92%.结论 纹状体内注射6-OHDA后第7天,通过行为学方法能确定早期PD动物模型.     

Experimental Study on Heterograft of Glomus Cells of Carotid Body for Hemiparkinsonian Rats

Parkinson disease(PD) is a neuro- degenerativedisorder commonly found in middle- aged or olderpeople,which ischaracterized by the loss ofmelanin-containing neurons in the substantia nigra pars com-pacta(SNpc) and a reduction in dopamine in stria-tum.After several years of treatment by dopamineprecursor,L- dihydroxyphenylalanine (L - DOPA ) ,and due to the reduced effectof L- DOPA and dyski-nesia,some PD patients need to be switched to othertherapeutic approaches such as neural transplant…     

Experimental Study on Heterograft of Glomus Ccl ls of Carotid Body for Hemioarkinsonian Rats

Summary: To observe the effects of heterograft of glomus cells of carotid body on hemiparkinsonian rat models, rats with unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the right dopamin ergic neurons of substantia nigra received intrastriatal glomus cells heterograft. Apomorphine-induced rotation was monitored for 30 rmin at various time points after grafting. The striata were cut and ex-amined for dopamine content by HPLC and for immunohistochemical staining of tyrosine hydroxylase positive neurons (TH ) at the end of the experiments. The results showed that apomorphine-induced rotational behavior was significantly reduced for 12 weeks and the dopamine contents were signifi cantly elevated after grafting (P＜0.01), and TH cells survived better. The present study demon strates that intrastriatal heterograft of glomus cells within carotid body in rats with 6-OHDA-elicited lesions could reduce apomorphine-induced rotational behavior and elevate the dopamine contents and numbers of TH cell surviving within striatum, and can serve as a new and effective alternative for Parkinson disease.     

Dynamic expression of bFGF and TGFbeta2 in glomus cell grafts of carotid body in rat model of Parkinson disease.

To investigate the changes in the expression of basic fibroblast growth factor (bFGF) and transforming growth factor beta 2 (TGFbeta2) in glomus cell grafts of carotid body in the rat model of 6-hydroxydopamine-induced Parkinson disease, immunohistochemical staining of bFGF and TGFbeta2 in the sections of striate body was done on the 2nd, 4th and 12th week after transplantation. The results showed that on the 2nd week after transplantation, bFGF and TGFbeta2 were not detectable in the glumous cell grafts. On the 4th week after graft, bFGF and TGFbeta2 immunoreactivity was increased within the grafts and at the graft-host interface but was restricted only to astrocytes. In the striatum surrounding the graft, bFGF was expressed persistently, while TGFbeta2 showed transient expression. It was suggested that the transient expression of TGFbeta2 was likely due more to the trauma imposed by the graft procedure than to an intrinsic. The deficiency in astrocytic bFGF early after graft may be responsible for the poor survival of grafted glomus cells of carotid body.     

Therapeutic effect of human amniotic epithelial cell transplantation into the lateral ventricle of hemiparkinsonian rats

Background Human amniotic epithelial cells （HAECs） are able to secrete biologically active neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3, both of which exhibit trophic activities on dopamine neurons. Previous study showed that when human amniotic epithelial cells were transplanted into the striatum of 6-hydroxydopamine （6-OHDA）-induced Parkinson disease rats, the cells could survive and exert functional effects. The purpose of this study was to investigate the survival and the differentiation of human amniotic epithelial cells after being transplanted into the lateral ventricle of Parkinson＇s disease （PD） rats, and to investigate the effects of grafts on healing PD in models. Methods The Parkinson＇s model was made with stereotactic microinjection of 6-hydroxydopamine （6-OHDA） into the striatum of a rat. The PD models were divided into two groups： the HAECs group and the normal saline （NS） group. Some untreated rats were taken as the control. The rotational asymmetry induced by apomorphine of the HAECs group and the NS group were measured post cell transplantation. The expression of nestin and vimentin in grafts were determined by immunohistology. Ten weeks after transplantation the density of tyrosine hydroxylase positive cells in the substantia nigra of the HAECs group, NS group and the untreated group was determined. The differentiation of grafts was determined by TH immunohistology. High performance liquid chromatography （HPLC） was used to determine monoamine neurotransmitter levels in the striatum. Results The rotational asymmetry induced by apomorphine of the HAECs group was ameliorated significantly compared to the NS group two weeks after transplantation （P 〈0.01）. The grafts expressed nestin and vimentin five weeks after transplantation. TH immunohistochemistry indicated that the TH positive cells in the substantia nigra of the HAECs group increased significantly compared to the NS group （P 〈0.01）. Tyrosine hydroxylase （TH） positive cells in the substantia nigra of the HAEC group and the NS group were decreased compared to the untreated group （P 〈0.01）. Dopamine and DOPAC levels in the striatum of the HAECs group increased significantly compared to the NS group （P 〈0.05）. Homovanillic acid （HVA） levels in the striatum of the HAECs group increased significantly compared to the NS group （P 〈0.01）. In addition dopamine, DOPAC, and HVA levels in the striatum and dopamine levels in the cerebrospinal fluid of the HAECs group and the NS group were decreased compared to the untreated group （P 〈0.05）. Conclusions Human amniotic epithelial cells could be used to ameliorate the rotational asymmetry induced by apomorphine of the PD models. This could have been due to the increased content of dopamine and its metabolic products, DOPAC and HVA, in the striatum in the PD models.     

Dynamic expression of bFGF and TGFβ2 in glomus cell grafts of carotid body in rat model of parkinson disease

Summary To investigate the changes in the expression of basic fibroblast growth factor (bFGF) and transforming growth factor beta 2 (TGF32) in glomus cell grafts of carotid body in the rat model of 6-hydroxydopamine-induced Parkinson disease, immunohistochemical staining of bFGF and TGFβ2 in the sections of striate body was done on the 2nd, 4th and 12th week after transplantation. The results showed that on the 2nd week after transplantation, bFGF annd TGFβ2 were not detectable in the glumous cell grafts. On the 4th week after graft, bFGF and TGFβ2 immunoreactivity was increased within the grafts and at the graft-host interface but was restricted only to astrocytes. In the striatum surrounding the graft, bFGF was expressed persistently, while TGFβ2 showed transient expression. It was suggested that the transient expression of TGFβ2 was likely due more to the trauma imposed by the graft procedure than to an intrinsic. The deficiency in astrocytic bFGF early after graft may be responsible for the poor survival of grafted glomus cells of carotid body. CAO Xuebing, male, born in 1968, Associate Professor This project was supported by a grant from the foundation of Youth Sciences and Technology Chengguang Planning of Wuhan (No. 20015105047).     

神经干细胞移植对帕金森病鼠旋转行为的影响

目的 :观察神经干细胞定向移植后对帕金森病 (Parkinsondisease,PD)鼠的旋转行为的影响。方法 :建立PD模型大鼠以及体外培养神经干细胞 ,然后将神经干细胞悬液立体定向移植到PD模型鼠黑质纹状体区 ,测定PD模型鼠旋转行为的变化 ,并与对照组相比。结果 :神经干细胞移植后PD模型鼠旋转行为比对照组明显减少。结论 :神经干细胞移植能减轻 6 羟基多巴胺对黑质纹状体多巴胺能神经元的损伤。     

多巴胺合成酶相关基因治疗帕金森病的实验研究

目的探讨重组多巴胺合成酶基因在体内外的表达、生成蛋白的活性及对帕金森病模型大鼠的治疗作用。方法在腺相关病毒介导下,将多巴胺合成酶相关基因酪氨酸羟化酶(TH)基因、芳香族氨基酸脱羧酶(AADC)基因与GTP环化水解酶Ⅰ(GCH-Ⅰ)基因导入COS7细胞,分别通过原位杂交与免疫组化的方法检测目的基因的转录与表达,通过高压液相分析检测酶活性。将携带目的基因的运载细胞移植到帕金森病模型大鼠纹状体,每周进行诱发旋转行为的检测。10周后高压液相分析脑内多巴胺含量,以进一步评估复合基因的治疗效果。结果重组多巴胺合成酶基因在体外可以表达并被正确修饰,具有联合酶促催化功能。当把表达TH、AADC与GCH-Ⅰ基因的细胞共培养时,在底物左旋酪氨酸存在的情况下,可以生成大量多巴胺。而且,把携带治疗基因的细胞移植到大脑后,经检测三基因移植组脑内多巴胺的生成量较单基因移植组多,且前者与后者相比,其行为学改善更为明显。但是三基因移植组与双基因组相比,脑内多巴胺生成量差异无显著意义。结论在帕金森病基因治疗策略中,应根据多巴胺能神经元的损伤程度来选择加入基因的组合量。复合基因治疗的效果明显好于单基因。     

大鼠胚肾组织促帕金森病移植神经元存活的实验研究

目的：探讨大鼠胚肾组织对帕金森病移植神经元的促存活作用。方法：用神经毒剂6-OHDA单侧损毁SD大鼠左侧中脑被盖腹侧区(VTA)和黑质致密部(SNC)建立动物模型：实验分3组：A组将胚鼠腹侧中脑组织(VM)预置于胚肾组织培养液中：B组将VM组织预置于胶质细胞源性神经营养因子(GDNF)营养液中；C组将VM组织预置于DMEM营养液中。6h后移植，于移植后1、4、7d将动物处死，采用原位末端标记法(TUNEL)结合流式细胞术及免疫组织化学方法对3组移植神经元；凋亡和存活情况进行定量分析。结果：根据TUNEL、酪氨酸羟化酶(TH)染色和流式细胞仪检测结果，A、B移植组较C移植组移植神经元凋亡率降低，凋亡细胞数减少，纹状体移植针道和针道周围都可见大量TH阳性细胞，而且TH阳性神经元数量明显增多，另外，A移植组和B移植组TH阳性神经元和凋亡细胞数比较也存在统计学差异。结论：胚肾组织分泌物对帕金森病移植神经元具有促存活作用；胚肾组织培养液较单一生长因子作用好。     

偏侧帕金森病猴模型的评价

目的通过测定行为、纹状体多巴胺转蛋白、多巴胺及黑质纹状体多巴胺能神经元评价应用ＭＰＴＰ制备的偏侧帕金森病猴模型。方法经右侧颈总动脉注射ＭＰＴＰ制备偏侧帕金森病猴模型,应用９９ｍＴｃ－ＴＲＯＤＡＴ－１ＳＰＥＣＴ显像观察纹状体多巴胺转运蛋白功能;采用微透析结合高效液相一电化学法检测纹状体细胞外液多巴胺（ＤＡ）及其代谢产物（ＤＯＰＡＣ、ＨＶＡ）含量;免疫组化法观察黑质纹状体酪氨酸羟化酶（ＴＨ）阳性神经元及纤维。结果ＭＰＴＰ注射侧多巴胺转运蛋白密度低于未注射侧,左右侧纹状体感兴趣区的比值为１．１０～１．１８。与未注射侧相比,ＭＰＴＰ注射侧纹状体细胞外液ＤＡ、ＤＯＰＡＣ、ＨＶＡ分别降低７０％、３４％和３８％,黑质和纹状体ＴＨ阳性细胞及纤维为对侧的１０％～１５％。结论应用ＭＰＴＰ制备的偏侧帕金森病猴模型与人类早期帕金森病有一定的相似性。微透析和ＳＰＥＣＴ在体检测多巴胺转运蛋白和多巴胺是研究帕金森病的有效手段。