TH基因修饰的神经干细胞移植治疗帕金森病的实验研究

目的 探讨TH基因修饰的神经干细胞脑内移植对帕金森病(PD)的治疗作用。方法 构建pN：ATH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，将表达TH的神经干细胞植入：PD大鼠纹状体内，测定：PD大鼠旋转行为改善，DA和DOPAC含量变化，以及TH在纹状体的表达。结果 TH基因修饰的神经干细胞移植8周时能显著降低PD大鼠旋转行为，增加纹状体DA和DOPAC含量，TH在纹状体内的表达增加，疗效好于单纯神经干细胞移植组。结论 TH基因修饰的神经干细胞移植对PD大鼠有明显的治疗作用，可望为PD治疗提供新的途径。     

基因修饰的神经干细胞脑内移植后TH表达和神经递质的变化

目的：探讨经TH基因修饰的神经干细胞脑内移植后，PD模型动物脑内TH表达和神经递质的变化。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，观察移植后TH的表达情况以及DA和DOPAC含量变化。结果：TH基因修饰的神经干细胞移植2月内，TH在纹状体内的表达增加，纹状体DA和DOPAC含量增高，好于单纯神经干细胞移植组和对照组，但比正常水平低，结论：TH基因修饰的神经干细胞，脑内移植能增加纹状体内TH的表达以及DA和DOPAC含量，为其脑内移植治疗PD提供了理论基础。     

转基因星形胶质细胞脑内移植治疗帕金森病大鼠的研究

目的　评价酪氨酸羟化酶 (TH)基因转染的星形胶质细胞移植入帕金森病 (PD)模型大鼠脑内后对旋转行为改善作用。方法　用pcDNA3 1/TH质粒转染原代培养的星形胶质细胞 ,采用免疫组化及RT PCR方法检测到TH表达后 ,将转基因的星形胶质细胞移植入PD模型大鼠脑纹状体内 ,观察PD大鼠的旋转行为变化情况。结果　移植后共观察 12周 ,转基因细胞移植组PD大鼠 (n =10 )的旋转行为明显改善 (P <0 0 5 ) ,改善情况在 2～ 8周最显著 ,对照组大鼠 (n =10 )的旋转行为无变化。结论　转基因的星形胶质细胞脑内移植后可短期改善PD大鼠的旋转行为 ,星形胶质细胞有可能作为有效的载体细胞。     

酪氨酸羟化酶cDNA移植治疗帕金森病的实验研究

帕金森病(PD)的主要病因是黑质多巴胺能神经元损伤,表达的酪氨酸羟化酶(TH)减少。本文在应用6-羟多巴胺(6-OHDA)制备偏侧PD大鼠模型的前提下,首次将TH cDNA移植到PD大鼠纹状体内,模型动物的旋转行为明显改善,用免疫组化法及PCR法证实了外源性THcDNA可以进入脑细胞内,并表达出有生物活性的TH。     

微囊化牛视网膜色素上皮细胞移植治疗伴Lewy小体形成帕金森病的实验研究

目的观察微囊化牛视网膜色素上皮细胞(RPE)移植对伴Lewy小体形成帕金森病(PD)大鼠的治疗作用。方法用蛋白酶体选择性抑制剂lactacystin制作伴Lewy小体形成的PD模型;将微囊化牛RPE植入大鼠纹状体,移植分生理盐水对照组(NS)、RPE、空微囊(APA)及微囊化RPE(RPE-APA)四组;观察移植后旋转行为变化、纹状体中多巴胺(DA)和高香草酸(HVA)及酪氨酸羟化酶(TH)含量的变化。结果微囊化RPE的旋转行为在移植后第1周开始改善(改善程度为21.3%,与空微囊组相比P<0.05),第4周改善更加明显(改善程度为57.89%,与第1周相比P<0.05),并一直持续到第14周(改善程度为59.47%,与空微囊组相比P<0.05)。行为学改善的大鼠,纹状体内DA和HVA含量的变化同其行为学改善相符合。行为学有改善大鼠囊内细胞TH染色呈弱阳性,微囊周边的纹状体可见TH阳性纤维密度较空微囊组高。结论微囊化牛RPE脑内移植对伴Lewy小体形成PD大鼠有治疗作用。     

深部脑刺激丘脑底核治疗帕金森病大鼠脑中钙调蛋白的变化

目的 研究慢性高频电刺激帕金森病(Parkinson's disease,PD)大鼠丘脑底核治疗PD的机制.方法 通过6-羟基多巴立体定向注射至大鼠中脑前脑束建立偏侧PD动物模型,对造模成功的PD大鼠模型丘脑底核进行高频电刺激,观察高频电刺激对PD大鼠行为的影响,并用蛋白印迹法检测纹状体酪氨酸羟化酶(TH)、钙调蛋白Calbindin-28和突触前膜囊泡蛋白(synaptophysin)的表达情况.此外,对接受慢性高频电刺激的6-羟基多巴损伤纹状体双侧PD大鼠的脑片进行免疫组化染色,检测黑质Calbindin-28和突触前膜囊泡蛋白的表达情况.结果 (1)高频电刺激偏侧PD大鼠丘脑底核可使阿朴吗啡诱导的旋转行为减少31%;(2)偏侧PD大鼠损伤侧纹状体内已无TH表达,慢性高频电刺激同侧丘脑底核对纹状体TH缺失无逆转,但增加健纹状体内TH表达量78.6%±9.5%;(3)偏侧PD大鼠损伤侧纹状体内Calbindin-28表达量增加75.4%±15.0%,慢性高频电刺激使其表达量减少43.0%±7.1%;(4)慢性高频电刺激亦显著抑制双侧PD大鼠黑质Calbindin-28表达:假手术大鼠、PD大鼠以及慢性高频电刺激后PD大鼠黑质致密部Calbindin-28阳性神经元分别为74.5±10.2、75.7±15.6和33.1±7.8;(5)慢性高频电刺激未影响黑质和纹状体内突触前膜囊泡蛋白的表达.结论 深部脑刺激治疗PD的机制与调节黑质和纹状体内钙调蛋白Calbindin-28和TH表达量有关.     

人胎盘底蜕膜间充质干细胞抗炎特性对帕金森病模型大鼠的神经保护作用

目的 研究人胎盘底蜕膜间充质干细胞(hPDB-MSCs)抗炎特性对帕金森病(PD)模型大鼠多巴胺能神经元的影响. 方法 体外培养hPDB-MSCs,6-羟基多巴(6-OHDA)制备大鼠PD模型并按照随机数字表法分为模型组与移植组,每组32只.hPDB-MSCs尾静脉移植观察各组大鼠行为学改变.免疫组化检测移植后3d、1周、2周及4周各组大鼠损伤侧黑质酪氨酸羟化酶(TH)、离子钙接头蛋白(Ibal)表达.实时荧光定量PCR检测各时间点各组大鼠损伤侧黑质抗炎因子人白细胞介素-10 (hIL-10)、人转化生长因子-β(hTGF-β)及肿瘤坏死因子-α(TNF-α)表达. 结果 hPDB-MSCs移植后1周、2周、4周,移植组大鼠阿朴吗啡诱导的平均旋转圈数明显低于模型组,差异有统计学意义(P＜0.05).免疫组化结果显示移植组大鼠损伤侧黑质部位TH阳性细胞数较模型组明显增加,1周、2周、4周时差异有统计学意义(P＜0.05).移植组大鼠损伤侧黑质部位Ibal阳性细胞则明显减少,4周时最为显著.mRNA水平,移植组大鼠hIL-10及hTGF-β表达均较模型组增加,而TNF-α表达量则逐渐降低. 结论 hPDB-MSCs尾静脉移植后能够通过抗炎机制抑制PD模型大鼠多巴胺能神经元丧失,改善PD模型大鼠症状.     

GFP转基因小鼠神经干细胞移植治疗大鼠帕金森病的实验研究

目的观察GFP(绿色荧光蛋白)转基因小鼠胚胎神经干细胞植入帕金森病大鼠纹状体后的存活、分化情况及治疗作用。方法建立PD模型大鼠及体外培养神经干细胞,然后将GFP转基因小鼠神经干细胞定向植入帕金森病大鼠毁损侧纹状体内,于移植后不同时间诱发旋转行为,并与对照组相比,观察症状的改善,并用酪氨酸羟化酶(TH)免疫组织化学染色方法检测移植GFP转基因小鼠神经干细胞的存活及分化状况。结果 GFP转基因小鼠神经干细胞脑内移植后,帕金森病大鼠的旋转行为明显改善。移植后2至4周时可检测到成片或散在的TH免疫阳性细胞。结论 GFP转基因小鼠神经干细胞移植至帕金森病大鼠纹状体后,可分化为多巴胺能神经元并能改善旋转症状。     

脑缺血条件下骨髓基质细胞分化成少突胶质细胞的研究

目的 观察体外培养的人胚胎骨髓基质细胞(MSCs)移植入全脑缺血大鼠胼胝体内能否定向分化成少突胶质谱系细胞.方法 无菌人胚胎股骨骨髓体外分离培养,应用免疫荧光流式细胞仪鉴定MSCs细胞表型,MSCs用Brdu标记后在立体定向仪下移植入脑缺血大鼠胼胝体内,用免疫荧光双染色及激光共聚焦显微镜观察BrdU-A285、BrdU-O4、BrdU-CNPase双阳性表达情况.结果 传代3次的人胚MSCs CD44、CD29阳性表达细胞占95%以上,而CD34.阳性表达细胞仅0.14%;移植点附近可见BrdU标记的MSCs移植入脑缺血大鼠胼胝体内存活.免疫荧光双染色显示,部分BrdU阳性细胞表达O4,但无BrdU-A285、BrdU-CNPase双阳性细胞.结论 人胚MSCs移植入脑缺血大鼠胼胝体内能部分定向分化成少突胶质谱系细胞,但分化延迟.     

雪旺细胞和神经干细胞共移植治疗大鼠帕金森病的实验研究

目的 研究雪旺细胞（SCs）和神经干细胞（NSCs）共移植治疗大鼠帕金森病（PD）的疗效。方法 18只PD模型的SD大鼠随机平均分成3组：对照组、NSCs组及共移植组。PBS、NSCs及SCs加NSCs分别植入PD大鼠右侧纹状体内。NSCs来源于孕14～16d胎鼠中脑腹侧的脑组织．SCs来源于1～3dSD新生鼠的坐骨神经。结果 移植10周后NSCs组及共移植组中纹状体区都出现TH染色阳性的神经元，与NSCs组相比，共移植组中的神经元体积及细胞核均较大。细胞数量多（P〈0．05）。与对照组相比，NSCs组及共移植组中大鼠的行为学均有明显的好转（P〈0．01），但NSCs组和共移植组相比，行为学的改变无明显差异（P〉0．05）。结论 SCs和NSCs共移植能有效治疗PD模型大鼠。     

Therapeutic effects of intranigral transplantation of mesenchymal stem cells in rat models of Parkinson's disease

Stem cell transplantation is a promising tool for the treatment of neurodegenerative disorders, including Parkinson's disease (PD); however, the therapeutic routes and mechanisms of mechanical approaches to stem cell transplantation must be explored. This study tests the therapeutic effect of transplantation of rat bone marrow mesenchymal stem cells (MSCs) into the substantia nigra (SN) of the PD rat. 5‐Bromo‐2‐deoxyuridine‐labeled rat MSCs were transplanted into the SN of the 6‐hydroxydopamine‐injected side of PD rat brains. The behavioral changes in PD rats were examined before and 4 and 8 weeks after MSC transplantation. The expression of tyrosine hydroxylase (TH) in the SN and the striatum and the survival and differentiation of MSCs were assessed by immunohistochemical and double immunofluorescence techniques. Abnormal behavior of PD rats was significantly improved by the administration of bone marrow MSCs, and the number of TH‐positive cells in the SN and the optical density of TH‐positive fibers in the striatum were markedly increased. Transplanted MSCs can survive and migrate in the brain and differentiate into nestin‐, neuron‐specific enolase‐, and GFAP‐positive cells. Our findings suggest that transplantation of rat bone marrow MSCs into the SN of PD rats may provide therapeutic effects. © 2016 Wiley Periodicals, Inc.     

Therapeutic benefit of TH-engineered mesenchymal stem cells for Parkinson's disease

The present study was designed to assess the potential of marrow stromal cells (MSCs) to deliver therapeutic genes to the brain and result in biologically significant functional recovery. The tyrosine hydroxylase (TH) gene was transfected to MSCs with an adeno-associated virus (AAV) vector. MSCs expressing TH gene were transplanted into the striatum of Parkinson's disease (PD) rat. The asymmetric rotation of these models after apomorphine administration was detected every week after transplantation. Six weeks after grafting, animals were sacrificed. Some brains were sectioned to do TH immunohistochemistry. The others were used to detect the dopamine levels by high-performance liquid chromatograph and electrochemical detection (HPLC-ECD). The results showed that MSCs multiply rapidly and formed fibroblast colony-forming units in primary culture. The gene expression efficiency was about 75%. The rounds of asymmetric rotation after apomorphine administration decreased after TH-engineered MSCs were grafted. Histological examination showed that TH gene was expressed around the transplantation points. The dopamine level in the lesioned striatum of rats injected with TH-MSCs was significantly greater than that in rats treated with LacZ-MSCs (P < 0.05). All the data demonstrated that MSCs could readily be genetically engineered. Therefore, MSCs could be useful gene delivery vehicles of gene therapy for Parkinson's disease.     

微囊化猪视网膜色素上皮细胞移植治疗帕金森病的实验研究(英文)

目的研究微囊化后的猪视网膜色素上皮细胞(retinal pigment epithelial,RPE)对帕金森病大鼠模型的移植疗效。方法原代培养RPE 并传代,高效液相色谱法测定培养液上清中多巴胺(dopamine, DA)和高香草酸(homovanillic acid, HVA)的含量,ELISA法检测脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)和胶质细胞源性神经营养因子(glial-derived neurotrophic factor, GDNF)的含量。用高压静电成囊装置制备海藻酸钠-多聚赖氨酸-海藻酸钠微囊化细胞。6-羟基多巴胺(6-hydroxydopamine, 6-OHDA)毁损内侧前脑束 (medial fore-brain bundle,MFB)建立 SD 大鼠帕金森病模型。立体定向移植 RPE+ 微囊,检验旋转实验、免疫组化和脑内生化的变化。结果 RPE 培养上清液中DA、HVA、BDNF、GDNF 的含量稳定,微囊化后细胞长期存活,活性没有明显变化。6-OHDA毁损MFB建立大鼠帕金森病模型的成模率为83%。移植微囊化的RPE后有效率为33%。结论猪 RPE 体外培养生长旺盛,持续分泌 DA、BDNF 和 GNDF,微囊化不影响其分泌功能。RPE 移植对帕金森病大鼠有一定的治疗作用。     

Transfer of the von Hippel-Lindau gene to neuronal progenitor cells in treatment for Parkinson's disease

Neuronal progenitor cells (NPCs) may provide dopaminergic neurons for the treatment of Parkinson's disease (PD). However, transplantation of NPCs into the striatum by current methods has had limited success. It is possible to reverse the symptoms of PD in model rats but difficult to reverse them in humans because the number of dopaminergic neurons generated from NPCs is low. We transduced the von Hippel-Lindau (VHL) gene into NPCs isolated from embryonic rat brain. The NPCs with the transduced VHL gene efficiently differentiated into tyrosine hydroxylase-positive neurons in vitro. NPCs with the transduced VHL gene, which were labeled in advance with bromodeoxyuridine, were transplanted into the striatum of a rat model of PD. Numerous bromodeoxyuridine-tyrosine hydroxylase double-labeled cells were seen close to the transplant site, showing that the transplanted cells efficiently generated new dopaminergic neurons within the host striatum. Moreover, all of the animals with NPCs with VHL showed a remarkable decrease in apomorphine-induced rotations. These findings show that NPCs with the VHL gene can efficiently generate dopaminergic neurons and that a sufficient number of dopaminergic neurons can develop from them to reverse the symptoms of PD in humans. VHL gene transduction provides a new therapeutic approach for treatment of PD.     

Human neural progenitor cells over-expressing IGF-1 protect dopamine neurons and restore function in a rat model of Parkinson's disease

Growth factors such as glial cell line-derived neurotrophic factor (GDNF) have been shown to prevent neurodegeneration and promote regeneration in many animal models of Parkinson's disease (PD). Insulin-like growth factor 1 (IGF-1) is also known to have neuroprotective effects in a number of disease models but has not been extensively studied in models of PD. We produced human neural progenitor cells (hNPC) releasing either GDNF or IGF-1 and transplanted them into a rat model of PD. hNPC secreting either GDNF or IGF-1 were shown to significantly reduce amphetamine-induced rotational asymmetry and dopamine neuron loss when transplanted 7 days after a 6-hydroxydopamine (6-OHDA) lesion. Neither untransduced hNPC nor a sham transplant had this effect suggesting GDNF and IGF-1 release was required. Interestingly, GDNF, but not IGF-1, was able to protect or regenerate tyrosine hydroxylase-positive fibers in the striatum. In contrast, IGF-1, but not GDNF, significantly increased the overall survival of hNPC both in vitro and following transplantation. This suggests a dual role of IGF-1 to both increase hNPC survival after transplantation and exert trophic effects on degenerating dopamine neurons in this rat model of PD.     

The contribution of fMRI to elucidate the pathophysiology of primary headaches

This study was designed to assess the potential therapeutic efficacy of gene-modified mesenchymal stem cells (MSCs), MSCs-TH and MSCs-GDNF, in PD rats. Fifty-nine PD rat models were divided into five groups and then the gene-modified MSCs were transplanted into the striatum of rats according to the design. Apomorphine-induced rotational behavior in rats was observed weekly; rats which received both MSCs-TH and MSCs-GDNF showed the most significant improvement compared with those in other groups (P < 0.01). Three weeks later, immunohistochemistry analysis found TH-positive cells and GDNF-positive cells in striatal. Eight weeks later, PD rats were killed. HPLC and ELISA results showed DA and GDNF content in striatum of rats which received both MSCs-TH, and MSCs-GDNF was considerably higher compared with those of other groups (P < 0.01),respectively. In conclusion, our results suggest that combined transplantation of MSCs expressing TH and GDNF can lead to remarkable therapeutic effects in a rat model of PD.     

Therapeutic effects of differentiated bone marrow stromal cell transplantation on rat models of Parkinson's disease

This study was to explore curative effect of bone marrow stromal cells (BMSCs) differentiated into nestin-positive cells transplantation on rat Parkinson's disease (PD) model. The PD rats were selected and allocated randomly into three groups. BMSCs with differentiation, BMSCs without differentiation and physiological saline were injected into right striatum of PD rat. The rotation test and immunofluorescence double staining were done. Frequency of rotation was significantly less in differentiated or non-differentiated BMSCs groups than that in normal saline group. Brdu/GFAP- and Brdu/NSE-positive cells appeared except BrdU/TH-positive cells. BMSCs differentiated had better effect than that of BMSCs without differentiated and physiological saline group.     

Delayed gene therapy of glial cell line-derived neurotrophic factor is efficacious in a rat model of Parkinson's disease

Gene transfer of glial cell line-derived neurotrophic factor (GDNF) in rodent models of Parkinson's disease (PD) has been shown to protect against neurodegeneration either prior to or immediately after neurotoxin-induced lesions; however, the nigrostriatal pathway was largely intact when gene delivery was completed in these models, which may not accurately reflect the clinical situation encountered with Parkinson's patients. In this study, replication-incompetent adenoviral vectors encoding the rat GDNF gene were administered into the striatum 4 weeks following 6-hydroxydopamine (6-OHDA) injection in the unilateral striatum, more closely resembling fully developed PD. Apomorphine-induced rotational behavior testing was performed every week following 6-OHDA injection. At the 10th week after gene transfer, the striatal dopamine concentrations were measured by HPLC with an electrochemical detector and the number of tyrosine hydroxylase (TH)-positive dopamine neurons in the substantia nigra (SN) was determined by immunohistochemistry. Injection of 6-OHDA into the striatum produced stable increases in rotation, which reached a plateau between 4 and 5 weeks post-injection. The number of TH-positive neuron in the SN and dopamine levels in the striatum was significantly lower in the 6-OHDA group compared to the normal group. Gene transfer of GDNF, but not beta-galactosidase, significantly increased the number of TH-positive neurons and dopamine levels, with a subsequent behavioral recovery between 5 and 10 weeks following GDNF transduction. These findings demonstrate that adenovirus-mediated gene transfer of GDNF is efficacious even in the late stages of 6-OHDA-induced PD rats. They also provide further evidence on the effectiveness of GDNF-based gene therapy for experimental Parkinson's disease.     

Intrastriatal transplantation of mouse bone marrow-derived stem cells improves motor behavior in a mouse model of Parkinson's disease

Strategies of cell therapy for the treatment of Parkinson's disease (PD) are focused on replacing damaged neurons with cells to restore or improve function that is impaired due to cell population damage. In our studies, we used mesenchymal stromal cells (MSCs) from mouse bone marrow. Following our novel neuronal differentiation method, we found that the basic cellular phenotype changed to cells with neural morphology that express specific markers including those characteristic for dopaminergic neurons, such as tyrosine hydroxylase (TH). Intrastriatal transplantation of the differentiated MSCs in 6-hydroxydopamine-lesioned mice led to marked reduction in the amphetamine-induced rotations. Immunohistological analysis of the mice brains four months post transplantation, demonstrated that most of the transplanted cells survived in the striatum and expressed TH. Some of the TH positive cells migrated toward the substantia nigra. In conclusion, transplantation of bone marrow derived stem cells differentiated to dopaminergic-like cells, successfully improved behavior in an animal model of PD suggesting an accessible source of cells that may be used for autotransplantation in patient with PD.     

A role for tumor necrosis factor alpha in death of dopaminergic neurons following neural transplantation

Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease (PD). We have examined the temporal release profile of an inflammatory cytokine, tumor necrosis factor-alpha (TNFalpha), following transplantation of fetal mesencephalic tissue into the rat striatum. The amounts of TNFalpha released in vivo when added to cultures of embryonic DA neurons, significantly reduced the survival of DA neurons in vitro, and this cell death could be prevented by the inclusion of an antibody to the TNFalpha receptor type 1. Inclusion of this antibody in cell suspensions during transplantation also increased the survival of transplanted fetal DA neurons by approximately 250%. Use of this therapeutic antibody approach may offer significant improvements to neural transplantation as a treatment for PD.