纹状体内移植胚胎干细胞来源的神经前体细胞可升高帕金森病小鼠纹状体内多巴胺含量

目的探讨胚胎干细胞来源的神经前体细胞移植治疗帕金森病(Parkinson's disease,PD)的疗效及作用机制。方法以小鼠腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)建立PD小鼠模型;于体外将小鼠胚胎干细胞(embryonic stem cells,ESCs)向神经前体细胞(neural precursor cells,NPC)方向分化,经PCR与免疫荧光染色分析鉴定后将高纯度的NPC移植入PD小鼠纹状体内,2周和4周分别取纹状体制作冰冻切片并进行免疫荧光染色,用高效液相色谱法测定纹状体多巴胺的含量;对移植后死亡小鼠,取其脑进行常规石蜡切片、HE染色。结果移植的NPC可在移植部位存活,并分化为表达酪氨酸羟化酶(TH)的多巴胺能神经元;高效液相色谱法检测显示,PD小鼠纹状体内多巴胺含量明显降低,移植NPC 2周后的PD小鼠纹状体内多巴胺含量较未移植小鼠明显升高,但仍低于正常对照小鼠;移植4周后PD小鼠纹状体内多巴胺含量与未移植小鼠接近;有18.75%的PD小鼠移植NPC后形成畸胎瘤而死亡。结论 ESCs来源的NPC移植因可补充病变处多巴胺含量而对PD有一定治疗作用。     

小鼠脑室内注射神经干细胞裂解液促进谷氨酸盐诱导的兴奋性神经元损伤的修复

先前的研究已经证实,阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液具有改善抑郁症样模型大鼠的行为学障碍、上调其海马和大脑皮质神经生长因子(nerve growth factor,NGF)和脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)的表达、增加海马神经干细胞(neural stem cells,NSCs)/神经前体细胞(neural progenitor cells)增殖的效果。该研究的目的在于探讨神经干细胞裂解液的无细胞滤液(cell-free filtrate ofneural stem cell lysates,FNSCL)脑室内注射促进谷氨酸盐诱导的成年小鼠兴奋性神经元损伤修复的可能性。成年小鼠谷氨酸单钠(monosodiumglutamate,MSG,2.0g/(kg·d))灌胃,连续10日,造成兴奋性神经元损伤模型。自孕15 d的昆明种小鼠取胎脑,分离、培养神经干细胞,免疫细胞化学法检测巢蛋白(nestin)抗原,制备神经干细胞裂解液的无细胞滤液。MSG+NSCs组动物在MSG灌胃后接收脑室内NSCs移植,MSG+FNSCL组动物在MSG灌胃...     

骨髓间充质干细胞黑质内移植对帕金森病大鼠的治疗作用

目的探索骨髓间充质干细胞（BMSCs）移植到帕金森病（Parkinson’s disease,PD）大鼠毁损侧黑质内,PD模型大鼠的姿势不对称性和黑质及纹状体内酪氨酸羟化酶（tyrosinehy droxylase,TH）表达的改变,以及BM—SCs在大鼠脑内的存活、分化情况。方法黑质、前脑内侧束两点法注射6一羟多巴胺（6-OHDH）并行为学分析筛选PD模型大鼠。将PD模型大鼠随机分为移植组和对照组。BMSCs移植术后4周和8周,观察大鼠姿势不对称性,免疫组织化学及免疫荧光显色方法检测黑质和纹状体酪氨酸羟化酶（tyrosine hydroxylase,TH）的表达变化以及BMSCs在大鼠体内的存活、迁移及分化情况。结果BMSCs黑质内移植可使PD模型大鼠的转动频率由（10．62±2．97）r／min降至（4．65±1．08）r／min（P〈0．01）,显著增加毁损侧黑质TH阳性细胞数量和纹状体内TH阳性纤维密度。BMSCs在大鼠黑质内可以存活至少8周,部分细胞分化为神经干细胞、神经元和神经胶质细胞。结论黑质内移植BMSCs对PD模型大鼠有一定的治疗作用。     

NAAGS基因修饰神经干细胞在创伤性颅脑损伤治疗中的研究

目的：探讨移植NAAG合酶（NAAG synthetase,NAAGS）基因修饰的神经干细胞（Neural Stem Cells,NSCs）能否促进创伤性颅脑损伤大鼠神经功能的恢复。方法：利用电穿孔转染大鼠NSCs,通过脑立体定向仪分别将PBS（模型组）、NSCs（NSCs组）、转基因NSCs（NAAGS＋NSCs组）移植到创伤性颅脑损伤（Traumatic Brain Injury,TBI）大鼠局部损伤灶边缘,通过NSS评分评价移植后大鼠神经功能的变化以及用TUNEL法检测NSCs的凋亡情况,并采用放射免疫法分析脑组织中促炎因子水平。结果：Nss评分结果显示NAAGs＋NSCs组和NSCs组在第7、14、21天神经功能评分均低于模型组（P〈0．05）;NAAGS＋NSCs组在第14和21天神经功能评分低于NSCs组（P〈0．05）;在各时间点细胞移植组比模型组的神经细胞凋亡数明显减少;转基因NSCs移植能明显降低TBI脑组织中促炎因子水平。结论：转基因NSCs移植后可以合成NAAGS促进TBI大鼠神经功能的恢复。     

神经干细胞移植对脑损伤大鼠整合素表达的影响

目的探讨神经干细胞（NSCs）移植对大鼠创伤性脑损伤（TBI）整合素（integrin）表达的影响。方法从E14大鼠胚胎分离NSCs,进行原代培养及传代培养;对NSCs进行诱导分化;采用免疫细胞化学技术对NSCs和其分化为神经元的表型进行鉴定。采用改良的Feeney法制备创伤性脑损伤模型。利用脑立体定位仪和微量注射泵进行NSCs脑内移植。采用免疫组织化学技术、免疫印迹技术和RT—PCR技术检测在移植后不同时间脑组织损伤区整合素的表达。结果在培养基中,NSCs呈球团状悬浮生长,Nestin表达阳性。用含10％胎牛血清的培养基对NSCs进行体外诱导分化后第2d,多数细胞伸出突起,以后突起逐渐延长,分支增加。分化后第5d,部分细胞呈βⅢ-微管蛋白阳性。整合素阳性产物主要表达于细胞膜,呈棕黄色。在对照组及移植组均可见阳性细胞表达。在不同时间点,NSCs移植组移植点及其周围脑组织中整合素的mRNA表达均显著高于对照组（P〈O．01）。整合素的蛋白表达结果和tuRNA表达结果相一致。结论移植NSCs至TBI大鼠损伤脑组织,在移植点周围脑组织中整合素的表达显著增加。     

干细胞移植对鼠脑缺血再灌注损伤后STAT3和bcl-2蛋白表达的影响

目的：通过检测神经于细胞移植后Wistar大鼠脑缺血再灌注损伤后局部STAT3蛋白和bcl—2蛋白表达水平的变化,来研究移植入的神经千细胞对损伤后神经细胞的凋亡抑制作用和可能的机制。方法：SPF级SD大鼠20只,以线栓法建立大脑中动脉缺血再灌注模型,随机分为2组,模型组（对照组）10只,干细胞移植组（治疗组）10只。取孕14天的Wistar胎鼠脑皮质神经干细胞培养至第三代,于损伤后的24小时立体定向注射植入成年Wistar大鼠脑缺血损伤局部。于移植后48小时处死鼠取脑,免疫组化检测bcl-2蛋白和westernblot法检测STAT3蛋白表达的变化差异。结果：移植入神经干细胞后,实验组局部STAT3蛋白和bcl-2蛋白的表达都比对照组明显的增强,差异具有统计学意义（P〈0．01）。结论：移植入的神经干细胞通过上调STAT3蛋白和bcl-2蛋白的表达,发挥局部神经元的抗凋亡作用,减轻局部缺血再灌注损伤,保护神经功能。     

小鼠脊髓损伤模型的建立及神经干细胞移植后运动功能恢复情况的研究

目的制作小鼠脊髓损伤打击模型,观察神经干细胞(NSCs)移植对脊髓损伤小鼠运动功能恢复及Nestin表达的影响。方法将50只小鼠随机分为空白组(5只)、模型组(15只)、对照组(15只)、治疗组(15只),运用改良Allen's法制备小鼠T10脊髓损伤模型并立即在损伤节段进行NSCs移植,于损伤后1、3、7、14、21d进行BBB评分,并通过免疫荧光法及荧光定量PCR检测Nestin的表达情况。结果所有脊髓打击后小鼠均出现双后肢瘫痪,但随时间延长运动功能可有不同程度恢复,NSCs移植14d后治疗组较模型组及对照组BBB评分显著增高(P0.05),且治疗组Nestin表达量也高于模型组及对照组。结论成功建立了小鼠脊髓损伤打击模型;移植的外源性神经干细胞在脊髓损伤处存活并促进损伤后小鼠运动功能的恢复。     

骨髓间充质干细胞源神经细胞移植治疗帕金森病大鼠模型

目的探讨骨髓间充质干细胞（mesenchymal stemcells,MSCs）源神经细胞脑内移植对帕金森病（Parkinson s disease,PD）大鼠的治疗作用。方法贴壁培养法分离、培养大鼠骨髓MSCs,脑匀浆上清诱导第3代MSCs向神经细胞分化,采用免疫细胞化学法鉴定诱导分化后细胞的性质,激光共聚焦显微镜检测诱导前后细胞Ca2＋浓度变化,6只PD大鼠行纹状体内MSCs源神经细胞移植作为细胞移植组,6只PD大鼠作为对照组。细胞移植术后4周检测PD大鼠的行为变化,观察移植细胞在脑内的分布情况。结果倒置显微镜下可见MSCs呈纺锤形和多角形,有1～2个核仁,MSCs经脑匀浆上清诱导后其胞体折光性增强,发出数个细长突起,互相交织成网,有的似轴突。诱导后细胞表达神经元特异性标志物神经元特异性烯醇化酶（NSE）和神经丝蛋白（NF）,胞质Ca2＋荧光强度显著增强,可推测诱导后的细胞为MSCs源神经细胞,将BrdU标记的MSCs源神经细胞移植到PD大鼠纹状体治疗4周后,可见细胞散在分布于注射侧脑组织,有少量细胞可迁移到对侧脑组织,PD大鼠的旋转行为得到显著改善。结论MSCs源神经细胞移植治疗帕金森病大鼠可使其旋转行为得到改善。     

诱导多能干细胞在帕金森病治疗中的应用进展

帕金森病（Parkinson's disease, PD）是由于黑质中多巴胺能神经元（dopaminergic neurons, DAns）的病变导致多巴胺含量降低而引起的一种神经退行性疾病,其发病机制尚不明确,而且临床缺乏有效的早期诊断和治疗手段。诱导多能干细胞（induced pluripotent stem cells, iPSCs）的出现为神经系统疾病特别是神经退行性疾病的治疗带来了希望。基于iPSCs的细胞模型可以广泛开展PD发病机制的研究,同时以iPSCs来源的DAns、神经干细胞（neural stem cells, NSCs）等的细胞移植治疗,更是未来PD治疗最有希望的手段。从基于iPSCs的不同基因突变类型的细胞模型与不同分化程度的细胞移植治疗两个方面介绍诱导多能干细胞在PD研究中的进展,旨在分析诱导多能干细胞在帕金森病方面的应用及不足。     

大鼠胚胎神经干细胞纹状体内移植后特性

观察大鼠胚胎神经干细胞移植入成年大鼠纹状体后的存活、迁移和分化状况。自14天胎鼠脑室下区分离获得神经干细胞,利用无血清培养基培养扩增并进行鉴定。经4～5代的扩增后,以BrdU标记的神经干细胞通过脑立体定位注射移植入成年大鼠纹状体内,然后分别于移植后2周、4周、6周和8周时做脑冰冻切片,通过免疫组织化学和免疫荧光方法检测移植细胞的数量、定位和分化情况。8周后移植细胞的检出率约16%;移植细胞向周围宿主组织有广泛的迁移表现,尤以沿着白质束向头尾方向的迁移最为显著,最远向后侧达到内囊;纹状体中移植细胞主要分化为神经元和星形胶质细胞。星形胶质细胞数量最多,主要位于移植区与宿主组织临界部位,而神经元处于移植区中央。培养的大鼠胚胎神经干细胞可以作为移植替代治疗神经退行性疾病研究的供体细胞源,而移植中的迁移现象值得注意。     

神经干细胞在治疗脑损伤中的应用

神经干细胞（neural stem cells,NSCs）是中枢神经系统中既具有自我更新能力又能分化为神经系统各类细胞的细胞群。在体外一定条件下,NSCs能保持增殖能力,经定向诱导能分化为具有成熟神经细胞特征的各类细胞。NSCs移植治疗研究显示,植入的NSCs能分化为移植部位的神经细胞,并融入、整合该部位,重建受损神经网络,在一定程度上缓解病症。近年来,激活体内内源NSCs治疗神经损伤也逐渐得到广泛关注。因此,NSCs在治疗神经损伤中的应用研究已成为当前神经生物学基础理论和临床应用研究的热点。本文简要介绍了最近关于NSCs在治疗脑损伤中的应用研究进展。     

Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson''s disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.     

Experimental Study on Trace Marking and Oncogenicity of Neural Stem Cells Derived from Bone Marrow

It has been well accredited that the neural stem cells (NSCs) derived from bone marrow stroma cells (BMSCs) can be used as the therapeutic application. However, their efficacy and safety in therapeutic application are uncertain. In this experiment, the trace marking and oncogenicity of NSCs derived from BMSCs (BMSCs-D-NSCs) were studied. The BMSCs were harvested by gradient centrifugation and cultured in "NSCs medium" in vitro. The verified CD133/Nestin-positive BMSCs-D-NSCs were then transplanted into nude mice to detect the oncogenicity, into the right lateral cerebral ventricle or right caudae putamen and substantia nigra to examine, whether the symptoms were improved in Parkinson's Disease (PD) models after transplantation, by both SPECT image assay of dopamine transporter (DAT) in corpus striatum and its average standard uptake value (SUVave) in corpus striatum and thalamus. Tissue samples and surviving model animals were studied at 1, 3, and 6 months post-transplantation. Before transplantation, the cells were labeled with BrdU or rAAV-GFP for the pathological sections, and with Feridex for the in vivo trace by MRI assay. The concanavalin A (ConA) agglutination test, stop-dependence test with soft agar, karyotype analysis of chromosome G zone in BMSCs-D-NSCs, and the nude mouse neoplasia test were also performed. The BrdU, rAAV-GFP or Feridex can be used as trace markers of BMSCs-D-NSCs during transplantation. The transplanted BMSCs-D-NSCs displayed neither toxicity nor neoplasia up to 6 months in vivo, but could play an important role in improving the symptoms of the animals with degenerative diseases like PD.     

PET imaging a MPTP-induced mouse model of Parkinson's disease using the fluoropropyl-dihydrotetrabenazine analog [18F]-DTBZ (AV-133)

Parkinson's disease (PD) is characterized by the loss of dopamine-producing neurons in the nigrostriatal system. Numerous researchers in the past have attempted to track the progression of dopaminergic depletion in PD. We applied a quantitative non-invasive PET imaging technique to follow this degeneration process in an MPTP-induced mouse model of PD. The VMAT2 ligand (18)F-DTBZ (AV-133) was used as a radioactive tracer in our imaging experiments to monitor the changes of the dopaminergic system. Intraperitoneal administrations of MPTP (a neurotoxin) were delivered to mice at regular intervals to induce lesions consistent with PD. Our results indicate a significant decline in the levels of striatal dopamine and its metabolites (DOPAC and HVA) following MPTP treatment as determined by HPLC method. Images obtained by positron emission tomography revealed uptake of (18)F-DTBZ analog in the mouse striatum. However, reduction in radioligand binding was evident in the striatum of MPTP lesioned animals as compared with the control group. Immunohistochemical analysis further confirmed PET imaging results and indicated the progressive loss of dopaminergic neurons in treated animals compared with the control counterparts. In conclusion, our findings suggest that MPTP induced PD in mouse model is appropriate to follow the degeneration of dopaminergic system and that (18)F-DTBZ analog is a potentially sensitive radiotracer that can used to diagnose changes associated with PD by PET imaging modality.     

Survival and neural differentiation of adult neural stem cells transplanted into the mature inner ear

The cochlear sensory epithelium and spiral ganglion neurons (SGNs) in the adult mammalian inner ear do not regenerate following severe injury. To replace the degenerated SGNs, neural stem cell (NSC) is an attractive alternative for substitution cell therapy. In this study, adult mouse NSCs were transplanted into normal and deafened inner ears of guinea pigs. To more efficiently drive the implanted cells into a neuronal fate, NSCs were also transduced with neurogenin 2 (ngn2) before transplantation. In deafened inner ears and in animals transplanted with ngn2-transduced NSCs, surviving cells expressed the neuronal marker neural class III beta-tubulin. Transplanted cells were found close to the sensory epithelium and adjacent to the SGNs and their peripheral processes. The results illustrate that adult NSCs can survive and differentiate in the injured inner ear. It also demonstrates the feasibility of gene transfer to generate specific progeny for cell replacement therapy in the inner ear.     

Treatment of Aganglionic Megacolon Mice via Neural Stem Cell Transplantation

To explore a potential methodology for treating aganglionic megacolon, neural stem cells (NSCs) expressing engineered endothelin receptor type B (EDNRB) and glial cell-derived neurotrophic factor (GDNF) genes were transplanted into the aganglionic megacolon mice. After transplantation, the regeneration of neurons in the colon tissue was observed, and expression levels of differentiation-related genes were determined. Primary culture of NSCs was obtained from the cortex of postnatal mouse brain and infected with recombinant adenovirus expressing EDNRB and GDNF genes. The mouse model of aganglionic megacolon was developed by treating the colon tissue with 0.5 % benzalkonium chloride (BAC) to selectively remove the myenteric nerve plexus that resembles the pathological changes in the human congenital megacolon. The NSCs stably expressing the EDNRB and GDNF genes were transplanted into the benzalkonium chloride-induced mouse aganglionic colon. Survival and differentiation of the implanted stem cells were assessed after transplantation. Results showed that the EDNRB and GDNF genes were able to be expressed in primary culture of NSCs by adenovirus infection. One week after implantation, grafted NSCs survived and differentiated into neurons. Compared to the controls, elevated expression of EDNRB and GDNF was determined in BAC-induced aganglionic megacolon mice with partially improved intestinal function. Those founding indicated that the genes transfected into NSCs were expressed in vivo after transplantation. Also, this study provided favorable support for the therapeutic potential of multiple gene-modified NSC transplantation to treat Hirschsprung’s disease, a congenital disorder of the colon in which ganglion cells are absent.