人神经干细胞的分离、克隆和动物脑内移植及转基因表达

目的分离和克隆人神经干细胞,并在体外和体内分析其生物学特征.方法我们联合采用四步法从人胚胎前脑分离制备多潜能神经干细胞,并使用重组腺病毒相关病毒载体(rAAV)将LacZ基因和胶质细胞起源的神经营养因子(GDNF)基因转移到神经干细胞.结果二株人神经干细胞被成功建立.这些克隆化后的神经干细胞在细胞培养中和移植到新生小鼠脑内后能发育分化成神经元、少枝胶质细胞和星形胶质细胞.在rAAV转导基因后,神经干细胞可在体外和体内表达转基因产物.结论这种具有转基因表达能力的神经干细胞为神经系统疾病的进一步治疗研究提供了有潜在价值的细胞资源.     

携带cdc2-siRNA重组腺相关病毒对C型尼曼-皮克病小鼠神经元细胞骨架损伤的保护作用

目的 评估经小脑注射携带cdc2-siRNA的重组腺相关病毒(rAAV)对C型尼曼-皮克病(NPC)小鼠神经元细胞骨架损伤是否具有保护作用.方法 将携带cdc2-siRNA重组腺相关病毒(rAAV) 注射入 2周龄npc-/-小鼠的小脑,采用免疫组织化学及免疫印迹方法观察该病毒对 npc-/-小鼠神经元细胞骨架损伤的保护作用.结果 (1)携带cdc2-siRNA的rAAV明显减少npc-/-小鼠轴突球状体的数量;(2)携带cdc2-siRNA的rAAV有效抑制磷酸化的神经丝(由SMI31识别)及磷酸化的Tau蛋白(由PHF-1识别)的表达. 结论小脑注射携带cdc2-siRNA的rAAV对npc-/-小鼠神经元细胞骨架损害具有保护作用.     

骨骼肌重塑中的钙调神经磷酸酶信号转导通路***☆

背景：骨骼肌重塑是骨骼肌对多种刺激因素所产生的形态结构与代谢机能的适应性变化。近几年关于钙调神经磷酸酶(CaN )/ (NFATS)在骨骼肌重塑中的作用备受关注。 目的：探讨钙调神经磷酸酶在骨骼肌从无氧转向有氧的代谢重塑、肌纤维类型转化以及在骨骼肌肥大过程中的信号转导作用。 方法：由第一作者用计算机检索ISI Web of knowledge数据库(1998/2010),检索词为 “calcineurin,skeletal muscle,hypertrophy,NFAT,myofiber type”,语言设定为英文。从钙调神经磷酸酶信号系统在骨骼肌代谢、肌纤维类型转换和肌肉肥大中的作用方面进行总结,对其调节机制、肌肉重塑等方面进行介绍。 结果与结论：共检索到186篇文章,按纳入和排除标准对文献进行筛选,共纳入33篇文章。结果表明CaN/NFATS信号激活有助于Ⅰ型肌纤维分化,提高线粒体有氧代谢能力,但骨骼肌对耐力运动的适应并不绝对依赖CaN。CaN/NFATS转导通路有可能通过转录激活utrophin A来调控骨骼肌的肥大反应。由此可知钙调神经磷酸酶参与骨骼肌代谢、纤维转化和肥大的重塑过程,调节骨骼肌对刺激产生适应性应答反应。     

磷脂酰肌醇-3激酶在人骨骼肌纤维的表达

目的 研究磷脂酰肌醇-3激酶在肌病患者骨骼肌纤维的表达及其意义.方法 选择Duchenne型肌营养不良(Duehenne muscular dystrophy, DMD)9例、Becker假肥大型肌营养不良(Becker muscular dystrophy, BMD)6例、先天性肌营养不良(congenital muscular dystrophy, CMD)3例、多发性肌炎(polymyositis, PM)5例,开放式骨骼肌活检,连续冰冻切片,苏木精-伊红(hematoxylin and eosin, HE)、三磷酸腺苷酶(adenosin triphosphatase, ATPase)组织化学染色;抗-磷脂酰肌醇-3激酶(phosphatidylinositol 3-kinase,PI3K)、抗-神经细胞粘附分子(neural cell adhesion molecule, NCAM)、抗-结蛋白(desmin)单克隆抗体免疫组织化学染色,病理分析.结果 [1]PI3K在再生早期小径肌纤维膜上表达,伴随再生肌纤维的成熟PI3K表达消失;[2]PI3K在幼儿肌纤维膜上表达显著;[3]PI3K在动静脉平滑肌上表达显著.结论 PI3K可能在人骨骼肌纤维再生早期起重要作用.     

mdx小鼠骨骼肌组织成肌、成脂、成骨基因的特异性表达**

背景：干细胞移植是治疗肌营养不良症的有效方法之一,但移植的干细胞在病理骨骼肌中成肌表达较低。 目的：通过比较mdx小鼠和C57小鼠的骨骼肌形态及成肌、成脂、成骨基因表达的差异,探讨mdx小鼠骨骼肌病理改变的可能机制。 方法：取mdx小鼠与C57小鼠的骨骼肌组织行冰冻切片,苏木精-伊红染色和Vonkossa染色观察两种小鼠肌肉组织的形态特征;提取mdx小鼠和C57小鼠骨骼肌组织总RNA,real-time PCR检测成肌、成脂、成骨相关基因的表达。 结果与结论：mdx小鼠骨骼肌有肌纤维坏死和再生,伴有轻度脂肪、纤维结缔组织增生,Vonkossa染色可见钙结节沉积,而C57小鼠的骨骼肌细胞形态清晰,核位于细胞周边。与C57小鼠比较,mdx小鼠肌肉组织成骨、成脂基因表达有不同程度的上调(P < 0.05),而成肌基因表达下调(P < 0.05)。dystrophin基因缺失及成肌基因表达下调、成骨和成脂基因上调是造成mdx小鼠肌肉组织变性坏死的原因。     

骨骼肌25000蛋白分布于Ⅰ型肌纤维

目的:探讨骨骼肌25 000蛋白分布与肌纤维类型的关系。方法:分别对正常人及重症肌无力患者骨骼肌相邻的连续冰冻切片进行ATPase染色及25 000蛋白的免疫组化染色,阐明25 000蛋白在骨骼肌的分布及与骨骼肌纤维类型的关系。用免疫印迹方法观察以Ⅰ型纤维为主的比目鱼肌和以Ⅱ型纤维为主的肱三头肌中25 000蛋白表达的差异。结果:相邻冰冻切片ATPase染包和25 000蛋白免疫组化的结果表明,无论是正常人还是MG患者的骨骼肌,25 000蛋白均存在于胞质内,并主要分布在Ⅰ型纤维;在相同免疫染色条件下,该蛋白在MG骨骼肌的染色强度明显低于正常人。免疫印迹显示,在相同的骨骼肌总蛋白量中,比目鱼肌的25 000蛋白表达水平明显高于肱三头肌,进一步证实了25 000蛋白主要分布在Ⅰ型纤维。结论:25 000蛋白主要在骨骼肌Ⅰ型纤维中表达,MG患者骨骼肌的易疲劳和收缩无力可能与该蛋白在Ⅰ型纤维中低表达有关。     

成肌调节因子Myf-5在失神经骨骼肌萎缩不同时段的表达

背景：成肌调节因子Myf-5是参与肌肉发生过程分子调控、启动和维持骨骼肌细胞生长发育的重要基因,可能与失神经骨骼肌萎缩的发生有关。 目的：观察不同部位、不同时段骨骼肌失神经支配后成肌调节因子Myf-5基因的表达情况。 设计、时间及地点：随机对照动物实验,于2008-03/04在山西医科大学完成。 材料：选择健康8周龄雄性SD大鼠24只,随机分成4组,即假手术组(有神经支配)、去神经2 d组、去神经7 d组、去神经28 d组,每组6只。 方法：假手术组不切断坐骨神经,仅做假手术。去神经组右下肢后部中段切断坐骨神经1 cm以上,分别于去神经第2,7,28天用脊椎脱臼法处死大鼠,分离出右小腿的胫骨前肌、比目鱼肌、腓肠肌、跖肌标本。 主要观察指标：用反转录-聚合酶链反应技术检测各组肌肉Myf5 mRNA表达情况,抗Myf-5 多克隆抗体免疫组织化学染色(ABC 法),测量灰度值。 结果：去神经骨骼肌早期,Myf-5的mRNA在去神经支配后第2,7,28天均表达上调(P < 0.05)。Myf-5 抗体阳性染色细胞核数在SD大鼠骨骼肌失神经28 d时的肌卫星细胞中最多。 结论：Myf-5在大鼠失神经骨骼肌萎缩早期不同肌肉表达均为上调。大鼠骨骼肌失神经支配后早期肌卫星细胞中Myf-5表达上调。     

Mdx小鼠骨骼肌纤维化及Spp1基因表达

目的通过观察Duchenne型肌营养不良的模型鼠mdx小鼠骨骼肌中纤维化情况,研究Spp1基因在mdx小鼠及其对照鼠中不同时期的表达,探讨在mdx小鼠中Spp1与肌纤维化的关系。方法选取雄性C57BL/10Sc Sn-Dmdmdx/JNju鼠为实验组,雄性C57BL/6Sc Sn小鼠为对照组,根据年龄分为2w组、4w组、8w组、12w组。每组选取6只,3只用于冰冻切片的苏木精-伊红染色及马松(masson)染色,3只用于基因芯片及q RT-PCR。结果 2w及4w时mdx小鼠无明显结缔组织增生,8w时,mdx小鼠可见轻度结缔组织增生;12w时,mdx小鼠结缔组织增生程度较8w稍加重,仍为小片状区域的纤维化,对照组小鼠不同时期均未见纤维化;mdx小鼠2w与12w股四头肌基因芯片表达谱对比,其中Spp1基因在mdx小鼠2w与12w相比fold-change值为-15.1354,变化明显;Spp1在mdx小鼠股四头肌不同时期表达量比较:8w组较4w组明显升高,12w组较8w组表达量下降,但仍高于4w组,8w组与12w组Spp1表达量较同期对照组明显升高,差异具有统计学意义。结论 mdx小鼠早期(2w~4w)肌纤维化表现不明显,8w时骨骼肌内可见少量结缔组织增生,随后缓慢进展。2 Spp1基因在mdx小鼠成熟期(8w~12w)表达量明显增加,推测其在mdx小鼠肌纤维化中发挥一定作用。     

Ezrin蛋白在肌病患者骨骼肌中的表达及意义

目的 研究Ezrin蛋白在肌病患者骨骼肌中的表达及意义.方法 取肌纤维再生活跃的假肥大型肌营养不良(DMD,9例)和多发性肌炎(PM,5例)患者的骨骼肌标本,冰冻连续切片,进行HE染色及抗-Ezrin、抗-神经细胞黏附分子(NCAM)单克隆抗体免疫组化染色,观察被检肌的病理改变和Ezrin蛋白的表达.结果 DMD、PM患者被检肌HE染色所见再生肌纤维直径较小、核位于中央、胞浆嗜碱性;NCAM染色再生肌纤维深染;再生肌纤维Ezrin呈阳性表达,伴随肌纤维成熟Ezrin表达逐渐减弱,成熟肌纤维无Ezrin表达;成肌细胞Ezrin呈阳性表达.结论 Ezrin蛋白与DMD、PM肌病患者骨骼肌纤维再生可能存在密切关系.     

成年小鼠嗅球神经干细胞的培养和鉴定

目的 建立完善的成年小鼠嗅球神经千细胞分离培养和鉴定方法,探索新的成年神经干细胞种子来源. 方法 用无血清方法 分离培养成年小鼠嗅球来源的神经干细胞;用克隆培养、5-溴2-脱氧尿嘧啶核昔(BrdU)整合的方法 检验培养细胞的干细胞特性;用免疫荧光细胞化学的方法 检测BrdU、神经干细胞标记物巢蛋白(nestin)和SOX2、分化的细胞标记物Tuj1、胶质纤维酸性蛋白(GFAP)、04的表达. 结果 从成年小鼠嗅球能够分离、培养出具有自我更新、增殖能力的神经球.构成神经球的细胞呈nestin和SOX2阳性,它们分化后产生TuJ1阳性的神经元、GFAP阳性的星形胶质细胞、04阳性的少突胶质细胞. 结论 成年小鼠嗅球存在神经干细胞,其能够在体外进行培养、增殖、分化.是神经干细胞的新的种子来源.     

Fiber-type-dependent expression of adenovirus-mediated transgene in mouse skeletal muscle fibers

We show the efficient transduction and expression of the lacZ gene in the skeletal muscle of adult C57BL/10ScSn mice after adenovirus-mediated gene transfer. Of the myofibers in the tibialis anterior muscle 62% were β-galactosidase positive after injection of the lacZ gene under the control of the chicken β-actin promoter and the cytomegalovirus enhancer. The transduced gene was preferentially expressed in type IIA and IIX fibers, which were richer in oxidative enzymes than type IIB fibers. Received: 14 August 1997 / Revised, accepted: 18 March 1998     

Full-length dystrophin cDNA transfer into skeletal muscle of adult mdx mice by electroporation

We showed that a LacZ expression plasmid (pCAG-lacZ) injection followed by electroporation increased the expression of the LacZ gene in the skeletal muscles of adult mdx mice up to ninefold higher as compared with simple intramuscular DNA injection. When full-length mouse dystrophin plasmid (pCAG-dys) and pCAG-lacZ were co-transfected by electroporation, 56% of dystrophin-positive fibers were stained for beta-galactosidase activity suggesting most of these myofibers are not revertants but transfected ones. Our data indicate that electroporation in vivo could introduce large full-length dystrophin cDNA into skeletal muscle of adult mdx mice.     

Variables affecting the transduction efficiency of adenovirus vectors in bovine aortic endothelial cells

Features and kinetics of Adenovirus (Ad)-mediated gene transfer to endothelial cells (EC) are not ultimately determined. We tested variables pertinent to the efficiency of Ad-mediated gene transfer to bovine aortic endothelial cells (BAEC) including: (1) Ad-vectors with different promoters, (2) kinetics of transduction efficiency of LacZ gene to BAEC, (3) the concentration and volume of vector-containing medium, (4) the period of incubation time of Ad vectors with BAEC, (5) the duration of transgene expression. An Ad5-LacZ vector with a cytomegalovirus (CMV) promoter transduced the LacZ gene to the cells more efficiently than vectors with the Rous sarcoma virus (RSV) promoter. However, both vectors exhibited a dose-dependent relationship between the vector multiplicity of infection (moi) and the percentage of LacZ-expressing cells. The higher moi of both vectors achieved nearly 100% of transduction efficiency in cultured BAEC. Although the Ad-CMV-LacZ vector better transduced the LacZ gene to BAEC than Ad-RSV-LacZ, a long period of vector exposure to BAEC could overcome the slightly difference in transduction efficiency between the two vectors. These results indicate that both Ad vectors are efficient for gene transfer to endothelial cells, and higher moi of vectors or a longer period exposure of vectors to EC can facilitate efficient transduction of foreign gene into EC in culture.     

Aquaporin 4 expression in human skeletal muscle fiber types

Introduction: Aquaporins (AQPs) are a family of transmembrane proteins involved in the maintenance of osmotic gradients. AQP4 is abundant in skeletal muscle, where it seems to be associated with glycolytic metabolism. We investigated the pattern of expression of AQP4 in normal human myofibers relative to the main forms of myosin heavy chain (MHC). Methods: Six normal human muscle biopsies were analyzed by double immunofluorescence for co‐expression of AQP4 and slow or fast MHC. Results: A high percentage (64–99%) of MHC‐fast positive fibers showed immunoreaction for AQP4. Immunoreactivity for AQP4 was also present in MHC‐slow positive fibers, but with a higher variability (5–72%) among biopsies. Discussion: The expression pattern of AQP4 in human myofibers is highly variable among different patients and cannot be predicted for single fibers depending on MHC type expression. Other factors, possibly related to muscle activity, may modulate AQP4 expression. Muscle Nerve 57 : 856–859, 2018     

Transduction of murine cerebellar neurons with recombinant FIV and AAV5 vectors

Our data demonstrate that vectors derived from recombinant feline immunodeficiency virus (rFIV) and adeno-associated virus type 5 (rAAV5) transduce cerebellar cells following direct injection into the cerebellar lobules of mice. Both recombinant viruses mediated gene transfer predominantly to neurons, with up to 2500 and 1500 Purkinje cells transduced for rAAV5 or rFIV-based vectors, respectively. The vectors also transduced stellate, basket and Golgi neurons, with occasional transduction of granule cells and deep cerebellar nuclei. rAAV5 also spread outside the cerebellum to the inferior colliculus and ventricular epithelium, while rFIV demonstrated the ability to undergo retrograde transport to the physically close lateral vestibular nuclei. Thus, AAV5 and FIV-based vectors show promise for targeting neurons affected in the hereditary spinocerebellar ataxias. These vectors could be important tools for unraveling the pathophysiology of these disorders, or in testing factors which may promote neuronal survival.     

Laser microdissection-based expression analysis of key genes involved in muscle regeneration in mdx mice

We have used the mdx mice strain (C57BL/10ScSn-mdx) as an experimental subject for the study of reiterative skeletal muscle necrosis-regeneration with basement membrane preservation. In young mdx muscle, by means of Hematoxylin-Eosin staining, different types of degenerative-regenerative groups (DRG) can be recognized and assigned to a defined muscle regeneration phase. To evaluate the expression of known key-regulatory genes in muscle regeneration, we have applied Laser Capture Microdissection technique to obtain tissue from different DRGs encompassing the complete skeletal muscle regenerative process. The expression of MyoD, Myf-5 and Myogenin showed a rapid increase in the first two days post-necrosis, which were followed by MRF4 expression, when newly regenerating fibers started to appear (3-5days post-necrosis). MHCd mRNA levels, undetectable in mature non-injured fibers, increased progressively from the first day post-necrosis and reached its maximum level of expression in DRGs showing basophilic regenerating fibers. TGFbeta-1 mRNA expression showed a prompt and strong increase following fiber necrosis that persisted during the inflammatory phase, and progressively decreased when new regenerating fibers began to appear. In contrast, IGF-2 mRNA expression decreased during the first days post-necrosis but was followed by a progressive rise in its expression coinciding with the appearance of the newly formed myofibers, reaching the maximum expression levels in DRGs composed of medium caliber basophilic regenerating myofibers (5-7 days post-necrosis). mdx degenerative-regenerative group typing, in conjunction with laser microdissection-based gene expression analysis, opens up a new approach to the molecular study of skeletal muscle regeneration.     

Neuron-Specific Transduction in the Rat Septohippocampal or Nigrostriatal Pathway by Recombinant Adeno-associated Virus Vectors

Viral vector-mediated gene transfer in brain can provide a means for gene therapy and functional studies. However, robust and persistent transgene expression in specific populations of the adult brain has been difficult to achieve. In an attempt to produce localized and persistent transduction in rat brain, we compared recombinant adeno-associated virus (rAAV) vectors incorporating either the immediate early cytomegalovirus (CMV) promoter or the neuron-specific enolase (NSE) promoter. Transduction in hippocampus resulting from the NSE promoter-containing construct was more efficient and persistent than that resulting from the CMV promoter-containing construct. Most hippocampal cells transduced with the NSE promoter had multipolar neuron morphology. Neurons with glutamatergic morphology were transduced weakly. In order to produce a local supply of neurotrophic factor to cells that degenerate under certain disease and experimental conditions, the NSE promoter was utilized to drive expression of brain-derived neurotrophic factor (BDNF) in medial septum or substantia nigra. In this construct, the NSE promoter drives dicistronic expression of BDNF and an enhanced version of green fluorescent protein (GFP). We estimated 3000–15,000 GFP-positive cells per injection of rAAV into septum or substantia nigra, a transduction ratio of 5–20 infectious virus particles per transduced cell. This frequency may be sufficient for trophic factor gene therapy as well as for investigating specific protein function in “topical (i.e., localized) transgenic” animals produced by rAAV.     

Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic delivery to the neonatal rat and minipig striatum. The efficiency of GFP expression and the phenotype of GFP-positive cells were assessed within the forebrain at different time points up to 12 months after surgery. Both rAAV1-GFP and rAAV5-GFP delivery resulted in transduction of the striatum as well as striatal input and output areas, including large parts of the cortex. In both species, rAAV5 resulted in a more widespread transgene expression compared to rAAV1. In neonatal rats, rAAV5 also transduced several other areas such as the olfactory bulbs, hippocampus, and septum. Phenotypic analysis of the GFP-positive cells, performed using immunohistochemistry and confocal microscopy, showed that most of the GFP-positive cells by either serotype were NeuN-positive neuronal profiles. The rAAV5 vector further displayed the ability to transduce non-neuronal cell types in both rats and pigs, albeit at a low frequency. Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity to study effects of genetic manipulation in this non-primate large animal species. Finally, we generated an atlas of the Göttingen minipig brain for guiding future studies in this large animal species.     

Myosin expression in hypertrophied fast twitch and slow tonic muscles of normal and dystrophic chickens.

Disruption of the development program of myosin gene expression has been reported in chicken muscular dystrophy. In the present report, the relationship between muscular dystrophy and the ability of muscle to respond to an increased work load with a transition in the myosin phenotype has been investigated. Hypertrophy of slow tonic anterior latissimus dorsi (ALD) and fast twitch patagialis (PAT) muscles was induced by overloading for 35 days and myosin expression was analyzed by electrophoresis and immunocytochemistry. Normal and dystrophic chicken ALD muscles have nearly identical proportions of SM-1 and SM-2 isomyosins and both exhibit an age-related repression of the SM-1 isomyosin which is enhanced and accelerated by overloading. Immunocytochemistry with anti-myosin heavy chain (MHC) antibodies demonstrates the appearance of nascent myofibers in overloaded ALD muscles from both normal and dystrophic chickens. A minor fast twitch fiber population is also identified which doubles in number with overloading in normal ALD muscles. There are only half as many fast twitch fibers in control dystrophic ALD muscles and this number does not increase with overloading. In contrast to ALD muscles, the isomyosin profile of normal and dystrophic PAT muscles is quite different. There is significantly more FM-3 and significantly less FM-1 isomyosin in the dystrophic PAT muscle. However, both normal and dystrophic PAT muscles exhibit an overload-induced accumulation of the FM-3 isomyosin. Immunocytochemistry reveals that, unlike the normal PAT muscle, the dystrophic PAT muscle contains a population of myofibers which express slow MHCs. As in the ALD muscle, overload-induced hypertrophy is associated with a repression of the SM-1 MHC in these fibers. Nascent myofiber formation does not occur in either normal or dystrophic overloaded PAT muscles.