神经管过表达BRE基因促进体节形成和分化

 摘要 目的：探讨BRE 基因对鸡胚胎早期体节发育的作用。方法：显微注射法将含有BRE基因的质粒注射入HH10期鸡胚神经管,活体胚胎细胞电穿孔方法转染半侧神经管,以另一侧为正常对照侧,原位杂交及荧光免疫组化法观察过表达BRE基因后体节的发育。结果：BRE基因在鸡胚表达始于神经板,而后高表达在脑泡、神经管及体节。过表达BRE基因后,神经管形态及PAX7 和FGF8表达与对照侧相比无明显异常,而实验侧体节PAX7标记发现生皮生肌节较大,FGF8表达与对照侧相比无明显异常。结论：过表达BRE基因对体节的形成和分化有促进作用,其机制可能与PAX7相关。     

高温致金黄地鼠神经管畸形中细胞凋亡的组织学观察

为研究细胞凋亡与高温致神经管畸形的关系，采用核固红－结晶紫染色方法和电镜技术，观察高温后胚胎发育变化。结果显示，高温后８ｈ胚胎发育迟滞，神经管延迟闭合或不闭合，胚胎各段尤期是头部的神经上皮和周围间充质的细胞凋亡比对照组增多，其超微结构改变为染色质浓缩，边聚，核膜皱缩，微绒毛消失并出现异常的包质突起；１６ｈ后细胞凋亡明显增多，胞质内出现了大量空泡，凋亡小体增多；     

BDNF、NGF对AD模型鼠脑移植区胚胆碱能神经元发育生长的调控

目的探讨脑源性神经营养因子（ＢＤＮＦ）、神经生长因子（ＮＧＦ）对ＡＤ模型鼠脑植入胚基底前脑后行为改善和移植区胚胆碱能神经元发育生长的影响。方法用使君子酸损毁ＳＤ大鼠基底大细胞核制成ＡＤ模型,分成ＢＤＮＦ、ＮＧＦ、ＢＤＮＦ＋ＮＧＦ及单纯移植对照４组。在额、顶叶皮质植入同种胚基底前脑细胞悬液,前３组含相应神经营养因子,移植后每２ｄ向侧脑室灌注相应神经营养因子共７次。移植后１、２．５和４个月时行避暗回避试验和跳台试验,最后脑切片经ＡＣｈＥ组织化学、ＣｈＡＴ和ＬＮＧＦＲ免疫组织化学等染色,对移植区中的神经元及纤维作定量分析和图像处理。结果移植后应用神经营养因子的动物较对照组行为改善明显迅速,ＢＤＮＦ＋ＮＧＦ组行为改善又较ＢＤＮＦ组或ＮＧＦ组明显。形态学证实,应用神经营养因子动物移植区中ＡＣｈＥ阳性神经元数、１６９００μｍ２网格中ＡＣｈＥ阳性纤维交点数及ＡＣｈＥ阳性神经元面积均优于对照组,ＢＤＮＦ＋ＮＧＦ组的结果又好于ＢＤＮＦ组或ＮＧＦ组。结论ＢＤＮＦ与ＮＧＦ一样能促进植入胚基底前脑的ＡＤ模型鼠行为改善和移植区胚胆碱能神经元的发育生长,ＢＤＮＦ和ＮＧＦ联合使用比单独使用一种因子更为有效。     

人脂肪间充质干细胞静脉移植修复脊髓损伤:相关因子及行为学评价

背景:研究证实脂肪间充质干细胞在体外经丹参等诱导剂诱导后可分化为神经元样细胞,因此有可能成为治疗脊髓损伤新的种子细胞。目的:探讨脂肪间充质干细胞尾静脉移植后,对急性闭合性脊髓损伤大鼠行为学及损伤脊髓组织中各因子表达的影响。方法:无菌条件下体外分离培养人脂肪间充质干细胞,传至第4代,将细胞收集并制成浓度为1×109L-1细胞悬液。盐水对照组、细胞移植组大鼠建立脊髓损伤模型,造模成功后1周,细胞移植组经尾静脉注射1mL干细胞悬液,盐水对照组同法注射等体积的生理盐水,模型对照组不做任何处理。结果与结论:与模型对照组和盐水对照组比较,细胞移植组大鼠后肢运动功能明显恢复,BBB评分明显升高(P0.05);胶质纤维酸性蛋白阳性表达明显减少(P0.05),神经元特异性烯醇化酶、巢蛋白的阳性表达均明显升高(P0.05)。移植后3d及1周,在损伤区及临近的脊髓节段可见经荧光染料标记的脂肪间充质干细胞,主要聚集在受损伤脊髓节段1cm范围内,呈不均匀分布。提示急性闭合性脊髓损伤大鼠经尾静脉移植人脂肪间充质干细胞后,其行为学得到改善,受损脊髓节段局部神经元细胞分化明显增多,修复速度加快。     

局部注射骨髓间充质干细胞治疗大鼠脊髓损伤:运动功能有改善吗?

背景:目前研究多为骨髓间充质干细胞的体外培养及细胞移植对颅内疾病的治疗,对植入细胞在损伤脊髓中的成活、分化、迁移、结构重建等了解有限。目的:探讨局部骨髓间充质干细胞移植在脊髓损伤修复中的作用和骨髓间充质干细胞替代治疗的可行性。方法:成年健康雌性SD大鼠随机分为细胞移植组和对照组,建立SD大鼠脊髓横断损伤模型,伤后即刻分别向损伤区局部移植大鼠骨髓间充质干细胞悬液或无钙镁磷酸缓冲液。在术前和术后1d,1周,2周,3周,4周和8周进行BBB评分,观测大鼠的运动功能,并于移植后1周免疫组织化学染色法观察BrdU标记的骨髓间充质干细胞在脊髓损伤处的存活情况,移植后4周进行损伤脊髓的大体观察和组织学检测。结果与结论:移植后第1～8周细胞移植组BBB评分均髙于对照组;术后1周免疫组织化学染色结果显示在细胞移植组大鼠脊髓远端检测到BrdU阳性细胞,术后4周脊髓损伤处发现有神经纤维。证实通过损伤后立即局部注射的方式将骨髓间充质干细胞移植进大鼠脊髓损伤区,细胞可在损伤区存活;存活的骨髓间充质干细胞可分化为神经元,在损伤局部形成神经元通路,从而促进脊髓神经纤维传导功能的恢复,并促进高位脊髓损伤后大鼠后肢运动功能恢复。     

hCG对T细胞表型及功能亚群的作用

分别用ＣＤ３、ＣＤ４及ＣＤ８分子作为检测Ｔ细胞表型亚群的指标，以ＩＦＮγ和ＩＬ－４作为检测ＣＤ４Ｔ细胞中Ｔｈ１与Ｔｈ２功能亚群的指标，观察了ｈＣＧ对Ｔ细胞各类亚群的作用。结果证明一定剂量范围内的ｈＣＧ对上述各Ｔ细胞功能亚群均有显著抑制作用。用抗ｈＣＧ各亚单位的单抗封闭ｈＣＧ后作上述试验，证明抗ｈＣＧβ（１０号）及抗完整ｈＣＧ（１７号）单抗可阻断上述抑制作用，而抗ｈＣＧα单抗（４号）则无此作用，阐明了ｈＣＧ分子上参与此抑制作用的表位所在。     

人骨髓间充质干细胞移植老年性痴呆大鼠认知能力和海马超微结构的变化

背景:因发病机制不明,目前尚无治愈老年性痴呆的有效方法。现临床上主要是采用药物治疗,而骨髓间充质干细胞的替代治疗尚处于基础研究阶段,其海马移植后对老年性痴呆认知能力的影响未见报道。目的:探讨人骨髓间充质干细胞移植对老年性痴呆大鼠认知能力和海马超微结构的影响。方法:老年雄性Wistar大鼠30只,制备自然衰老痴呆模型,造模后随机分为3组,选取双侧海马为移植区,分化细胞移植组注射定向神经细胞诱导分化的人骨髓间充质干细胞悬液4μL(2×105个细胞),干细胞移植组注射等量常规培养的人骨髓间充质干细胞,模型组注射等量生理盐水。通过Y迷宫试验测定大鼠的学习、记忆能力,透射电镜观察海马区超微结构。结果与结论:与移植前大鼠学习、记忆分数比较,移植后12周模型组均显著下降(P0.01),干细胞移植组均有所提高(P0.05),分化细胞移植组均显著提高(P0.01)。移植后12周与模型组比较,干细胞移植组、分化细胞移植组大鼠学习、记忆分数均显著提高(P0.01)。电镜观察模型组大鼠海马区神经细胞可见明显损伤,干细胞移植组损伤减轻,分化细胞移植组多数神经细胞结构正常。证实骨髓间充质干细胞移植可以提高老年性痴呆大鼠的认知能力,且定向神经诱导分化的骨髓间充质干细胞移植治疗效果优于未分化的骨髓间充质干细胞,提示骨髓间充质干细胞减少海马组织神经细胞变性坏死可能是其改善老年性痴呆大鼠认知功能障碍的作用机制之一。     

兔骨髓间充质干细胞构建心脏电传导通路的潜能

背景:自体干细胞移植到心脏是目前治疗心力衰竭的一个研究方向,但自体骨髓间充质干细胞移植针对心脏传导系统方面的研究相对较少。目的:评价兔骨髓间充质干细胞是否具有治疗心脏传导阻滞方面疾病的潜能。方法:获取兔骨髓间充质干细胞并利用5-氮胞苷诱导为心肌样细胞。14只新西兰大白兔开胸手术后,将左心耳左心室前壁进行缝合(实验组8只,对照组6只)。术后1个月将实验组兔经5-氮胞苷诱导4周的自体骨髓间充质干细胞采用DAPI标记后,开胸直视下注射入左心室前壁-左心耳左心室缝合瘢痕区-左心耳;对照组予以培养基替代细胞。细胞移植后1个月再次开胸暴露心脏,在左心耳、左室前壁分别插入电极,进行心脏房室旁道的电生理检测,观察骨髓间充质干细胞是否在左心房左心室缝合瘢痕区形成房室旁道。结果与结论:细胞移植到兔左心耳左心室缝合区后,实验组有2只兔子在行电生理刺激时心电图提示有房室旁道形成。移植后心脏冰冻切片在荧光显镜下能观察到移植的细胞在左心室和缝合区存活,而对照组则未有上述表现。骨髓间充质干细胞表达Cx43并与心肌细胞形成间隙连接的特点结合在细胞移植组中形成房室旁道的心脏电生理检测表现,提示骨髓间充质干细胞具备能够用于治疗心脏传导系统阻滞相关疾病的可能。     

骨髓间充质干细胞旁分泌作用与脑缺血后的细胞凋亡

背景:骨髓间充质干细胞移植治疗脑缺血的机制之一是骨髓间充质干细胞的旁分泌作用,而目前对于这一机制的研究报道较少。目的:观察骨髓间充质干细胞旁分泌作用对脑缺血后细胞凋亡的抑制作用并探索相关机制。方法:体外培养大鼠骨髓间充质干细胞,建立大鼠大脑中动脉缺血模型。24只SD大鼠随机数字表法分为4组,每组6只。细胞移植给药组:大鼠纹状体内移植骨髓间充质干细胞后给予ERK1/2抑制剂U0126;非移植给药组:注射等量的PBS后给予U0126;细胞移植对照组:移植骨髓间充质干细胞后给予溶剂对照;非移植对照组:注射等量的PBS后给予溶剂对照。7d后通过Westernblot检测血管内皮细胞生长因子、磷酸化ERK1/2蛋白的表达;TUNEL染色检测梗死区周围及皮质区细胞凋亡情况。结果与结论:细胞移植组较非移植组大鼠纹状体内血管内皮细胞生长因子蛋白的表达明显增高,磷酸化ERK1/2表达增强,细胞凋亡数明显减少;经U0126处理后,血管内皮细胞生长因子的表达没有变化,而随着磷酸化ERK1/2的表达受到抑制,细胞凋亡数明显增高。提示骨髓间充质干细胞在大脑纹状体内可以旁分泌血管内皮细胞生长因子,并通过激活ERK1/2抑制了脑梗死区细胞的凋亡。     

缺损运动神经元的大鼠脊髓内胚胎脊髓移植物的存活与生长

ＳＤ大鼠于出生２４ｈ内行左侧坐骨神经钳压术，造成左侧腰段脊髓前角运动神经元缺损。５￣１２周后，作为脊髓移植的受体鼠。供体为胚龄１３￣１４ｄ的ＳＤ大鼠胚胎，取其脊髓腹侧组织块作为移植物，植入受体鼠左侧腰段脊髓的背外侧部。将受体鼠右侧带神经的Ｍｕ长伸肌移放到脊椎旁，神经的断端插入胚胎移植物处。术后动物存活６￣８周，行组织学（包括电镜）、ＡＣｈＥ组织化学、ＣｈＡＴ免疫组织化学和ＨＲＰ逆行追踪等观察。结果     

Morphological analysis of the role of the neural tube and notochord in the development of somites

The differentiation of avian somites and skeletal muscles, which themselves are derived from somites, was studied in ovo after the isolation of the unsegmented segmental plate from the notochord and/or neural tube by surgical operations at the level of the segmental plate. In each experiment, the newly formed somites had a normal histological structure, with an outer epithelial somite and core cells in the somitocoeles. Thereafter, the three derivatives of the somites (dermatome, myotome and sclerotome) reacted differently to the different operations. When the somites developed without the notochord, only somitocoele cells showed massive cell death, and muscles developed regardless of the presence or absence of the neural tube. On the contrary, no cell death was observed in any part of the somites that were formed with the neural tube or the notochord present, and muscle cells developed. However, in those embryos that retained only the notochord, striated muscles developed only in the lateral body wall. In each of the experimental operations, the surface ectoderm always covered the somites, and, regardless of the state of sclerotome and/or myotome differentiation, the dermatome always survived. These histological observations indicate that the first step in somite formation is independent of axial structures. The results further suggest that the notochord may produce diffusible factors that are necessary for the survival and further development of sclerotomal cells, and that both the neural tube and notochord can support muscle differentiation. However, it is likely that each structure has a relationship to the development of epaxial muscles and hypaxial muscles respectively. Furthermore, an intimate relationship may also exist between the surface ectoderm and the development of the dermatome.     

The eventful somite: patterning, fate determination and cell division in the somite

The segmental somites not only determine the vertebrate body plan, but also represent turntables of cell fates. The somite is initially naive in terms of its fate restriction as shown by grafting and rotation experiments whereby ectopically grafted or rotated tissue of newly formed somites yielded the same pattern of normal derivatives. Somitic derivatives are determined by local signalling between adjacent embryonic tissues, in particular the neural tube, notochord, surface ectoderm and the somitic compartments themselves. The correct spatio-temporal specification of the deriving tissues, skeletal muscle, cartilage, endothelia and connective tissue is achieved by a sequence of morphogenetic changes of the paraxial mesoderm, eventually leading to the three transitory somitic compartments: dermomyotome, myotome and sclerotome. These structures are specified along a double gradient from dorsal to ventral and from medial to lateral. The establishment and controlled disruption of the epithelial state of the somitic compartments are crucial for development. In this article, we give a synopsis of some of the most important signalling events involved in somite patterning and cell fate decisions. Particular emphasis has been laid on the issue of epithelio-mesenchymal transition and different types of cell division in the somite.     

Somitogenesis in cultured embryos of the Japanese quail,Coturnix coturnix japonica

The ability of unsegmented paraxial mesoderm from Japanese quail embryos to form somites was studied by culturing pieces of embryos, containing the segmental plates, on an agar medium. In the first experiments, two explants were prepared from each donor embryo. Both explants contained a segmental plate and neural tube, but only one contained notochord. The explants containing notochord formed 11.4 ± 2.1 somites, while the explants without notochord formed 11.1 ± 1.3 somites. It was concluded that explants containing Japanese quail segmental plates readily form somites in culture and that the continued presence of the notochord is not required for these somites to form. In a second series of experiments, one explant from each donor embryo contained neural tube and notochord along with the segmental plate, while the corresponding explant did not contain axial structures. The results, which were similar to those obtained in the first experiments, indicated that neither neural tube nor notochord is required for somitogenesis in vitro. Additional experiments demonstrated that bilateral symmetry extends to the unsegmented somite mesoderm, where there was a strong tendency for each segmental plate of a given embryo to form the same number of somites. It was also shown that over a three-fold range of segmental plate length, there was only a slight tendency for shorter segmental plates to make fewer somites. It was estimated that Japanese quail embryos having five to 21 pairs of somites have segmental plates that represent 11.3 ± 2.9 prospective somites each.     

The role of the notochord in vertebral column formation

The backbone or vertebral column is the defining feature of vertebrates and is clearly metameric. Given that vertebrae arise from segmented paraxial mesoderm in the embryo, this metamerism is not surprising. Fate mapping studies in a variety of species have shown that ventromedial sclerotome cells of the differentiated somite contribute to the developing vertebrae and ribs. Nevertheless, extensive studies in amniote embryos have produced conflicting data on exactly how embryonic segments relate to those of the adult. To date, much attention has focused on the derivatives of the somites, while relatively little is known about the contribution of other tissues to the formation of the vertebral column. In particular, while it is clear that signals from the notochord induce and maintain proliferation of the sclerotome, and later promote chondrogenesis, the role of the notochord in vertebral segmentation has been largely overlooked. Here, we review the established role of the notochord in vertebral development, and suggest an additional role for the notochord in the segmental patterning of the vertebral column.     

Cells of all somitic compartments are determined with respect to segmental identity.

Development of somite cells is orchestrated by two regulatory processes. Differentiation of cells from the various somitic compartments into different anlagen and tissues is regulated by extrinsic signals from neighboring structures such as the notochord, neural tube, and surface ectoderm. Morphogenesis of these anlagen to form specific structures according to the segmental identity of each somite is specified by segment-specific positional information, based on the Hox-code. It has been shown that following experimental rotation of presomitic mesoderm or newly formed somites, paraxial mesodermal cells adapt to the altered signaling environment and differentiate according to their new orientation. In contrast, presomitic mesoderm or newly formed somites transplanted to different segmental levels keep their primordial segmental identity and form ectopic structures according to their original position. To determine whether all cells of a segment, including the dorsal and ventral compartment, share the same segmental identity, presomitic mesoderm or newly formed somites were rotated and transplanted from thoracic to cervical level. These experiments show that cells from all compartments of a segment are able to interpret extrinsic local signals correctly, but form structures according to their original positional information and maintain their original Hox expression in the new environment.     

The development of the avian vertebral column

Segmentation of the paraxial mesoderm leads to somite formation. The underlying molecular mechanisms involve the oscillation of ”clock-genes” like c-hairy-1 and lunatic fringe indicative of an implication of the Notch signaling pathway. The cranio-caudal polarity of each segment is already established in the cranial part of the segmental plate and accompanied by the expression of genes like Delta1, Mesp1, Mesp2, Uncx-1, and EphA4 which are restricted to one half of the prospective somite. Dorsoventral compartmentalization of somites leads to the development of the dermomyotome and the sclerotome, the latter forming as a consequence of an epithelio-to-mesenchymal transition of the ventral part of the somite. The sclerotome cells express Pax-1 and Pax-9, which are induced by notochordal signals mediated by sonic hedgehog (Shh) and noggin. The craniocaudal somite compartmentalization that becomes visible in the sclerotomes is the prerequisite for the segmental pattern of the peripheral nervous system and the formation of the vertebrae and ribs, whose boundaries are shifted half a segment compared to the sclerotome boundaries. Sclerotome development is characterized by the formation of three subcompartments giving rise to different parts of the axial skeleton and ribs. The lateral sclerotome gives rise to the laminae and pedicles of the neural arches and to the ribs. Its development depends on signals from the notochord and the myotome. The ventral sclerotome giving rise to the vertebral bodies and intervertebral discs is made up of Pax-1 expressing cells that have invaded the perinotochordal space. The dorsal sclerotome is formed by cells that migrate from the dorso-medial angle of the sclerotome into the space between the roof plate of the neural tube and the dermis. These cells express the genes Msx1 and Msx2, which are induced by BMP-4 secreted from the roof plate, and they later form the dorsal part of the neural arch and the spinous process. The formation of the ventral and dorsal sclerotome requires directed migration of sclerotome cells. The regionalization of the paraxial mesoderm occurs by a combination of functionally Hox genes, the Hox code, and determines the segment identity. The development of the vertebral column is a consequence of a segment-specific balance between proliferation, apoptosis and differentiation of cells. Accepted: 25 May 2000     

The generation of vertebral segmental patterning in the chick embryo

We have carried out a series of experimental manipulations in the chick embryo to assess whether the notochord, neural tube and spinal nerves influence segmental patterning of the vertebral column. Using Pax1 expression in the somite-derived sclerotomes as a marker for segmentation of the developing intervertebral disc, our results exclude such an influence. In contrast to certain teleost species, where the notochord has been shown to generate segmentation of the vertebral bodies (chordacentra), these experiments indicate that segmental patterning of the avian vertebral column arises autonomously in the somite mesoderm. We suggest that in amniotes, the subdivision of each sclerotome into non-miscible anterior and posterior halves plays a critical role in establishing vertebral segmentation, and in maintaining left/right alignment of the developing vertebral elements at the body midline.     

Urokinase expression during the epithelial-mesenchymal transformation of the avian somite.

Early events in the morphogenesis of the axial skeleton involve an epithelial-mesenchymal transformation of the somites. Cells of the ventromedial wall of the somite (the sclerotome) migrate to regions surrounding the notochord and neural tube and condense to form the cartilage model of the vertebrae. Urokinase activity in the axial region of the quail embryo trunk was found to increase during these stages. In situ hybridization localized urokinase mRNA expression in this region and suggests an important role for this protease in the process of cell migration and matrix remodeling during development of the axial skeleton.     

The ventralizing effect of the notochord on somite differentiation in chick embryos

The dorso-ventral pattern formation of the somites becomes manifest by the formation of the epithelially organized dorsal dermomyotome and the mesenchymal ventrally situated sclerotome. While the dermomyotome gives rise to dermis and muscle, the sclerotome differentiates into cartilage and bone of the axial skeleton. The onset of muscle differentiation can be visualized by immunohistochemistry for proteins associated with muscle contractility, e.g. desmin. The sclerotome cells and the epithelial ventral half of the somite express Pax-1, a member of a gene family with a sequence similarity to Drosophila paired-box-containing genes. In the present study, changes of Pax-1 expression were studied after grafting an additional notochord into the paraxial mesoderm region. The influence of the notochord and the floor-plate on dermomyotome formation and myotome differentiation has also been investigated. The notochord is found to exert a ventralizing effect on the establishment of the dorso-ventral pattern in the somites. Notochord grafts lead to a suppression of the formation and differentiation of the dorsal somitic derivatives. Simultaneously, a widening of the Pax-1-expressing domain in the sclerotome can be observed. In contrast, grafted roof-plate and aorta do not interfere with dorso-ventral patterning of the somitic derivatives.     

Rostro-caudal polarity in the avian somite related to paraxial segmentation

Segmental organization of the vertebrate body is one of the major patterns arising during embryonic development. Somites that play an important role in this process show intrinsic patterns of gene expression and differentiation. The somites become polarized in all three dimensions, rostrocaudal, mediolateral and dorsoventral, the quadrants giving rise to several tissue components. The timing of polarization was studied by means of antibodies against HNK-1, tenascin and neurofilament. Whole mounts and serial sections of quail and chick embryos show that somites are already polarized at the moment of their segregation from the segmental plate. The rostral hemisomite carries the HNK-1 epitope preferentially, while the caudal hemisomite stains more strongly for tenascin. HNK-1-stained areas in the segmental plate strongly relate to the notochordal sheath, suggesting that axial structures determine the fate of paraxial structures. Neural crest cells were only seen to colonize the rostral part of a somite after they had differentiated into HNK-1 positive cells. Their colonization pattern seems to be guided by the segmental organization of the somite. Moreover, this somite organization probably dictates the organization of both sensory and motor fibres converging towards the segmental dorsal root ganglia, justifying a shift in the connections between neural tube and somites. This segmental shift takes place over one quarter of a somite length in a rostral direction.