臂丛损伤再生中GAP-43 mRNA及其蛋白的表达

目的：分析臂丛损伤后脊髓前角运动神经元表达GAP-43 mRNA及其蛋白的变化规律，探讨神经损伤再生的机制。方法：建立3种臂丛损伤模型：右C₇前根撕脱（A组）；右C₇前根撕脱＋同侧C5-T₁后根断离（B组）；右C₇前根撕脱+右C₅C₆间脊髓半横断（C组）。用荧光定量RT-PCR方法检测术后14 d时 C₇前角GAP-43 mRNA的表达量。用免疫组化方法检测术后1、 3、 7、14 d脊髓前角GAP-43免疫阳性运动神经元的表达。结果：对照组C₇前角GAP-43 mRNA呈低表达，损伤组GAP-43 mRNA表达显著上调。损伤组术后1 d、3 d时均未见C₇前角 GAP-43免疫阳性神经元，术后7 d各损伤组GAP-43免疫阳性神经元开始出现，14 d时免疫阳性神经元数目达到高峰。3组间比较，C组表达量最高，B组最低，A组居中。结论：臂丛损伤诱导运动神经元GAP-43 mRNA及其蛋白表达上调，GAP-43合成增加是神经元蛋白重组所致，与轴索再生和神经功能重建有关。     

臂丛损伤后脊髓前角运动神经元超微结构改变

目的探讨大鼠臂丛损伤导致脊髓前角运动神经元死亡的机制。方法成年雄性SD大鼠24只,其中对照组6只,损伤组18只。建立3种臂丛损伤模型:右C7前根撕脱(A组);右C7前根撕脱+同侧C5～T1后根离断(B组);右C7前根撕脱+右C5与C6之间脊髓半横断(C组)。术后14d取C7节段脊髓,采用尼氏染色方法和透射电镜技术,观察脊髓前角运动神经元的存活率及其超微结构改变。结果术后2周A组脊髓前角运动神经元的存活率最高,B组居中,C组最低。3个臂丛损伤组C7前角均可见凋亡特征性改变:运动神经元内核染色质聚集靠边,核固缩、碎裂、核膜皱褶并内陷,并有染色质团块形成的凋亡小体。细胞体积缩小,胞浆内细胞器密集,线粒体轻度肿胀,核周粗面内质网减少,游离核糖体增多,胞浆内可见较多的空泡。神经元胞体周围的有髓神经纤维和无髓神经纤维呈轻度肿胀,髓鞘的板层结构消失。结论臂丛损伤诱导脊髓运动神经元死亡途径中存在凋亡和坏死两种机制,运动神经元可形成凋亡小体。     

臂丛损伤脊髓运动神经元与神经根GAP-43 mRNA表达

目的：探讨臂丛根性撕脱伤后脊髓腹角运动神经元胞体及其神经根GAP-43 mRNA的表达变化及其影响因素,为臂丛损伤的修复治疗提供理论依据.方法：本实验创立三种臂丛根性撕脱伤模型：C7前根撕脱（Ⅰ组）;C7前根撕脱＋切断同侧C5～T1后根（Ⅱ组）;C7前根撕脱＋C5和C6之间作同侧脊髓半横断（Ⅲ组）.术后2周按CBS评分标准检查动物神经缺失症状,用SYBR Green荧光定量RT-PCR方法检测脊髓腹角运动神经元胞体及其神经根GAP-43 mRNA的表达改变.结果：根据CBS评分标准,对照组计为0分,Ⅰ组计分较低、Ⅲ组计分最高.对照组C7神经元胞体和C7神经根中GAP-43 mRNA表达量相近,但三种损伤组术后2周神经元胞体内GAP-43 mRNA表达均上调,而神经根内表达却下调.结论：（1）臂丛根性撕脱伤后脊髓腹角运动神经元胞体GAP-43 mRNA表达受突触前机制的调控;（2）臂丛损伤2周时神经元胞体内GAP-43 mRNA表达呈现高峰期,此时进行神经移位术将显著提高神经修复的效果.     

臂丛根性撕脱伤激活脊髓表达磷酸化CaMKⅡ的时程及定位研究

目的研究大鼠臂丛根性撕脱伤后磷酸化CaMKⅡ的时间和空间定位。方法将SD大鼠(6～8周龄)在手术显微镜下做臂丛根性撕脱伤手术后,应用Western blot方法测定相应脊髓节段(C7、C8)磷酸化CaMKⅡ在1、2、3 d时间点及正常大鼠的表达水平。同时应用免疫荧光染色方法检测目标蛋白在1 d时间点大鼠脊髓节段的表达水平及空间定位。结果正常大鼠脊髓相应节段检测不到磷酸化CaMKⅡ,在臂丛根性撕脱伤后1～2 d对应脊髓节段的磷酸化CaMKⅡ表达明显升高,撕脱伤后3d明显下降并接近正常水平;免疫荧光结果显示:臂丛根性撕脱伤后1 d,损伤侧脊髓前角运动神经元和中间神经元明显表达磷酸化CaMKⅡ,对侧脊髓前角运动神经元和中间神经元也有表达,但强度较撕脱侧明显减弱。结论臂丛根性撕脱伤诱导磷酸化CaMKⅡ在损伤节段1 d时高表达,然而在3 d时逐步下降到正常水平。     

臂丛根性撕脱伤激活脊髓表达磷酸化CaMKⅡ的时程及定位研究

目的研究大鼠臂丛根性撕脱伤后磷酸化CaMKⅡ的时间和空间定位。方法将SD大鼠（6-8周龄）在手术显微镜下做臂丛根性撕脱伤手术后,应用Western blot方法测定相应脊髓节段（C7、C8）磷酸化CaMKⅡ在1、2、3d时间点及正常大鼠的表达水平。同时应用免疫荧光染色方法检测目标蛋白在1d时间点大鼠脊髓节段的表达水平及空间定位。结果正常大鼠脊髓相应节段检测不到磷酸化CaMKⅡ,在臂丛根性撕脱伤后1～2d对应脊髓节段的磷酸化CaMKⅡ表达明显升高,撕脱伤后3d明显下降并接近正常水平;免疫荧光结果显示：臂丛根性撕脱伤后1d,损伤侧脊髓前角运动神经元和中间神经元明显表达磷酸化CaMKⅡ,对侧脊髓前角运动神经元和中间神经元也有表达。但强度较撕脱侧明显减弱。结论臂丛根性撕脱伤诱导磷酸化CaMKⅡ在损伤节段1d时高表达,然而在3d时逐步下降到正常水平。     

RNA干扰技术抑制nNOS基因对根性撕脱伤脊髓运动神经元的影响

目的研究神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)基因在臂丛神经根撕脱伤中的作用地位。方法设计、筛选并构建针对nNOS基因的siRNA表达载体,建立C5～T1神经根撕脱伤SD雄性大鼠模型,于撕脱后14d脊髓鞘内给予nNOS的siRNA干预,3d后用NADPH-d酶组化反应结合中性红染色评估撕脱伤脊髓运动神经元的nNOS基因表达水平和创伤神经元的存活率。结果臂丛C5～T1根性撕脱伤17d后,C7节段损伤侧前角运动神经元的存活率在siRNA组、siRNA序列对照组和生理盐水组分别为(80.51±9.16)%、(88.26±2.95)%和(89.13±3.47)%;C7节段损伤侧前角运动神经元的nNOS阳性率为(18.10±6.63)%、(42.21±2.29)%和(39.46±2.41)%。nNOS的siRNA能显著减低撕脱伤诱导的nNOS蛋白在前角运动神经元内的表达水平。虽然与对照组相比,nNOS-siRNA组撕脱伤后运动神经元存活率有下降趋势,nNOS的siRNA但并未对撕脱伤导致的运功神经元的死亡造成显著影响。结论鞘内应用siRNA技术能下调撕脱伤后大鼠脊髓运动神经元内nNOS蛋白的表达,nNOS基因有可能发挥维持撕脱伤后运动神经元存活的作用。     

葛根素对臂丛根性撕脱后脊髓MMP-9蛋白表达的影响

目的　探讨大鼠臂丛根性撕脱后脊髓基质金属蛋白酶-9（MMP-9）蛋白的表达变化以及葛根素对其表达的影响。 方法　成年雄性SD大鼠174只，随机分为正常组、模型组、葛根素低、中、高处理组。模型组和葛根素处理组行右侧C5~C7脊神经前根撕脱术，术后腹腔注射给药。Western Blot法检测C5~C7节段脊髓MMP-9蛋白的表达。 结果　与正常组比较，臂丛神经根性撕脱后1 d时MMP-9蛋白表达达高峰，3 d降至接近正常，1周后下降至较低水平。葛根素处理组脊髓组织MMP-9蛋白表达在1 d时比模型组显著降低。 结论　MMP-9可能参与臂丛根性撕脱后早期损伤反应，且葛根素可能有助于减轻该种损伤。     

幼年大鼠臂丛根性损伤模型的构建及其评价

目的：构建幼年大鼠臂丛损伤动物模型并对该模型进行评价。方法：选取20只幼年（P18）SPF级SD大鼠，手术撕脱实验组大鼠左侧臂丛根（C5～C7），造成大鼠臂丛根性撕伤，观察大鼠行为学和脊髓前角运动神经元的组织病理学变化。结果：实验组SD大鼠行为学出现明显异常，并且脊髓前角运动神经元在数量上较对照组显著减少；透射电镜显示实验组脊髓前角运动神经元超微结构也出现明显变化。结论：该方法可成功构建幼年SD大鼠臂丛根性撕脱伤动物模型，为分娩性臂丛损伤研究提供了一个科学可行的实验平台。     

大鼠臂丛前根撕脱延期再植回对运动神经元存活的作用

目的：建立大鼠臂丛前根撕脱延期再植回模型，研究脊髓前角运动神经元的存活与再生。方法：采用FB逆行示踪法结合NADPH—d组化法与中性红复染，计数阳性神经元．结果：前根撕脱延期3、7、14d再植回组，动物存活3周及6周，脊髓前角运动神经元的存活率分别为79％和75％、58％和51％、57％和50％；NOS阳性神经元的表达率分别为18％和13％、30％和8％、20％和7％。仅做前根撕脱组动物，脊髓前角运动神经元存活率为41％和29％，NOS阳性神经元的表达率为55％和58％。结论：前根撕脱延期再植回能促进脊髓前角运动神经元的存活与再生。     

大鼠坐骨神经压榨损伤后早期降钙素基因相关肽的变化

目的：研究大鼠坐骨神经压榨损伤后早期降钙素基因相关肽(CGRP)的动态变化及与神经再生的关系。方法：SD大鼠坐骨神经压榨损伤后分别存活1d到21d,免疫组化技术观察CGRP分布和含量的变化。结果：(1)1d组神经CGRP大量堆积,压榨近端明显多于远端,随即下降,21d组基本消失。(2)1d组背根节、脊髓后角和前角CGRP开始增高,并分别在3～5d、5～7d和7d组达峰值,随后渐降,21d组脊髓前角CGRP阳性运动神经元仍明显高于假手术组和对照侧。结论：神经压榨损伤后CGRP表达变化呈明显的时空模式,可能参与了神经元保护并介导了损伤信号的传导。     

Co-ordination of cough and swallow in vivo and in silico

Coughing and swallowing are airway-protective behaviours. The pharyngeal phase of swallowing prevents aspiration of oral material (saliva, food and liquid) by epiglottal movement, laryngeal adduction and clearing of the mouth and pharynx. Coughing is an aspiration-response behaviour that removes material from the airway. Co-ordination of these behaviours is vital to protect the airway from further aspiration-promoting events, such as a swallowing during the inspiratory phase of coughing. The operational characteristics, primary strategies and peripheral inputs that co-ordinate coughing and swallowing are unknown. This lack of knowledge impedes understanding and treatment of deficits in airway protection, such as the co-occurrence of dystussia and dysphagia common in Parkinson's and Alzheimer's diseases, as well as stroke.     

ALA和HMME水凝胶栓剂的制备和体内外释药研究

目的 制备光敏剂5-氨基酮戊酸(ALA)和血卟啉单甲醚(HMME)水凝胶栓剂,评价其对直肠肿瘤组织的光敏剂递送效率.方法 将皮下移植人直肠癌细胞SW837的BALB/c小鼠随机分为水凝胶栓剂直肠局部给药组、皮肤局部给药组、瘤内注射给药组和静脉注射给药组.用荧光光谱仪测量直肠壁、皮肤和皮下肿瘤中原卟啉(PpⅨ)和HMME的浓度,荧光光谱系统测定相应的光敏剂分布情况.结果 ALA水凝胶栓剂直肠局部给药组的PpⅨ浓度分别是皮肤局部给药组的9.76倍(1 h)和5.80倍(3 h),差异均具有统计学意义(均P＜0.05).皮肤局部给药后2h,ALA在肿瘤组织内达到最大穿透深度(3～6 mm).而HMME水凝胶栓剂直肠局部给药后,直肠壁中的HMME浓度极低,且皮肤局部给药后的最大肿瘤穿透深度不足2 mm.结论 与皮肤相比,ALA更易穿透黏膜屏障,以水凝胶栓剂形式直肠局部给药有望成为ALA用于光动力疗法治疗直肠癌的一种给药方式.     

Incorporation and Disappearance of Sulfate in Different Regions of Mouse and Rat Costal Cartilage in vivo and in vitro

A rapid and simple method for liquid scintillation counting of ³³S-sulfate in biological tissues is described. Sulfate incorporation per mg dry weight into selected lateral, medial and intermediate regions of ribs was studied using costal cartilage from rats and mice. In vitro, cartilage pieces embracing the osteochondral junction had 2–4 times larger incorporation rate than the remaining homogeneous parts of the ribs, both if entire rib cages or stripped and diced cartilage were incubated. After in vivo injection to rats and mice the incorporation rates of sulfate into the region of the osteochondral junction was 2.6 times that found in “resting” cartilage in rats and 4.4 times in mice. The ³⁵S-sulfate uptake in the ribs per mg dry cartilage diminished in the caudal direction. Growth of the chest cage was mainly a longitudinal growth of the bony segments. Marked differences in the disappearance rates of previously incorporated sulfate were found in different regions along the rib. Rapid sulfate disappearance was found in the bony segment which, however, had incorporated only one seventh of the amount per mg taken up by “resting” cartilage. The “resting” cartilage segments exhibited a steady slow disappearance rate for sulfate and the osteochondral junction region consisting of several differently active tissues showed a rapid sulfate disappearance in the beginning followed by a slow clearance after 2 weeks similar to that of “resting” cartilage. The necessity of careful selection of costal cartilage samples with respect to regional differences is emphasized.     

Characteristics of Tremor in Normal Subjects and in the Diagnosis and Treatment of Parkinsonism

Tensotremorography was used to record voluntary forces and to study the characteristics of the involuntary and voluntary components of isometrically recorded hand strength. The frequency ranges for changes in the spectral density of oscillations recorded here supported the existence of two suprasegmental systems associated with voluntary control and continuous regulation of force maintaining or holding a posture. Cross-correlation analysis of hand force maintained in conditions of visual feedback in normal conditions and in conditions of central disorders of the movement control system is presented.     

Expression of EpCam and villin in Barrett's esophagus and in gastric cardia

In the current study we aimed to clarify the potential of EpCAM and villin as in vivo biomarkers for both Barrett esophagus (BE)-associated neoplasia and BE versus cardiac mucosa. Immunohistochemical staining in BE with various degrees of intraepithelial neoplasia (IN), Barrett carcinoma (BC) and in normal cardiac mucosa (CM) revealed a lack of EpCam and villin in squamous esophageal epithelium. All specimens of IN and BC showed EpCam with varying staining intensities. In 57% of CM samples a weak signal was detected; the remainder displayed strong EpCam expression. Villin was found in 97% of BE specimens and in all those with IN; 37% of BC and 75% of CM specimens were~also positive. We conclude that expression of EpCam and villin differs only between squamous epithelium and BE. Determination of these proteins does not allow discrimination between different degrees of neoplasia or between esophageal intestinal metaplasia and cardiac mucosa.     

Expression of desmin and myoglobin in rhabdomyosarcomas and in developing skeletal muscle

Immunohistochemical staining for desmin and myoglobin was investigated in 35 rhabdomyosarcomas from young people and in skeletal muscle from 16 human fetuses of known gestational age. Twenty-nine of the rhabdomyosarcomas expressed desmin but six undifferentiated or poorly-differentiated tumours were desmin negative. Of the desmin positive cases, most undifferentiated or poorly-differentiated sarcomas expressed desmin alone (12/35). Tumours with increasing rhabdomyoblastomic differentiation co-expressed myoglobin (9/35) and well-differentiated examples also contained cross-striations (7/35). Skeletal muscle from fetuses aged 8 weeks or less consisted mainly of primitive desmin negative round cells. As the cells began to differentiate they quickly expressed desmin and, at approximately 10 weeks, myoglobin was expressed and cross-striations were seen. The combined results strengthen the view that desmin (within a strictly defined context of round cell tumours in young people) is a reliable marker for rhabdomyoblastic differentiation. Support is also given to the notion that very primitive rhabdomyosarcomas may be desmin-negative, although the difficulties of establishing firm diagnoses for some of these tumours is emphasized.     

Fine structure of bovine morulae and blastocysts in vivo and in vitro

 The ultrastructure of bovine morulae and blastocysts developed from in vitro-matured and -fertilized oocytes in a serum-supplemented medium was compared with that of morulae and blastocysts collected non-surgically from superovulated cows. In the in vivo-derived morulae, two characteristic cells types could be identified by the electron-density of their cytoplasm and by their ultrastructural features. One type appeared light in color with low electron-dense cytoplasm. These cells were located in the peripheral layer of the cluster of blastomeres, possessed numerous cellular organelles such as mitochondria and Golgi apparatus and had microvilli projecting into the perivitelline space. The other cell type was distinguished by cytoplasm that stained more densely than that of the lighter-appearing cells. The darker-appearing cells generally possessed fewer organelles than the lighter cells, but many lysosome-like structures were present in the cytoplasm. The in vitro-developed morulae also contained two types of cells similar to those observed in the in vivo morulae. However, most of the in vitro-developed cells possessed numerous lipid droplets and contained fewer lysosome-like structures than the cells of the in vivo-derived morulae. The blastocysts, both in vivo and in vitro, showed a clear differentiation of trophoblast cells and inner cell mass (ICM)-cells. In the in vivo-derived blastocyst, the apical membrane of trophoblast cells was covered with large, numerous microvilli and well-developed junctional complexes were observed. Lipid droplets were present in the cytoplasm of trophoblast and ICM-cells but were not abundant. In vitro-developed blastocysts showed less well-developed junctional complexes between trophoblast cells, less well-developed apical microvilli on the trophoblast cells, and contained large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM-cells. The zonae pellucidae of in vitro-developed embryos were thinner than that of the in vivo-derived embryos. This study demonstrates conspicuous differences in the ultrastructural features between the in vivo-derived and in vitro-developed embryos, suggesting that the ultrastructure may reflect the various physiological anomalies observed in previous studies. Accepted: 29 October 1998