大鼠脑组织NT-3融合蛋白的表达、纯化及抗体制备

目的克隆大鼠脑组织神经营养素3(NT-3)基因,原核诱导表达并纯化,制备高效价抗体,为研究其对周围神经损伤修复的影响提供实验基础。方法用RT-PCR法扩增目的基因,依次克隆入pMD-18T载体及亚克隆入pRSET-A原核表达载体。导入大肠杆菌BL21经IPTG诱导表达,SDS-PAGE电泳鉴定后用镍柱纯化。免疫家兔制备特异性抗体。结果RT-PCR得到777 bp的片断,酶切鉴定及测序证实与GeneBank中大鼠NT-3序列一致,成功构建了原核表达载体pRSET-A-NT-3。导入BL21诱导得到一条约34 ku的新蛋白带。免疫家兔获得1∶64000的高效价特异性抗大鼠NT-3血清。     

NT-3基因修饰施万细胞与神经干细胞联合移植治疗全横断脊髓损伤的实验研究

为探索一种治疗急性脊髓损伤的新策略 ,本实验先用核荧光标记培养的神经干细胞 (NSCs) ;再用含神经营养素 3基因的腺病毒 (AdvNT 3)感染培养的施万细胞 (SCs) ,简称NT 3 SCs。然后建立大鼠全横断脊髓损伤模型 ,向损伤处分别移植NSCs、SCs +NSCs和NT 3 SCs +NSCs。所有动物存活 6 0d后 ,进行爬网格测验和BBB评分。接着在脊髓横断处远端组织内注射荧光金 (FG) ,动物再存活 7d。在处死动物前检测皮质运动诱发电位 (CMEP)和皮质体感诱发电位 (CSEP)。最后灌注固定动物 ,取材和切片并行形态学观察。结果显示 :1 NSCs能在损伤…     

部分去背根猫备用背根节和脊髓Ⅱ板层NT-3及其mRNA的表达变化

采用免疫组化和原位杂交技术探讨了部分去背根猫备用背根节 (L6 )和 L3、L5脊髓 II板层 NT-3及其 m RNA的表达变化。结果发现 ,正常组 NT-3及其 m RNA阳性产物主要分布于背根节的大型神经元和少数中、小型神经元。部分去背根后 ,3 d和10 d两时相 NT-3 m RNA大型神经元阳性数明显减少 ,而 NT-3阳性大型神经元数术后 10 d时方明显减少 (P<0 .0 1) ;NT-3及其 m RNA阳性小型细胞数在术后两时相均较正常组者增多 (P<0 .0 1) ;而在中型神经元只有 NT-3阳性神经元数有增加。相对地 ,在脊髓 板层 ,两时相 NT-3阳性神经元及胶质细胞百分数均较正常者明显增加 (P<0 .0 1) ,且以 3 d组者为最明显 ,但均未见 NT-3 m RNA阳性信号。结果表明 ,部分去背根不仅导致背根节各类神经元中 NT-3的表达发生了变化 ,且对 板层 NT-3阳性神经元及胶质细胞数量也有明显影响。提示 NT-3可能在脊髓 板层可塑性中发挥作用     

周围神经损伤后慢性失神经及其对神经再生的影响

正人类周围神经损伤后,由于神经再生距离较长、再生速度较慢,长时间后往往在损伤远侧段发生由轴突溃变导致的一系列改变,称之为慢性失神经(chronic denervation)。慢性失神经不仅涉及远侧段神经,还会影响所支配的肌肉和皮肤以及近侧段神经,导致损伤后神经修复失败,伤者的感觉和运动功能恢复不理想,有的还会出现神经病理性痛。因此,     

部分背根切断对备用背根节NT-3表达的影响

目的　探讨部分去背根后备用背根节 (L6 )各类细胞NT 3及其mRNA的含量变化。　方法　对成年雄性猫行单侧部分背根切断术 (切除一侧L1 ～L5,L7～S2 DRG ,保留L6 为备用根 )。取正常组一侧和术后 3d及 7d组手术侧的L6 DRG制作 2 0 μm厚冰冻切片 ,分别用NT 3抗体及NT 3cRNA探针行免疫组织化学及原位杂交染色。观察NT 3及其mRNA在DRG各类细胞的分布 ,测定NT 3及其mRNA在神经元和卫星细胞的光密度值 ,所得数据用q检验进行统计分析。　结果　部分去背根后 ,各时相备用背根节大神经元内NT 3的光密度值较正常者进行性减少 ,(P <0 0 5 ) ,而NT 3mRNA的光密度值术后 3d减少 ,7d回升至近正常者水平。比较之 ,小神经元和卫星细胞NT 3及其mRNA的光密度值进行性增多 (P <0 0 5 )。　结论　部分背根切断对备用背根节各类细胞NT 3表达的影响不同 ,其功能意义可能与NT 3参与脊髓Ⅱ板层可塑性有关     

神经上皮干细胞蛋白在体外培养的大鼠肌卫星细胞中的表达

目的：探讨大鼠脑创伤后神经元型及诱生型一氧化氮合酶及其一氧化氮的神经毒性作用机制。方法：雄性Wistar大鼠250只，随机分成①假于术组，②创伤对照组，③氨基胍(AG)治疗组，④7-硝基吲哚(7-NI)治疗组，⑤AG和7-NI联合治疗组，用Marmarou方法造成大鼠重型弥漫性颅脑创伤，采用免疫组织化学检测nNOS和iNOS蛋白表达情况，     

失神经萎缩肌组织成分对成肌细胞性能影响的实验研究

目的：探讨长期失神经萎缩骨骼肌组织成分对体外培养成肌细胞的性能影响。方法：①获取长期失神经支配肌萎缩模型大鼠的腓肠肌，无菌条件下清洗、剪碎、匀浆、过滤，收集上清液；②收集Schwann细胞的体外培养基；③体外培养C2C12成肌细胞系，扩增传代并分组：A．对照组，常规培养，无任何处理因素；B．添加Schwann细胞培养基；C．添加萎缩肌匀浆液。倒置显微镜观察3组细胞的形态特征，免疫荧光检测C2C12细胞MyoD和Myogenin的差异表达。结果：倒置显微镜下，各组细胞贴壁良好，A、C组形态无差异，未见分化肌管；B组于培养48h后细胞变纤细，可见肌管形成。荧光染色证实，培养24h后，3组均出现MyoD阳性细胞，C组阳性细胞数量明显多于A、B组；72h时，3组均可见Myogenin阳性细胞，B组阳性细胞数量明显多于A组，C组阳性细胞数量稀少。结论：长期失神经萎缩肌组织成分能够促进成肌细胞的增殖与活化，但抑制活化成肌细胞的进一步分化成熟。     

糖尿病大鼠额叶皮质神经营养素-3的表达及其意义

糖尿病是一种严重危害人体健康的常见病,糖尿病视网膜病变和糖尿病肾病早已受到了人们的广泛关注。近年来,糖尿病脑病也引起了人们的重视[1]。神经营养素3(NT-3)广泛分布在中枢神经系统内,具有维持神经元存活、诱导其轴突生长和促进损伤神经元的修复及阻止神经元损伤后死亡的功能。本实验观察糖尿病大鼠额叶皮质神经元NT-3表达的改变,探讨糖尿病脑病的机制,为临床上糖尿病及糖尿病脑病的诊断和治疗提供一定的实验依据。1材料和方法1·1动物和试剂健康雄性SD大鼠30只,清洁级,体重200~250 g,浙江大学医学院动物实验中心提供。链脲佐菌素(S…     

NGF、BDNF和NT-3在正常成年大鼠主要脑区表达的免疫组织化学研究

目的研究神经生长因子(NGF)﹑脑源性神经营养因子(BDNF)﹑神经营养素-3(NT-3)在正常成年大鼠主要脑区的表达。方法将正常成年大鼠用4%多聚甲醛灌注固定后取脑制成20μm厚的冰冻切片,应用上述3种因子的抗体进行免疫组织化学染色。结果3种因子的表达既有共同点也有不同点:共同点是在主要脑区都能表达,不同点是在单个细胞中,BDNF主要表达于胞浆边缘;NGF在大部分神经元的整个细胞都表达,但少量神经元则在胞核无表达;NT-3则与BDNF相似。结论3种因子在正常成年大鼠主要脑区都能表达。     

灵芝孢子对大鼠脊神经腹根切断后脊髓运动神经元存活及其NT-3、NOS表达的影响

目的探讨灵芝孢子（萌动激活赤灵芝孢子）对大鼠脊髓受损伤运动神经元存活和表达神经营养素-3（NT-3）及一氧化氮合酶（NOS）的影响.方法对单侧腹根切断后的大鼠胃饲不同剂量的灵芝孢子,计算受损伤运动神经元存活率;用免疫组织化学及原位杂交方法检测NT-3的表达;用酶组织化学方法检测NOS的活性.结果腹根切断后35 d,对照组运动神经元存活率为47.32%,灵芝孢子低、中、高剂量组的运动神经元存活率分别为67.11%、72.67%和81.67%;腹根切断后高剂量组存活的运动神经元NT-3和NOS的表达都高于对照组. 结论灵芝孢子促进大鼠脊髓前角受损伤的运动神经元存活,其存活率与用药剂量有关;灵芝孢子促进大鼠脊髓存活的运动神经元表达NT-3和NOS.     

Caspase-3 mRNA在子痫前期患者胎盘中的表达及其意义

目的检测半胱天冬氨酸蛋白酶-3（Caspase-3）mRNA在子痫前期患者胎盘中的表达,以探求Caspase-3在子痫前期发病机制中的意义。方法用半定量逆转录聚合酶链反应（RT-PCR）技术检测正常妊娠（10例）,子痫前期（22例）剖宫产后胎盘中Caspase-3基因在转录水平的表达。结果子痫前期组胎盘中Caspase-3 mRNA表达量较正常妊娠组显著升高（P〈0.01）。结论Caspase-3在子痫前期的发病中起着重要作用,可能与Caspase-3表达的增加有关。     

大鼠脊髓半横断损伤后大脑皮质初级运动区caspase-3的表达

目的探讨大鼠脊髓半横断损伤（hSCI）后大脑皮质初级运动区（M1）神经细胞的凋亡情况。方法成年健康Wistar大鼠T8-9脊髓左侧半横断术后，用RT-PCR和免疫组织化学法检测各个时间点caspase-3mRNA的表达量以及阳性细胞的分布。结果手术组中左、右两侧M1区的caspase-3mRNA表达量与假手术组比较，在6h即显著上升，1d达到高峰，3d、7d、14d组下降，而在21d又略有上升；6h组M1区表达量右侧明显高于左侧。免疫组织化学显示阳性细胞主要集中在M1的多形细胞层、锥体层。结论大鼠脊髓半横断损伤后M1细胞凋亡在早期最为剧烈，不仅导致支配损伤侧的M1区细胞凋亡，同样也导致支配未损伤侧脊髓的M1区神经细胞凋亡。     

针刺对脊髓全横断SD大鼠BDNF基因表达的影响

目的探讨电针刺对脊髓全横断成年SD大鼠脑源性神经营养因子(brain-derived neurotrophic fac-tor,BDNF)mRNA在横断脊髓上端、下端、大脑皮质和比目鱼肌中的表达及变化的影响。方法建立40只SD大鼠脊髓全横断损伤模型,雌雄不限,随机分为针刺1d、3d、7d、14d和手术1d、3d、7d、14d组,每组5只,选"足三里 悬钟"及"三阴交 伏兔"2组穴位隔天交替针刺。采用RT-PCR技术检测各时间段脊髓横断上、下端、大脑皮质、比目鱼肌四部位中BDNF mRNA的表达。结果针刺14d组比目鱼肌中BDNF mRNA较手术14d组显著增加(P<0.05)。结论电针刺可促进BDNF在损伤部位所支配的肌肉-靶组织中的表达。     

Muscles Involved in Naris Dilation and Nose Motion in Rat

In a number of mammals muscle dilator nasi (naris) has been described as a muscle that reduces nasal airflow resistance by dilating the nostrils. Here we show that in rats the tendon of this muscle inserts into the aponeurosis above the nasal cartilage. Electrical stimulation of this muscle raises the nose and deflects it laterally towards the side of stimulation, but does not change the size of the nares. In alert head‐restrained rats, electromyographic recordings of muscle dilator nasi reveal that it is active during nose motion rather than nares dilation. Together these results suggest an alternative role for the muscle dilator nasi in directing the nares for active odor sampling rather than dilating the nares. We suggest that dilation of the nares results from contraction of muscles of the maxillary division of muscle nasolabialis profundus. This muscle group attaches to the outer wall of the nasal cartilage and to the plate of the mystacial pad. Contraction of these muscles exerts a dual action: it pulls the lateral nasal cartilage outward, thus dilating the naris, and drags the plate of the mystacial pad rostrally to produce a slight retraction of the vibrissae. On the basis of these results, we propose that muscle dilator nasi of the rat should be re‐named muscle deflector nasi, and that the maxillary parts of muscle nasolabialis profundus should be referred to as muscle dilator nasi. Anat Rec, 298:546–553, 2015. © 2014 Wiley Periodicals, Inc.     

Evolution of the Muscles of Facial Expression in a Monogamous Ape: Evaluating the Relative Influences of Ecological and Phylogenetic Factors in Hylobatids

Facial expression is a communication mode produced by facial (mimetic) musculature. Hylobatids (gibbons and siamangs) have a poorly documented facial display repertoire and little is known about their facial musculature. These lesser apes represent an opportunity to test hypotheses related to the evolution of primate facial musculature as they are the only hominoid with a monogamous social structure, and thus live in very small groups. Primate species living in large groups with numerous social relationships, such as chimpanzees and rhesus macaques, have been shown to have a complex facial display repertoire and a high number of discrete facial muscles. The present study was designed to examine the relative influence of social structure and phylogeny on facial musculature evolution by comparing facial musculature complexity among hylobatids, chimpanzees, and rhesus macaques. Four faces were dissected from four hylobatid species. Morphology, attachments, three‐dimensional relationships, and variation among specimens were noted and compared to rhesus macaques and chimpanzees. Microanatomical characteristics of the orbicularis oris muscle were also compared. Facial muscles of hylobatids were generally gracile and less complex than both the rhesus macaque and chimpanzee. Microanatomically, the orbicularis oris muscle of hylobatids was relatively loosely packed with muscle fibers. These results indicate that environmental and social factors may have been important in determining morphology and complexity of facial musculature in the less social hylobatids and that they may not have experienced as strong selection pressure for mimetic muscle complexity as other, more social primates. Anat Rec, 294:645–663, 2011. © 2011 Wiley‐Liss, Inc.     

Hormonal Treatment Effects on the Cross‐sectional Area of Pubococcygeus Muscle Fibers After Denervation and Castration in Male Rats

We explore the interaction of muscle innervation and gonadal hormone action in the pubococcygeus muscle (Pcm) after castration and hormone replacement. Male Wistar rats were castrated and the Pcm was unilaterally denervated; after 2 or 6 weeks, the cross‐sectional area (CSA) of Pcm fibers was assessed. Additional groups of castrated rats were used to examine the effects of hormone replacement. At 2 weeks post surgeries, rats were implanted with Silastic capsules containing either dihydrotestosterone (DHT), estradiol benzoate (EB) or both hormones, and the CSA of Pcm fibers was assessed after 4 weeks of hormone treatment. At 2 weeks post surgeries, gonadectomy without hormone replacement resulted in reductions in the CSA of Pcm fibers, and denervation combined with castration increased the magnitude of this effect; further reductions in CSA were present at 6 weeks post surgeries, but again denervation combined with castration increased the magnitude of this effect. Hormone replacement with DHT resulted in hypertrophy in the CSA of nondenervated muscles compared to those of intact normal males, but this effect was attenuated in denervated muscles. Hormone replacement with EB treatment prevented further castration‐induced reductions in CSA of nondenervated muscles, but denervation prevented this effect. Similar to that seen with treatment with EB alone, combined treatment with both DHT and EB prevented further reductions in CSA of Pcm fibers in nondenervated muscles, but again denervation attenuated this effect. Thus, while hormone replacement can reverse or prevent further castration‐induced atrophy of Pcm fibers, these effects are dependent on muscle innervation. Anat Rec, 300:1327–1335, 2017. © 2017 Wiley Periodicals, Inc.     

Effect of Unilateral Damage to Sciatic Nerve on Phenotype of Lumbrical Muscles in Experimental and Contralateral Legs in Rats

Expression of slow myosin in fast lumbrical muscle during unilateral damage to the sciatic nerve was studied in rats. Four weeks after unilateral excision of sciatic nerve segment or its crushing, the content of slow fiber in the lumbrical muscle of the contralateral leg tended to increase (detected using monoclonal antibodies against slow myosin). The extrapyramidal system seems to modulate the phenotype of muscle fibers via motoneurons of the spinal cord, while the compensatory activation of this system manifests in pronounced “deceleration” of both the fast and slow muscles in the hind legs.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 5, pp. 589–591, May, 2005     

人前臂肌群的构筑学研究

研究了甲醛固定的成年男性尸体5具（10侧）前臂肌群的构筑学特性。结果表明：前臂肌均为羽肌和半羽肌，羽状角5．4°～16．3°之间。在腕屈伸肌系统中，腕的屈肌和伸肌的生理横切面积（CSA）分别为13．54cm2和14．82cm2,这表明腕的屈肌和仲肌的肌力配布相当。在指屈伸肌系统中，指的屈肌和伸肌的CSA分别为25．82cm2和7．68cm2，屈肌是伸肌的3倍多，表明手指的屈肌力量明显高于伸肌。根据构筑特性分析，前臂各肌肌纤维的肌节长度明显不同，范围在2．07～2．77um，其中屈肌的肌节长度平均为2．32um，伸肌的肌节长度平均为2．60um．伸肌明显高于屈肌，这提示伸肌的缩短速度和位移幅度明显比屈肌大。本文对肌构筑与肌功能的关系作了讨论。     

Denervation and Castration Effects on the Cross‐Sectional Area of Pubococcygeus Muscle Fibers in Male Rats

The number of fibers in skeletal muscles changes little through life; however, the cross‐sectional area of its fibers is modified as result of denervation and in some muscles by castration. The pubococcygeus muscle (Pcm) participates in micturition and ejaculatory processes and its fibers cross‐sectional area is reduced in castrated rats, but denervation effects remained unknown. Here, we used a model in which unilateral denervation of this muscle in gonadally intact and castrated male rats, allowed us to explore the neural and gonadal hormone effects on the cross‐sectional area of its fibers. Denervation significantly reduced the mean cross‐sectional area values; likewise, the percentage distribution of its fibers. We found that castration had a greater effect than denervation. Castration resulted in a lack of fibers from 2,000 to 3,999 μm², while in denervation it was from 2,500 to 3,999 μm². It was interpreted that the castration effect was due to a lack of the direct gonadal hormone effect on muscle fibers, and to a reduction of the indirect hormonal action in its neuromuscular complex. In denervated Pcm of gonadally intact animals these effects were present; however, in denervated but castrated animals these were absent. Thus, combined surgeries resulted in the lowest mean cross‐sectional area values with a restricted fiber distribution from 500 to 1,499 μm². In conclusion, the study in this important muscle showed that cross‐sectional area of its fibers depends on neural and direct/indirect gonadal hormone effects. Anat Rec, 296:1634–1639, 2013. © 2013 Wiley Periodicals, Inc.     

骨性面神经管的应用解剖学

为适应耳科行乳突根治术和面神经减压术、面神经修补术等需要,本文用50例成人锯开的颞骨标本对骨性面神经管的鼓室段(水平段)、锥段、乳突段(垂直段)进行了观察:测量了其长度、管径、壁厚及其与颈静脉窝、前庭窗的距离:探讨了其与临床的关系。