周围神经修复过程的超微结构研究

为了解周围神经损伤后的修复过程,观察了大鼠坐骨神经冷冻损伤当时至４周后的超微结构。发现冷冻损伤后３天,除变性神经纤维外,有新生轴突,皆由雪旺细胞包裹着。１周皇一神经纤维已较丰富,以无髓纤维为多,遥髓纤维罕见,且髓鞘较薄,雪量细胞和巨噬细胞皆吞噬变性的神经纤维。此后,形态正常的雪旺细胞和神经纤维渐多,被吴噬的变性物质渐少,直至损伤４周后,神经组织恢复到基本正常,但仍可见少量被吞噬的髓样结构。这提示,     

酸性磷酸酶在大鼠脉络丛上皮细胞内的亚细胞定位

研究大鼠脑室脉络丛上皮细胞内酸性磷酸酶(Acid phosphatase,AcPase)的超微结构分布.取成年SD大鼠侧脑室、第三脑室和第四脑室的脉络丛组织,按硝酸铅法进行酸性磷酸酶电镜酶细胞化学染色显示.透射电镜下ACPase阳性反应物仅见于脉络丛上皮细胞内的初级和次级溶酶体上.通过延长孵育时间和改变固定条件(戊二醛浓度为0.5%)在脉络丛上皮细胞内的高尔基复合体扁囊和囊泡中也观察到了ACPase阳性反应物.各脑室脉络丛上皮细胞酸性磷酸酶的分布未见明显差异.文中讨论了脉络丛上皮细胞内ACPase的分布特征及其功能.     

脑室内注射链脲佐菌素大鼠海马神经元和突触的超微结构异常

目的:观察侧脑室内重复注射小剂量链脲佐菌素(streptozocin,STZ)阿尔茨海默氏病(alzheimer disease,AD)模型大鼠的学习记忆和海马神经元的超微结构改变.方法:采用Morris水迷宫进行行为学测试;取海马CA1区组织制成超薄切片,透射电镜观察.结果:模型组大鼠Morris水迷宫平均游泳时间及平均游泳距离明显延长.海马CA1区神经元出现程度不等的退行性变.细胞核形态不规则.部分核内染色质浓集;胞浆内脂褐素颗粒增多,高尔基囊泡扩张.严重者出现早期细胞凋亡改变.神经毡内可见有髓鞘轴突胞质内脂褐素颗粒明显增多,突触结构异常,突触小泡聚集增多,分布无序.突触后膜胞质面上的致密斑不规则增厚,或断断续续不完整.结论:侧脑室内注射STZ可引起大鼠海马神经元和突触的超微结构病变,导致大鼠学习记忆能力减退.     

阴道毛滴虫吞噬精子过程的超微结构观察

本文报告应用透射电镜观察体外阴道毛滴虫吞噬、消化人精子全过程的超微结构变化。虫体与精子共育１５～３０ｍｉｎ后，虫体粘附、包绕和吞噬精子，在胞质中形成吞噬泡（食物泡）；共育４５～７５ｍｉｎ，次级溶酶体（消化泡）中的精子被消化；９０～１０５ｍｉｎ后，消化过程结束，细胞质中多见残体（排泄泡）和空泡；１２０～１５０ｍｉｎ，虫体出现自噬现象或摄食。在吞噬与消化精子阶段，虫体的粗面内质网较丰富、高尔基体发达、氢化酶体数量较多。本研究进一步证实阴道毛滴虫具有较强的吞噬与消化的潜在能力，提示滴虫性阴道炎可能影响女性受孕的几率     

血管性痴呆小鼠海马神经元超微病理特征研究

目的：探讨血管性痴呆(VD)小鼠海马神经元超微病理特征及其变化的意义。方法：采用双侧颈总动脉线结、反复缺血—再灌注法,制作小鼠VD动物模型。取两组小鼠海马组织制成超薄切片,透射电镜观察。结果：(1)假手术组：海马神经元和神经毡结构均正常。(2)VD模型组：海马神经元核肿胀、有局部凹陷现象;核周体细胞器明显减少,多聚核糖体稀散;仅残留受到破坏的高尔基体和粗面内质网、线粒体变性。髓鞘层裂;轴突中神经微管结构模糊。神经毡中可见树突和轴突不同程度水肿,尤其是树突呈现极度扩张形成不规则的“气球”,其中含有许多大小不等的薄膜空泡。突触数量显著减少,可见穿孔和异形的突触;有的突触小泡破裂,呈片状均质化。结论：海马神经元的超微结构病变导致神经元功能异常、突触可塑性降低,可能在VD的病因中起重要作用。     

盼生安对人肝癌细胞超微结构的影响

目的：以氟尿嘧啶(5-Fu)作为阳性对照观察了盼生安对BEL-7402肝癌细胞超微结构的影响,为该药物的抑癌作用提供形态学依据。方法：用透射电镜观察经不同剂量药物处理后细胞超微结构的变化。结果：经盼生安处理后,胞浆空泡化,细胞器中线粒体严重空泡变性,其次是高尔基体和粗面内质网的空泡化,核糖体减少。胞内次级溶酶体,多泡小体,自噬体及髓样小体增多。5-Fu对肝癌细胞形态的影响不如盼生安明显。结论：盼生安主要导致显著的线粒体超微结构改变,胞浆空泡化,抑制了肿瘤细胞的能量代谢和蛋白质合成系统而使细胞功能减退,代谢缓慢最终使细胞退变死亡。     

沙眼衣原体结构的电子显微学研究

沙眼衣原体（Chlamydia trachomatis,Ct）,可导致沙眼、感染性致盲,且是引起泌尿生殖道感染等性传播疾病的主要病原体之一。本文利用常温常规电镜制样方法和电子断层成像技术,体外对临床上分离得到的沙眼衣原体进行了研究。研究发现,此种衣原体在发育过程中,胞质发生了一系列的变化,首先RB胞质浓缩变小形成具有类核的IB,随后发育成高度致密且体积较小的EB。伴随此过程,膜也发生了变化：首先RB的膜易皱缩,EB的膜则较坚固;其次RB发育成EB时,多余外膜会形成囊泡;此外,其外膜结构随着RB发育为EB而变厚并更稳定。     

鼠重组腺病毒介导的骨形成蛋白-9转染大鼠牙囊干细胞的实验研究

目的：探讨鼠重组腺病毒介导的骨形成蛋白9基因转染大鼠牙囊干细胞的可行性及其转染后的成骨作用,以获得可用于牙周骨组织再生工程的基因修饰的种子细胞。方法：取大鼠下颌骨,解剖显微镜下体外分离培养纯化鉴定牙囊干细胞,腺病毒介导的骨形成蛋白9基因转染第三代牙囊干细胞,并设立空白对照组,绿色荧光病毒组（GFP组）。通过观察细胞形态及生长曲线变化,荧光显微镜及RT—PCR检测转染后骨形成蛋白9基因mRNA的表达,碱性磷酸酶及钙茜素红染色测定转染后牙囊干细胞的成骨活性。结果：与未转染对照组比较,转染组细胞转染2周后形成钙化结节,停滞期延长,数量轻度下降,倍增时间延长。牙囊干细胞转染骨形成蛋白9基因后12h后即有荧光表达,转染3,6,9,12d后骨形成蛋白9mRNA均呈阳性表达且逐渐增强,未转染对照组呈阴性。转染组碱性磷酸酶活性随转染时间的延长呈升高趋势,ALP染色及茜素红钙结节染色为阳性：未转染对照组碱性磷酸酶染色及钙茜素红染色均呈弱阳性表达,转染组显著高于未转染组。结论：鼠重组腺病毒介导的RBMP-9基因可以成功地转染大鼠牙囊干细胞,转染后牙囊干细胞高表达骨形成蛋白9,且具有明显的成骨作用。     

呼肠病毒与细胞凋亡

从SARS患者生前咽拭子和尸检肺组织分离病毒阳性的培养细胞超薄切片中,在电子显微镜下不仅发现了呼肠病毒,而且还查见具有超微结构形态特征的凋亡细胞。凋亡早期的感染细胞,其细胞核染色质浓缩、边集显著,胞浆内尚可查见病毒包涵体的残迹。多数凋亡细胞胞浆内未查见呼肠病毒及其包涵体。在早期,凋亡细胞胞核形状变化不大,核染色质呈不规则的边集、浓缩,或呈平台状。继而,凋亡细胞胞核形状高度不规则,染色质浓缩、致密,可见核孔残迹,但细胞轮廓尚保存完好,外周可查见含核质凋亡小体。到后期,凋亡细胞高度皱缩．核固缩并碎裂成细小的片块或颗粒,胞质稀少,胞体呈芽球状,细胞膜外围可查见大小不等、形状多样的含核质凋亡小体。此研究结果提示：与SARS相关呼肠病毒所诱导的细胞凋亡有可能在SARS的发病机制中起重要作用。     

激光照射对急性脊髓损伤后脊髓再生的促进作用

建立大鼠皮质脊髓背侧束全横断模型,利用弱激光照射脊髓受损部位的皮肤,观察激光照射对急性脊髓损伤后脊髓再生的促进作用.30只SD大鼠,随机分成对照组和照射组.照射组:急性皮质脊髓背侧束全横断后15 min进行连续14 d采用弱激光照射脊髓受损部位的皮肤.对照组:急性皮质脊髓背侧束全横断后未行弱激光经皮照射治疗.术后两组分别于第3,7,14 d分别取材,用苏木精-伊红染色法(HE)染色和免疫荧光标记染色观察.实验发现,脊髓损伤后14 d,照射组的空洞及瘢痕形成面积小于对照组,有统计学差异(p<0.05);对照神经胶质酸性组蛋白(GFAP)和硫酸软骨素(CS)表达强烈分布紧密,照射组GFAP和CS56表达达微弱且分布稀疏;对照组少量神经微丝蛋白(NF)在损伤区周围,神经生长相关蛋白(GAP43)形态肿大变形分布紊乱,照射组大量成纤维丝状的NF分布在损伤区周围,并与GAP43相伴行.结果表明,脊髓损伤急性期采用弱激光照射脊髓受损部位的皮肤,可减少脊髓损伤后空洞形成,并促进轴突再生.     

Creutzfeldt-Jakob病二例电镜观察

本实验用透射电镜观察了二例Ｃｒｅｕｔｚｆｅｌｄｔ－Ｊａｋｏｂ病（ＣＪＤ）的尸检及活检脑组织。其中一例为死后６天尸检脑组织,电镜下可见神经元及神经胶质细胞明显变性,核染色质块状凝集,核碎裂,可见细胞碎片。另一例为穿刺活检脑组织,因取材过少光镜病理检查只发现脑组织水肿,不能明确诊断,电镜观察二例均少见神经元,神经胶质细胞多见且变性,最突出的表现为脑组织的海绵状界膜空泡变性,有髓神经纤维髓鞘解离并有部分溶解,轴索溶解消失并呈空泡样变性。本实验表明：对临床上表现为进行性痴呆,脑电图广泛异常的病人的组织活检,送检组织过少,一般病理检查不能明确诊断时,电镜检查具有重要意义,即使是死亡６天后的尸检仍可为ＣＪＤ这种疑难和罕见病例的确诊提供有力的证据。     

Myelin Sheath as a Dielectric Waveguide for Signal Propagation in the Mid‐Infrared to Terahertz Spectral Range

The myelin sheath enables dramatic speed enhancement for signal propagation in nerves. In this work, myelinated nerve structure is experimentally and theoretically studied using synchrotron‐radiation‐based Fourier‐transform infrared microspectroscopy. It is found that, with a certain mid‐infrared to terahertz spectral range, the myelin sheath possesses a ≈2‐fold higher refraction index compared to the outer medium or the inner axon, suggesting that myelin can serve as an infrared dielectric waveguide. By calculating the correlation between the material characteristics of myelin and the radical energy distribution in myelinated nerves, it is demonstrated that the sheath, with a normal thickness (≈2 µm) and dielectric constant in nature, can confine the infrared field energy within the sheath and enable the propagation of an infrared signal at the millimeter scale without dramatic energy loss. The energy of signal propagation is supplied and amplified when crossing the nodes of Ranvier via periodic relay. These findings provide the first model for explaining the mechanism of infrared and terahertz neurotransmission through myelinated nerves, which may promote the development of biological‐tissue label‐free detection, biomaterial‐based sensors, neural information, and noninvasive brain–machine interfaces.     

Magnetic Assembly of a Multifunctional Guidance Conduit for Peripheral Nerve Repair

Nerve growth conduits are designed to support and promote axon regeneration following nerve injuries. Multifunctionalized conduits with combined physical and chemical cues, are a promising avenue aimed at overcoming current therapeutic barriers. However, the efficacious assembly of conduits that promote neuronal growth remains a challenge. Here, a biomimetic regenerative gel is developed, that integrates physical and chemical cues in a biocompatible “one pot reaction” strategy. The collagen gel is enriched with magnetic nanoparticles coated with nerve growth factor (NGF). Then, through a remote magnetic actuation, highly aligned fibrillar gel structure embedded with anisotropically distributed coated nanoparticles, combining multiple regenerating strategies, is obtained. The effects of the multifunctional gels are examined in vitro, and in vivo in a 10-mm rat sciatic nerve injury model. The magneto-based therapeutic conduits demonstrate oriented and directed axonal growth, and improve nerve regeneration in vivo. The study of multifunctional guidance scaffolds that can be implemented efficiently and remotely provides the foundation to a novel therapeutic approach to overcome current medical obstacles for nerve injuries.     

Peripheral Nerve Regeneration with 3D Printed Bionic Scaffolds Loading Neural Crest Stem Cell Derived Schwann Cell Progenitors

Peripheral nerve injuries are one of the most common types of traumatic damage to the nervous system. Treatment of peripheral nerve injuries aims to promote axon regrowth by imitating and improving the microenvironment for sciatic nerve regeneration. In this study, regeneration efficiency and behavior of peripheral nerves are compared under three treatment strategies: 1) transplantation of Schwann cell progenitors induced from purified neural crest stem cells; 2) implantation of a multiscale scaffold based on high-resolution 3D printing; and 3) implantation of this bionic scaffold loading Schwann cell progenitors. The results of structural, electrophysiological, and behavioral tests demonstrate that the three treatment strategies result in different degrees of regeneration. The purified neural crest stem cells differentiate into functional Schwann cells and promote axon regeneration. The multifunctional 3D printed scaffold promotes oriented growth and myelination, and the myelinated nerve regrows with increased density and without visible scaffolds after six months. For the regeneration, scaffold treatment produces better performance than cell graft alone. Finally, it is shown that implantation of multiscale scaffolds preloaded with neural crest stem cell derived Schwann cell progenitors is the best strategy to promote peripheral nerve regeneration with improved anatomy and function among the three different strategies.     

Aligned PCL Fiber Conduits Immobilized with Nerve Growth Factor Gradients Enhance and Direct Sciatic Nerve Regeneration

Topographical guidance and chemotaxis are crucial factors for peripheral nerve regeneration. This study describes the preparation of highly aligned poly(ε‐caprolactone) (PCL) fiber conduits coated with a concentration gradient of nerve growth factor (NGF) (A/G‐PCL) using a newly designed electrospinning receiving device. The A/G‐PCL conduits are confirmed in vitro to enhance and attract the neurite longitudinal growth of dorsal root ganglion (DRG) neurons toward their high‐concentration gradient side. In vivo, the A/G‐PCL conduits are observed to direct a longitudinal stronger attraction of axons and migration of Schwann cells in 15 mm rat sciatic nerve defects. At 12 weeks, rats transplanted with A/G‐PCL conduits show satisfactory morphological and functional improvements in g‐ratio, total number, and area of myelinated nerve fibers as well as the sciatic function index, compound muscle action potentials, and muscle wet weight ratio as compared to aligned PCL fibers conduits with uniform NGF (A/U‐PCL). The performance of A/G‐PCL is similar to that of autografts. Moreover, mRNA‐seq and RT‐PCR results reveal that Rap1, MAPK, and cell adhesion molecules signaling pathways are closely associated with axon chemotactic response and attraction. Altogether, by combining structural guidance with axon chemotaxis, the NGF‐gradient/aligned PCL fiber conduits represent a promising approach for peripheral nerve defect repair.     

透射电镜神经组织制样包埋剂比较

为探讨两种常用包埋剂Epon 812和Eponate 12对神经组织透射电镜样品的影响,本文采用多聚甲醛-戊二醛溶液对wistar大鼠进行全身灌流固定后,取材海马、脊髓及坐骨神经,按常规透射电镜样品技术制作超薄切片.电镜观察显示,经Eponate 12包埋后的样品,在切片的透明度及超微结构的清晰度上均优于Epon 812包埋的超薄切片样品.由于Eponate 12的浸透效果好,有效地避免了有髓神经纤维髓鞘上出现孔洞结构的制样问题.本实验小组的实验结果表明:对于神经组织,尤其是周围神经组织的超薄切片样品,使用Eponate 12作为包埋剂,效果会更好一些.     

Measurement of the repeat period of myelin sheath using ultrathin frozen sections

The myelin sheath of peripheral nerves was observed by transmission electron microscopy (TEM) using plastic-embedded sections and ultrathin frozen sections. Repeat distances of myelin sheaths were measured in high-powered electron micrographs. The ultrathin frozen sections showed a longer repeat distance than the plastic-embedded sections. The ultrathin frozen sections were thought to contain fewer artefacts, as they had not been subject to dehydration and embedding. It is known that broken myelin sheaths are often observed under conventional TEM. It is thought that these procedures cause contraction and partial destruction of the myelin sheath.     

Regenerating axons and their growth cones observed by scanning electron microscopy

A predenervated sciatic nerve segment, which had been treated by repeated freezing and thawing to kill Schwann cells, was grafted to the original sciatic nerve in the rat. Three to five days later, the graft was chemically fixed and treated by KOH-collagenase digestion, a treatment which selectively removes almost all non-cellular elements including the collagen fibrils and basal laminae from the tissue, thus making it possible to observe regenerating axons by scanning electron microscopy. Debris of degraded Schwann cells and myelin sheaths remained in the form of "columns", and several thick (2-3 microns in diameter) and thin (less than 1 micron in diameter) axons ran singly or in bundles on such "cell debris columns." Thick axons have an almost straight contour, while there were various swellings at intervals along the thin axons. In most cases, the growing tips of regenerating axons were swollen as growth cones ranging from 2 microns to 5 microns in diameter. Growth cones exhibited fusiform to polygonal variations in structure and had only a few filopodial processes on the surface.