激光共聚焦扫描显微镜和透射电镜观察大鼠纹状体内谷氨酸能突触连接的对比观察

目的 探讨使用激光共聚焦扫描显微镜 (Laser scanning confocal microscope,LSCM)观察大鼠纹状体内谷氨酸能突触连接的方法的可行性.方法 12只正常大鼠分为两组,6只大鼠进行纹状体中等棘刺神经元的CM-DiI 单细胞标记,然后Ⅰ型囊泡膜谷氨酸转运体(vesicular glutamate transporter 1,VGluT1 )免疫荧光标记,LSCM层扫后三维重建,观察VGluT1阳性位点在中等棘刺神经元树突上的分布.另外6只大鼠用TEM观察不对称性突触在纹状体神经元树突上的分布.对两种方法的结果进行比较.结果 用LSCM 和TEM方法观察到的纹状体神经元上谷氨酸能突触连接分布情况一致,没有统计学差异.但LSCM更具优越性的是,可以对图像进行三维重构,从而有利于对神经元之间突触连接的空间分布观察和定量分析.结论 神经细胞荧光标记技术结合LSCM观察是考察纹状体神经元上谷氨酸能突触连接的有效方法.     

激光扫描共聚焦显微镜在花粉研究中的应用

激光扫描共聚焦显微镜（Laser scanning confocal microscope,LSCM）是普通光学显微镜、激光、计算机及其相应的软件技术结合的产物,能实现连续光学切片和生物三维结构重组及动态分析.作为一种先进的技术手段,LSCM技术已经为花粉生物学的研究提供了有价值的新资料,大大地推动了植物生殖生物学的发展.本文综述了LSCM技术在花粉粒形态（形状、大小及三维重建）、花粉的内部结构（如细胞骨架）、花粉的遗传学、花粉的发育、花粉的萌发、花粉自发荧光特性、孢粉研究等方面中的应用,并指出了LSCM的不足之处,最后提出了LSCM技术在花粉研究中的应用展望.未来LSCM技术应该与多光子技术、活细胞工作站技术相结合使用,才能更准确地进行花粉动态研究的实时监测.     

增强型绿色荧光蛋白标记兔间充质干细胞体外复合小肠黏膜下层的生长特性

目的利用绿色荧光蛋白（EGFP）标记兔来源的骨髓间充质干细胞（BMSC）,观察其作为种子细胞复合SIS基质体外培养的生长特性,为复合物回植体内时间提供参考。方法通过物理和化学方法制备小肠黏膜下层（SIS）;取新西兰大白兔分离BMSC,体外培养增殖至P3代,行转染增强型EGFP,转染前后根据增殖速度,绘制生长曲线。然后将转染后的第3代间BMSC,以30×106的浓度接种于1 cm×1 cm大小的SIS支架材料上培养。观察细胞转染后的生长特性,检测复合后的细胞总数,荧光显微镜观察复合后细胞形态。结果 BMSC经转染后生长增殖速度未受明显影响。细胞DNA检测显示复合物培养第7天培养结果显示细胞增殖状态最佳差异有统计学意义（P〈0.05）。荧光显微镜观察显示复合物培养7天细胞增殖良好差异有统计学意义（P〈0.05）,第10天后呈现老化状态。结论增强型的EGFP转染不影响间充质干细胞传代以及增殖。间充质干细胞-SIS基质复合物体外培养7 d回植较为合适。     

动脉平滑肌细胞内钙瞬变与自发性瞬时外向电流同步记录技术(英文)

本文旨在建立一种可同步观察细胞内信号分子和细胞膜离子通道变化之间相互关系的实时研究方法。联合应用激光扫描共聚焦显微镜(laser scanning confocal microscopy,LSCM)显微成像技术和全细胞穿孔膜片钳技术,在急性酶分离的小鼠脑动脉平滑肌细胞上同步记录自发性瞬时外向电流(spontaneous transient outward currents,STOCs)和细胞内的钙瞬变。在全细胞模式膜片钳记录平滑肌细胞膜钾电流的同时,LSCM可准确记录到胞浆内出现的钙瞬变。此技术对于从分子水平揭示细胞内信号转导过程和离子通道相关疾病的机制有重要意义。     

新型数字化高灵敏度荧光显微镜及其在生物学中的应用

尽管普通荧光显微镜已广泛应用于生物医学领域,其性能上还存在一些不足,为了克服它的弱点,借助于像增强器和ＣＣＤ,将倒置生物显微镜改造为数字化高灵敏度荧光显微镜,并增加动态图像获取功能。改进后的荧光显微镜具有以下优点：（１）灵敏度极高,可探测微弱的荧光图像;（２）运用图像融合技术,实现荧光准确定位;（３）适合于观察切片和活细胞;（４）可研究细胞荧光图像的动力学特征;（５）能够给出图像上各点的坐标和对应的发光强度,具有定量显示特点;（６）图像处理软件功能完善,操作方便。利用该荧光显微镜,以吖啶橙为荧光标记物观察正常细胞和凋亡细胞的不同状态,获得良好的实验效果。     

杂交鹅掌楸悬浮细胞高效摄取具有良好生物相容性的超微介孔氧化硅纳米颗粒

采用扫描电子显微镜(SEM)和激光共聚焦显微镜(LSCM)系统研究了超微介孔氧化硅纳米颗粒(MSNs)和杂交鹅掌楸悬浮细胞共孵育的互作特征.将MSNs通过异硫氰酸荧光素(FITC)标记后,再利用LSCM观察其被植物细胞摄取的情况.结果表明,5~15nm的MSNs可以通过内吞途径有效将FITC分子载入完整植物细胞.然而,在无MSNs作为载体的情况下,游离FITC分子则无法进入完整植物细胞.SEM观察结果进一步表明,MSNs容易聚集在完整植物细胞的表面,而且通过硅元素含量对比分析,直接证明了MSNs可以被完整植物细胞摄取到其内部.其次,利用荧光素二乙酸酯染料分析和MSNs共培养24h后的植物细胞的活性,其结果表明,这些细胞仍然保持很好的活力,而且也没有观察到明显的细胞死亡.最后发现,这些和MSNs共培养后的鹅掌楸细胞仍可以通过胚胎发育实现植株再生.综上所述,在植物生物学中,这种超微MSNs作为纳米载体可以应用到细胞的体外外源基因转染.     

自组装IKVAV多肽纳米纤维支架凝胶及其对嗅鞘细胞的作用

旨在观察自组装IKVAV多肽纳米纤维支架凝胶对鼠嗅鞘细胞(OECs)的作用。通过调整IKVAV溶液pH值并加入培养液触发多肽自组装为支架凝胶, 用原子力显微镜检测IKVAV分子可以自组装成编织状纳米纤维(直径为3~5 nm)。采用原代分离培养方法获得OECs单细胞悬液后, 使用差速贴壁法两次纯化OECs且在第12天通过免疫染色计数OECs纯度为85%。将IKVAV多肽纳米纤维支架凝胶与OECs复合培养, 倒置显微镜下观察OECs生长良好, Calcein-AM/PI活、死细胞染色表明活细胞数达95%。CCK-8法间接细胞计数证实IKVAV多肽可促进OECs的黏附, 对OECs增殖没有影响。由此可见IKVAV多肽可以自组装成纳米纤维支架凝胶且对OECs有良好的生物相容性及黏附作用, 可作为神经组织工程支架材料。     

下颌下腺脱细胞基质材料的生物相容性及毒理学研究

为探讨下颌下腺脱细胞基质支架材料的生物相容性,应用3%TritonX-100对SD大鼠的下颌下腺组织进行脱细胞处理,制备脱细胞基质支架材料,将该材料的浸提液注入小鼠体内进行全身急性毒性试验,观察小鼠全身反应.将该材料植入Wistar鼠肌内进行体内植入试验,不同时间观察支架材料与组织反应.用传代培养的第2代下颌下腺细胞与支架材料体外复合培养,第7 d时进行MTT检测,观察支架材料对细胞增殖的影响.全身急性毒性试验结果显示,实验组与对照组无显著性差别(P＞0.05),体内植入试验2、4、8 W时光镜下表现与对照组基本相似,MTT检测结果,细胞相对增长率为91.66%,支架材料的毒性为0级.结果可见,经3%TritonX-100脱细胞处理后所制备的下颌下腺生物衍生支架材料具有良好的生物相容性,对机体无毒害作用.     

颌下腺导管细胞与胶原海绵细胞相容性的实验研究

为探讨胶原海绵对颌下腺 (submandibulargland ,SMG)导管细胞的细胞相容性 ,采用HE染色光镜观察及免疫组化观察SMG导管细胞接种于胶原海绵后 ,细胞的生长情况。光镜下可见接种后第 1d细胞数量较少 ,分散于胶原海绵支架中间 ,第 7d细胞数量明显增加 ,免疫组织化学染色抗IV型胶原抗体染色呈阳性 ,说明细胞与支架材料之间已经有细胞外基质产生。胶原海绵具有良好的细胞相容性 ,是一种理想的支架材料。与胶原海绵复合培养 ,颌下腺导管细胞仍可保持良好的增殖能力。     

胶原蛋白-壳聚糖支架的构建及其与骨髓基质干细胞复合的实验研究

目的:构建一种组织工程神经支架,并观察体外培养的骨髓基质干细胞在其内部的生长情况,为后续种子细胞的移植提供阶段性实验数据.方法:以Ⅰ型胶原蛋白和壳聚糖为原料通过冷冻干燥技术制备神经支架,扫描电镜观察其内部结构,测量其孔径大小、孔隙率等指标.将体外培养的骨髓基质干细胞与Ⅰ型胶原蛋白-壳聚糖神经支架复合,共培养2天;扫描电镜观察细胞在支架内部的生长情况.结果:构建的神经支架均为圆柱状,内部为纵向平行排列的孔径均匀的微管样结构,细胞紧密贴附在支架微孔内壁上,细胞生长状况良好.结论:Ⅰ型胶原蛋白-壳聚糖支架具有良好的内部三维结构和生物相容性,可与细胞复合后用于修复周围神经缺损.     

BK通道在细胞膜上的单分子定位及空间分布

目的：研究大电导、钙离子和电压激活的钾离子通道（BK通道）在HEK293细胞膜上的单分子定位及其总体空间分布情况。方法：分别用mEos2、Dronpa等荧光蛋白标记BK通道的α亚基和辅助性β2亚基,将这些质粒在HEK293细胞内瞬时转染以表达通道蛋白,然后用激光共聚焦荧光显微成像、全内反射荧光显微成像、光敏定位荧光成像等技术观察BK通道的亚细胞定位及单分子分布,并用电生理实验技术检测荧光蛋白对BK通道有影响。结果：激光共聚焦荧光显微成像和全内反射荧光显微成像技术只能在亚细胞水平定位通道蛋白,BK通道在细胞膜上聚集并形成不规则的蛋白簇,它的仅亚基和β2亚基在细胞膜上完全共定位;光敏定位荧光成像技术成功定位BK通道蛋白簇里面的单分子,虽然α和β2亚基紧紧靠在一起,它们之间依然存在空间距离;BK通道的质膜表达和功能特性不受荧光蛋白的影响。结论：BK通道蛋白簇里面包含大量的α和β2亚基的蛋白单分子,它们紧密地聚集在一起,但是并没有完全共定位,在分子水平上揭示了BK通道α和p亚基功能耦合的结构基础,为以后研究大分子蛋白质间的相互作用机制提供了很好的分子模型,光敏定位荧光成像技术作为一种全新的单分子荧光成像手段,在基因表达、信号通路、蛋白质相互作用等许多重要生命活动的研究中发挥重要作用。     

Imaging of single fluorescent molecules using video-rate confocal microscopy

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nipkow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled with fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecomposition lifetimes of the fluorophores were approximately 10 s for TMR and approximately 2 s for IC5 under the incident laser power of 0.5 W/mm(2). Both the fluorescence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm(2). 2D sliding movement of single kinesin molecules along microtubules on glass surface and 3D Brownian motion of individual kinesin molecules in viscous solution could be observed using this microscopy. These results indicated that this method could be applicable to the study of single molecular events in living cells at real time.     

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.     

生物衍生松质骨支架材料的Micro—CT量化分析

目的：在骨组织工程中,如何制备出理想的支架材料一直是研究重点;目前主要的有天然生物支架材料、人工合成有机材料和无机材料等;生物衍生骨即天然生物支架材料的一种,由于其与天然骨在形态结构上较为相似,是近年来研究较多的支架材料之一;既往形态学研究局限于在二维层面,对于其三维结构参数分析较少。故本实验主要运用Micro-CT对生物衍生松质骨的三维结构参数进行分析,量化评价其作为骨组织工程支架材料的结构参数。方法：截取新鲜猪松质骨,经脱脂脱蛋白部分脱钙及去抗原处理后,制作成生物衍生骨支架;应用Micro．CT扫描,重建三维图并量化分析其结构参数,统计软件SPSS分析各参数间的相关性。结果：经Micro．CT扫描,得到二维CT图和三维重建图。各三维结构参数的值分别为：BV／TV（20．48±5．14）％;BS／BV（41．66±5．39）1／ram;Porosity（79．52±5．14）％;Tb．Th（0．10±0．01）mm;Tb．N（1．99±0．47）l／mm;Tb．Sp（0．32±0．05）mm;Tb．Pf（2．03±4．70）1／mm;SMI（1．28±0．35）;DA（1．60±0．23）;Corm．Dn（158．53±106．09）I／mm3。各参数间相关系数具有统计学意义的为：（1）Porosity与BS／BV、Tb．Th;（2）BV／TV与BS／BV、Tb．Th;（3）BS／BV与Coma．Dn、Porosity、BV/TV、;（4）Tb．Th与Porosity、BV厂IV、Conn．Dn;（5）DA与Corm．Dn;（6）Conn．Dn与Bs／BV、Tb．Th、DA。结论：Micro-CT扫描、量化分析是评价支架材料结构参数的理想方法;也证明生物衍生骨支架符合骨组织工程对支架材料的三维结构要求,尤其在孔径大小、孔隙率、表面积体积比等三维结构参数,此外,也可为其他支架材料的制备在三维结构上的要求提供参考依据。     

用激光共聚焦显微镜鉴别可疑笔迹与印章

本文采用激光共聚焦显微镜鉴别可疑笔迹和印章及其它的文件。法医在鉴别可疑文件时通常都是凭经验和用普通光学显微镜,找出其中的一些物理特征。但还是有许多文件的笔迹顺序无法确定,尤其是墨水写的笔迹,为解决法医的这一问题,我们用激光共聚焦显微镜鉴别了72例不同铅笔和圆珠笔写的肉眼难于鉴别的交叉笔顺和其他的一些文字文件。在激光共激光共聚焦显微镜下大多数笔迹和印章都能发出荧光,因此很容易鉴别其笔顺和印章的特征,必要时还可以进行笔顺的三维图象构建,以帮助鉴别。结论：激光共聚焦显微镜可以更准确地鉴别可疑笔迹和印章。印章和笔迹的交叉也很容易分辨出来。     

Engineering of osteoinductive grafts by isolation and expansion of ovine bone marrow stromal cells directly on 3D ceramic scaffolds

In this work, we investigated whether osteoinductive constructs can be generated by isolation and expansion of sheep bone marrow stromal cells (BMSC) directly within three-dimensional (3D) ceramic scaffolds, bypassing the typical phase of monolayer (2D) expansion prior to scaffold loading. Nucleated cells from sheep bone marrow aspirate were seeded into 3D ceramic scaffolds either by static loading or under perfusion flow and maintained in culture for up to 14 days. The resulting constructs were exposed to enzymatic treatment to assess the number and lineage of extracted cells, or implanted subcutaneously in nude mice to test their capacity to induce bone formation. As a control, BMSC expanded in monolayer for 14 days were also seeded into the scaffolds and implanted. BMSC could be isolated and expanded directly in the 3D ceramic scaffolds, although they proliferated slower than in 2D. Upon ectopic implantation, the resulting constructs formed a higher amount of bone tissue than constructs loaded with the same number of 2D-expanded cells. Constructs cultivated for 14 days generated significantly more bone tissue than those cultured for 3 days. No differences in bone formation were found between samples seeded by static loading or under perfusion. In conclusion, the culture of bone marrow nucleated cells directly on 3D ceramic scaffolds represents a promising approach to expand BMSC and streamline the engineering of osteoinductive grafts.     

MSCs在自组装多肽纳米纤维支架中的三维培养及脂向分化研究

拟采用自组装多肽RADA16水凝胶建市MSCs的三维培养体系,以观察MSCs在RADA16中的黏附、形态及脂向分化情况.将第3代细胞置于RADA16水凝胶中进行三维培养.实验组细胞-材料复合物以脂向诱导液诱导,对照组以DMEM培养液培养,然后进行形态学观察,油红O染色及Western blot检测.荧光染色显示,绝大多数MSCs在RADA16水凝胶中能存活,且在不同的三维平面上生长.经诱导后,细胞内有大量油红O染色阳性的脂滴聚集,Western blot分析结果显示有脂肪细胞特异的蛋白PPARγ表达,且表达量随诱导时间的延长而增加.以上结果表明,RADA16水凝胶支架为MSCs的黏附、生长提供良好的三维环境,经定向诱导后支架内的MSCs可向脂肪组织方向分化.     

用气溶胶法和摇床法建立铜绿假单胞菌生物膜体外模型

目的模拟体内环境,体外建立细菌生物膜模型,为进一步深入研究细菌生物膜生物学特点提供基础。方法将粘附载体置于气溶胶法和摇床法模拟体内细菌生物膜形成的微环境中,将铜绿假单胞菌株培养3d后,取出标本分别进行通过FITC—ConA染色及SYT09／PI染色,然后分别进行荧光显微镜检测及激光共聚焦检测,观察细菌生物膜的形成情况;进行电子显微镜扫描观察形成的细菌生物膜的形态特点。结果在气溶胶的微环境下,FITC—ConA染色后在荧光显微镜观察到明亮成片状的细菌生物膜;SYT09／PI染色后在激光共聚焦检测,观察到片状,层叠如积云状,棉絮样的细菌生物膜;在电子显微镜扫描观察到大量细菌成团聚集,团状丛生突出表面,具有立体结构的细菌生物膜。在摇床法的微环境下,用3种检测方法都观察到成流线状的细菌生物膜。结论运用气溶胶法、摇床法可成功建立分别模拟体内呼吸系统及循环、泌尿系统的微环境下生物膜形成模型。     

Highly efficient uptake of ultrafine mesoporous silica nanoparticles with excellent biocompatibility by Liriodendron hybrid suspension cells

The characteristics of the interactions co-cultures of ultrafine mesoporous silica nanoparticles (MSNs) and the Liriodendron hybrid suspension cells were systematically investigated using laser scanning confocal microscope (LSCM) and scanning electron microscopy (SEM). Using fluorescein isothiocyanate (FITC) labeling, the LSCM observations demonstrated that MSNs (size, 5-15nm) with attached FITC molecules efficiently penetrated walled plant cells through endocytic pathways, but free FITC could not enter the intact plant cells. The SEM measurements indicated that MSNs readily aggregated on the surface of intact plant cells, and also directly confirmed that MSNs could enter intact plant cells; this was achieved by determining the amount of silicon present. After 24 h of incubation with 1.0mg mL-1 of MSNs, the viability of the plant cells was analyzed using fluorescein diacetate staining; the results showed that these cells retained high viability, and no cell death was observed. Interestingly, after the incubation with MSNs, the Liriodendron hybrid suspension cells retained the capability for plant regeneration via somatic embryogenesis. Our results indicate that ultrafine MSNs hold considerable potential as nano-carriers of extracellular molecules, and can be used to investigate in vitro gene-delivery in plant cells.