心肌细胞传感器及其在生物医学中的应用

传统心肌细胞电生理的研究,因其对细胞的穿刺损害作用,难以实现长时程测量,同时也难以实现对细胞群体的多位点同时测量.该研究采用微机械加工技术开展了基于细胞传感器(cell-based biosensor)的微电极阵列(microelectrode array, MEA)和光寻址电位传感器(light addressable potentiometric sensor, LAPS)等生物传感器技术,在传感器芯片表面培养了心肌细胞,并对其电生理特性进行了传感测量.该技术可对多个细胞同时进行长期、无损检测,在药物筛选、环境检测等生物医学领域具有良好的应用前景.     

TAS焊接技术在传感器等器件制造中的应用

本文介绍了采用Ar H_2混合气体保护自动点焊(TAS焊)新工艺,实现了压力传感器、电感传感器等器件的封装,文中对传感器结构及焊接特点,TAS焊接设备及工艺作了阐述。     

电喷雾电离-四极杆飞行时间质谱法研究三种β_2-受体激动剂的质谱裂解特征

采用电喷雾电离 -四极杆飞行时间质谱 ( ESI-Qq TOFMS)联用技术 ,对三种β2 -受体激动剂克仑特罗( Clenbuterol)、沙丁胺醇 ( Salbutamol)和盐酸 SPFF进行碰撞诱导解离 ( Collision induced dissociation,CID)研究。将三种药物标准品用甲醇溶解至 0 .1μg/m L直接进样 ,选择正离子方式进行检测。以质子化准分子离子 [M+H]+ 作为内标物 ,对碎片离子进行了准确质量测定 ,确认了这些碎片离子的元素组成 ,探讨了该类化合物的质谱裂解规律。研究发现 :它们的 ESI-MS2质谱分别生成脱去 H2 O分子、叔丁氨基等的碎片离子 ,其中叔丁氨基碎片离子 ( m/z74)为三种药物共有的碎片离子 ,这些特征可为 β2 -受体激动剂类药物的体内代谢转化和定量分析研究提供有力的依据。     

陶瓷微板热隔离半导体气体传感器阵列设计与实现

具有高浓度、挥发性、强氧化性的液氯、氮氧化物等危化品泄露、爆炸的安全检测一直是一个难点问题,要求检测传感器具有较宽量程和抗腐蚀设计。基于Al N陶瓷的微热板半导体气体传感器阵列设计,采用耐腐蚀的Al N陶瓷为衬底,物理化学性能稳定的Pt膜作为信号和加热器电极,经杂化修饰的In-Nb复合半导体氧化物为敏感材料,结合柔性光刻剥离工艺和激光微加工工艺,制备了陶瓷微板热隔离气体传感器阵列。为验证传感器阵列热结构设计的合理性,进行了有限元热仿真分析,优化了设计结构。经静态气敏测试分析,传感器阵列对浓度体积比500×10~(-6)的Cl_2和100×10~(-6)的NO_2两种气体的气敏响应时间分别为30 s和60 s左右,灵敏度最高分别为275倍和4倍,且在0~500×10~(-6)和0~100×10~(-6)检测范围均具有良好的气敏特性,对Cl_2等高浓度宽量程危化品气体检测具有良好的应用前景。     

细胞重组：机遇与挑战

人诱导性多能干（iPS）细胞能够分化成很多种类的祖细胞,这为临床和研究提供了新的机遇。这些细胞系可以用于疾病模型的建立、药物筛选以及毒性测试、帮助解决传统方法的局限性,也可以建立人类各种复杂疾病的细胞模型,对应用于更加个性化的医药研究领域也很有潜力。这些细胞系具有重新塑造临床研究领域的潜力,但是研究人员也面临着很多问题和挑战,也需要去发现新的应用。     

基于集成芯片的细胞生理参数自动分析仪的硬件设计

基于集成芯片的多功能细胞生理自动分析仪需要对微电极阵列传感器(Microelectrode Array Sensor, MEAS),光寻址电位传感器(Light-Addressable Potentiometric Sensor,LAPS)和细胞电阻抗传感器(Electric Cell Substrate Impedance Sensor, ECIS)3类传感器的输出信号进行检测.文中介绍了针对MEAS设计的专用前置放大电路;针对LAPS和ECIS,以AD5933集成阻抗转换芯片为核心,设计了电压衰减(增益)电路、电压偏置电路、频率扫描电路和偏压扫描电路,信号的放大采用了微弱电流采样技术.利用MSP430单片机编程实现了对整个系统的控制及与PC机的通讯.最后完成了对硬件系统的测试,结果表明文中的设计基本达到了系统检测的要求.     

细胞膜色谱在线HPLC-IT-TOF MS联用系统筛选分析射干中次野鸢尾黄素的抗EGFR活性作用

中药射干具有多种药理作用并得到广泛研究,但对其抗肿瘤活性组分的研究较少。本研究建立了一种用于筛选射干中抗肿瘤活性组分的方法。利用高表达表皮生长因子受体(EGFR)细胞制备细胞膜色谱柱,并通过十通切换阀与高效液相色谱-离子阱-飞行时间质谱(HPLC-IT-TOF MS)构建成二维在线联用系统。利用吉非替尼验证该系统的适用性,分子模拟对接实验研究活性组分与受体的相互作用方式,并利用MTT实验研究所筛选组分对EGFR细胞的体外抑制作用。结果表明,利用该系统可从射干总提取物中筛选出能够作用于EGFR的活性成分,并鉴定为次野鸢尾黄素;次野鸢尾黄素与EGFR的相互作用方式与阳性药吉非替尼相似;在浓度为0.4~50μmol/L范围内,吉非替尼、次野鸢尾黄素对高表达EGFR细胞的体外抑制作用具有剂量依赖性。EGFR细胞膜色谱在线HPLC-IT-TOF MS法有望成为从中草药中发现潜在抗肿瘤候选药物的有效方法。     

沙丁胺醇药物残留检测方法综述

沙丁胺醇药物是一种人工合成的β肾上腺素能受体激动剂(又称β受体兴奋剂)。因其具有营养再分配作用,在食品安全、体育运动等方面存在药物滥用问题,从而引起药物残留,进而对人体健康造成严重危害。基于对沙丁胺醇药物残留检测的需要,其检测方法就显得至关重要。本文回顾了近三十多年沙丁胺醇药物残留检测方法的研究使用情况,并根据不同检测方法分为光谱法、色谱法、质谱法、色-质联用法、免疫分析法、生物传感器及其他分析法共七种类型进行叙述说明,以期对研究沙丁胺醇及新型或其他β兴奋剂的相关部门及研究人员有所借鉴。     

小檗碱对TLR4蛋白表达和小胶质细胞活化的作用

目的研究小檗碱对Toll样受体-4(TLR4)蛋白表达和小胶质细胞活化的作用,探讨小檗碱调控脑缺血后炎症反应的可能机制。方法给予10μM小檗碱预处理小胶质细胞BV2,再经200ng/ml脂多糖(LPS)刺激24h后,分别用免疫印迹和免疫荧光染色方法检测TLR4、诱导型一氧化氮合酶(iNOS)和肿瘤坏死因子-α(TNF-α)蛋白的表达变化。结果免疫印迹结果显示,LPS刺激BV2细胞活化后,TLR4蛋白表达显著升高,iNOS和TNF-α表达明显增多。而小檗碱显著抑制了TLR4蛋白的表达升高,并降低了iNOS和TNF-α的蛋白水平。免疫荧光结果显示,LPS刺激BV2细胞后,在TLR4蛋白表达较高的细胞中,iNOS的表达水平也较高;小檗碱同时降低了两种蛋白的表达。结论小檗碱抑制了LPS刺激小胶质细胞活化后炎症因子的产生,该作用可能与降低TLR4蛋白的升高有关。     

基于碳点的小型自校正比率荧光光纤铬(VI)传感器

荧光比率传感探针检测方法能够克服环境(如样品基质、猝灭剂等)和仪器等外部因素影响, 得到更加可靠的检测结 果。 本文合成用于组成比率型荧光纳米探针的两种荧光碳点(CDs), 在 365 nm 紫外光的激发下, 这两种碳点分别在 440 nm 和 570 nm 处具有荧光发射峰, 且对 Cr(VI)具有相反的响应。 通过水凝胶将比率荧光探针包覆在设计的光纤尖端上, 能够实 现 Cr(VI)浓度的在线检测。 利用飞秒激光微加工系统制作的齿形光纤结构, 有利于荧光探针的激发和传感器荧光的收集。 为进一步降低传感器成本、缩减传感器体积, 采用低成本的 LED 灯珠作为激发光源。 采用比率荧光探针包覆, 克服 LED 灯珠 光源稳定性差的缺点, 使传感器可以实现自校正。 实验表明,即使光源强度波动较大, 该传感器连续 7 次测量的相对标准偏差 (RSD)仅为 3. 1% 。 在 0~ 200 μM 范围内, 该传感器对 Cr(VI)具有良好的线性关系, 检出限(LOD)为 0. 9 μM。 该传感器能够 应用于实际样品中 Cr(VI)的检测, 说明该传感器的具有实用性和可靠性。     

Ultrastructure and function of hepatic fat-storing and pit cells

The present paper reviews the literature on the ultrastructure and function of sinusoidal fat-storing cells and pit cells in the mammalian liver. Ultrastructurally, fat-storing cells are characterized by the presence of cytoplasmic fat droplets, well developed rough endoplasmic reticulum; a Golgi complex; multivesicular bodies; one or two centrioles; and few, rather small, lysosomes. These lysosomes are sometimes associated with fat droplets. Fat-storing cells may bear a cilium and project characteristic cytoplasmic processes into the space of Disse. These processes contain microtubules and filaments. Fat-storing cells are the main storage site of retinol esters in the mammalian body. Moreover, these cells have the potential of synthesizing several connective tissue components including the collagens type I, III, and IV; fibronectin; laminin; heparan sulfate; chondroitin sulfate; and dermatan sulfate. Pit cells are polarized cells, with most organelles localized at one site of the nucleus near the cytocentre. They are characterized electron microscopically by the presence of dense cytoplasmic granules with a specific ultrastructure, by rod-cored vesicles, and by multivesicular bodies. It has recently been shown that pit cells have natural killer activity to certain tumor cells and have many features in common with large granular lymphocytes. They therefore may act in the liver as a first line of defense against neoplasia, metastasis, and viral infections.     

Fine structure of hepatic sinusoids and sinusoidal cells in disease

Liver sinusoids are special capillaries that are limited by fenestrated endothelial cells, without a genuine basement membrane, surrounded by perisinusoidal cells storing vitamin A, and harbouring Kupffer cells and pit cells, resident macrophages, and large granular lymphocytes, respectively. Each nonparenchymal cell and parenchymal cell of the liver interacts with all others and with the extracellular matrix. Therefore, the functional ability of each cell is constantly being modified by the metabolic activity of the others. Human liver biopsies (132), needle or surgical, perfusion-fixed with glutaraldehyde and processed for transmission electron microscopy (TEM), and occasionally for scanning electron microscopy (SEM), were examined. The study included liver diseases (such as alcoholic liver diseases, benign and malignant liver tumors, cholestasis of various origins, fulminant hepatitis, acute rejection after orthotopic liver transplantation, Budd-Chiari syndrome), as well as general or extrahepatic diseases (such as diabetes, hemochromatosis, hypervitaminosis A, various hematological disorders), and normal controls. Ultrastructural abnormalities are described and illustrated under two different headings: (1) elementary lesions of sinusoidal cells (endothelial, Kupffer, perisinusoidal and pit cells), nonsinusoidal cells (in the space of Disse and/or in the lumen), the extracellular matrix; and (2) the major pathological entities including perisinusoidal fibrosis, capillarization of sinusoids, sinusoidal dilatation, and peliosis. In the discussion, an overview of the major abnormalities reported in the literature is presented, and some specific questions regarding (1) perisinusoidal fibrosis in liver with normal histology, (2) the overload of perisinusoidal cells with lipids in non-hypervitaminosis A intoxication and (3) the etiological relationship of sinusoidal dilatation, peliosis, perisinusoidal fibrosis, or sinusoidal tumors with drugs and toxic compounds are discussed. In the event that lesions are not specific to any diagnosis, the knowledge of the ultrastructure of sinusoids is extremely useful from the perspective of the liver as an ecosystem.     

Phagocytosis of apoptotic cells by liver: a morphological study

The present review deals with the morphological features of the removal of apoptotic cells by liver. The engulfment of cells undergoing apoptosis can be considered a specialized form of phagocytosis, playing a major role in the general tissue homeostasis in physiological and pathological conditions. In fact, defects of phagocytosis of apoptotic cells might have deleterious consequences for neighboring healthy cells, i.e., pathogenesis of inflammatory disease or dysregulation of the immune system. Phagocytosis of apoptotic cells by liver is a complex phenomenon, involving multiple molecular mechanisms of recognition (i.e., lectin-like receptors and receptors for externalized phosphatydilserine) of both parenchymal (hepatocytes) and nonparenchymal (Kupffer and endothelial cells) liver cells, often operating in cooperation. The data discussed in the present review are drawn from studies of phagocytosis of apoptotic cells in the liver, carried out with in vivo and in situ adhesion experiments as well as in vitro assays. Our results indicate that the three main liver cell types (hepatocytes, Kupffer, and endothelial cells) are able to recognize and internalize apoptotic cells by means of specific receptors (galactose and mannose-specific receptor; receptor for phosphatydilserine) and by cytoskeletal reorganization that favors the engulfment of the apoptotic cells. The "flags" for the identification of apoptotic cells by the liver are modifications of the surface of dead cells, i.e., sugar residues and phosphatydilserine exposition. Vitronectin receptor is not involved in such a recognition. The adhesions between modified cell surfaces of apoptotic cells and phagocytes generate cytoplasmatic signaling pathways that drive apoptotic cells to their final fate within the phagocytes (i.e., lysosomal digestion).     

Comparative characterization of human fetal neural stem cells and induced neural stem cells from peripheral blood mononuclear cells

Human-induced neural stem cells (iNSCs) transplantation is a potential treatment of neurodegeneration diseases. However, whether the reprogrammed cells have the same characterizations as human fetal neural stem cells needs further exploration. Here we isolated human fetal neural stem cells from aborted 12-week fetal brains and compared with iNSCs reprogrammed from human peripheral blood mononuclear cells in gene expression, proliferation ability, differentiation capacity, and the responses to tumor necrosis factor-α. We found that iNSCs and NSCs both expressed neural stem cell markers Nestin, SOX1, and SOX2. However, only iNSCs can be patterned into dopaminergic neurons and motor neurons. Furthermore, both iNSCs and NSCs can differentiate into oligodendrocyte progenitor cells. In addition, a low dose of tumor necrosis factor-α did not inhibit the proliferation and differentiation of iNSCs and NSCs. In conclusion, iNSCs have properties similar to, and even better than, fetal neural stem cells and may be suitable for disease modeling and transplantation.     

Ultrastructure of the seminiferous epithelium and intertubular tissue of the human testis

The ultrastructural features of the human testis are reviewed with emphasis upon the process of spermatogenesis and the cytology of the Leydig cells. The seminiferous epithelium is structurally partitioned by the Sertoli cells into basal and adluminal compartments via the specialized tight junctions between the Sertoli cells. Spermatogonia reside in the basal compartment, and, via a series of cell divisions, produce the primary spermatocytes, which at the commencement of their development move into the adluminal compartment, and thus the lengthy process of meiotic maturation is initiated. The fine structure of primary spermatocytes is described together with the complex transformation of the spermatids into spermatozoa during the process of spermiogenesis. Earlier studies of the organization of the human seminiferous epithelium showed that germ cells at different developmental stages formed identifiable collections termed cell associations or stages, but since several stages were seen in a single tubule cross-section, this gave the impression of an extremely irregular pattern of spermatogenic development. When the topographic arrangement of germ cells was re-examined with the aid of computer modelling, a highly ordered distribution was revealed, conforming to a helical pattern based on the geometry of spirals. Thus spermatogenesis in the human testis is subjected to a precise regulation in keeping with the ordered arrangement of the germ cells seen in other mammalian species. The intertubular tissue of the human testis is composed of loose connective tissue containing blood vessels, occasional lymph capillaries, macro-phages, mast cells, and the Leydig cells which occur either as single cells or form small clusters. The Leydig cell cytoplasm contains an abundant supply of smooth endoplasmic reticulum and mitochondria with tubular cristae, both features being characteristic of steroidogenic cells. Human Leydig cells contain large Reinke crystalloids of variable size and number, but their function remains obscure. The frequent occurrence of paracrystalline inclusions within the cytoplasm of the human Leydig cell suggests that these elements are precursors of the Reinke crystalloids.     

剪切力对动脉血管平滑肌细胞增殖分化的影响

目的：探讨剪切力作用下人脐动脉血管平滑肌细胞的增殖分化的变化。方法：利用改进的灌流及流动培养装置,通过蠕动泵提供稳定的剪切力,同时提供静态培养所需的其它条件,建立体外人脐动脉血管平滑肌流动培养模型。分别对体外培养的人脐动脉血管平滑肌细胞加载3,4,5,8,10dyn／cm^2的定长流剪切力24h,同时以静态培养的细胞为对照组。相差倒置显微镜观察玻片上细胞的数量并作细胞计数,α—actin免疫组化染色．结果：细胞记数显示,同对照组相比,七刀应力各组的增殖能力都有所下降,5dyn／cm^2切应力作用下,细胞增殖缓慢最为明显．α—actin免疫组化染色提示切应力各组的分化程度较对照组高。结论：初步研究表明,剪切力对人脐动脉血管平滑肌细胞的增殖有一定抑制作用,并且可能促进了细胞的分化。     

Study of the conditions necessary for propane-jet freezing of fresh biological tissues without detectable ice formation

The performance of a commercial double-propane-jet freezer (Balzers QFD 101) has been assessed, for rapid freezing of fresh tissues in freeze-etch work. Samples of diaphragm muscle and intestinal villi were frozen between copper sheets, with a spacer to give 20–30μm thickness of tissue. Fracture cuts were made with the Balzers BAF 400 freeze-etch microtome within 5–10μm of a freezing face (i.e. a tissue face in contact with the copper sheets of the frozen sandwich). After some modifications to the QFD 101, replicas showing no evidence of ice were obtained of muscle cells, although for intestinal epithelial cells some evidence of ice formation was found. Infiltration with 5% glycerol or dimethylsulphoxide improves the depth of good freezing. Results and problems arising from such infiltration are briefly discussed.     

MRT letter: A novel tegumental gland in female imagoes of the neotropical termite Cornitermes cumulans (Isoptera,termitidae, syntermitinae)

In general, the exocrine glands of social insects are structures involved in the chemical communication associated with social life. Here, we report the discovery of an unknown tegumental gland that is present in the female imagoes of Cornitermes cumulans and occurs next to the well‐developed tergal glands that have previously been described. The tegumental glands release their secretion in the intersegmental membrane and are composed of bicellular units, a secretory cell and a canal cell, that are closely located to the epidermal cells in the inferior part of the eighth and ninth tergites. The ultrastructure of the glandular cells showed abundant smooth endoplasmic reticulum, suggesting that the secretion may be pheromonal, although its function is still unknown. These exocrine structures are facing the tergal glands, and we hypothesized that they act synergistically with the tergal glands to generate short‐range attraction during tandem behavior. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

Planar patch-clamp force microscopy on living cells

Here we report a new combination of the patch-clamp technique with the atomic force microscope (AFM). A planar patch-clamp chip microstructured from borosilicate glass was used as a support for mechanical probing of living cells. The setup not only allows for immobilizing even a non-adherent cell for measurements of its mechanical properties, but also for simultaneously measuring the electrophysiological properties of a single cell. As a proof of principle experiment we measured the voltage-induced membrane movement of HEK293 and Jurkat cells in the whole-cell voltage clamp configuration. The results of these measurements are in good agreement with previous studies. By using the planar patch-clamp chip for immobilization, the AFM not only can image non-adhering cells, but also gets easily access to an electrophysiologically controlled cellular probe at low vibrational noise.     

Differentiation of Leydig cells in the Mongolian gerbil

Information on postnatal Leydig cell (LC) differentiation in the Mongolian gerbil has been unavailable. Therefore, current investigation was designed to examine LC lineage differentiationin this rodent, from birth to adulthood. Gerbil testes at 1 day, 1–7 weeks (w), 2 and 3 months of age were conventionally processed by light and transmission electron microscopy. Immunocytochemistry for specific markers of steroidogenic enzymes, namely 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and 11β‐hydroxysteroid steroid dehydrogenase 1 (11β‐HSD1) and also for androgen receptor (AR) was performed. The establishment of adult Leydig cell populations (ALC) during testis maturation in the gerbil follows the pattern previously described in other mammalian species, with the four progressive stages of differentiation. The LC progenitors were identified at second w by 3β‐HSD expression; the first newly formed ALC were recognized at fourth w whereas immature ALC appeared at fifth w. The latter were recognized by abundance of cytoplasmic lipid, in addition to expression of 11β‐HSD1 and intense nuclear AR immunoreaction. Mature ALC in gerbil exhibited irregular eccentric nuclei and a cytoplasmic canaliculus in the perinuclear area. Only one third of mature ALC in adult gerbils showed a high expression of 11β‐HSD1 and AR expression was highly variable among them. In conclusion, the process of differentiation of ALC population in gerbil follows the pattern previously established for other rodents. However, the resulting mature ALC are strikingly different due their singular asymmetric morphology and presence of a cytoplasmic canaliculus and as well as their functional heterogeneity. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.