Cell-attached膜片上,咖啡因对猪冠状动脉平滑肌细胞K_(Ca)的调节作用

目的:研究咖啡因对猪冠状动脉平滑肌细胞KCa通道的调控机理,以期揭示胞内钙库RyR激活后,局部钙离子浓度升高和KCa的关系。方法:采用急性酶分离方法,应用膜片钳单通道电流记录技术记录大脑皮层神经元猪冠状动脉平滑肌细胞上KCa通道电流活动。电流信号经放大、滤波及A/D、D/A转换后输入微机进行采样和储存。实验数据应用CLAMP9.0软件系统进行数据采集及分析。结果:在cell-attached膜片上咖啡因对KCa通道有明显的作用,咖啡(0.1-5.0 mM)可以增加通道的开放概率(NPo),呈现出浓度依赖性,开放时间延长,关闭时间也随之缩短,而对电流幅值无明显影响,开放概率的增加是通过明显缩短平均关闭时间实现的(n=8,P<0.05);洗去药物后通道活性可以一定程度的恢复到对照水平,再加入一定浓度咖啡因(如1.0mM)可再次激活KCa,激活程度与洗脱前较接近。结论:在细胞贴附式构型上咖啡因浓度依赖性地激活KCa,有饱和性。可能是通过影响胞内信号转导过程而调控KCa活性。

Integrated of Proximity-Coupling High-Order Modes Patch Antenna with Solar Cells for Sustainable Communication

An integration of single-layer proximitycoupling patch antenna and solar cells with bandwidth enhancement and optical energy harvesting is proposed for sustainable communication.For this purpose,many dual-function components are selected for designing the miniaturized solar cell antenna.On the one hand,by greatly affecting the current flow of the rectangular patch,vias and proximity-coupling are introduced to control the resonance modes frequency and matching,respectively,for wideband application,and the radiation performance property can be achieved by high-order mode.On the other hand,vias and proximity-coupling are beneficial to complete direct-current(DC)loop of solar cell and improve compatibility of DC-RF(radio frequency),whereas a high-order mode is beneficial to increase the area of collected light energy.To prove the working principle,fabricated and manufactured solar cell antenna.The measured and simulated results illustrate that the solar cell antenna gain is raised to as high as 9.27 d Bi in4.37 to 5.06 GHz applied to fifth generation communication(5G).

An Ultrastructural Study of Phagocytosis and Transport of Formalized Campylobacter jejuni By M Cells into Mouse Peyer's Patches

Suspension of formalized Campylobacter jejunt (2×10 CFU/ml)was injected into a bothend-ligated intestinal loop of ileum which contained some Peyer's patches from non-immunized adult mice after laparotomy under anesthesia.After 1-hour post inoculation, the specimen was taken out and prepared for TEM and SEM observation.The results showed that bacteria adhered specifically on the surface of M cells of Peyer's patches.The microvilli and microfolds of the M cells fused to capture the bacteria and to form a large endocytotic vesicle in the cytoplasm of the M cell,then transported inward,and, at last.released into the central cavity between lymphocytes.Occasionally, we found some of them were phagocytosed by lymphocytes.

The role of the hedgehog/patched signaling pathway in epithelial stem cell proliferation: from fly to human

The hedgehog-patched (hh-ptc) intercellular signaling pathway has recently been shown to control the proliferation of epithelial stem cells in both Drosophila and vertebrates. Mutant and ectopic expression analyses in Drosophila suggest that the HH protein diffuses from the signaling cells to promote the proliferation of nearby ovarian somatic stem cells by antagonizing the suppression of its receptor PTC towards the CI transcription factor in the stem cells. Consequently, the transcription of CIdependent genes leads to stem cell proliferation. This regulatory pathway appears to function also in vertebrates,where defects in ptc cause basal cell carcinoma, tumors of epidermal stem cell origin. Basal cell carcinoma can also be induced by ectopic expression of Sonic hedgehog (shh) or Glil, the vertebrate homolog of ci. These studies suggest the conservation of the hh signaling pathway in controlling epithelial stem cell divisions among different organisms.

Induction of pancreatic duct cells of neonatal rats into insulin-producing cells with fetal bovine serum: A natural protocol and its use for patch clamp experiments

AIM： To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study. METHODS： The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine ceils with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic β-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels （KATP）, voltage-dependent potassium channels （Kv）, and voltage-dependent calcium channels （KcA） in β-cells were identified by patch clamp technique. RESULTS： After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly （MTT value from 0.024 ±0.003 to 3.028±0.003, insulin secretion from 2.6±0.6 to 3.1±0.8 mIU/L）. Then MTT value and insulin secretion increased quickly from d 5 to d 10 （MTT value from 0.028 ±0.003 to 0.052±0.008, insulin secretion from 3.1±0.8 to 18.3±2.6 mIU/L）, then reached high plateau （MTT value 〉0.052±0.008, insulin secretion 〉18.3±2.6 mIU/L）. In contrast, for the isolated adult pancreatic islet cells, both insulin release and MTT value were stable in the first 4 d （MTT value from 0.029±0.01 to 0.031±0.011, insulin secretion from 13.9±3.1 to 14.3±3.3 mIU/L）, but afterwards they reduced gradually （MTT value 〈0.031 ±0.011, insulin secretion 〈8.2±1.5 mIU/L）, and the pancreatic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic β-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from KATP, Kv, and KCA. CONCLUSION： Islet cells differentiated from neonatal pancreatic duct cells with the new natural protocol are more advantageous in performing patch clamp study over the isolated adult pancreatic islet cells.

A Biodegradable Knitted Cardiac Patch for Myocardium Regeneration Using Cardiosphere-Derived Cells(CDCs)

In order to regenerate myocardium and provide appropriate mechanical support after a heart attack,jersey,tuck and rib stitch structures were knitted from polylactic acid(PLA)yarns to fabricate a cardiac patch,which mimicked the mechanical properties of myocardium in both directions.Cardiosphere-derived cells(CDCs) were seeded on these PLA patch fabrics,and using scanning electron microscopy(SEM) characterization and an MTT assay the cells proliferated and attached successfully to the PLA fabrics.Based on the results,the rib stitch structure is the most promising candidate for fabricating cardiac patches due to its high elasticity and its ability to promote cell proliferation.

Interaction between enteric epithelial cells and Peyer's patch lymphocytes in response to Shigella lipopolysaccharide: Effect on nitric oxide and IL-6 release

AIM： TO investigate the effect of interaction between enteric epithelial cells and lymphocytes of Peyer＇s patch on the release of nitric oxide （NO） and IL-6 in response to Shigella lipopolysaccharide （LPS）. METHODS： Human colonic epithelial cells （Caco-2） were mixed cocultured with lymphocytes of Peyer＇s patch from wild-type （C57 mice） and inducible NO synthase knockout mice, and challenged with Shigella F2a-12 LPS. Release of NO and raiL-6 was measured by Griess colorimetric assay and enzyme-linked immunosorbent assay （ELISA）, respectively. RESULTS： In the absence of LPS challenge, NO was detected in the culture medium of Caco-2 epithelial cells but not in lymphocytes of Peyer＇s patch, and the NO release was further up-regulated in both cocultures with lymphocytes from either the wild-type or iNOS knockout mice, with a significantly higher level observed in the coculture with iNOS knockout lymphocytes. After Shigella F2a-12 LPS challenge for 24-h, NO production was significantly increased in both Caco-2 alone and the coculture with lymphocytes of Peyer＇s patch from the wild-type mice but not from iNOS knockout mice. LPS was found to stimulate the release of mIL-6 from lymphocytes, which was suppressed by coculture with Caco-2 epithelial cells. The LPS-induced mIL-6 production in lymphocytes from iNOS knockout mice was significantly greater than that from the wild-type mice. CONCLUSION： Lymphocytes of Peyer＇s patch maintain a constitutive basal level of NO production from the enteric epithelial cell Caco-2. LPS-induced mIL-6 release from lymphocytes of Peyer＇s patch is suppressed by the cocultured epithelial cells. While no changes are detectable in NO production in lymphocytes from both wild-type and iNOS knockout mice before and after LPS challenge, NO from lymphocytes appears to play an inhibitory role in epithelial NO release and their own mIL-6 release in response to LPS.

马钱子碱抑制豚鼠心室肌细胞钠电流的作用研究

目的本研究旨在分析马钱子碱(brucine)对分离的单个豚鼠心室肌细胞钠电流(INa+)的作用,并探索其抗心律失常的可能机制。方法共选取24只健康的成年豚鼠作为实验对象,雌雄不拘,随机分为4组,每组6只:第1组是未接受任何处理的对照组,之后通过微量加样器使培养皿中brucine药物的终浓度分别达到3μmol/L、10μmol/L、30μmol/L。采用急性酶解法分离获得单个豚鼠心室肌细胞,通过全细胞膜片钳技术测量离子通道电流,以检测不同浓度的brucine对豚鼠心室肌细胞INa+的影响。结果正常的对照组没有明显的INa+电流峰值的变化,而brucine 3μmol/L组给药前后基本不影响INa+电流峰值,当brucine的浓度增加到10μmol/L时,给药前后INa+电流峰值显著下降(P<0.05),在30μmol/L组给药前后INa+电流峰值大幅度减少(P<0.01),并且这种抑制作用呈浓度依赖性。正常对照组及brucine 3μmol/L剂量组中,电流-电压曲线(Ⅰ~Ⅴ曲线)并未受到显著的影响;然而,10μmol/L、30μmol/L剂量组与对照组比较,INa+的Ⅰ~Ⅴ曲线上移,无平行移动,且曲线形状不变。结论Brucine能够通过抑制心室肌细胞钠通道电流发挥抗心律失常作用。

吡罗昔康贴剂溶出度的测定及不同测定方法结果的评估

目的:采用桨碟法、转筒法、扩散池法测定吡罗昔康贴剂的溶出度,并对溶出结果进行评价,以期选择能更加准确反映吡罗昔康贴剂释药过程的测定方法,为科学准确评价药品质量提供参考。方法:建立检测吡罗昔康的液相色谱测定方法,分别使用桨碟法、转筒法、扩散池法考察吡罗昔康贴剂的24 h溶出曲线,分别使用f 1差异因子法、f 2相似因子法、Weibull模型拟合对溶出曲线进行比较,评估不同测定方法的结果。结果:吡罗昔康在1~150μg·mL^(-1)范围内线性关系良好(r=1.000),准确度100.9%(n=9),精密度1.7%(n=9),样品溶液在72 h内稳定。溶出曲线比较结果表明,吡罗昔康贴剂的溶出曲线更符合Weibull模型。在相同溶出介质和温度的条件下,桨碟法和转筒法差异不大,存在相互替代的可能性,扩散池法与其他2种方法均存在显著性差异。结论:扩散池法的装置更加符合吡罗昔康贴剂在实际使用中的溶出过程,为该药物的质量评价提供更多的参考。

Isolation of Protoplast and Ion Channel Recording in Plasma Membrane of Suspension Cells of Populus euphratica

The authors used suspension cells of Populus euphratica to isolate protoplast in the present study. Protoplasts were successfully obtained after 4 hours incubation in enzyme solution containing 1 0% cellulase “onozuka” R\|10, 0\^01% pectolyase Y\|23,0\^15% macerozyme R\|10 and 0\^1% hemicellulase at 25℃. Outward and inward single channels in plasma membrane were observed using cell\|attached recording of patch\|clamp technique. In this study, single channel records showed that more than one species of channel were obtained. These attempts in protoplast isolation and ion channel recording offers the opportunity to characterize cellular mechanisms of salt tolerance in tree species.

心肌补片:细胞来源、完善策略及最佳制作方法分析

背景:心肌补片是一种修复损伤心肌的有效方式,但目前使用哪种细胞制作心肌补片和如何使心肌补片在体内发挥最大的治疗效果还存在争议。目的:通过综述心肌补片的细胞来源和完善心肌补片的策略来寻找出制作心肌补片的最佳方法。方法:由第一作者应用计算机以“cell sheet,cell patch,cardiomyocytes,cardia progenitor cells,fibroblasts,embryonic stem cell,mesenchymal stem cells”等为英文检索词检索PubMed和Web of Science数据库;以“心肌补片,生物3D打印,心肌”为中文检索词检索中国知网和万方数据库,经过入组筛选后,最终纳入94篇文献进入结果分析。结果与结论:①心肌补片的细胞来源主要分为3类:分别是体细胞、单能干细胞和多能干细胞。心肌补片的细胞来源丰富,但是并不是所有的细胞都适合制作心肌补片,例如,成纤维细胞和骨骼肌母细胞制成的心肌补片会有致心律失常的风险,间充质干细胞在体内的作用时间短并且存在伦理方面的问题。随着诱导式多功能干细胞的发现,为制作心肌补片提供了一个可靠的细胞来源。②心肌补片的制作方法有两种:一种是使用细胞片技术,另一种是使用生物3D打印技术。细胞片技术可完整保留细胞外基质成分,可以最大程度地模拟细胞在体内的生长循环,但是想要通过细胞片技术获得具有三维结构的心肌补片还存在困难。而生物3D打印技术可以通过计算机个性化设计获得具有三维结构的心肌补片。③完善心肌补片的策略主要包括:多种细胞共同培养后制作成心肌补片、改进生物3D打印技术中的墨水配方和支架成分、提高心肌补片的治疗效果、抑制移植后的免疫排斥反应和完善干细胞的分化培养方案。④目前还没有制作心肌补片的最佳细胞来源和制作方法,单靠某一种细胞或者某一种技术所获得的心肌补片常无法达到预期的治疗效果,因此研究者们在制作心肌补片之前需要根据预期的治疗效果来选择合适的策略制作心肌补片。

Dual human iPSC-derived cardiac lineage cell-seeding extracellular matrix patches promote regeneration and long-term repair of infarcted hearts

Human pluripotent stem cell-derived cardiovascular progenitor cells (hCVPCs) and cardiomyocytes (hCMs) possess therapeutic potential for infarcted hearts;however, their efficacy needs to be enhanced. Here we tested the hypotheses that the combination of decellularized porcine small intestinal submucosal extracellular matrix (SIS-ECM) with hCVPCs, hCMs, or dual of them (Mix, 1:1) could provide better therapeutic effects than the SIS alone, and dual hCVPCs with hCMs would exert synergic effects in cardiac repair. The data showed that the SIS patch well supported the growth of hCVPCs and hCMs. Epicardially implanted SIS-hCVPC, SIS-hCM, or SIS-Mix patches at 7-day post-myocardial infarction significantly ameliorated functional worsening, ventricular dilation and scar formation at 28- and 90-day post-implantation in C57/B6 mice, whereas the SIS only mildly improved function at 90-day post-implantation. Moreover, the SIS and SIS-cell patches improved vascularization and suppressed MI-induced cardiomyocyte hypertrophy and expression of Col1 and Col3, but only the SIS-hCM and the SIS-Mix patches increased the ratio of collagen III/I fibers in the infarcted hearts. Further, the SIS-cell patches stimulated cardiomyocyte proliferation via paracrine action. Notably, the SIS-Mix had better improvements in cardiac function and structure, engraftments, and cardiomyocyte proliferation. Proteomic analysis showed distinct biological functions of exclusive proteins secreted from hCVPCs and hCMs, and more exclusive proteins secreted from co-cultivated hCVPCs and hCMs than mono-cells involving in various functional processes essential for infarct repair. These findings are the first to demonstrate the efficacy and mechanisms of mono- and dual-hCVPC- and hCM-seeding SIS-ECM for repair of infarcted hearts based on the side-by-side comparison.

手动膜片钳检测盐酸罗哌卡因及其右旋异构体对HEK293细胞hERG电流的影响

目的研究比较盐酸罗哌卡因和盐酸罗哌卡因右旋异构体对高表达hERG钾通道的HEK293细胞hERG电流的影响。方法用手动膜片钳检测转染后hERG钾通道稳定表达的HEK293细胞电流,多菲莱德做阳性药,将盐酸罗哌卡因和盐酸罗哌卡因右旋异构体依次稀释成30.00、10.00、3.33、1.11、0.37μmol·L^(-1),依次作用于细胞,记录电流变化,计算抑制率。结果盐酸罗哌卡因0.37、1.11、3.33、10、30μmol·L^(-1)对电流Iherg-tail的抑制率分别为(6.12±0.30)%、(13.04±1.20)%、(19.21±0.33)%、(35.56±0.66)%、(65.37±4.17)%,IC_(50)为19.482μmol·L^(-1)(n=15)。盐酸罗哌卡因右旋异构体0.37、1.11、3.33、10.00、30.00μmol·L^(-1)对电流Iherg-tail的抑制率分别为(4.13±3.43)%、(7.34±5.60)%、(9.49±2.75)%、(16.60±0.87)%、(31.36±1.45)%,IC_(50)>30μmol·L^(-1)(n=15)。阳性对照药品多菲莱德0.00185、0.00556、0.01667、0.05000、0.15000μmol·L^(-1)对电流Iherg-tail的抑制率分别为(7.81±2.77)%、(19.67±1.88)%、(57.16±4.39)%、(89.71±3.55)%、(99.66±0.89)%、IC_(50)为0.015μmol·L^(-1)(n=15)。结论和阳性对照药品多菲莱德比较,盐酸罗哌卡因对hERG通道为弱抑制作用,盐酸罗哌卡因右旋异构体对hERG通道为无明显抑制作用。

K^+channels inhibited by hydrogen peroxide mediate abscisic acid signaling in Vicia guard cells

A number of studies show that environmental stress conditions increase abscisic acid (ABA) and hydrogen peroxide (H2O2) levels in plant cells. Despite this central role of ABA in altering stomatal aperture by regulating guard cell ion transport, little is known concerning the relationship between ABA and H2O2 in signal transduction leading to stomatal movement. Epidermal strip bioassay illustrated that ABA- inhibited stomatal opening and ABA-induced stomatal closure were abolished partly by externally added catalase (CAT) or diphenylene iodonium (DPl), which are a H2O2 scavenger and a NADPH oxidase inhibitor respectively. In contrast, internally added CAT or DPI nearly completely or partly reversed ABA-induced closure in half-stoma. Consistent with these results, whole-cell patch-clamp analysis showed that intracellular application of CAT or DPI partly abolished ABA-inhibited inward K+ current across the plasma membrane of guard cells. H2O2 mimicked ABA to inhibit inward K+ current, an effect which was reversed by the addition of ascorbic acid (Vc) in patch clamping micropipettes. These results suggested that H2O2 mediated ABA-induced stomatal movement by targeting inward K+ channels at plasma membrane.

Effects of Lizhong Tang on cultured mouse small intestine interstitial cells of Cajal

AIM:To investigate the effects of Lizhong Tang,an herbal product used in traditional Chinese medicine,on mouse small intestine interstitial cells of Cajal(ICCs).METHODS:Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues.The ICCs were morphologically distinct from other cell types in culture and were identified using phase contrast microscopy after verification with anti c-kit antibody.A whole-cell patch-clamp configuration was used to record potentials(current clamp) from cultured ICCs.All of the experiments were performed at 30-32 ℃.RESULTS:ICCs generated pacemaker potentials,and Lizhong Tang produced membrane depolarization in current-clamp mode.The application of flufenamic acid(a nonselective cation channel blocker) abolished the generation of pacemaker potentials by Lizhong Tang.Pretreatment with thapsigargin(a Ca 2+-ATPase inhibi-tor in the endoplasmic reticulum) also abolished the generation of pacemaker potentials by Lizhong Tang.However,pacemaker potentials were completely abolished in the presence of an external Ca 2+-free solution,and under this condition,Lizhong Tang induced membrane depolarizations.Furthermore,When GDPβ-S(1 mmol/L) was in the pipette solution,Lizhong Tang still induced membrane depolarizations.In addition,membrane depolarizations were not inhibited by chelerythrine or calphostin C,which are protein kinase C inhibitors,but were inhibited by U-73122,an active phospholipase C inhibitors.CONCLUSION:These results suggest that Lizhong Tang might affect gastrointestinal motility by modulating pacemaker activity in interstitial cells of Cajal.

Cardiogenic differentiation of mesenchymal stem cells on elastomeric poly (glycerol sebacate)/collagen core/shell fibers

AIM: To facilitate engineering of suitable biomaterials to meet the challenges associated with myocardial infarction. METHODS: Poly (glycerol sebacate)/collagen (PGS/ collagen) core/shell fibers were fabricated by core/ shell electrospinning technique, with core as PGS and shell as collagen polymer; and the scaffolds were characterized by scanning electron microscope (SEM), fourier transform infrared spectroscopy (FTIR), contact angle and tensile testing for cardiac tissue engineering. Collagen nanofibers were also fabricated by electrospinning for comparison with core/shell fibers. Studies on cell-scaffold interaction were carriedout using cardiac cells and mesenchymal stem cells (MSCs) co-culture system with cardiac cells and MSCs separately serving as positive and negative controls respectively. The co-culture system was characterized for cell proliferation and differentiation of MSCs into cardiomyogenic lineage in the co-culture environment using dual immunocytochemistry. The co-culture cells were stained with cardiac specific marker proteins like actinin and troponin and MSC specific marker protein CD 105 for proving the cardiogenic differentiation of MSCs. Further the morphology of cells was analyzed using SEM.RESULTS: PGS/collagen core/shell fibers, core is PGS polymer having an elastic modulus related to that of cardiac fibers and shell as collagen, providing natural environment for cellular activities like cell adhesion, proliferation and differentiation. SEM micrographs of electrospun fibrous scaffolds revealed porous, beadless, uniform fibers with a fiber diameter in the range of 380 ± 77 nm and 1192 ± 277 nm for collagen fibers and PGS/collagen core/shell fibers respectively. The obtained PGS/collagen core/shell fibrous scaffolds were hydrophilic having a water contact angle of 17.9 ± 4.6° compared to collagen nanofibers which had a contact angle value of 30 ± 3.2°. The PGS/collagen core/shell fibers had mechanical properties comparable to that of native heart muscle with a young's modulus of 4.24 ± 0.7 MPa, while that of collagen nanofibers was comparatively higher around 30.11 ± 1.68 MPa. FTIR spectrum was performed to confirm the functional groups present in the electrospun scaffolds. Amide Ⅰ and amide Ⅱ of collagen were detected at 1638.95 cm -1 and 1551.64 cm -1 in the electrospun collagen fibers and at 1646.22 cm -1 and 1540.73 cm -1 for PGS/collagen core/shell fibers respectively. Cell culture studies performed using MSCs and cardiac cells co-culture environment, indicated that the cellproliferation significantly increased on PGS/collagen core/shell scaffolds compared to collagen fibers and the cardiac marker proteins actinin and troponin were expressed more on PGS/collagen core/shell scaffolds compared to collagen fibers alone. Dual immunofluorescent staining was performed to further confirm the cardiogenic differentiation of MSCs by employing MSC specific marker protein, CD 105 and cardiac specific marker protein, actinin. SEM observations of cardiac cells showed normal morphology on PGS/collagen fibers and providing adequate tensile strength for the regeneration of myocardial infarction. CONCLUSION: Combination of PGS/collagen fibers and cardiac cells/MSCs co-culture system providing natural microenvironments to improve cell survival and differentiation, could bring cardiac tissue engineering to clinical application.

Whole-cell recordings of calcium and potassium currents in acutely isolated smooth muscle cells

AIM： To record calcium and potassium currents in acutely isolated smooth muscle cells of mesenteric arterial branches in rats. METHODS： Smooth muscle cells were freshly isolated by collagenase digest and mechanical trituration with polished pipettes. Patch clamp technique in whole-cell mode was employed to record calcium and potassium currents. RESULTS： The procedure dissociated smooth muscle cells without impairing the electrophysiological characteristics of the cells. The voltage-gated Ca^2＋ and potassium currents were successfully recorded using whole-cell patch clamp configuration. CONCLUSION： The method dissociates smooth muscle cells from rat mesenteric arterial branches. Voltage-gated channel currents can be recorded in this preparation.

Effects of Atractylodes Macrocephala on the Cytomembrane Ca^(2+)-activated K^+ Currents in Cells of Human Pregnant Myometrial Smooth Muscles

The study examined the inhibitory effect of Atractylodes macrocephala （AM） on the uterine contraction during premature delivery and explored its electrophysiological mechanism by studying the effects of AM on the Ca^2＋-activated K^＋ currents of pregnant human myometrial smooth muscle cells with or without the treatment with intedeukin-6. Single cells were acutely isolated from pregnant human myometrial smooth muscles. Whole-cell Ca^2＋-activated K^＋ currents were recorded by using an Axopatchl-D amplifier. The cells were divided into three groups： group A in which AM was added into perfusate, group B, in which interleukin-6 was added into perfusate） and group C in which AM was added into perfusate after addition of interleukin-6. IL-6 10 ng/mL inhibited BKca by 36.9%±13.7% as compared with control （P〈0.01）. AM at 2 mg/mL raised BKca by 36.7%±22.6% or 45.2%±13.7% with or without the treatment of IL-6, respectively （P〈0.01）. It is concluded that AM was able to enhance the BKca of pregnant human myometrial smooth muscle cells treated or untreated with interleukin-6 and its effect on the BKca IL-treated cells was stronger that its effect on BKca of untreated cells. Our results suggested that AM can help to maintain the membrane potentials and the resting status of pregnant human myometrial smooth muscle cells.

Tyrosine hydroxylase-positive cells and dopaminergic neuronal function in human embryonic stem cells: An electrophysiological validation

BACKGROUND： Induced differentiation strategies and cytochemical properties of human embryonic stem ceils （hESCs） have been investigated. However, the electrophysiological functions of tyrosine hydroxylase （TH）-positive cells dedved from hESCs remain unclear. OBJECTIVE： To investigate the differentiation efficiency of TH-positive cells from hESCs in vitro using modified four-step culture methods, including embryoid body formation, and to examine the functional characteristics of the differentiated TH-positive cells using electrophysiological techniques. DESIGN, TIME AND SETTING： Neuroelectrophysiology was performed at the Reproductive Medicine Center and Stem Cell Research Center, Peking University Third Hospital, and the Neuroscience Research Institute and Department of Neurobiology, Peking University, from September 2004 to August 2008. MATERIALS： The hESC line, PKU-1.1, a monoclonal cell line derived from a pre-implantation human blastocyst in the Reproductive Medical Center of Peking University Third Hospital. The patch clamp recording system was provided by the Neuroscience Research Institute and Department of Neurobiology, Peking University. METHODS： The hESC line was induced to differentiate into TH-positive cells in vitro using a modified four-step culture method, including the formation of embryoid body, as well as the presence of sonic hedgehog and fibroblast growth factor 8. The cell karyotype was assessed by G-banding karyotype analysis techniques and specific markers were detected immunocytochemically. Whole-cell configuration was obtained after obtaining a tight seal of over 1 GΩ. Ionic currents were detected by holding the cells at -70 mV and stepping to test voltages between -80 and 40 mV in 10-mV increments in voltage-clamp configuration. MAIN OUTCOME MEASURES： We measured the cell karyotype, specific cell markers, and the electrophysiological properties of the voltage-gated ion channels on the cell membrane of TH-positive dopaminergic cells differentiated from our hESCs line in vitro. RESULTS： The differentiated cells had a consistent appearance, and the majority of cells （〉 90%） expressed TH and β-tubulion, as well as the neural progenitor marker, nestino Cell karyotype analysis demonstrated that all of the hESCs had a stable and normal karyotype （46, XX） after differentiation. In addition, patch clamp recording showed that the 10 recorded TH-positive cells exhibited a fast inward current when the test voltage depolarized to -30 mV, and a delayed outward current when the test voltage depolarized to -10 mV. The peak of inward current was obtained at voltage between 10 mV and 0 mV, while the peak of outward current was obtained at 40 mV. The average peak of inward current density was （ -50.05 ± 15.50） pA/pF, and the average peak of outward current density was （41.98 ± 13.55） pA/pE CONCLUSION： More than 90% of the differentiated hESC-derived cells induced by the modified four-step culture method exhibit dopaminergic neuronal properties, including general electrophysiological functional properties, such as functional potassium and sodium channels.

Prion Protein Binds to Aldolase A Produced by Bovine Intestinal M Cells

Microfold (M) cells are a kind of intestinal epithelial cell in the follicle-associated epithelium (FAE) of Peyer’s patches. They can transport antigens and microorganisms to lymphoid tissues. Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder in cattle. It is linked to variant Creutzfeldt-Jakob disease in humans. Although it is thought that M cells transport the BSE agent, the exact mechanism by which it crosses the intestinal barrier is not clear. We have bovine intestinal epithelial cell line (BIE cells), which can differentiate into the M cell type in vitro after stimulation, and which is able to transport the BSE agent. We show here that M cells are able to incorporate large numbers of PrP coated magnetic particles into intracellular vesicles, which we collected. The results of 2-DE show a specific protein associated with the PrP-coated particles, compared with non-coated particles. This protein was identified as aldolase A, a glycolytic pathway enzyme, using LC-MS/MS analysis. Aldolase A was synthesized and secreted by BIE cells, and increased during M cell differentiation. In the villi of the bovine intestine, aldolase A was detected on the surface of the epithelium and in the mucus droplet of goblet cells. In the FAE of bovine jejunal and ileal Peyer’s patches, aldolase A was localized on the surface and the apical part of the M cells. The binding of rbPrP to aldolase A was clearly detected and inhibited by pre-treatment of anti-aldolase A antibody. Aldolase A was co-stained with incorporated PrPSc in M-BIE cells. These results suggest that bovine M cells and goblet cells synthesize aldolase A, and that aldolase A may have the ability to bind PrP and associate with PrP in cellular vesicles. Therefore, aldolase A-positive M cells may play a key role in the invasion of BSE into the body.