SIET（self-referencing ion electrode technique,自参考离子选择性电极技术）是电生理学研究的新手段,可以在植物抗逆研究中无损地获得植物细胞、组织、器官微区内离子流动态变化信息,而离子选择性微电极的制备及性能测试的标准化是SIET系统对植物活细胞、活体组织原位离子流测试的前提。该文以钾离子选择性微电极为例,详细讨论了离子选择性微电极的拉制、硅烷化、灌充等制备过程,研究了微电极内阻等电极参数的测量方法,测试了微电极的能斯特响应斜率、检测范围、响应时间等参数,讨论了制备过程中微电极性能的影响因素。离子选择性微电极使用WD-2型微电极拉制仪由无导液丝的TW150-3型硼硅酸盐玻璃毛细管拉制成形,其尖端直径为1～9μm,干燥后用5%硅烷试剂在150℃温度下做硅烷化处理,再灌充入内充液与LIX（liquid ion exchanger,液态离子交换剂）而制成。研究表明：LIX成分是影响微电极内阻的重要因素,灌充LIX后的钾离子选择微电极（LIX长度为150～210μm）内阻达到108～109Ω,明显高于灌充LIX前;微电极在0.01～500mmol/L K＋浓度范围内具有很好的线性关系,R2=0.9998,能斯特斜率为53.095mV/dec;微电极对1和100mmol/L KCl溶液的平均响应时间t95%小于1s。研究结果表明,离子选择性玻璃微电极的制备过程是影响微电极性能的关键,微电极尖端尺寸、内阻、响应时间等参数对微电极的应用影响显著。该研究可为离子选择性微电极的制备及其在SIET系统中的应用提供参考。

An self-referencing ion electrode technique provides a novel electrophysiological tool which can non-invasively measure the dynamic influxes and effluxes of ions from cells and organs in vivo. In fact, the foundation of this technique is the fabrication and performance test of an ion selective microelectrode (ISME). In this paper, the K+ISMEs with good performances were obtained. We elaborated the procedure to prepare the glass micropipettes and to fill the pipettes with internal filling solution and liquid ion exchangers (LIX) of potassium, and then estimated the performance of these ion selective microelectrodes. Measurement of tip size, measuring method of resistance, testing of detection range, Nernstian slope, and response time, were described in detail. Ion selective microelectrodes were calibrated before and after experiments using two or more different kinds of concentrations of K+within its operating range based directly on the potentiometric analysis. The procedure for ion se

目的 探讨载肝细胞生长因子(HGF)的聚乳酸-氧-羧甲基壳聚糖(PLA-O-CMC)纳米粒子对大鼠肝细胞移植后移植肝细胞生存的影响.方法 Seglen原位两步灌流法分离大鼠肝细胞,培养24 h后分别将5ml常规胶原大鼠肝细胞悬液(5.0×107个细胞,下同)(A组)、5 ml PLA-O-CMC纳米粒子大鼠肝细胞(B组)、5 ml载HGF的PLA-O-CMC纳米粒子大鼠肝细胞(C组)移植到大鼠腹腔内,5 ml PLA-O-CMC纳米粒子大鼠肝细胞腹腔移植加每日静脉注射HGF 10 μg/kg ×7 d(D组).观察各组培养1d和腹腔肝细胞移植后移植肝细胞的超微结构变化.观察移植前培养液和受体腹腔渗液丙氨酸转移酶(ALT)和门冬氨基转移酶(AST)水平变化.流式细胞仪检测培养和移植肝细胞凋亡率、坏死率及存活率.结果 移植肝细胞的存活时间以C组最长,达7d,术后3～7 d移植肝细胞的电镜变化A组核质浓缩最为明显,C组内质网最为丰富.移植后1d时腹腔渗液ALT A组＞C组(1 002.68 ±229.37) U/L比(770.62 ±120.77) U/L,P＜0.05].移植后1、3、5d,活率B、C、D组＞A组(P＜0.05),其中5d时C组[(59.51±1.59)％]＞D组[(55.98±2.27)％,P＜0.05];移植后3、5d凋亡率A组＞B、C、D组(P＜0.05);移植后1、5d,坏死率A组＞B、C、D组.结论 载HGF的PLA-O-CMC纳米粒子培养

Objective To evaluate the hepatocyte growth factor (HGF) loaded polylactic acid-Ocarboxymethylated chitosan (PLA-O-CMC) nanoparticles on the survival of the transplanted hepatocytes after hepatocyte transplantation.Methods Two-steps in situ collagenase perfusion method described by Seglen was used to isolate rat hepatocytes.Respectively 5ml rat free hepatocyte suspension (5.0 × 107 cells) (Group A),5 ml rat hepatocytes co-cultured with PLA-O-CMC nanoparticles (5.0 × 107 cells) (Group B) and 5ml rat hepatocytes co-cultured with HGF loaded PLA-O-CMC nanoparticles (5.0 × 107 cells) (Group C) were transplanted into the abdominal cavity of SD rats at 48 h after D-Gal injection.As Group B,plus intravenous injection HGF 10 μg/kg everyday as comparison (Group D).The pathological changes and ultramicrostructure of hepatocyte culured 1d or transplanted hepatocyte of each group were observed.The alanine transaminase (ALT) and aspartate aminotransferase (AST) of culture solution and

目的探讨大鼠脐带间充质干细胞(UMSC)对原代大鼠肝细胞及肝卵圆细胞增殖与功能的影响。方法采用2-乙酰氨基芴加肝脏三分之二切除术建立肝卵圆细胞增殖模型,通过原位二步胶原酶灌流法分离到单个肝脏细胞,再经过Percoll密度梯度离心分离到肝卵圆细胞。原代大鼠肝细胞/肝卵圆细胞各分为3组:UMSC组、原代大鼠肝细胞组/肝卵圆细胞组及UMSC与原代大鼠肝细胞共培养组/UMSC与肝卵圆细胞共培养组,分别于第1、3、6、8天通过MTT法检测各组细胞增殖能力,于第3天通过ELISA法检测各组细胞培养上清液中白蛋白含量。结果 UMSC与原代大鼠肝细胞共培养组在第3、6、8天的A值均比相应时间点的UMSC组A值与原代大鼠肝细胞组A值之和大(P0.05);UMSC与肝卵圆细胞共培养组在第3、6、8、10天的A值均比相应时间点的UMSC组A值与肝卵圆细胞组A值之和大(P0.05)。UMSC无白蛋白分泌能力,UMSC与原代大鼠肝细胞共培养组培养上清液中白蛋白水平为(266.21±50.44)ng/mL,较原代大鼠肝细胞组(130.79±22.10)ng/mL高(P=0.013);UMSC与肝卵圆细胞共培养组培养上清液中白蛋白水平((49.64±3.56)ng/mL)较肝卵圆细胞组(13.54±1.53)ng/mL高(P=0.000)。结论 UMSC在体外可以促进原代大鼠肝细胞及肝卵圆细胞的存活和增殖,并可增强其分泌白蛋白的作用。

Objective To explore the effects of UMSC on proliferation and function of primary rat hepatocytes and hepatic oval cells in vitro . Methods Rat model for hepatic oval cell proliferation was established by 2-acetylaminofluorenne and two third partial hepatectomy (2-AAF/PH).Liver cells were isolated by two steps collagenase perfusion via portal vein in site,and then separated to hepatic oval cells by density gradient centrifugation of Percoll. Primary rat hepatocytes/hepatic oval cells were respectively divided into three groups:UMSC group,primary rat hepatocytes/hepatic oval cells group and UMSC co-culture with primary rat hepatocytes/hepatic oval cells group.Cells proliferation rate of each group was measured by MTT on days 1 ,3,6,8,and concentration of albumin in culture medium was measured by ELISA on day 3.Results OD value of UMSC co-culture with primary rat hepatocytes group was significantly higher than the sum of OD values of UMSC group and primary rat hepatocytes group on days 3

目的 探讨丹参酚酸B盐(Sal B)对内皮素-1(ET-1)诱导的大鼠肝星状细胞(HSC)收缩以及对细胞骨架的影响.方法 采用肝脏原位灌流酶消化法、Nycodenz密度梯度离心法分离大鼠HSC.培养细胞分为对照组、ET-1组、SalB组和Y-27632组.ET-1组细胞予以10-8 mol/L ET-1刺激,SalB组或Y-27632组细胞在ET-1刺激前分别予以10-5 mol/L Sal B或105 mol/L Y-27632预处理30 min.用胶原凝胶收缩法观察HSC收缩情况.用甘油胶电泳和Odyssey荧光成像系统检测肌球蛋白轻链2 (MLC2)的磷酸化水平.用FITC-鬼笔环肽特异性染肌动蛋白丝,观察Sal B对HSC中肌动蛋白骨架的影响.两组间比较用t检验,多组间比较用方差分析,Q检验. 结果 对照组凝胶面积为76.89％±3.84％,ET-1组凝胶与对照组相比明显收缩,面积为37.10％±5.10%(q=25.51,P＜ 0.01).104mol/L Sal B或10-5 mol/L Y-27632预处理能显著抑制HSC收缩,凝胶面积分别为67.01％±4.14％和77.28％±2.00％,与ET-1组相比,q值分别为16.97、25.76,P值均＜0.01,差异均有统计学意义.在基础状态下MLC2磷酸化水平为(0.35±0.05) mol PO4/mol MLC2; ET-1刺激后,MLC2磷酸化水平迅速上升,在ET-1刺激5min时为(0.87±0.04) molPO4/mol MLC2,30min时达到高峰[(0.96±0.04)mol POJmol MLC2].SalB能显著抑制ET-1诱导的MLC2磷酸化,可使ET-1刺激30min时MLC2磷酸化水平下降63.1％(q=26.67,P＜0.01),并且使肌动蛋白丝解聚,细胞骨架松弛. 结论 Sal B能有效抑制ET-1诱导的大鼠HSC收缩.这种效应是基于SalB抑制ET-1诱导的MLC2磷酸化以及抑制HSC中肌动蛋白纤维的聚合.

Objective To investigate the effects of salvianolic acid B (Sal B) on endothelin-1 (ET1)-induced contraction and cytoskeleton reorganization of rat hepatic stellate cells (HSCs).Methods HSCs were collected from Sprague-Dawley rats by in situ perfusion with pronase E and isolated by density-gradient centrifugation with Nycodenz.Cells were treated with ET-1,with or without Sal B or Y-27632 (a specific inhibitor of rho-associated protein kinases) pretreatment.HSC contraction was evaluated by collagen gel contraction assay.Cytoskeletal reorganization in response to ET-1 was evaluated by detecting changes in phosphorylation of myosin light chain 2 (MLC2) using glycerol-urea PAGE and the Odyssey Infrared Imaging System.Changes in actin stress fiber polymerization were detected by FITC-labeled phalloidin.Differences between the various cell treatment/pretreatment groups were statistically analyzed.Results Compared to the untreated control cells,the lattice area of ET-1-treated cells showed si