羟基磷灰石/钛酸钡压电陶瓷涂层的体外细胞毒性

背景:为优化羟基磷灰石的生物活性,前期实验利用等离子喷涂技术在羟基磷灰石表面等离子喷涂制备了压电陶瓷涂层,但此种新型材料的细胞毒性还不明确。目的:评价羟基磷灰石/钛酸钡压电陶瓷涂层的体外细胞毒性。方法:将第3代比格犬骨髓间充质干细胞分别接种于羟基磷灰石/钛酸钡压电陶瓷试件与羟基磷灰石试件上,接种5 d后,扫描电镜观察材料上细胞黏附情况。分别以羟基磷灰石/钛酸钡压电陶瓷试件浸提液、羟基磷灰石试件浸提液、含5%二甲基亚砜和体积分数15%胎牛血清的低糖DMEM溶液、只含体积分数15%胎牛血清的低糖DMEM培养基培养第3代比格犬骨髓间充质干细胞,培养的1,3,5 d采用CCK-8法检测各组细胞毒性。结果与结论:骨髓间充质干细胞在羟基磷灰石/钛酸钡压电陶瓷试件与羟基磷灰石试件表面增殖旺盛,达到复层生长,伪足与细胞之间连接很紧密,胞体丰富,表明两组材料具有良好的细胞相容性。CCK-8细胞毒性实验显示,羟基磷灰石/钛酸钡压电陶瓷试件与羟基磷灰石试件组细胞相对增殖率均在80%以上,毒性分级为1级,无细胞毒性。     

胰腺导管腺癌中胰腺星形细胞的研究进展

胰腺星形细胞(pancreatic stellate cells,PSC)是胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)肿瘤微环境中最重要的成分,在PDAC发生发展中具有非常关键的作用.目前大量研究关注于PSC与胰腺癌细胞(pancreatic cancer cells,PCC)之间的相互作用及PSC在PDAC微环境中发挥的作用.PSC在许多情况下发生活化,如乙醇、氧化应激和高血糖等.PDAC早期即可出现PSC的活化,PCC可以诱导刺激PSC发生活化,活化的PSC可以产生大量胶原纤维,形成适宜PCC生长的间质微环境,促进PCC的增殖,减少化疗药物对肿瘤细胞的杀伤作用.另外,PSC还可以与间质中各种细胞成分如内皮细胞和各种免疫细胞相互作用,在血管生成、免疫逃逸和神经侵犯等方面协助肿瘤进展.因此,阐明PSC与肿瘤细胞以及其他间质成分之间复杂的相互作用至关重要.     

人截短型凋亡诱导因子的表达对HeLa细胞的促凋亡作用

目的：观察人截短型AIF基因的表达对HeLa细胞的促凋亡作用。方法：用RT-PCR法分段克隆全长人AIF基因，经改造截去其N-端线粒体定位信号及部分Spacer区域（1-120位氨基酸）的编码序列，从而获得人截短型AIF（AIF△1-120）基因。将其克隆入pIRES2-EGFP绿色荧光蛋白（EGFP）共表达载体，用脂质体法转染HeLa细胞，通过荧光显微镜观察。免疫组织化学检测，间接免疫荧光检测，电镜观察等方法。检测目的基因在转染细胞中的表达，以及对转染细胞的形态及生长状况的影响。结果：成功地构建了人截短型AIF（AIF△1-120)基因的真核表达载体。转染HeLa细胞后，可检测到人截短型AIF分子的表达，随着转染后时间的延长，可观察到表达人截短型AIF分子的HeLa细胞呈现典型的凋亡特征。结论：人截短型AIF基因的表达可诱导HeLa细胞凋亡。     

多功能细胞传感器集成芯片的设计及实验研究

为了实现对细胞的生长凋亡等状态和电兴奋细胞的胞外电场及胞外离子代谢的同步检测,本研究提出了将具有对应功能的叉指电极(IDE)、微电极阵列(MEA)、光寻址电位传感器(LAPS)集成在单片硅基底上,设计了细胞多功能检测的集成芯片。在集成芯片的设计上,各功能模块采用多通道布局,并且优化了电极尺寸、电极间排布、电极表面特性处理等方面的设计,以减少电极之间干扰并提高性能。在加工方面利用SiO2层同时作为MEA、IDE的绝缘层和LAPS的保护层,采用微加工技术将三种传感器融合加工于同一硅基底上。器件的评估结果显示,3种传感器的性能和相应的单独设计芯片性能相近,且满足生物相容性的要求。本集成芯片成功弥补了传统细胞传感器检测参数单一的缺陷,建立了细胞多生理参数检测的细胞传感器平台。     

基于细胞3D打印技术的肿瘤药物筛选细胞芯片研究

现有的药物筛选评价技术中,动物筛选模型存在种属差异和周期长等缺点,高通量筛选和细胞筛选模型则与体内环境差异大,药物筛选准确率低。细胞3D打印技术为在体外构建仿真的组织器官模型提供了可能,当其与细胞芯片技术结合则为体外构建高效准确的药物筛选模型提供了新的技术空间。本研究构建了含有多个叉指电极（IDEs）阵列的细胞芯片,用细胞3D打印技术在芯片上组装了卵巢癌细胞HO-8910和人肝间充质干细胞HMSC-H组织模型,并通过对组织模型内细胞阻抗变化的检测,反映细胞生长、贴附、增殖、凋亡的过程及药物对细胞活性的影响等,最终基于该模型检测了抗癌药物顺铂和环磷酰胺对肿瘤细胞的杀伤和肝毒性。结果显示：支架微丝直径和孔径约为200～300 μm,肿瘤细胞和肝细胞在三维结构里生长良好;DMEM作为电解液,芯片在10.4 Hz可准确检测到三维结构中细胞增殖引起的阻抗变化,20 h后阻抗升高69.6%;基于该筛选模型,能同步检测到药物的抗肿瘤作用和肝毒性,并筛选出需要肝的二次代谢产物才能产生抗肿瘤性的药物环磷酰胺。     

低剂量X线全身照射对鼠肺标记瘤细胞清除的影响

低剂量Ｘ线全身照射对鼠肺标记瘤细胞清除的影响安徽医科大学病理生理教研室核医学教研室△（合肥２３００３２）汪思应张宝霆程洁金敖兴△黄帼△魏道严△本室曾研究了低剂量电离辐射对肿瘤生长尤其是转移的影响。为进一步探讨其临床应用前景，本文利用Ｂ１６黑色素...     

长春新碱诱导的自噬性细胞凋亡对线粒体膜电位的影响

目的：了解长春新碱（VCR）诱导的自噬性细胞凋亡是否与线粒体跨膜电位（△Ψm）改变有关及其可能的机制。材料与方法：以正常人的肝细胞系为体外模型，应用碘化丙啶（PI）／Rhodamine123(Rh123)双重染色检测△Ψm，通过亚GI期细胞和透射电镜鉴定细胞凋亡。结果与结论：VCR可明显诱导线粒体△Ψm下降，也可以诱导L－02细胞自噬性凋亡。线粒体△Ψm下降可能是VCR诱导自噬性细胞凋亡的关键环节。     

人可溶性粘附素5截短体的克隆、表达及生物活性测定

目的：克隆和转染人可溶性粘附素5截短体（sCadherin5△），并检测其生物活性。方法：RT—PCR扩增Cadher-in5△cDNA，并克隆人pMSCV逆转录病毒载体，构建pMSCV／sCadherin5△，转化感受态大肠杆菌XL—blue菌株，提取、纯化和酶切质粒，测序分析sCadherin5△，转染NIH3T3细胞，mRNA和蛋白质水平检测NIH3T3细胞表达和分泌sCadherin5△，MTT方法检测sCadherin5△生物活性。结果：PCR产物约为1128bp，构建了pMSCV／sCadherin5△质粒载体，序列测定的结果与GeneBank中Cadherin5胞膜外区121bp至1248bp之间的序列一致。pMSCV／sCadherin5△转染的NIH3T3细胞表达和分泌sCadherin5△，sCadhefin5△抑制HUVEC细胞体外增殖。结论：克隆、表达和分泌了具有生物活性的人sCadherin5△。     

PcDNA3-hBMP2转染骨髓基质细胞及其稳定表达

目的：通过观察转染hBMP-2基因的骨髓基质细胞的生长特性的变化和检测目的基因在受体细胞中表达，探讨骨髓基质细胞用作hBMP-2基因的受体细胞的可能性。方法：在脂质体介导下将hBMP-2基因导入体外培养的骨髓基质细胞中，用G418筛选获得阳性细胞，分别应用原位杂交技术和酶联免疫吸附方法检测目的基因在受体细胞内的存在与表达。结果：在mRNA水平可检测到目的基因在阳性细胞中进行了转录，在培养基中，用ELISA的方法能检测到目的基因在骨髓基质细胞中进行了表达，并分泌到培养基中。从细胞的生长曲线上，可知细胞的倍增时间并没有因为目的基因的导入而改变，其生长时间也与正常的细胞相近。结论：骨髓基质细胞可以用作hBMP-2基因的受体细胞，表达并分泌hBMP-2蛋白质，也可作为骨组织工程学中的种子细胞。     

姜黄素对前列腺癌细胞LNCaP增殖的影响

目的:姜黄素对前列腺癌细胞株LNCap增殖和凋亡的影响。 方法:用不同剂量的姜黄素分别处理LNCap细胞，显微镜下观察细胞形态；MTT法检测细胞生长情况；流式细胞技术（FCM）检测细胞凋亡率；然后检测姜黄素处理LNCap细胞后培养液中总前列腺特异抗原（PSA）的变化，并用免疫印迹Western blotting技术检测雄激素受体（AR）的表达。 结果:姜黄素能够抑制前列腺癌细胞LNCap的增殖和生长，40 μmol/L作用24 h最强，细胞存活率为对照的40%；姜黄素诱导LNCap细胞凋亡，细胞形态呈凋亡特征，且40 μmol/L作用最强，凋亡率为9.23%；姜黄素抑制LNCap细胞PSA表达，且40 μmol/L姜黄素处理细胞24 h对PSA表达的抑制作用最强，PSA含量仅为对照的20%；Western blotting检测结果显示姜黄素抑制AR的表达，并且对AR表达的抑制程度依赖于姜黄素的浓度。 结论:姜黄素抑制LNCap细胞增殖，诱导细胞凋亡，且表现出时间和剂量依赖性。姜黄素抑制LNCap细胞PSA与AR 受体的表达。     

在石蜡切片中进行微切割-PCR-银染的方法

目的：探索在石蜡切片中应用微切割－ＰＣＲ－银染技术,检测大肠癌的微卫星不稳定性和杂合性缺失。方法：用微切割技术在２８例石蜡切片中提取淋巴细胞和间质细胞、癌旁粘膜腺管、不典型地生腺管、腺瘤腺管和腺癌细胞,每例至少包含３种类型细胞,经蛋白酶Ｋ消化后,直接用于ＰＣＲ扩增,产物进行８％变异聚丙烯酰胺凝胶电泳－银染。共进行了ＴＧＦ－βＲⅡ（Ａ）１０、ｈＭＳＨ２（Ａ）２６－Ｂａｔ２６,ｈＭＳＨ３（Ａ）８,ｈＭＳＨ６（Ｃ）８４个微卫星位点的检测。结果：ｈＭＳＨ３（Ａ）８和ｈＭＳＨ６（Ｃ）８位点在２８例标本全部扩增出目的片段,ＴＧＦ－βＲⅡ（Ａ）１０和ｈＭＳＨ２（Ａ）２６位点则分别有２３例、２２例扩增出目的片段,且ｈＭＳＨ２（Ａ）２６位点有５例癌细胞呈阳性,其中１例同时有腺癌细胞阳性。结论：微切割－ＰＣＲ－银染技术应用于石蜡切     

冰冻切片免疫过氧化物酶染色法在抗细胞性单克隆抗体筛选和鉴定中的应用

本文介绍一种快速准确地筛选和鉴定抗细胞性单克隆抗体(单抗)的冰冻切片免疫过氧化物酶染色技术。它既能筛选融合后的大量单抗(一般在3小时以内能筛选200～400孔杂交瘤细胞上清液);又可根据组织固有结构及细胞分区分布的特性,作为全面鉴定单抗的重要方法。我们运用此法从12次融合中,筛选并经克隆化获得291个单抗,进一步全面鉴定后得到抗T细胞等101个单抗。此外还讨论了消除非特异性染色的体会。     

FITC标记单克隆抗体检测icIL-1ra的方法研究

目的 建立用单克隆抗体技术检测细胞IL-1ra的方法。方法 制备异硫氰酸荧光素(FITC)标记的抗人IL-1ra单克隆抗体，使其进入U937细胞内与icIL-1ra结合，然后用流式细胞仪(FACS)检测荧光强度，并比较U937细胞在刺激前后荧光强度的变化情况是否与icIL-1ra表达的变化情况相一致。结果 在一定剂量范围内，经PMA分化和LPS刺激的U937细胞的icIL-1ra表达量增加，其检测荧光强度相应增加。结论 用单克隆抗体检测细胞内icIL-1ra是一种快速、准确的方法，本实验方法能够满足实际研究工作中对icIL-1ra定量检测的要求。     

Evaluation of rapid antibody dissociation techniques

Two new antibody dissociation techniques, citric acid treatment and chloroquine diphosphate (CDP) treatment at 37 C, were evaluated for their usefulness in investigating the red cells of patients with a positive direct antiglobulin test (DAT). The techniques were compared to the ZZAP and CDP-22 C procedures for (a) removal of coating antibody prior to performing warm autoadsorptions, and (b) removal of antibody so antigen typings can be performed. Twenty patient or donor antibodies were used to sensitize red cells in vitro. Complete removal of antibody was obtained by the CDP-37 C treatment in 19 cases (95%), by ZZAP treatment in 17 cases (8 5%), by the citric acid method in 10 cases (50%), and by the CDP-22 C method in three cases (15%). The CDP-37 C treatment left all red cell antigens investigated intact, and the citric acid technique left all but Kell system antigens intact. In addition, patient red cells treated by the citric acid technique could be used to adsorb warm-reacting autoantibodies from autologous serum in 15 to 30 minutes at room temperature. Investigation of a positive DAT may be facilitated by using the citric acid and CDP-37 C treatments.     

A Simple Method to Detect Complement Receptors using Baker's Yeast: YC Rosettes

A technique is described to identify complement-receptor-bearing cells, using serum-treated baker's yeast as a ligand. The method consists of incubation of heat-killed baker's yeasts with fresh AB normal serum, freezing, thawing, and washing of the particles, followed by mixing with the cells. Serum is required to coat the yeasts for the rosette formation. Experiments designed to establish the serum factors responsible for the attachment of the particles to cells show that heat inactivation, chelating agents, or anti-C3 treatment prevent rosette formation. This is taken as evidence that yeasts (Y) are coated with complement (C) to compose the reagent for the YC rosette technique. The application of this technique to twenty-five normal individuals demonstrated that a mean of 11.6 per 100 lymphocytes (±4.3) form rosettes; absolute number: 275 (±160) rosette-forming lymphocytes per mm³. Either AB or autologous fresh serum can be used to coat the yeasts. A combined technique for YC plus E rosettes can be performed allowing the identification and enumeration of four populations of lymphocytes: (a) those having receptors for sheep erythrocytes, (b) complement-receptor-bearing lymphocytes, (c) those having both receptors (D lymphocytes), and (d) non-rosette-forming non-phagocytic cells.     

Karyotyping of human synaptonemal complexes by cenM-FISH

The purpose of this work was to adapt the recently described centromere-specific multicolour (cenM-) FISH technique to human meiotic cells, and evaluate the usefulness of this multiplex fluorescence method for karyotyping human synaptonemal complex (SC), previously analysed by immunocytogenetic approaches. The results obtained demonstrate that cenM-FISH is a reliable one-single-step method, which allows for the identification of all SC present in pachytene spreads. Moreover, when cenM-FISH is applied after immunocytogenetic analysis, the number and distribution of MLH1 foci per chromosome can be established and recombination analysis for each chromosome can be performed easily.     

A quick and simple method for phenotyping IgG-sensitized red blood cells

Positive (IgG) direct antiglobulin test (DAT) reactivity ranging from weakly positive to 2+ can be eliminated using a simple "blocking" technique with anti-IgG. This method can be used for antigen typing DAT-positive red blood cells that require the antiglobulin technique.     

A simplification of the enzyme-linked immunospot technique. Increased sensitivity for cells secreting IgG antibodies to Haemophilus influenzae type b capsular polysaccharide.

A simplified enzyme-linked immunospot (ELISPOT) technique is described for the detection of cells secreting antibodies to tetanus toxoid (TT), diphtheria toxoid (DT) or Haemophilus influenzae type b capsular polysaccharide (PRP). By combining the cell suspension with the enzyme-linked secondary antibodies in one incubation, the second incubation and washing procedure could be omitted from the original technique. The simplified assay had the same sensitivity for anti-TT and anti-DT spot-forming cells as the ordinary ELISPOT assay. The IgG anti-PRP spots were, however, improved both in quality and in quantity (median: 40% more spots), while the detection of IgM and IgA anti-PRP spot-forming cells was the same in the two techniques. This simplified technique can probably also be used to save time in other antigen systems and should be considered when designing ELISPOT assays for the detection of polysaccharide-specific antibody-secreting cells.     

Double immunofluorescence microscopic technique for accurate differentiation of extracellularly and intracellularly located bacteria in cell culture.

A double immunofluorescence staining technique is described for differentiation between cell-attached (extracellular) and ingested (intracellular) bacteria by HEp-2 cells in cell culture monolayers. This method is based upon the observation that membranes of viable mammalian cells are impermeable for antibodies but are rendered permeable by treatment with fixatives. Consequently, extracellular bacteria can be stained by specific rhodamine-labeled antibodies before fixation, and intracellular bacteria can be visualized by treatment with specific fluorescein-labeled antibodies after fixation. The accuracy and simplicity of this method is demonstrated with HEp-2 cell culture monolayers as target cells and an isogenic pair of Yersinia enterocolitica, one of which is phagocytosis resistant and the other of which is phagocytosis sensitive. Furthermore, it is shown that this staining technique is also applicable for studying the interaction of bacteria with macrophages and fibroblasts.     

大鼠心脏穿刺采血方法的改进

目的:介绍一种大鼠活体重复采血方法。方法:大鼠腹腔麻醉后,仰卧位沿剑突下斜行向上刺入2.5~3.0cm入心,抽血。60只大鼠,每间隔10天采血2.0ml,共4次。测定其Hb、RBC、WBC、Hct、ALT、AST、TP、BUN等指标,同时心脏切片行病理学检查。结果:采用本法进行大鼠心脏取血,成功率97.8%。各次标本的实验室指标均无显著性差异(P>0.05)。20只大鼠第4次采血后剖胸,未见胸腔脏器出血、粘连、血肿等并发症。心脏切片未见心肌组织学损伤。结论:该技术操作简便,定位准确,可重复采血,动物存活率高。尤其用于自身对照,可减少动物用量,提高实验准确性。