A549/DDP细胞的抗凋亡特性与其pHi和[Ca2+]i变化相关

以l临床用药剂量(30μmol/L)的顺铂处理敏感的人肺腺癌细胞A549和抗顺铂的A549/DDP细胞,在相同条件下培养.对培养不同时间的两株细胞DNA琼脂糖凝胶电泳分析的结果表明,前者在培养12h后即呈现DNA梯子,而后者在培养48h后却未见凋亡特征.用流式细胞计检测其凋亡峰也获得相似的结果.当测定与凋亡相关的生物化学和生物物理变化时发现对顺铂敏感的A549细胞的线粒体膜电势显著降低,胞内更加酸化且胞内钙离子浓度升高;而抗顺铂药物的A549/DDP细胞的线粒体膜电势和pH值却维持在相对较高的水平,而其胞内钙离子浓度随培养时间的延长下降.这些结果表明,抗顺铂的人肺腺癌A549/DDP细胞的抗凋亡特性与其胞内的相对碱化和较高的线粒体膜电势,以及胞内钙离子浓度的降低相关.这些特性可能参与了A549/DDP的顺铂耐药性的调节.     

隧道纳米管——一种新的细胞间连接

细胞间通讯和物质交换是多细胞生物发育、组织修复和细胞生存的重要环节.最近发现细胞间存在隧道纳米管(tunneling nanotubes,TNTs)连接,并以此进行长距离通讯. TNTs是一种细胞间长距离的物理连接,能够在相连的细胞之间实现远程、定向的通讯.研究表明,不同细胞间TNTs在结构、形成机制和功能特性上存在相当大的差异.尽管这一新的研究领域取得了进展,但仍迫切需要进一步探索.本文综述TNTs的结构形态特征、生物功能、形成机制及其在疾病传播和疾病治疗中的应用前景,并讨论由TNTs介导的电耦联的研究现状.     

膜脂物理状态的变化与肺腺癌细胞A549的顺铂耐药性

用荧光探剂DPH分别标记药物敏感的肺腺癌细胞A549和抗顺铂药物的肺腺癌细胞A549/DDP, 对其膜脂物理状态的变化研究结果表明, 对顺铂药物敏感的A549的DPH各向异性值(P)为0.162, 而抗顺铂药物的A549/DDP者为0.194, 统计分析表明二者具有明显的差异. 当用可探测细胞膜脂双层不同层次的荧光探剂2-AS, 7-AS和12-AS进一步测定不同层次的膜脂质分子的流动性变化时, 结果表明: 分别反映膜表层和中层的2-AS和7-AS的各向异性值, 对敏感性的A549细胞分别为0.134和0.144, 具抗药性的A549/DDP细胞则分别为0.171和0.178. 而反映膜深层脂质分子变化的12-AS的各向异性值二者却无显著差异. 这提示, 两种细胞膜脂流动性的变化主要反映在膜的表层和中层. 同时, 用MC540荧光探剂标记两种细胞膜在FCM (flow cytometry)上测定反映膜脂质分子头部堆积程度差异的二维散点图及频数分布直方图的结果分析也表明, A549/DDP细胞膜脂质分子的有序性增加, 即流动性降低. 用气相色谱测量两株细胞膜脂肪酸的不饱和度, A549/DDP细胞膜脂肪酸的不饱和度明显低于A549细胞, 进一步肯定了上述结果. 结果提示, 在药物长期作用下, 膜脂物理状态的变化亦可能是肿瘤细胞具有抗药性的原因之一.     

A549/DDP细胞的抗凋亡特性与其pHi和［Ca2+］i变化相关

以临床用药剂量(30 μmol/L)的顺铂处理敏感的人肺腺癌细胞A549和抗顺铂的A549/DDP细胞,在相同条件下培养.对培养不同时间的两株细胞DNA琼脂糖凝胶电泳分析的结果表明，前者在培养12 h后即呈现DNA梯子，而后者在培养48 h后却未见凋亡特征.用流式细胞计检测其凋亡峰也获得相似的结果.当测定与凋亡相关的生物化学和生物物理变化时发现对顺铂敏感的A549细胞的线粒体膜电势显著降低，胞内更加酸化且胞内钙离子浓度升高；而抗顺铂药物的A549/DDP细胞的线粒体膜电势和pH值却维持在相对较高的水平，而其胞内钙离子浓度随培养时间的延长下降.这些结果表明，抗顺铂的人肺腺癌A549/DDP细胞的抗凋亡特性与其胞内的相对碱化和较高的线粒体膜电势,以及胞内钙离子浓度的降低相关.这些特性可能参与了A549/DDP的顺铂耐药性的调节.     

miR-126增加非小细胞肺癌细胞A549对顺铂的敏感性

miR-126在多种恶性肿瘤中存在表达下调并显示抑癌基因的功能,然而其在肿瘤敏感性中的作用仍不明确.为了探讨miR-126在非小细胞肺癌细胞A549对顺式铂氨(cis-diammine dichloroplatoum, cisplatin, CDDP)敏感性中的作用及可能机制,本研究用MTS法检测非小细胞肺癌细胞A549及其衍生的CDDP耐受细胞A549/DDP对CDDP的敏感性.结果表明,A549/DDP细胞对CDDP的耐受性是A549细胞的4.05倍(P=0.0078)|用qRT-PCR检测发现,相比于A549细胞,A549/DDP细胞中miR-126的表达下调了8.45倍(P=0.0063),而survivin和Bcl-2的表达明显上调|通过MTS、qRT-PCR及Western印迹实验发现,miR-126 mimics使A549/DDP细胞中miR-126的表达上调了12.63倍(P=0.0013),并明显增加A549/DDP细胞对CDDP的敏感性及下调survivin和Bcl-2的表达;相反,miR-126 inhibitor能明显增加A549细胞对CDDP的耐受性及增加survivin和Bcl-2的表达.本研究结果提示,miR-126在非小细胞肺癌CDDP耐受细胞中的表达下调,上调miR-126的表达能增加耐药细胞对CDDP的敏感性. miR-126是逆转肺癌CDDP耐受的可能潜在靶标.     

沉默MDR1基因表达逆转肺癌A549/DDP细胞对顺铂的耐药性

本研究以A549/DDP细胞为实验对象,利用shRNA(short hairpin RNA)沉默MDR1基因,逆转人肺癌A549/DDP细胞对顺铂的耐药性。构建3种靶向MDR1基因重组干扰载体,稳定转染A549/DDP细胞,qRT-PCR检测MDR1 mRNA表达水平,Western blotting检测MDR1蛋白表达水平,MTT法检测细胞对顺铂的敏感性。结果显示成功构建了3种靶向MDR1的重组表达载体p2.1-1、p2.1-2和p2.1-3。3种干扰表达载体均能有效沉默A549/DDP细胞MDR1基因表达,其中p2.1-3对MDR1沉默效果最好,对mRNA和蛋白的沉默效率分别为51.47%和53.24%。转染p2.1-3的细胞对顺铂的IC50由(72.08±7.00)μmol/L降至(31.89±3.39)μmol/L,逆转率达到(67.60±5.70)%。这些结果表明靶向MDR1的重组干扰载体均能够有效抑制MDR1表达,其中p2.1-3干扰效果最佳并且能逆转A549/DDP细胞对顺铂的耐药性。     

合成的哌啶并噻吩类化合物抑制肺癌细胞生长的初步研究

为了发现新的可用于肺癌治疗的小分子药物,从实验室现有的小分子化合物库中,选取13个未见报道的新型哌啶并噻吩类化合物,对其抑制肺癌A549细胞生长的作用进行初步评价。通过体外培养A549细胞,应用WST-1法检测小分子化合物(终浓度为10μmol/L)处理后细胞存活率的变化,发现化合物11(噻吩并[2,3-c]哌啶-3-甲酰胺-2-[(3-甲氧基-萘-2-羰基)-氨基]-6-苄基-,盐酸盐)能显著抑制A549细胞的生长,抑制率可达到(71.34±0.96)%。对小分子化合物结构与活性关系的分析发现苯并结构可能是增强化合物对A549细胞生长的抑制作用的关键结构基团。随后,进一步分析了化合物11(终浓度分别为1、2.5、5、10μmol/L)对A549细胞和正常人胚肺成纤维MRC-5细胞生长的抑制作用。结果表明,随着该化合物浓度增加,对A549细胞生长的抑制率逐渐增大,用GraphPad Prism软件计算出该化合物的IC50为2.407μmol/L;而该化合物对MRC-5细胞生长的抑制率显著低于对A549细胞生长的抑制率,当作用终浓度为10μmol/L时,其对MRC-5细胞生长的抑制率也只有30.41%。这些研究结果初步显示化合物11是一种新的较为理想的治疗肺癌药物先导化合物的候选分子。     

利用CRISPRi技术沉默MRP1基因增强A549/DDP细胞对顺铂的敏感性*

目的: 采用CRISPRi技术沉默人肺癌A549/DDP细胞MRP1基因表达,观察细胞对顺铂敏感性的变化。方法: 采用生物信息学软件分析MRP1启动子序列,设计合成3对sgRNA干扰片段,定向克隆到pSPgRNA载体中,构建靶向MRP1的干扰表达载体,分别与dCas9表达载体共转染A549/DDP细胞。实验共分为5组,分别为A549/DDP细胞组,Scrambled组,sgRNA-MRP1-1组,sgRNA-MRP1-2组和sgRNA-MRP1-3组,每组设置3个复孔,处理48 h后进行后续实验。通过qRT-PCR和Western blot检测MRP1 mRNA和蛋白表达水平,MTT法检测细胞对药物的敏感性,激光共聚焦显微镜下观察细胞的形态变化。结果: 成功构建了sgRNA-MRP1-1、sgRNA-MRP1-2和sgRNA-MRP1-3 3种干扰载体,分别与dCas9表达载体共转染A549/DDP细胞后,均能显著降低细胞MRP1基因表达(P<0.01),其中sgRNA-MRP1-2干扰效果最好,MRP1 mRNA水平降低了72%,蛋白水平降低了53%。将sgRNA-MRP1-2转染细胞后,细胞对顺铂的敏感性显著增加,IC₅₀值由74.26±3.71降低至34.29±2.51,细胞形态由梭形变为椭圆形,染色质高度凝聚、边缘化,产生凋亡小体。结论: 成功构建3种靶向MRP1的干扰表达载体,均能有效沉默A549/DDP细胞中MRP1基因表达,可增强细胞对顺铂的敏感性。     

RNA干扰沉默缺氧诱导因子1α逆转肺癌细胞耐药性

目的：观察RNA干扰沉默缺氧诱导因子1α（HIF-1α）对肺癌细胞耐药性的影响。方法：构建靶向HIF-1α小干扰RNA基因,并转染到人肺腺癌耐顺铂细胞株A549／DDP细胞中。逆转录聚合酶链反应RT—PCR）检测细胞的HIF-1α、多药耐药基因-（MDR-1）以多药耐药相关蛋白基因（MRP）mRNA变化,免疫细胞化学法观察干扰后HIF-1α、P-糖蛋白以及MRP蛋白的变化。MTT法检测不同浓度的顺铂作用下细胞死亡率。结果：HIF-1αsiRNA组中H1F-1α、MDR—1、MRPmRNA水平显著降低（P〈0．05）。且蛋白水平也显著下降（P〈0．05）。HIF-1αsiRNA组细胞死亡率较未转染组均明显增高（P〈0．05）,转染siRNA阴性组不影响肿瘤细胞的耐药性。结论：HIF-1αsiRNA可显著降低A549／DDP细胞中H1F-1α、MDR-1、MRP表达,从而起到逆转肺腺癌A549／DDP细胞的耐药作用。     

隧道纳米管:一种新型的细胞间通讯连接方式

多细胞生物体的细胞间存在着细胞连接、细胞通讯等多种形式的相互作用,这对于个体的生长发育是至关重要的.最近在哺乳动物细胞间发现了一种新型的细胞间通讯连接方式,根据其形态及结构特征,这种细长的膜管被命名为隧道纳米管(tunneling nanotubes,TNTs),TNTs一直处于形成和断裂的持续变化状态之中,在相互连接的细胞间形成了复杂的网络结构,并且可以作为细胞间交流的通道,从而在广泛的生理过程中发挥着重要的作用.TNTs在动物细胞间很可能是一种普遍存在的生物学现象.     

Vesicle traffic through intercellular bridges in DU 145 human prostate cancer cells

We detected cell-to-cell communication via intercellular bridges in DU 145 human prostate cancer cells by fluorescence microscopy. Since DU 145 cells have deficient gap junctions, intercellular bridges may have a prominent role in the transfer of chemical signals between these cells. In culture, DU 145 cells are contiguous over several cell diameters through filopodial extensions, and directly communicate with adjacent cells across intercellular bridges. These structures range from 100 nm to 5 microm in diameter, and from a few microns to at least 50-100 microm in length. Time-lapse imagery revealed that (1) filopodia rapidly move at a rate of microns per minute to contact neighboring cells and (2) intercellular bridges are conduits for transport of membrane vesicles (1-3 microm in diameter) between adjacent cells. Immunofluorescence detected alpha-tubulin in intercellular bridges and filopodia, indicative of microtubule bundles, greater than a micron in diameter. The functional meaning, interrelationship of these membrane extensions are discussed, along with the significance of these findings for other culture systems such as stem cells. Potential applications of this work include the development of anti-cancer therapies that target intercellular communication and controlling formation of cancer spheroids for drug testing.     

小麦类根瘤中胞间细菌的运动及其对细胞壁的影响

用电子显微镜和光学显微镜观察了小麦类根瘤,以探讨小麦类根瘤中胞间细菌的运动及其对细胞壁的影响.结果表明:(1)小麦类根瘤由薄壁细胞、分生细胞和侵染细胞组成,它们中有许多胞间隙,其中一些还含有大量细菌;它们的胞间层常常彼此分离,形成间隙,间隙中有时也有细菌存在;(2)小麦类根瘤中没有侵入线,细菌运动主要在胞间进行;具有细菌的胞间隙和胞间层大小不等、形状各异,其细胞壁还常常出现不同程度的变化,变化的大小一般与它们中的细菌有关,且随细菌数量的增加而增加.     

Endogenous lectins as mediators of tumor cell adhesion

Endogenous carbohydrate-binding proteins have been found in various normal tissues and cells. Although lectins with different sugar-binding specificities have been described, the most prevalent ones are those that bind beta-galactosides. The ability of some normal and malignant cells to bind exogenous carbohydrate-containing ligands suggested that lectinlike activity is associated with the cell surface and that carbohydrate-binding proteins might mediate intercellular recognition and adhesion. We found that extracts of various cultured murine and human tumor cells exhibit a galactoside-inhibitable hemagglutinating activity. This activity was associated with two proteins of molecular weights of 34,000 and 14,500 daltons, which were purified by affinity chromatography by using immobilized asialofetuin. That these lectins are present on the cell surface was indicated by the binding of monoclonal antilectin antibodies to the surface of various tumor cells and by the immunoprecipitation of 125I-labeled lectins from solubilized cell-surface iodinated cells by polyclonal antilectin antibodies. That these cell surface lectins are functional was demonstrated by the ability of the galactose-terminating asialofetuin to enhance cell aggregation and of asialofetuin glycopeptides to block this homotypic aggregation as well as to suppress cell attachment to substratum, and by the inhibition of both asialofetuin-induced cell aggregation and cell attachment to substratum by the binding of monoclonal antilectin antibodies to the cell surface. These findings implicate cell surface lectins as mediators of cell-cell and cell-substratum adhesion. Some of these cellular interactions might be important determinants of tumor cell growth and metastasis.     

Impaired spermatogenesis in the finch hybrid L. castaneothorax by L. punctulata: Transmission electron microscopy and genetic analysis

As part of a project to determine whether there is any correlation between the form of hybrid sterility and the genetic relatedness of the parental species, we studied a male intrageneric hybrid between two finch species (Lonchura custaneothorax × L. punctuluta), and compared the ultrastructural basis of hybrid sterility in this species with that reported by Swan [1985] for an intergeneric bird hybrid. In the latter study the sterility appeared to have an autoimmune basis, due to lack of Sertoli-Sertoli tight junctions. In the hybrid examined in the present study, lanthanum tracing showed that the junctions were tight. There was no testicular immune reaction; the parental species were almost identical in chromosomal constitution, having only a small inversion difference on chromosome 5, and only two structural protein differences could be detected through examination of the variation at 38 protein loci. Nevertheless, the hybrid appeared sterile and had the following ultrastructural testicular features. Intercellular bridges where present were usually abnormal in structure; centrioles in a centriole pair were arranged in parallel. Many spermatocytes and spermatids degenerated and were phagocytosed by Sertoli cells. Some spermatids progressed to mature testicular spermatozoa in sperm bundles, but commonly had multiple (2–4) axonemes or disrupted doublets and accessory fibers. The multiple axonemes present in most spermatids inserted separately into the base of the nucleus and the multiple centrioles were capable of organizing separate neck structures. We conclude that these cytological abnormalities were caused by genic effects and discuss why they appeared to be restricted to the germ line.     

Source—sink characteristics of carbon transport in Chara hispida

Abstract Rates of uptake of ¹⁴C-labelled inorganic carbon were measured for whole Chara hispida plants, detached parts of the shoot and isolated (split-chamber technique) apices, lateral branchlets and rhizoid—node complexes. The rates of inorganic carbon uptake by the rhizoid—node complex expressed per gram fresh weight whole plant were three to four orders of magnitude less than the uptake for the whole plant. Up to 70% of the carbon taken up by the rhizoid—node complex was translocated to the shoot. After 12 h exposure to ¹⁴C-labelled inorganic carbon the concentration of ¹⁴C was greater in apices than in uppermost or central internodal cells and in all lateral branchlets, regardless of whether label was supplied to the whole plant or isolated rhizoid—node complexes. Measurement of inorganic carbon uptake by detached internodal cells and detached and isolated apices and lateral branchlets showed that lateral branchlets had the greatest rates of inorganic carbon uptake. During 12 h exposure to ¹⁴C, isolated lateral branchlets translocated to the attached shoot 55% of the labelled carbon taken up; for isolated apices this value was only 13%. It is concluded that it is highly unlikely that the rhizoid of Chara hispida could acquire a significant fraction of the whole plant requirement for inorganic carbon and that apices are sink regions for photosynthate while lateral branchlets are source regions.     

Rat myometrial smooth muscle cells show high levels of gap junctional communication under a variety of culture conditions

Summary Gap junctional communciation was examined in rat myometrial smooth muscle cells cultured under a variety of conditions. As a functional measure of gap junctional communication, donor cells were microinjected with the fluorescent dye, Lucifer yellow, and the transfer of dye from donor cells to primary neighbor cells was monitored by fluorescence microscopy. In a myometrial smooth muscle cell line established from midgestation (Day 10) rats, high levels of dye transfer, in excess of 90%, were observed in primary cultures and at Passages 1 and 10. A slight decrease in dye transfer to 75% was observed at Passage 5. Similarly, high levels of dye transfer were observed in a smooth muscle cell line established from the myometrium of a late-gestation (Day 19) rat under subconfluent as well as confluent culture conditions. Myometrial smooth muscle cell cultures established from sexually immature 19-day-old rats also exhibited high levels of dye transfer in primary cultures and at Passage 10. Treatment of primary myometrial smooth muscle cell cultures derived from immature 19-day-old rats with 17β-estradiol (50 ng/ml) and 4-pregnen-3,20-dione (150 ng/ml) for 48 h in vitro had no significant effect on the high levels of dye transfer. Thus, extensive dye transfer was observed in the rat myometrial smooth muscle cells under all culture conditions examined, regardless of sexual maturity or gestational stage of the animal, in vitro hormone treatment, or cell density.     

Efficient synthesis and protein conjugation of β-(1→6)-d-N-acetylglucosamine oligosaccharides from the polysaccharide intercellular adhesin

A wide variety of medically important biofilm forming bacteria produce similar polysaccharide intracellular adhesins (PIAs). The PIA structures consist of partially de-N-acetylated β-(1→6)-N-acetylglucosamine polymers. These exopolysaccharides are key components of the bacterial biofilm matrix. Here, we describe the efficient synthesis of PIA oligosaccharides using an acid reversion reaction of N-acetylglucosamine in HF·pyridine. The PIA oligosaccharides produced by this reaction can be purified to homogeneity by size exclusion chromatography. Chemistry was developed to conjugate the PIA oligosaccharides to bovine serum albumin using a new heterobifunctional linker containing a thiol and an N-methylhydroxylamine functional group. These glycoconjugates may serve as useful precursors for the development of defined conjugate vaccines against PIA producing bacterial strains.     

The role of catecholamines on intercellular coupling,myocardial cell synchronization and self ventricular defibrillation

Ventricular fibrillation (VF) is one of the most life threatening events. Although in humans VF is generally sustained (SVF) requiring artificial defibrillation, in various mammals and in some cases in humans VF terminates by itself, reverting spontaneously into sinus rhythm. Since VF is one of the main causes of sudden death, one of the important clinical problems today is if and how we can transform the fatal SVF into a self limited transient one (TVF).From electrophysiological studies carried out on anaesthetized open chest animals, we have found that TVF requires a high degree of intercellular coupling and synchronization.Cardiac myocytes are electrically coupled with adjacent cells. The intercellular coupling is a focus of low electrical resistance which allows rapid transmission of electrical impulses between cells. Any decrease in intercellular coupling decreases the ability of the heart for self defibrillation. The cell-to-cell coupling decreases with age, ischemia, VF and variations in physiological conditions probably due to an increase in intercellular resistance (Ri), widening in the internexal gaps, decrease in electrotonic space constant () etc. All of these factors are known to be affected by intracellular concentration of free Ca⁺⁺ ([Ca⁺⁺]).On the basis of studies carried out on various mammals at different ages, we hypothesized that the ability of the heart to defibrillate depends on the cardiac catecholamine level [CA], during VF. This hypothesis is supported by the facts, known from the literature, that increase in [CA] decreases intracellular free Ca⁺⁺ concentration, decreases Ri and increases . By these effects, increase in [CA] enhances intercellular coupling and intercellular synchronization, and thereby, according to our hypothesis, leads to spontaneous ventricular defibrillation — TVF.During VF the sympathetic activity is enhanced but in some cases the [CA] does not reach the level needed for TVF. In order to help the heart in its effort to elevate the [CA] during VF, we proposed to treat these cases with drugs which inhibit the reuptake of [CA]. The facts that administration of [CA] reuptake inhibitors, before the induction of VF, and/or intracoronary infusion of adrenaline, during VF, transforms SVF into TVF, emphasized the validity of our hypothesis.