细胞松弛素D、秋水仙素作用下大鼠肝癌细胞中黏弹性的研究

采用微管吮吸技术测定大鼠肝癌细胞的黏弹性;研究了秋水仙素、细胞松弛素D以及两者混合作用后对于肝癌细胞黏弹性的影响。结果表明:用CD处理癌细胞后发现癌细胞的弹性系数K1明显下降。与对照组相比:微丝骨架被CD抑制后,在加入Col后肝癌细胞的弹性系数K1显著降低;而微管骨架被col抑制后,在加入CD后肝癌细胞的弹性系数K1、K2和μ无明显变化。提示在微管骨架系统完整的情况下,微丝对肝癌细胞的黏弹性系数的的影响起主要作用。而微管骨架系统受到破坏后,微丝需借助于微管网络的作用来影响细胞的黏弹性。本研究对揭示癌细胞中骨架系统之间的交互作用对于细胞黏弹性的影响提高实验依据。     

细胞松弛素B促微丝解聚对DNA合成的作用

利用微丝（ＭＦ）解聚药物细胞松弛素Ｂ（ＣＢ）处理Ｇ０期小鼠Ｃ３Ｈ１０Ｔ１／２成纤维细胞,对Ｇ０至Ｓ期ＤＮＡ合成,胸腺嘧啶核苷激酶（ＴＫ）活性、ＴＫ基因表达、钙调素（ＣａＭ）水平和一些细胞周期早期基因的表达进行了观察。Ｇ０期细胞经３ｍｇ／ＬＣＢ处理２ｈ,促ＭＦ解聚增强了血清对Ｓ期细胞ＴＫ活性、ＴＫ基因表达和ＤＮＡ合成的刺激作用,并促进细胞提前进入Ｓ期。血清刺激Ｇ０期细胞进入晚Ｇ１期和Ｓ期时,ＣａＭ     

细胞松弛素D对小鼠乳腺腺癌细胞系(MA782/5S-8102)细胞的作用

本文研究了细胞松弛素D(CD)对小鼠乳腺腺癌细胞系(MA782/5S-8102)细胞的作用,统计了在0.4微克/毫升CD作用下的多核细胞形成率,并与HEp2细胞及正常婴兔肾原代培养细胞进行比较。结果表明:CD处理48小时后,肿瘤细胞形成大量的多核,≥3核的细胞数在MA782/5S-8102细胞中为37.05％,在HEp2细胞中为18.37％。正常婴兔肾原代培养细胞经同样处理后≥3核的细胞数只有0.059％。肿瘤细胞多核形成的比例会随CD处理时间的延长而显著增高,并出现排核现象。CD处理96、144小时后的多核细胞数,MA782/5S-8102细胞分别为47.90％,53.12％,HEp2细胞分别为43.94％,51.94％,而正常婴兔肾原代培养细胞则仅为0.26％和0.39％,亦未见排核现象,肿瘤细胞和正常细胞对CD的不同反应,有可能作为鉴别肿瘤细胞与正常细胞的一个简便易行的工具。     

细胞松弛素B对蚕豆气孔运动的影响

以蚕豆叶片下表皮条为材料,研究了微丝在气孔运动中的作用。利用肌动蛋白纤丝专一性抑制剂──细胞松弛素Ｂ（ＣＢ）预处理后,再用诱导气孔运动的因子处理表皮条,在显微镜下观测气孔孔径的变化。结果显示,用ＣＢ处理开放或关闭状态气孔,其开度均不发生变化;ＣＢ处理使微丝解聚,气孔运动被抑制;且ＣＢ处理后气孔的运动是可以恢复的。实验进一步表明,开放气孔经１０ｍｇ／Ｌ的ＣＢ预处理后,ＡＢＡ、Ｃａ２＋及暗诱导气孔关闭的作用均不同程度地受到抑制,推测微丝可能参与ＡＢＡ、Ｃａ２＋及暗诱导的气孔关闭过程;关闭气孔经１０ｍｇ／Ｌ的ＣＢ预处理后,Ｋ＋和（或）光诱导气孔开放的作用受到抑制,推测微丝可能参与光及Ｋ＋诱导的气孔开放过程。     

细胞松弛素D对棉铃虫核型多角体病毒复制和装配的影响

杆状病毒感染引起宿主细胞肌动蛋白骨架的构象变化 ,使之形成缆绳结构 .棉铃虫核型多角体病毒 (HaNPV)的衣壳蛋白也能使宿主昆虫的肌动蛋白发生凝聚 ,用细胞松弛素D抑制宿主肌动蛋白形成纤丝结构 ,病毒感染Hz AM1,空斑计数表明 ,0 1μg/ml细胞松弛素D可使棉铃虫核型多角体病毒的增殖下降 10 4倍 ,细胞松弛素D浓度增高到 0 5 μg/ml则测不到子代病毒粒子 .Western印迹分析表明 ,细胞松弛素D并不影响受染细胞中肌动蛋白的含量 .斑点印迹 (dotblot)也表明 ,病毒DNA的合成也没有受到影响 ,推测宿主细胞的肌动蛋白纤丝结构与病毒的复制有关 .在电子显微镜下观察超薄切片发现 ,在 0 5 μg/ml细胞松弛素D处理细胞中形成的病毒粒子形态与正常形态明显不同 ,提示细胞松弛素D抑制HaNPV的增殖是由于抑制病毒组装成完整有感染性的病毒粒子 .从而可以认为宿主昆虫细胞的丝状肌动蛋白对子代病毒的复制和组装是必需的 .     

细胞松弛素E对大鼠生精细胞发育的影响

本文采用微丝抑制剂——细胞松弛素E对大鼠生精细胞发育的影响作了形态学观察,特别对支持细胞骨架复合体的作用进行了较为详细的研究。结果表明睾丸内注射0.1ml,1000μmol/L～2000μmol/L,细胞松弛素E,6-14小时后,光镜下可见曲细精管上皮排列疏松,组合紊乱,有的生精上皮基底部出现双核和三核的圆形细胞和多核巨精子细胞,管腔内出现未成熟的精子;在第ⅤⅢ～Ⅸ期曲细精管上皮中,有许多第8、第9期的精子细胞顶体不指向基底方向,属定向不正的精子。电镜下,实验组动物可见一些面向第8-18期精子细胞顶体的支持细胞骨架复合体出现不同程度的缺如,有的断裂成小段;有的破坏仅发生在顶体上方;有的几乎全部丢失,并有类管球复合体的形成。另外,在高渗液处理下,可见精子细胞顶体和支持细胞间的间隙扩大。最后对微丝在精子发育中的作用进行了讨论。     

细胞松弛素B和鬼笔环肽对梨花粉萌发和花粉管生长的影响

以砂梨(Pyrus pyrifoliaNakai)品种今村秋(Imamuraaki)和丰水(Hosui)为材料,分别用光学显微镜和荧光显微镜观察了离体和半活体条件下微丝骨架解聚剂细胞松弛素B(cytochalasin B,CB)和稳定剂鬼笔环肽(phalloidin)对梨花粉萌发和花粉管生长的影响.结果表明:(1)低浓度(10μg/mL)鬼笔环肽能促进花粉萌发和花粉管生长,但高浓度对花粉萌发和花粉管的生长具有抑制作用;CB抑制花粉萌发和花粉管生长,且抑制效应随其浓度的增加而增强.(2)鬼笔环肽处理柱头后进行自花授粉,可明显促进自花花粉萌发和花粉管的生长,而CB处理柱头后异花授粉则抑制异花花粉萌发及其花粉管生长.可见,微丝骨架参与了梨花粉萌发和花粉管生长过程,并参与了梨自交不亲和反应的调节.     

细胞松弛素B和鬼笔环肽对梨花粉管钙离子浓度和尖端钙离子通道的影响

应用激光共聚焦显微镜和全细胞膜片钳技术研究了微丝骨架解聚剂细胞松弛素B(CB)和稳定剂鬼笔环肽(PD)对梨花粉管细胞内钙离子浓度动态变化和尖端质膜上钙离子通道的影响。结果显示:CB处理能促进花粉管内胞质钙离子[Ca2+]i浓度增加,同时还能激活质膜上的钙离子通道;而PD处理对花粉管内[Ca2+]i浓度及钙离子通道几乎没有影响。研究表明,微丝骨架的解聚激活了花粉管质膜上的钙离子通道,使得胞外钙离子大量流入,胞内钙离子浓度升高,从而抑制花粉管生长。     

利用细胞松弛素B分离柑橘及金柑植物微核的研究

采用细胞松弛素B(CB)处理结合蔗糖梯度超速离心方法分离柑橘和金柑亚原生质体的结果表明,柑橘和金柑原生质体的直径为20～30μm,采用蔗糖梯度超速离心分离亚原生质体,其适宜离心力是1400000×g;4',6-二脒基-2-苯基吲哚(DAPI)的染色效果最好,细胞核和细胞质可明显区分;愈伤组织经CB处理36h后再分离亚原生质体,可显著提高微原生质体的产量,分离的微原生质体直径为1.8～3.5μm,产量达到2～3×104个·g-1(FW)。     

海洋真菌Xylaria sp.SOF11中细胞松弛素类化合物的研究

从海洋真菌Xylaria sp.SOF11的液体发酵提取物中分离到3个化合物,通过1H及13C NMR,2D NMR等方法,分别鉴定为19,20-epoxycytochalasin Q(1)、18-deoxy-19,20-epoxycytochalasin Q(2)和19,20-epoxycytochala-sin C(3)。化合物1～3对两株肿瘤细胞MCF-7和NCI-H460显示较弱的细胞毒活性。     

低浓度CO暴露促进大鼠肝前体细胞增殖的体外研究

目的:观察低浓度一氧化碳暴露对营养相对匮乏情况下大鼠肝前体细胞系生长的影响并探索其作用机制。方法:分别在营养充足(10%胎牛血清,10%FBS)和营养相对匮乏(4%胎牛血清,4%FBS)条件下对大鼠肝脏前体细胞系WB-F344进行体外培养,观察细胞的生长情况;同时,在不同两组4%FBS培养基中分别加入无活性的一氧化碳释放分子iCORM-2和有活性的一氧化碳释放分子CORM-2,两者的浓度均为100μM,观察比较低浓度一氧化碳暴露是否有利于WB-F344的生长和增殖;利用吖啶橙染色观察不同组别培养细胞酸性自噬小体的形成情况,并进行Western-Blot,检测自噬相关蛋白LC3-Ⅰ和LC3-Ⅱ的转化情况,探讨其可能的作用分子机制。结果:在含有10%FBS的培养基中,WB-F344细胞生长良好,呈指数生长,并在第5天达到生长高峰;与其相比,4%FBS培养基对WB-F344的生长有明显抑制,细胞生长相对缓慢,并且相同时间点的细胞增殖数量明显不足。在进行浓度为100μM的一氧化碳分子暴露后,WB-F344细胞生长情况有明显改善,细胞增殖的峰值有显著增加。吖啶橙染色显示,4%FBS培养基条件下培养的WB-F344细胞,细胞内出现橘红色荧光,在暴露于低浓度的一氧化碳分子后,橘红色荧光的强度有明显增加。进一步的Western-Blot检测发现,营养相对匮乏的培养基(4%FBS)能使WB-F344细胞在生长过程产生LC3-Ⅰ向LC3-Ⅱ的转化,在暴露于低浓度的一氧化碳分子后,LC3-Ⅱ的量进一步增加,LC3-Ⅱ/LC3-Ⅰ的比值显著增大(P0.05);进一步的研究显示在此过程中伴随着p-AKT和mTOR蛋白的表达降低,同时,HO-1蛋白的表达增加。结论:低浓度一氧化碳暴露能促进WB-F344细胞产生自噬,有利于大鼠肝前体细胞系的生长和增殖,同时这和AKT/mTOR信号途径的抑制及HO-1的表达增加有关。     

D-gal致大鼠急性肝损伤过程中肝卵圆细胞增殖及迁延

目的用D-gal建立大鼠急性肝损伤模型,观察肝损伤后再生过程中肝卵圆细胞的增殖和迁延。方法建立大鼠急性肝损伤模型,于第1、3、7和14天分别取肝组织,分别行病理、免疫组织化学,观察卵圆细胞的分布迁移情况,并取第7天肝组织进行组织电镜观察汇管区新增生细胞超微结构。结果病理切片显示肝细胞变性坏死程度以第7天和第14天为主,出现新增生的细胞。免疫组化示随时间阳性细胞明显增多,分布于汇管区,并向小叶中心迁移,形成大量的胆小管,并有部分向坏死区迁移。透射电镜有新生内源性细胞,小于成熟的肝细胞,细胞器较少,有细胞紧密连接,以数个细胞排列成小胆管,与免疫组化一致。结论在大鼠急性肝损伤时HOC被活化、增殖,并向肝小叶中心迁移,全程参与了肝再生过程。     

Effects of Acrylamide on the Nervous Tissue Antioxidant System and Sciatic Nerve Electrophysiology in the Rat

To investigate the time-dependent effects of acrylamide (ACR) on the antioxidative status in rat nerve tissues, adult male Wistar rats were given ACR (40 mg/kg, i.p., 3 times/week) for 2, 4, 6 and 10 weeks, respectively. The time-dependent changes of the lipid peroxidation (malondialdehyde, MDA) and antioxidative status (glutathione, GSH; glutathione peroxidase, GSH-Px; glutathione reductase, GR; superoxide dismutase, SOD and anti-reactive oxygen species, anti-ROS) in nerve tissues were investigated. The electrophysiology indices (nerve conduction velocity, NCV; compound action potential duration, CAPD; compound action potential amplitude, CAPA; compound action potential latency, CAPL) in the sciatic nerve were determined using BL-420E Biologic Function Determining System. The results showed that MDA levels increased significantly (P < 0.05) in nerve tissues, while GSH levels markedly decreased (P < 0.05) in a time-dependent manner. SOD activity (in the spinal cord and sciatic nerve) and GR activity (in the sciatic nerve) increased significantly after 4 weeks ACR treatment (P < 0.01), but then decreased (P < 0.05). The anti-ROS activity in the sciatic nerve was markedly decreased at the end of week 6 and 10 (P < 0.01). The above indices changed most in the sciatic nerve. The levels of GSH, MDA and anti-ROS in rat sciatic nerve were in high correlation (P < 0.05, |r| > 0.80) with the electrophysiology indices according to the exposure time. Thus, ACR-induced neurotoxicity may be associated with the enhancement of lipid peroxidation and reduction of the antioxidative capacity. Depletion of neural GSH level might be one of the primary events in ACR-induced neuropathy. Ying-Jian Zhu and Tao Zeng––These authors contributed equally to this work.     

猴艾滋病病毒（SRV）的装配及释放与细胞内骨架系统关系的电镜观察

将猴艾滋病 Ｄ 型逆转录病毒（ Ｓｉｍｉａｎ Ａ Ｉ Ｄ Ｓｔｙｐｅ Ｄｒｅｔｒｏｖｉｒｕｓ , Ｓ Ｒ Ｖ１） 感染的 Ｒａｊｉ 细胞进行选择性抽提制备成“核骨架中间纤维”这一细胞内骨架系统,再结合常规电镜技术和 Ｄ Ｇ Ｄ 包埋去包埋剂电镜技术,对此病毒在宿主细胞内的装配和释放与这一骨架系统的关系进行了研究。结果表明 Ｓ Ｒ Ｖ１ 病毒核壳体的装配是在细胞核附近的胞质中进行的,其“装配中心”是结合在胞质骨架的中间纤维上,正在装配的和已装配好的病毒核壳体都是紧密结合在中间纤维上的。而在核内骨架系统中未观察到病毒粒子。这表明 Ｓ Ｒ Ｖ 的装配可能是依赖于胞质中的中间纤维作支架,且装配好的核壳体可能沿着中间纤维被运送至质膜内侧或胞质内病毒感染后形成的空泡膜上,然后出芽释放     

急纤虫营养细胞和休眠细胞的中间纤维-核骨架体系

利用生化分级抽提、DGD包埋—去包埋透射电镜术和SDS—PAGE凝胶电泳,研究了膜状急纤虫营养细胞和休眠细胞内中间纤维—核骨架体系的分化特征及其蛋白组成。观察到营养细胞中,位于细胞质不同区域的中间纤维形成网状,其网络的密度不同;核骨架中,核纤层位于细胞核周缘,薄层状,厚约50nm;核内骨架由较致密的纤维网络组成。休眠细胞内该结构体系依然存在,但位于细胞内不同层次的纤维网比营养细胞的同种结构要致密得多,这可能与纤毛虫脱分化时细胞大范围的收缩有关;休眠细胞的包囊壁中层壁存在相当于中间纤维的网络结构。SDS—PAGE电泳图谱显示,休眠细胞内该体系的蛋白组成发生了较明显的变化,其中保留了营养细胞的部分蛋白条带,丢失了部分条带,同时还产生了一些特异性条带。分析表明,膜状急纤虫的中间纤维—核骨架体系是细胞在营养条件下和休眠状态下都稳定存在的结构;而纤毛虫形成休眠细胞后中间纤维—核骨架体系及蛋白组成上的变化提示,细胞在休眠状态下,基因的表达水平与营养细胞是不同的。     

A Comparison of the Effects of Acrylamide and Experimental Diabetes on the Retrograde Axonal Transport of Proteins in the Rat Sciatic Nerve: Analysis by Two-Dimensional Polyacrylamide Gel Electrophoresis

Retrogradely transported proteins that accumulated for 18 h distal to a ligature on the sciatic nerve of rats were separated by two-dimensional polyacrylamide gel electrophoresis and visualised with silver stain. A total of 14 proteins were detectable in this way as being retrogradely transported. In rats rendered diabetic 14 days previously by a single intravenous dose of streptozotocin, the accumulation of four of those proteins was noticeably decreased. Administration of acrylamide to a cumulative dose of 150 mg.kg-1 or 350 mg.kg-1 decreased the accumulation of four and eight proteins, respectively. Three of the four protein changes were common to the diabetic and acrylamide-treated animals, and were present before signs of neuropathy could be detected. The results suggest that those alterations may play a causal role in the development of neuropathy.     

Effect of cytochalasin D on the adhesion of a neuroblastoma x glioma cell line (NG108-15) to laminin and plastic substrates

Summary Adhesion of the neuronal cell surface to its underlying substrate plays an important role in neurite outgrowth in vitro. I have investigated the adhesive basis for neurite outgrowth in the presence of cytochalasin D, a disruptor of actin-containing microfilaments, and in the presence of vinblastine, a depolymerizer of microtubules. Scanning electron microscopy shows that cytochalasin D does not alter the branching configuration of filopodia on a laminin substrate, although processes are shorter and tapered distally in the presence of the drug. Using a standard attachment assay for the neuroblastoma x glioma cell line (NG108-15) I show that vinblastine does not influence attachment of NG108-15 cells to either plastic or laminin. Cytochalasin D-treated cells normally attach to high concentrations of a laminin substrate (20 g/ml). However, when cell are seeded on a laminin substrate at lower concentrations (0.001–10 g/ml), or on YIGSR, a fragment of laminin, cytochalasin D increases cell attachment. Cytochalasin D increases attachment in a dose-dependent manner when cells are seeded on plain polystyrene plastic, so that the number of cells attached to plastic in 1 M cytochalasin D is similar to the number attached to laminin (20 g/ml). Combining low concentrations of cytochalasin D and laminin results in greater attachment than with either agent alone. Mild trypsinization of the cell surface reduces the CD-enhanced attachment to plastic, indicating that a protein on the cell surface may be involved. The effect of cytochalasin D appears to be cell specific since cytochalasin D does not affect the attachment of a fibroblast cell line (NIH 3T3) to laminin and plastic. I hypothesize that a molecular mechanism is involved in which cytochalasin D promotes attachment by interacting with the cell surface via the actin microfilament system.     

藤茶总黄酮对大鼠肝纤维化的防治作用

目的:观察藤茶总黄酮(Tengcha flavonoids,TCF)对大鼠肝纤维化的防治作用。方法:66只雄性SD大鼠被随机分为5组:正常对照组(10只),正常喂养;模型组(14只):采用四氯化碳(CCL4)皮下注射诱导大鼠肝纤维化模型;TCF治疗组分为低、中、高剂量组(每组14只):在造模的同时,每日分别予以TCF25mg/100g、50mg/100g、100mg/100g体重灌胃。于第12周末检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(CⅣ)的水平以及肝组织中超氧化物歧化酶(SOD)活性、丙二醛(MDA)的含量变化。结果:与模型组比较,TCF各剂量组血清中ALT、AST、HA、LN、PCⅢ、CⅣ的水平显著下降(P<0.05);SOD活性明显增高,MDA的含量明显降低(P<0.05)。结论:TCF对CCL4实验性肝纤维化大鼠有较好的防治作用,其作用机制可能与抗氧化、抗脂质过氧化有关。     

Acrylamide Influence on Activity of Acetylcholinesterase,Thiol Groups,and Malondialdehyde Content in the Brain of Swiss Mice

Acrylamide is a neurotoxin inhibiting neurotransmission in peripheral nerves. Less is known about acrylamide influence on the central nervous system. Here we measured acrylamide influence on the acetylcholinesterase activity in brain stem, hemispheres, and cerebellum of mice (males, Swiss strain) in relation to the thiol groups and malondialdehyde concentration. Acrylamide was injected intraperitoneally (20 and 40 mg/kg, i.e. 0.52 and 1.04 mg per animal). The brain structures were taken 24, 48, and 192 h after the injection. Acetylcholinesterase activity was significantly lower (p < 0.001 to p < 0.05) in all the structures. It was accompanied by the statistically significant (p < 0.001 to p < 0.05) increase in malondialdehyde concentrations in most of the studied structures time periods and ACR doses. –SH groups concentrations were significantly depleted in the right hemisphere (p < 0.01) after 24 h and in brain stem (p < 0.05) after 48 h. We suggest that neurotoxicity of acrylamide in brain is related to acetylcholinesterase inhibition and redox imbalance.