二巯基敌枯双生物作用的实验研究Ⅲ:时效关系的探讨

目的 :研究甲状腺激素干扰物二巯基敌枯双所致甲状腺的形态、血清 T4 和 TSH改变 ,探讨二巯基敌枯双生物作用的时效关系。方法 :Wistar大鼠 78只 ,随机分为正常对照组和实验组。正常对照组给予溶剂二甲基亚砜 ,实验组给予二巯基敌枯双的二甲基亚砜溶液 ,剂量为 5 0 mg/ kg体重。于实验组的给药第 1d、第 3d和第 5 d,分别放血处死两组动物。测定血清中 T4 ,TSH的浓度 ;取动物甲状腺称重 ,同时光镜下观察其形态学的变化。结果 :第 3d和第 5 d实验组血浆 TSH的浓度和对照组相比 ,显著增高 (P<0 .0 5 ) ,第 5 d实验组动物的甲状腺脏器系数显著高于对照组 (P<0 .0 5 ) ,组织学观察到该组甲状腺滤泡上皮局灶增生成增生斑。第 3d实验组甲状腺脏器系数与对照组比较尽管无差异 ,但组织学观察到该组甲状腺滤泡上皮已增生成复层。结论 :TSH、甲状腺的脏器系数和甲状腺的组织学观察在本实验中呈现明显的时间效应关系 ,而且最早出现生物学指标变化的时间是实验第 3d。     

Selective modulation of PKC isozymes by inflammation in canine colonic circular muscle cells

BACKGROUND & AIMS: Protein kinase C (PKC) is a key signaling molecule in excitation-contraction coupling in several types of smooth muscle cells. We investigated whether the attenuated contraction in inflamed colon cells is caused by alterations in the expression, distribution, and activation of specific PKC isozymes. METHODS: Kinase assays, immunofluorescence imaging, and Western immunoblotting were performed on single circular smooth muscle cells obtained from the normal dog colon as well as from colon with experimental colitis induced by mucosal exposure to ethanol and acetic acid, to determine the distribution, expression, and activation of PKC isozymes. RESULTS: Classical (alpha, beta, and gamma), novel (delta and epsilon), and the atypical PKC (iota, lambda, and zeta) isozymes were detected in colonic circular muscle cells. The expression of PKC alpha, beta, and epsilon isozymes was down-regulated, whereas that of PKC iota and lambda isozymes was up-regulated; other isozymes were not affected by inflammation. Acetylcholine (ACh) treatment translocated only the PKC alpha, beta, and epsilon isozymes from the cytosol to the membrane in normal cells; this translocation was absent in inflamed colon cells. Immunofluorescence imaging confirmed the translocation of PKC alpha from the cytosol to the membrane in response to ACh in normal cells. PKC inhibitors, chelerythrine, and myristoylated peptides to alpha, beta, and epsilon isozymes inhibited the contractile response to ACh in normal, but not in inflamed, cells. PKC iota and lambda did not participate in the contractile response to ACh. CONCLUSIONS: ACh-induced contraction is mediated by PKC alpha, beta, and epsilon isozymes in normal colonic circular muscle cells. Contractile dysfunction in inflamed colon cells is, in part, caused by decreased expression and impaired activation of specific PKC isozymes.     

Correlation of in vivo and in vitro functions of fresh and stored human platelets

BACKGROUND: Long-term storage of human platelets has been hindered by the loss of function of the platelets stored under current protocols. Novel preservation methods have encouraged examination of platelet function of cells preserved by cooling and freezing. The function of the platelets was assessed by using both in vitro assays and an in vivo rabbit bleeding model. STUDY DESIGN AND METHODS: Human platelets were stored in the presence or absence of 2 microM: cytochalasin B and 80 microM: EGTA/AM at 4 degrees C for 14 days or by freezing in the presence or absence of 5 percent DMSO. After the storage period, the platelets were resuspended in normal saline and infused into rabbits. Platelet function was assessed in vivo in a kidney bleeding model and in vitro by platelet-induced clot retraction and by platelet aggregation. RESULTS: Platelets stored at either 4 degrees C or -145 degrees C exhibited shorter survival times in the rabbit circulation than did fresh platelets. Platelets cooled to 4 degrees C, in both the presence or absence of cytochalasin B and EGTA/AM treatment, or frozen in the absence of DMSO were not effective in halting bleeding. However, frozen DMSO-treated platelets were as effective as fresh platelets in stopping bleeding. In vitro assays showed that cooled platelets treated with cytochalasin B and egtazic acid/AM and frozen DMSO-treated platelets retained 30 to 40 percent platelet function, while the cooled and frozen control samples exhibited no platelet-induced clot retraction. With thrombin as the agonist, only frozen DMSO-treated platelets exhibited a tendency to aggregate, although at only 22 percent of the aggregation function of fresh platelets. CONCLUSION: It is possible to freeze platelets and retain in vivo efficacy if the cryopreservative DMSO is included in the preparation. In vitro responses were greatly reduced by all of the storage protocols, but it may not be necessary to retain 100 percent in vitro function to have a platelet substitute or storage product that functions satisfactorily in vivo.     

中药水蛭对小白鼠胚胎发育的影响

妊娠小白鼠58只随机分组,至孕第7～11天给实验组孕鼠每天分别以500mg/kg(水蛭1组)及1000mg/kg(水蛭2组)的中药水蛭煎剂灌胃,至孕第18天记录各组的孕鼠和胎鼠体重,死胎、吸收胎及堕胎数。结果:水蛭1、2两组胎鼠体重下降,致畸作用显著,死胎、吸收胎比例升高;水蛭2组孕鼠体重下降,堕胎作用显著,与对照组比较,差异均有非常显著意义(P<0.01)。     

甲状腺激素干扰物二巯基敌枯双生物作用的实验研究I观察指标的探讨

目的 :研究二巯基敌枯双所致甲状腺的形态改变 ,探讨二巯基敌枯双是否属甲状腺激素干扰物。方法 :Wistar大鼠 60只 ,随机分为正常对照组和实验组。正常对照组给予溶剂二甲基亚砜 ,实验组给予二巯基敌枯双二甲基亚砜溶液 ,剂量为 5 0 mg/kg体重。于实验组的给药第 5 d、第 10 d和第 2 0 d,分别放血处死两组动物。测定血清中 T4,TSH的浓度 ;取动物甲状腺称重 ,常规 HE染色 ,光镜下观察。结果 :各时间点实验组血清 T4,TSH的浓度和对照组相比无显著变化 (P>0 .0 5 ) ;实验组动物的甲状腺脏器系数显著高于对照组 ,(P<0 .0 5 ) ;组织学观察到实验组在第 5 d甲状腺滤泡上皮增生 ,有的滤泡出现局灶性增生斑块 ,第 10 d复层化的甲状腺滤泡上皮细胞继续增生 ,并向间质突出 ,形成实心芽 ,第 2 0 d实心芽向继发滤泡分化 ,出现了滤泡腔。结论 :二巯基敌枯双引起甲状腺增生 ,提示具有甲状腺激素干扰物的可能性     

二巯基敌枯双生物作用的实验研究 Ⅱ：组织化学观察

目的：研究甲状腺激素干扰物二巯基敌枯双所致甲状腺组织化学的改变，并探讨所致甲状腺增生的最佳取材时间和敏感指标。方法：Wistar大鼠60只，随机分成正常对照组和实验组，每组各30只。正常对照组给予溶剂二甲基亚砜，实验组给予二巯基敌枯双的二甲基亚砜溶液，剂量为50mg/kg体重。于实验组的给药第5d、第10d和第20d，分别放血处死两组动物。用酶组织化学方法观察甲状腺琥珀酸脱氢醇(SDH)、甲状腺过氧化酶酶活性的变化(TPO)；用免疫酶法测定核增殖抗原(PCNA)在甲状腺中的阳性表达。结果：实验组各期甲状腺滤泡上皮内SDH和TPO酶活性明显增高(P＜0．05)，实验组的各期PCNA表达阳性细胞数也明显高于同期对照组(P＜0．05)。结论：甲状腺激素干扰物二巯基敌枯双所致甲状腺滤泡上皮内SDH和TPO酶活性增高是TSH刺激的结果，其最佳取材时间应是5d以前，而敏感指标应该是核增殖抗原(PC-NA）。     

农药二巯基敌枯双对FRTL-5细胞合成甲状腺球蛋白的影响

目的 观察农药二巯基敌枯双对FRTL-5细胞株合成甲状腺球蛋白的影响,并探讨其作用机制以及FRTL-5细胞株用于筛选环境甲状腺素干扰物的可能性.方法 FRTL-5细胞分别经0.1、1.0和10.0 μg/mL二巯基敌枯双染毒处理48 h后,用酶组织化学法测定培养液中甲状腺球蛋白的浓度;用活细胞爬片的酶细胞化学染色观察细胞内甲状腺过氧化物酶的表达强度,并用透射电镜观察最高浓度处理组细胞的超微结构.结果 随二巯基敌枯双浓度的增大,FRTL-5细胞培养液中甲状腺球蛋白的浓度逐渐降低,其中0.1、1.0 μg/mL浓度组与溶剂对照组相比差异有统计学意义(P＜0.05),10.0 μg/mL浓度组培养液中未能检出甲状腺球蛋白;酶细胞化学染色未见染毒组细胞甲状腺过氧化物酶活性改变,图像分析结果 显示其阳性细胞的积分光密度(IOD)值与对照组相比,差异无统计学意义(P＞0.05);细胞超微结构观察发现染毒组细胞出现粗面内质网扩张.结论 二巯基敌枯双可能是通过损伤甲状腺滤泡细胞的粗面内质网,从而影响甲状腺球蛋白的合成与分泌;FRTL-5细胞培养液中甲状腺球蛋白的浓度可以作为体外筛选环境甲状腺素干扰物的敏感指标之一.     

抗肿瘤药物的研究:N′,N″-二螺三哌嗪类化合物的合成

Prospidine和spirobromine是全苏化学药物研究院相继研究出的两种较好的N′,N″-二螺三哌嗪类抗肿瘤药物,不但抗肿瘤活性强,而且毒性低,因此研究和开发这类药物是很有意义的。     

Ethambutol-induced toxicity is mediated by zinc and lysosomal membrane permeabilization in cultured retinal cells

Ethambutol, an efficacious antituberculosis agent, can cause irreversible visual loss in a small but significant fraction of patients. However, the mechanism of ocular toxicity remains to be established. We previously reported that ethambutol caused severe vacuole formation in cultured retinal cells, and that the addition of zinc along with ethambutol aggravated vacuole formation whereas addition of the cell-permeable zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), reduced vacuole formation. To investigate the origin of vacuoles and to obtain an understanding of drug toxicity, we used cultured primary retinal cells from newborn Sprague-Dawley rats and imaged ethambutol-treated cells stained with FluoZin-3, zinc-specific fluorescent dye, under a confocal microscope. Almost all ethambutol-induced vacuoles contained high levels of labile zinc. Double staining with LysoTracker or MitoTracker revealed that almost all zinc-containing vacuoles were lysosomes and not mitochondria. Intracellular zinc chelation with TPEN markedly blocked both vacuole formation and zinc accumulation in the vacuole. Immunocytochemistry with antibodies to lysosomal-associated membrane protein-2 (LAMP-2) and cathepsin D, an acid lysosomal hydrolase, disclosed lysosomal activation after exposure to ethambutol. Immunoblotting after 12 h exposure to ethambutol showed that cathepsin D was released into the cytosol. In addition, cathepsin inhibitors attenuated retinal cell toxicity induced by ethambutol. This is consistent with characteristics of lysosomal membrane permeabilization (LMP). TPEN also inhibited both lysosomal activation and LMP. Thus, accumulation of zinc in lysosomes, and eventual LMP, may be a key mechanism of ethambutol-induced retinal cell death.     

化学物质诱发的小鼠肢体畸形动物模型的建立

目的：建立发生率稳定、畸形类型特异和易于获得的化学物质所致小鼠肢体畸形动物模型。方法：采用致突变性致畸物N—甲基—N’—硝基—N—甲基亚硝基胍(N—methyl-N‘=nitro-N-nitrosoguanidine，MNNG)作为受试物，观察不同剂量和不同给药时间的胎鼠畸形率、畸形类型及特征。结果：孕期第12天一次给予MNNG 40mg/kg时，胎鼠畸形类型主要为肢体畸形，肢体畸形率以活胎计为33.7％(3l／92)，以窝计为8l.8％(9／11)，肢体畸形占畸形胎鼠的构成比为l00％。畸形类型以短指(趾)和缺指(趾)最常见。四肢畸形的发生率和严重程度存在不对称性，依次为左后＞左前＞右后＞右前。掌跖骨缺失和骨化不全发生率较高，此外还有胫腓骨的缺失和骨化不全，特别是大体形态所见短肢是胫腓骨缺失所致。结论：成功建立了小鼠肢体畸形动物模型，为进一步研究肢体畸形的分子机制奠定了基础。