DMBA诱发金黄地鼠颊囊癌的实验研究

目的：探讨二甲基苯并蒽(DMBA)诱发金黄地鼠颊囊癌后DNA含量的变化和细胞增殖情况。方法：金黄地鼠60只，随机分成6组，1—5组用0．5％DMBA涂擦双侧颊囊，分别于6周、8周、10周、12周、16周处死动物；正常对照组16周处死。切取颊囊标本每份标本分成两份，一份行组织学观察；另一份则行流式细胞仪检测，测定细胞DNA含量及细胞周期。结果：正常粘膜和单纯增生组细胞DNA均为二倍体(diploid)，轻、中度不典型增生有16．7％(2／12)异倍体出现；重度不典型增生则有33．3％(3／9)异倍体出现；原位癌有54．1％(6／11)异倍体出现；侵袭癌有78．57％(11／14)异倍体出现，且随颊粘膜病变加重，DI值及S期增殖率随之升高，重度不典型增生组S期细胞增殖率为最高，占39．31％。结论：DNA异倍体的出现是细胞恶性生物学行为的标志。S期细胞增殖率为恶性肿瘤增殖的指标，有利于对口腔癌的准确诊断，从而为临床口腔癌的治疗、预后评估提供有益的参考。     

Is Intracytoplasmic Sperm Injection Necessary for Couples Undergoing In Vitro Fertilization–Embryo Transfer with Normal Semen Analyses but Failing Hamster Egg Penetration Assays?

Purpose: Our purpose was to assess whether in vitro fertilization (IVF)–embryo transfer (ET) candidate couples with basically normal semen analyses but failing zona-free hamster egg penetration assay (HEPA) scares benefit from intracytoplasmic sperm injection (ICSI). Methods: Twenty consecutive IVF candidate couples with normal–borderline semen analyses and failing HEPA scores were recruited. Mature oocytes obtained from each woman were randomly divided between ICSI (group I; n = 126 oocytes) and standard insemination techniques (group II; 138 oocytes). Fertilization (two pronuclei) and cleavage (2–4 cells) rates were assessed for both groups. Results: There were no statistically significant differences between the two groups with respect to (mean ± standard error of the mean) fertilization (group I, 63.1 ± 7.75; group II, 77.8 ± 4.7%) or cleavage (group I, 87.3 ± 2.4%; group II, 91.2 ± 3.5%) rates. Conclusions: ICSI is not beneficial for IVF-ET when sperm samples demonstrate a failing HEPA score but have normal or minimally compromised semen analysis parameters.     

Time-dependent effects of melatonin on immune measurements in male Syrian hamsters

Abstract: Adult, male Syrian hamsters received daily subcutaneous melatonin (25 μg) injections or vehicle injections at 08:00 or 17:00 hr for 11 weeks. Body weights were measured weekly throughout the experiment and testes weights, spleen weights, and serum was collected at the end of the experiment. The spleens were sectioned and immunocytochemically analyzed for immunoglobulin G and serum levels of interferon-gamma, interleukin-2, and interleukin-4 were determined in heterologous mouse assays. Melatonin injections at 17:00 hr, but not at 08.00 hr, increased body weights, decreased testes weights and serum testosterone levels, and had no effect on immunoglobulin G content in the spleen. Likewise, melatonin injections at 17:00 hr, but not at 08:00 hr, increased serum interferon-gamma levels, had no effect on interleukin-2 levels, and appeared to increase interleukin-4 levels. Since melatonin injections at 08:00 hr were ineffective in altering immune measurements and correlations between reproductive measures and immune measures were high, the most parsimonious explanation for these results is that melatonin injections at 17:00 hr depressed reproductive hormone levels and these depressed levels altered immune measures.     

2-DG uptake patterns related to single vibrissae during exploratory behaviors in the hamster trigeminal system

Stimulation of one or several whiskers activates discrete foci throughout the trigeminal (V) neuraxis. These foci contribute to patterns, corresponding to the patterns of vibrissae, that have been directly related to aggregates of cells and axon terminals in the “barrel” cortex. Here, we combine high-resolution, 2-deoxyglucose (2DG) mapping and cytochrome oxidase (CO) staining to determine whether the known pattern of V primary afferent projections is sufficient to deduce the functional activation of their targets during exploratory behavior. Four adult hamsters had all of their large mystacial vibrissae trimmed acutely, except for C3 on the left, and B2 and D4 on the right; in two others, the left C3 and right A1 and E4 whiskers were spared. After fasting overnight, 2DG was injected and the animals behaved freely in the dark for 45 minutes. The brainstem, thalamus, and cortices were sectioned, then processed for both CO staining and 2DG autoradiography. Image-processing microscopy was used to separate the autoradiographic silver grains from the histochemical staining. CO patches were patterned in a whiskerlike fashion in the full rostrocaudal extent of V nucleus principalis and in caudal portions of spinal V subnuclei interpolaris and caudalis, but absent in subnucleus oralis. 2DG silver grains were densest above those CO patches in the pattern corresponding to the active whiskers. There were no consistent 2DG foci in subnuclei oralis or rostral caudalais. In these same cases, prominent 2DG labeling was restricted to the appropriate barrels in the contralateral cortex. Only one case, however, displayed a clear and appropriate region of heightened 2DG uptake in contralateral ventroposteromedial thalamus (VPM) and the adjacent part of the reticular thalamic nucleus. Patterns of increased glucose utilization with single whisker stimulation are well matched to the CO patterns that mirror distributions of neurons associated with a vibrissa in the V brainstem complex, thalamus, and cortex. Single whiskers are represented by relatively homogeneous longitudinal columns of 2DG labeling in the V brainstem nuclei. The columns are not continuous through the axial extent of the V brainstem complex; rather, they occur separately within principalis, interpolaris, and caudalis. While whisker columns were consistently labeled in interpolaris and caudalis in all animals, the labeling was increasingly variable in principalis, barrel cortex, and VPM, respectively. This suggests that the behaving animal can and does significantly modulate activity in this major, synaptically secure pathway. © 1993 Wiley-Liss, Inc.     

Wound healing in normal and diabetic Chinese hamsters

Summary Wound healing was examined in normal and diabetic, non-ketotic Chinese hamsters by morphological and morphometric methods. Dermal, perforating wounds were made in the ears of the hamsters and the response to injury was evaluated in tissue biopsies. The response in normal hamsters was characterized by vascular and cellular migration and pronounced infiltration of polymorphonuclear leukocytes into the area closest to the wound (zone 1). The transition region (zone 2) between wounded and non-wounded tissue was infiltrated primarily by fibroblasts and capillaries. In wounds from diabetic hamsters, 8 h after injury, there was less cellular infiltration (fibroblasts 49%, polymorphonuclear leukoytes 48% of control) and vascular proliferation (47% of control). In the late phase of healing (16 h after injury) the vascular (87% of control) and polymorphonuclear leukocyte (103%) responses in diabetic wounds were not significantly different from control in zones 1 and 2. Wounds from diabetic hamsters also showed considerable oedema (143% of control) in zones 1 and 2, which was accompanied by vascular degeneration and necrosis. At 16 h the collagen content of diabetic wounds was also decreased (54% of control). Increased oedema with reduced vascular proliferation and cellular infiltration in the early healing period characterises the response to injury in the diabetic Chinese hamster.     

GABA binding in the brains of aggressive and non-aggressive female hamsters

Ovariectomized female hamsters were selected for agrressiveness of non-aggressiveness toward a drug-treated target hamster. Animals in the aggressive group were found to have significantly higher levels of GABA binding in their brain ‘midregions’ (including limbic, striatal and diencephalic structures). There were no between-group GABA binding differences in cortex or pons/medulla and no differences in dihydroalprenolol (DHA) binding in any of these three regions. The groups did not differ on a variety of other behavioral tests including measures of activity, emotionality, feeding, and hormonally primed sexual behavior. The differences in ‘midregion’ GABA binding therefore may relate to levels of aggressiveness specifically.     

In vitro cytotoxicity of polychlorinated biphenyls (aroclors 1016, 1242, 1254 and 1260) and their effect on phospholipid and neutral lipid composition of Chinese hamster ovary (CHO-K1) cells

Cytotoxicity of 4 Aroclors (1016, 1242, 1254 and 1260) was compared in Chinese hamster ovary (CHO-K1) cells in Ham's F-12 medium. When parameters of toxicity were cell numbers or tissue protein, 50% lethality occurred at Aroclor concentrations between 30 and 45 ppm. An in vitro clonal assay with CHO-K1 cells was a sensitive indicator of cytotoxicity of the polychlorinated biphenyls (PCBs). From EC₅₀ values (concentration that allowed 50% survival of formed colonies), cytotoxicity was lower with Aroclor 1016 (32 ppm) and higher with Aroclors 1254 (27 ppm) and 1260 (28 ppm). In cells exposed 24 h to a marginally cytotoxic dose (20 ppm) of each Aroclor, phospholipid (PL) thin-layer chromatography (TLC) showed an increase in phosphatidylcholine (PC) and a decrease in phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG). Neutral lipid (NL) TLC of cells given Aroclors 1242, 1254 or 1260 showed a 3–4-fold increase in triglyceride (TG) and a similar reduction in cholesteryl esters (CE); in contrast to Aroclor 1016 which produced no change in TG and a smaller (2-fold) reduction in CE. Cholesterol and free fatty acid fractions were unaffected by any of the Aroclors. The TG:PL ratio remained unchanged in cells given Aroclor 1016, but increased 3–4-fold with Aroclors 1242, 1254, or 1260. Compared to total values in the untreated controls, CHO-K1 cells contained less neutral lipid and more phospholipid only with Aroclor 1016.These results support the concept that differences in the behavior of Aroclor 1016 are related to its PCB composition. Changes in membrane PL and NL components, observed at marginally cytotoxic levels of each Aroclor, provided further evidence that the PCBs may affect membrane integrity and associated metabolic functions.     

The mutagenicity of dialkyl nitrosamines in the salmonella plate assay

N-nitrosodimethylamine (DMN) is not mutagenic in the standard Salmonella plate incorporation assay (Ames test) in the presence of an in vitro metabolic activation system (S-9) derived from rat liver. When the S-9 was derived from Aroclor- or phenobarbital-induced mouse or hamster liver or from uninduced hamster liver, mutagenic activity was observed. Increasing the amount of S-9 above the usual maximum level of 50 μ1 per plate increased the mutagenic response. Similarly, the mutagenicity of N-nitrosodiethylamine (DEN) and N-nitrosodi(n-butyl)amine (DBN) was greater in the presence of hamster liver S-9 than when mouse or rat liver was used. Data are also presented indicating that the ability of rat liver S-9 to mediate the mutagenic activity of DMN in the “preincubation” assay is due to the fact that the various components are present in this assay at several times the concentrations attained in the standard plate incorporation assay.     

Fish somatostatin sst3 receptor: comparison of radioligand and GTPgammaS binding, adenylate cyclase and phospholipase C activities reveals different agonist-dependent pharmacological signatures

1 The fish somatostatin receptor 3 (fsst3) is one of the few somatostatin (SRIF) receptors cloned from a non-mammalian species so far. Here we extended our earlier characterization of this receptor by investigating the guanine nucleotide sensitivity of agonist radioligand binding at the fsst3 receptor recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. Further, we measured somatostatin (SRIF) and cortistatin (CST) analogues stimulated GTPgammaS binding, inhibition of forskolin-stimulated adenylate cyclase (FSAC) and stimulation of phospholipase C (PLC) activities. The present transductional data were then compared with previous radioligand binding and/or second messenger features determined for fsst3 and/or human SRIF receptors (hsst2, hsst3 and hsst5). 2 The GTP analogue guanylylimidodiphosphate (GppNHp) inhibited binding of [125I]CGP 23996 and [125I][Tyr3octreotide by 72 and 83% suggesting preferential labelling of G-protein-coupled fsst3 receptors. By contrast, [125I]LTT-SRIF28 and [125I][Tyr10]CST14 binding was rather GppNHp insensitive (42 and 35% inhibition) suggesting labelling of both coupled and non-coupled receptor states. These results might explain the apparent higher receptor densities determined in saturation experiments with [125I]LTT-SRIF28 and [125I][Tyr10]CST14 (4470 and 4030 fmol mg(-1)) compared with [125I]CGP 23996 and [125I][Tyr3]octreotide (3420 and 1520 fmol mg(-1)). 3 SRIF14 (10 microm)-stimulated specific [35S]GTPgammaS binding by three-fold; SRIF28 and octreotide displayed full agonism, whereas most other ligands displayed 60-80% intrinsic activity compared with SRIF14. SRIF14 and SRIF28 inhibited forskolin-stimulated AC (FSAC) activity by 60%; all tested ligands except BIM 23056 inhibited FSAC with comparable high intrinsic activities. SRIF14 stimulated PLC activity five- to six-fold, as determined by measuring total [3H] IP(x) accumulation; it was rather insensitive to pertussis toxin (PTX, 100 ng ml(-1), 21% inhibition), which suggests the G(q)-family proteins couple to PLC activity. SRIF14, SRIF28 and [Tyr10]CST14 showed full agonism at PLC, whereas all other ligands behaved as partial agonists (20-70% intrinsic activity). BIM 23056, which showed weak partial or no agonism, antagonized SRIF14-induced total [3H]-IP(x) production (pK(B) = 6.83), but failed to block competitively agonist-stimulated [35S]GTPgammaS binding or agonist-induced inhibition of FSAC activity. 4 Comparison of the pharmacological profiles of fsst3 receptors established in GTPgammaS binding, FSAC inhibition and PLC stimulation resulted in low correlations (r = 0.410-0.594). Both rank orders of potency and rank orders of relative efficacy varied in the three second messenger experiments. Significant, although variable correlations were obtained comparing GTPgammaS binding and inhibition of FSAC activity with previously reported affinity profiles of [125I]LTT-SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996, [125I][Tyr3]octreotide (r = 0.75-0.83; 0.68-0.89). By contrast, the PLC stimulation and radioligand-binding profiles did not correlate. 5 Comparison of the functional data (GTPgammaS binding, FSAC inhibition, PLC stimulation) of fsst3 receptors with those of human sst2, sst3, sst5 receptors expressed in CCL39 cells resulted in highest correlation with the hsst5 receptor (r = 0.94, 0.97, 0.49) > hsst2 (0.80, 0.50, n.d.) > hsst3 (0.25, 0.19, 0.17). 6 In summary, fsst3 receptors expressed in CCL39 cells are involved in signalling cascades similar to those reported for mammalian SRIF receptors, suggesting SRIF receptors to be highly conserved in evolution. Binding and functional data showed highest similarity of fsst3 receptors with the human sst5 receptor subtype. Different affinities, receptor densities and GppNHp-sensitivities determined with the four radioligands (agonists) are assumed to results from ligand-specific states of the fsst3-ligand complex. The differences in the rank orders of potency and relative efficacy in the various signalling cascades may be explained by agonist-induced receptor trafficking.