Successful in vitro growth of rat two-cell embryos to blastocysts using a simple chemically defined medium

The aim of the present study was to formulate a simple chemically defined medium for the in vitro growth of rat two-cell embryos to blastocysts. Embryos from day 2 pregnant rats were retrieved and placed in paraffin oil-covered droplets of "rat two-cell embryo culture medium" (R2ECM) containing combinations of various serum supplements, glucose, L-glutamine, and cultured up to 96 h in a CO(2) incubator. Embryos cultured in the basic medium (R2ECM), as well as those supplemented either with fetal bovine serum (FBS) or male rat serum (MRS) did not develop beyond the two- to four-cell stage. In R2ECM with 0.3% bovine serum albumin (BSA) and 7.5 mM glucose, 44% of embryos reached the blastocyst stage by 96 h in culture, and the blastulation rate increased to about 83% when 1 mM of L-glutamine was added. To evaluate the effects of varying doses of glucose, two-cell embryos were cultured in R2ECM supplemented with 0.3% BSA, 1 mM L-glutamine, and 2.5, 5.0, or 7.5 mM of glucose. The percentage of embryos reaching the blastocyst stage for 2.5, 5.0, and 7.5 mM glucose was 64.6%, 65.3%, and 82.9%, respectively. The present study showed that the modified medium (R2ECM) is a simple chemically defined medium that is capable of supporting in vitro growth of rat two-cell embryos to blastocysts in high proportion (greater than 80%) without the need for change of medium within 96 h of culture.     

Treatment outcome and incidence of psychiatric disorders in dermatological out-patients

OBJECTIVE: Epidemiological studies have shown that the prevalence of psychiatric disorders among dermatological patients is high. We aimed at estimating the short-term incidence of psychiatric disorders among patients with skin disease. METHODS: The 12-item General Health Questionnaire (GHQ-12) was used to identify subjects free from psychiatric morbidity at their first dermatological visit. The GHQ-12 was administered again after 1 month during a computer-assisted telephone interview. RESULTS: A total of 277 subjects was included in the study. At the follow-up interview, 21 (7.6%) were found to have significant psychiatric morbidity. Only lack of improvement was associated with increased incidence of psychiatric morbidity (13.6%), with an odds ratio of 3.1 (95% confidence interval 1.2-7.8), after adjustment for gender, age, educational level and clinical severity. CONCLUSIONS: Physicians should devote special attention to the risk of psychiatric complications in patients who have not improved with treatment.     

培养临床药师提高合理用药水平

医院药学的发展机遇与挑战并存,科技进步、改革的深化,要求医院药学必须转变观念。建议医学院校的药学院(系)逐步改革现行的化学模式的药学教育制度,建立生物医学模式的药学教育制度,开设临床药师专业,开办临床药师专业硕士、博士学位班,制定临床药学管理办法,明确临床药师岗位职责,营造有利于临床药师发展的医院环境。     

手术室护士锐器刺伤的危害及防护对策

文章编号:1009-6493(2006)1B-0167-01手术室护理工作者是一个在特殊环境中从事特殊护理专业的群体。据有关资料报道,锐器刺伤是导致医务人员发生血源性传播疾病最主要的职业因素,我国是肝炎的高流行区,近年来艾滋病在我国的发病率呈倍增的趋势[1]。医院是这些疾病的聚集地,而手术室是具有独特接触血液和锐器伤的高发科室,做好手术室日常工作中的职业防护是十分重要的。1锐器刺伤危害性锐器刺伤可以传播血源性疾病,最常见、危害最大的是HBV、HCV、HIV。美国疾病控制中心(CDC)报道,医务人员锐器刺伤被感染HBV的几率是2%～40%,被感染HC…     

犬自体肺组织瓣修补食管壁部分缺损的可行性

目的:分析犬自体肺组织瓣修补食管壁部分缺损的可行性。方法:实验于2003-01/2004-11在中国医科大学附属第二医院动物实验室完成。选用健康成年杂种犬20只,按随机数字表法分为2组,即支架组和无支架组,每组10只。20只实验犬经右胸第5肋间进胸,于胸内中段食管处胸内食管侧壁制成长4cm,环1/2~2/3周径全层缺损。于相应部位选择适当的肺组织,制成带蒂类舌状肺组织瓣。两组均将肺组织瓣覆盖并缝合固定于食管缺损处,支架组于食管缺损内衬自扩性记忆合金支架(管腔直径2.0cm、长6.0cm)并固定。术后抗炎及营养支持治疗。观察实验犬术后情况,并于术后2,4,6,8,10和12周定期处死实验犬行组织学观察。结果:无支架组实验犬存活7只,其中1只犬存活>24个月;支架组存活6只。①实验犬术后一般情况:存活犬于术后均能正常经口进食,早期有进食后呕吐,再吃下呕吐食物的现象,以支架组明显。②组织学观察结果:术后2周,无支架组均可见替代物表面有胶原及炎性渗出物,边缘见1~2层鳞状上皮细胞;支架组除有无支架组基本表现外,可见支架固定良好,光镜下见网架压迫处有较多中性粒细胞浸润。4~6周,两组均可见替代物表面有新生的3~5层复层鳞状上皮细胞;支架组见支架已基本陷入黏膜层内。8~10周,两组均可见管腔表面有6~8层新生复层鳞状上皮细胞;支架组网架边缘瘢痕组织增生,支架完全被包裹,炎症较重的局部有细胞爬行中断现象或新生细胞层数较薄,多为一两层。结论:应用自体肺组织瓣修补食管壁部分缺损是可行的,但支架组支架对食管修补处组织刺激大,炎性反应重,瘢痕重,因此如何选择合适的支撑物是今后替代节段性食管缺损面临的重要问题。     

Tumor cell anti-oxidant defenses. Inhibition of the glutathione redox cycle enhances macrophage-mediated cytolysis

The basis of resistance to oxidative injury was studied in six murine tumor cell lines that differed 54-fold in their resistance to enzymatically generated H(2)0(2). The tumors varied 56.7-fold in their specific activity of catalase, 5.3-fold in glutathione peroxidase (GPO), 3.3-fold in glutathione reductase (GR), and 2.7-fold in glutathione. There was no correlation among the levels of the three enzymes, and tumor cell resistance to lysis by H(2)0(2). However, the logarithm of the flux of H(2)0(2) necessary to cause 50 percent lysis of the tumor cells correlated with their content of glutathione (r = 0.91). The protective role of glutathione was analyzed by blocking GR and GPO, the catalysts of the glutathione redox cycle. This was facilitated by the demonstration that the anti-neoplastic agent 1,3-bis-(2- chloroethyl)-l-nitrosourea (BCNU) was a potent inhibitor of GR in intact tumor cells. BCNU inactivated tumor cell GR with a 50 percent inhibitory dose of 11 μM and a t(l/2) of inhibition of 30 s. Complete inhibition of GR was attained with no effect on GPO or catalase. Tumor cells whose GR was inactivated by BCNU could be lysed by fluxes of H(2)0(2) to which they were otherwise completely resistant. They could be killed by phorbol myristate acetate (PMA)-stimulated, bacilli Calmette-Guerin-activated macrophages in numbers which were otherwise insufficient, and by nonactivated macrophages, which otherwise were ineffective. BCNU-treated target cells were also much more sensitive to antibody-dependent, macrophage-mediated cytolysis. However, such tumor cells were no more sensitive than controls to lysis by alloreactive T cells or by antibody plus complement. Next, we deprived tumor cells of selenium by passage in selenium-deficient mice. GPO was inhibited 85 percent in such cells, with no effect on GR or catalase. Tumor cells with reduced GPO activity were markedly sensitized to lysis by small fluxes of H(2)0(2) or by PMA-stimulated macrophages or granulocytes. In contrast, inhibition of catalase with aminotriazole had no effect on the sensitivity of three tumors to peroxide-mediated lysis, and had modest effects with two others. Thus, the oxidation-reduction cycle of glutathione serves as one of the major defense mechanisms of tumor cells against three related forms of oxidant injury: lysis by fluxes of H(2)0(2), by PMA-triggered macrophages, and by macrophages in the presence of anti-tumor antibody.     

同型半胱氨酸对大鼠血管平滑肌细胞增殖及胶原生成的影响

目的:观察同型半胱氨酸的浓度对Ⅰ,Ⅲ,Ⅳ和Ⅵ型胶原的产生以及大鼠血管平滑肌细胞增殖效应的影响。方法:实验于2005-02/2006-04在瑞金医院心内科实验室完成。①采用组织块贴壁法原代培养大鼠胸主动脉平滑肌细胞,用不同浓度的同型半胱氨酸(0.025,0.05,0.1,0.2,0.5,1.0,1.5,2.0,3.0,5.0 mmol/L)刺激大鼠血管平滑肌细胞,并设对照孔(未加同型半胱氨酸)和空白孔(未加细胞)作对照。②分别作用12,24,48,72h,以四甲基偶氮唑盐法检测同型半胱氨酸对平滑肌细胞的活性细胞数量及细胞增殖情况;以5溴-2脱氧尿苷法检测同型半胱氨酸对大鼠血管平滑肌细胞DNA合成的作用;流式细胞仪观察细胞周期的变化;酶联免疫法测定胶原的含量。结果:①细胞增殖情况:在0.025,0.05 mmol/L的同型半胱氨酸浓度刺激下,12～48h血管平滑肌细胞增殖增加明显。②细胞DNA合成:与四甲基偶氮唑盐结果类似,24～48h时,在浓度为0.025,0.05 mmol/L的同型半胱氨酸刺激下细胞合成DNA增加(24h:432±33,774±52;48h:582±32,816±62),其促增殖作用增强(P<0.05);随着浓度的增加,大鼠血管平滑肌细胞合成DNA开始下降,至5 mmol/L浓度时最低。③细胞周期的变化:24h时在浓度为0.025,0.05 mmol/L的同型半胱氨酸刺激下增殖的细胞S期的百分含量较对照组和同型半胱氨酸其他浓度组明显增加。④胶原的含量:细胞的Ⅰ型和Ⅳ型胶原分泌随着同型半胱氨酸浓度的升高呈现剂量依赖性的增高模式。Ⅲ型胶原有轻度的升高,无统计学上意义。高浓度的同型半胱氨酸会减少Ⅵ型胶原的分泌,并呈剂量依赖性(r2=0.41;P<0.001)。结论:较低浓度的同型半胱氨酸刺激大鼠血管平滑肌细胞的增殖加速;并可能通过改变纤维斑块中的胶原构成类型导致了斑块的不稳定性和易损性,最终加速了粥样斑块的发生和发展。     

Transfusion-associated Serratia marcescens infection: studies of the mechanism of action

The growth of two strains of Serratia marcescens in blood components was tested in this study. One of the strains had been implicated in the epidemic of transfusion-associated sepsis experienced in Denmark and Sweden in 1991. In whole blood with a final concentration of 100 colony- forming units per mL of S. marcescens, there was an immediate reduction of more than 95 percent of colony-forming units, but no reduction of the bacterial concentration if the blood had been white cell-reduced before inoculation. This is interpreted as an effect due to phagocytosis by white cells and as a lack of bactericidal effect of the plasma. A reduction to 10 percent of the original concentration, observed if the blood had a nominal content of white cells, was most likely due to phagocytosis. White cell reduction by filtration after inoculation further reduced the bacterial concentration of one of the strains tested, but, after a 1-week lag phase, growth accelerated to high concentrations by 6 weeks. In platelet-rich plasma prepared from S. marcescens-inoculated units, abundant growth was found after 24 hours, increasing to very high concentrations (10(12) colony-forming units/mL) during 10-day storage at 22 +/− 2 degrees C. Keeping the whole blood at ambient temperature for 20 hours before preparation of platelet-rich plasma caused only temporary reduction of bacterial concentration in the S. marcescens experiments, but resulted in a complete absence of bacteria in the platelet-rich plasma for 10 days in control experiments performed with Staphylococcus epidermidis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antitumor effects of hydrogen peroxide in vivo

Glucose oxidase, covalently coupled to polystyrene microspheres (GOL), produced H(2)0(2) at an average rate of 3.6 nmol/min per 10(9) beads under standard assay conditions. Injection of 1.3 × 10(10) to 1.1 × 10(11) GOL i.p. prolonged the survival of mice by 27 percent after injection of 10(6) P388 lymphoma cells in the same site, consistent with destruction of 97.6 percent of the tumor cells. Placing mice for several hours in 100 percent O(2), the probable rate-limiting substrate for GOL, afforded a 42 percent prolongation of survival from P388 lymphoma, consistent with destruction of 99.6 percent of the tumor cells. When the P388 inoculum was 10(5), 10(4), or 10(3) cells, GOL led to long-term survival (presumed cure) of 23 percent, 77 percent, and 92 percent of the mice, respectively, consistent with reduction of the injected tumor dose to less than 10 cells. Subcutaneous growth of 10(5) P388 cells (approximately 300 lethal dose to 50 percent of mice) was suppressed in 83 percent of mice by admixture of GOL with the tumor cell inoculum. GOL alone had no effect against a more peroxide-resistant tumor, P815 mastocytoma. However, P815 cell glutathione reductase could be inhibited in vivo by well-tolerated doses of the antitumor agent, 1,3-bis(2-chloroethyl)- 1-nitrosourea (BCNU). BCNU alone cured few mice with P815. Together, BCNU and GOL apparently cured 86 percent of mice injected with 10(6) P815 cells i.p. The protective effect of GOL was abolished by boiling it to inactivate the enzyme, by co-injection of catalase coupled to latex beads, or by delaying the injection of tumor cells for 3 h, by which time the beads had formed aggregates. Soluble glucose oxidase, in doses threefold higher than that bound to GOL, had no detectable antitumor effect. A single injection of preformed H(2)0(2) readily killed P388 cells in the peritoneal cavity, but only at doses nearly lethal to the mice. In contrast, GOL had very little toxicity, as judged by the normal appearance of the mice for over 400 d, gross and microscopic findings at autopsy, and various blood tests. GOL injected i.p. remained in the peritoneal cavity, where it was gradually organized into granulomata by macrophages, without generalized inflammation. Thus, an H(2)0(2)-generating system confined to the tumor bed exerted clear- cut antitumor effects with little toxicity to the host.     

The immune response of allophenic mice to the synthetic polymer L-glutamic acid, L-lysine, L-phenylalanine. II. Lack of gene complementation in two nonresponder strains

The genetic control of the immune response of inbred strains of mice to certain antigens has been demonstrated to be governed by a set of Ir genes linked to the major histocompatibility complex (H-2) of mice (1,2). Until recently, the control was thought to be governed by single, dominant genes, located within the I region of the H-2 complex. Merryman et al. (3) originally demonstrated that the immune response to the synthetic terpolymer L-glutamic acid, L-lysine, L-phenylaline (GL) is under dominant, H-2-linked Ir gene control (4-7). This was shown both by crossing two nonresponder parental strains to produce responder offspring in the F(1) generation, and by the analysis of appropriate recombinant stains of mice. The two complementing genes have been mapped in the IA and IC regions of the H-2 complex, and have been termed β and α, respectively (5,6). Thus, any strain of mouse may contain neither, one, or both genes. Only mice containing both genes are capable of responding to GL. It has been shown using F(1) hybrid and recombinant strains of mice, that the α- and β-genes can complement each other in either the cis (on the same chromosome) or in the trans (on different chromosomes) position (8). In this paper we report the results of studies aimed at answering the question of whether or not the α- and β- genes can complement each other when they are present in different lymphoid cells. To this end we have constructed allophenic mice composed of two nonresponder strains (A and C57BL/6), which show gene complementation in the F(1) generation. Allophenic mice are chimeras containing two cell types coexisting in a “normal” environment. The mice were tested for the specific cellular composition of the two parental cell types and were found to possess a complete range in the relative proportion of the two cell types. This report demonstrates that regardless of the mixture of cell types present in the allophenic mice, none of them were responders to GL. Thus no complementation of the α- and β-genes is seen when the two genes are present in different cells.