碱性成纤维细胞生长因子基因转移对成骨细胞生长特性的影响

目的:碱性成纤维细胞生长因子(Basic fibroblast growth factor,bFGF)对骨组织的损伤有修复作用,许多体内外实验均表明外源性植入碱性成纤维细胞生长因子能明显促进骨形成过程。观察碱性成纤维细胞生长因子基因转移对成骨细胞生长特性的影响。方法:实验于2003-01/07在解放军广州军区总医院医学实验科完成。成年雄性新西兰白兔,体质量1.5～2.5kg,采用聚乙烯亚胺(jetPEITM)介导真核分泌表达载体pcDNA3.1-bFGF及非分泌表达载体pEGFP-C3-bFGF转染原代培养的成骨细胞,消化收集转染成骨细胞以及未经转染的成骨细胞,利用血细胞计数器进行细胞计数,绘制细胞生长曲线,同时测定DNA浓度、碱性磷酸酶及骨钙素含量的变化。结果:①未转染的细胞在第8天进入稳定期,而经转染的细胞在第10天才进入稳定期。未转染细胞最终达到的细胞数量为(1.70±0.02)×109L-1,而经转染的细胞数量为(2.10±0.03)×109L-1,差异显著(P<0.05)。②经pcDNA3.1-bFGF转染的成骨细胞的DNA量最高,约为(255±20)mg/L,而经pEGFP-C3-bFGF转染的成骨细胞的总DNA量约为(225±20)mg/L,未经转染的成骨细胞的总DNA量为(100±10)mg/L。③pcDNA3.1-bFGF转染的成骨细胞培养至第9天后,碱性磷酸酶分泌量为(8.0±0.22)IU/mL;而未转染的成骨细胞碱性磷酸酶分泌量为(13.12±0.18)IU/mL。经pEGFP-C3-bFGF转染的成骨细胞所分泌的碱性磷酸酶与未经转染的细胞的分泌量相当,约为(12.56±0.24)IU/mL。④随着培养时间的延长,骨钙素分泌量的变化趋势与碱性磷酸酶相同,但重组质粒转染成骨细胞与未转染细胞最终分泌的骨钙素量差异无显著性(P>0.05)。结论:①经pcDNA3.1-bFGF及pEGFP-C3-bFGF重组质粒转染后,成骨细胞的生长特性有一定的变化,与添加外源碱性成纤维细胞生长因子的影响基本相同。②pcDNA3.1-bFGF转染的细胞分泌的碱性成纤维细胞生长因子能够促进细胞分裂增殖,降低碱性磷酸酶的表达,而pEGFP-C3-bFGF转染的细胞不能分泌表达蛋白,难以发挥其生物学功能。     

脊髓损伤后真皮多能干细胞移植时机的选择

目的:观察脊髓损伤后不同时间移植真皮多能干细胞对大鼠运动功能修复的影响。方法:实验于2006-06/2007-03在第三军医大学生理学教研室完成。①实验材料:实验动物2～4月龄SPF级SD大鼠,体质量(210±40)g,雌雄不限,由第三军医大学实验动物中心提供。真皮多能干细胞为第三军医大学防原医学系从SD大鼠真皮中提取和分离。②实验方法:将42只SD大鼠在L4水平制成脊髓全横断损伤模型。将动物随机分为对照组(n=6)、真皮多能干细胞移植组(n=36)。真皮多能干细胞移植组又分为6个时间点:损伤后1,4,7,10,14,21d移植组,每组6只。各移植组于伤处移植大鼠真皮多能干细胞,而对照组于损伤后7d注射等量磷酸盐缓冲液。③实验评估:分别于移植后1d、1周、4周、8周、12周对各组大鼠进行动物行为学和脊髓诱发电位检测。结果:42只实验大鼠均进入结果分析,无脱落。①动物行为学评分:4周以后各组动物行为学评分比较差异有显著性意义(P<0.05),移植组明显高于对照组(P<0.05),各移植组间比较,以损伤后7,10d移植组动物行为学评分改善最显著。②各组大鼠脊髓诱发电位检查:体感诱发电位和运动诱发电位潜伏期和波幅值于移植后8,12周后明显高于对照组(P<0.05),各移植组间比较,以损伤后7,10d移植组体感诱发电位和运动诱发电位潜伏期和波幅值改善最显著。结论:脊髓损伤后7～14d进行真皮多能干细胞移植可明显改善大鼠后肢的运动功能。     

“双重束缚”解析

目的:介绍了Bateson提出双重束缚概念的过程,阐述双重束缚概念的特征,并且论述了这一概念对心理治疗起到的积极作用以及概念本身存在的问题。资料来源:应用计算机在ProQuest数据库、EBSCOhost数据库中搜索1980-01/2006-05相关文章,检索的关键词为“doublebind”,“paradox”;在西南大学图书馆手工查找有关“双重束缚”资料。资料选择:对查找的资料进行筛选,纳入阐述双重束缚提出的历史、概念以及应用的文献。排除:①非实证研究和综述。②管理、文化和道德中双重束缚的研究。③重复性的研究。资料提炼:共收集49篇文章,选择与本文相关的28篇进行综述。另外还参考《理解人性》,《家庭治疗基础》两本书籍。资料综合:“双重束缚”是Bateson结合“再学习”和“Russellian矛盾”两个概念提出的。对“双重束缚”的各种定义均体现了这一概念的如下特征:矛盾性、命令性、不平等性、不可逃避性和长期性。“双重束缚”在心理治疗中有广泛的应用,但是各种对“双重束缚”的研究都存在着不足。结论:“双重束缚”的提出有其特定的历史背景,它对精神分裂、精神错乱的形成有一定的解释力。     

Eliminating Animal Facility Light-at-Night Contamination and Its Effect on Circadian Regulation of Rodent Physiology, Tumor Growth, and Metabolism: A Challenge in the Relocation of a Cancer Research Laboratory

Appropriate laboratory animal facility lighting and lighting protocols are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations. Our recent experience in relocating to a new laboratory facility illustrates the importance of these considerations. Previous studies in our laboratory demonstrated that animal room contamination with light-at-night (LAN) of as little as 0.2 lx at rodent eye level during an otherwise normal dark-phase disrupted host circadian rhythms and stimulated the metabolism and proliferation of human cancer xenografts in rats. Here we examined how simple improvements in facility design at our new location completely eliminated dark-phase LAN contamination and restored normal circadian rhythms in nontumor-bearing rats and normal tumor metabolism and growth in host rats bearing tissue-isolated MCF7(SR^–) human breast tumor xenografts or 7288CTC rodent hepatomas. Reducing LAN contamination in the animal quarters from 24.5 ± 2.5 lx to nondetectable levels (complete darkness) restored normal circadian regulation of rodent arterial blood melatonin, glucose, total fatty and linoleic acid concentrations, tumor uptake of O₂, glucose, total fatty acid and CO₂ production and tumor levels of cAMP, triglycerides, free fatty acids, phospholipids, and cholesterol esters, as well as extracellular-signal-regulated kinase, mitogen-activated protein kinase, serine–threonine protein kinase, glycogen synthase kinase 3β, γ-histone 2AX, and proliferating cell nuclear antigen.Abbreviation: 13-HODE, 13-hydroxyoctadecadienoic acid; γH2AX, histone 2AX; AKT, serine–threonine protein kinase; ERK1/2, extracellular signal-regulated kinase p44/46; GSK3β, glycogen synthase kinase 3β: LAN light at night; MEK, mitogen-activated protein kinase kinase, PCNA, proliferating cell nuclear antigen; SR^–, steroid-receptor–negativeRelocating laboratory animal research from one institution to another can be a daunting task for both scientists and animal care personnel with regard to control of lighting and elimination of light-at-night (LAN) contamination. Appropriate facility lighting and lighting protocols, as outlined in the Guide for the Care and Use of Laboratory Animals,³⁰ are essential for maintaining the health and wellbeing of laboratory animals and ensuring the credible outcome of scientific investigations.^16-18,22 The profound effect of light on circadian behavior and physiology is well established.^{2,3,5,9,11,12,16-18,22,29,32,44,46,49,52,55-58,64}Minor alterations in light intensity,¹¹ spectral quality,¹² and duration⁹ at any given time of day can alter or disrupt chronobiologic rhythms markedly in all mammals.^{6,17,26,44,55-59} Light information, which initially is detected by a small group of intrinsically photosensitive retinal ganglion cells containing the blue light-sensitive photopigment melanopsin,^6,26 is transmitted through the retinohypothalamic tract⁵⁹ to a central molecular clock located in the suprachiasmatic nucleus of the hypothalamus.^32,57 The suprachiasmatic nucleus, the activity of which is entrained by the light:dark cycle,^32,57 sends projections over a polysynaptic pathway to the pineal gland driving a series of molecular events leading to the production of the pineal neurohormone melatonin (N-acetyl-5 methoxytryptamine), primarily during the night.^29,46 The daily rhythmic melatonin signal contributes to the temporal coordination of normal behavioral and physiologic functions including the sleep–wake^23,46,66 and reproductive cycles,^51,55 immune function,^38,41,56 hormone levels,^{19,31,45,47,68} temperature regulation,²³ electrolyte balance,⁶⁹ neural protein synthesis,⁶⁵ and redox states.^24,53Dark-phase LAN exposure suppresses endogenous melatonin concentrations and may lead to various disease states,^42,58 including carcinogenesis,^7,8,16,18,33 and metabolic syndrome.^{17,34-37,39,70} Previous in vivo studies in our former laboratory (at the Bassett Research Institute, Cooperstown, NY) demonstrated that animal room LAN of as little as 0.2 lx (0.08 µW/cm²; rodent eye level) during an otherwise normal dark-phase suppressed normal physiologic nighttime melatonin levels, leading to markedly disrupted circadian regulation of physiology and metabolism in nontumor-bearing host animals^16,18 and a stimulation in metabolism and proliferation of both tissue-isolated MCF7 steroid-receptor–negative (SR^–) human breast cancer xenografts and syngeneic grafts of rodent hepatoma 7288CTC in rats.^7,17 This effect was mediated by melatonin receptor-mediated suppression of cAMP, leading to inhibition of tumor linoleic acid uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). These events culminated in downregulation of epidermal growth factor and insulin-like growth factor 1 pathways.^7,8,16-19,62Exposure to LAN is likely to exert pervasive and problematic effects on mammalian behavior and physiology in laboratory animal facilities around the world. During the past decade, improved facility design and better adherence to animal room lighting protocols certainly has helped to reduce the problem. In moving to our laboratory animal facility at Tulane University School of Medicine (New Orleans, LA), we discovered considerable preexisting LAN contamination that had to be eliminated before we could resume our human cancer research.The current study was performed to monitor the effects of the elimination of animal room LAN contamination over time on animal health and wellbeing, tumor growth, and metabolic profiles by assessing well-established circadian parameters in physiology and metabolism.^7,8,16-18 We measured light-induced suppression of melatonin, an accepted and sensitive marker of the effects of light on the circadian system in all mammals,^{2,3,5,9,11,12,15,16,18,20,21,29,44,46,49,52,55-58,64} before and after tumor implantation and growth. Tissue-isolated MCF7(SR^–) human breast cancer xenografts and 7288CTC rat hepatomas have been well-characterized over the years in our former light-tight facilities^7,8,16-18 and provided us with unique markers and measures of the extent to which LAN contaminated our new animal quarters. In tumor-bearing animals exposed to even minimal LAN, the latency-to-onset of tumor growth and proliferation rates of these tumors increase markedly in direct proportion to LAN intensity. As improvements were made to eliminate LAN contamination in the new location over the course of more than 20 generations of tumor passages, we measured the changes and reestablishment of normal rat and host–tumor circadian regulation. The information from this study may assist investigators and animal care personnel in addressing this important influence on the health and wellbeing of laboratory animals and consequent effects on the outcome of scientific investigations.     

Agonist-induced internalization of CC chemokine receptor 5 as a mechanism to inhibit HIV replication

The chemokine G protein-coupled receptor CC chemokine receptor 5 (CCR5) is used as an entry gate by CCR5-tropic and dual- or CCR5/CXC chemokine receptor 4-tropic strains of HIV to enter the human host cells. Thus, CCR5 antagonists (i.e., maraviroc) have been proven to be clinically effective by preventing the interaction between viral glycoprotein 120 and CCR5 and thus impeding viral entry into host cells. However, the emergence of HIV strains resistant to CCR5 antagonists has been reported in vitro and in vivo, where the virus has adapted to enter the cells via antagonist-bound CCR5. An alternative strategy that should obviate this mode of viral resistance would entail the ablation of the CCR5 portal for HIV entry from the cell surface through agonist-induced receptor internalization. Although this protective effect has been demonstrated clearly with natural CCR5 ligands, the chemoattractant properties of these chemokines have precluded them from further consideration in terms of drug development. Thus, we sought to explore the possibility of developing novel small molecules and selective CCR5 agonists devoid of eliciting chemotaxis. Indeed, the CCR5 agonists described herein were found to induce profound down-modulation of CCR5 (and not CXC chemokine receptor 4) from the cell surface and its sustained sequestration in the intracellular compartment without inducing chemotaxis in vitro. The bioactivity profile of these novel CCR5 agonists is exemplified by the compound (R)-2-(4-cyanophenyl)-N-(1-(1-(N,1-diphenylmethylsulfonamido)propan-2-yl)piperidin-4-yl)acetamide (ESN-196) that potently inhibits HIV-1 infection in human peripheral blood mononuclear cells and macrophages in vitro with potencies comparable to that of maraviroc and moreover demonstrates full activity against a maraviroc-resistant HIV-1 RU570 strain.     

Mesenteric panniculitis in a man with abdominal pain,fever and diarrhea

In a 64-year-old white male with abdominal pain, fever, diarrhoea and weight loss there were radiological signs of mesenteric panniculitis: thickening of the small bowel loops, an infiltrate above the bladder and in the lower right abdomen an ileum loop with a spiculated border, and streaky strands emanating from the mesenteric fat in the lower abdomen. The clinical signs resolved spontaneously with conservative treatment in 3 weeks and after 3 months the radiological abnormalities had disappeared completely. The pathogenesis of mesenteric panniculitis is unknown. The prognosis is favourable. In advanced cases of the disease, anti-inflammatory or immunosuppressive medication may be beneficial.     

双波长紫外分光光度法测定贝母中腺苷和胸苷的含量

将4种贝母的甲醇提取物经薄层色谱法粗分离后,直接用双波长紫外分光光度法测定其含量。结果表明,该方法线性关系好,腺苷和胸苷标准曲线的相关系数均为0.9999,同时也发现平贝、炉贝和伊贝中腺苷都占核苷总量60%以上,而浙贝中仅占约40%,提示贝母生药的抗凝血活性可能与贝母中核苷类化合物的种类和含量的差别有关。     

头序木叶中三萜化学成分的研究

从头序木（ＡｒａｌｉａｄａｓｙｐｈｙｌａＭｉｑ）叶中分离到６个三萜化合物，用化学方法和波谱（ＵＶ，ＩＲ，ＭＳ，１ＨＮＭＲ，１３ＣＮＭＲ，１３ＣＤＥＰＴ，ＨＭＱＣ，ＨＭＢＣ）分析，确定其中４种化合物的结构，分别为齐墩果酸（Ｉ），１６β羟基１８βＨ齐墩果酸（Ｉ），齐墩果酸２８ＯβＤ吡喃葡萄糖甙（ＩＩ）和１６β羟基１８βＨ齐墩果酸２８ＯβＤ吡喃葡萄糖甙（ＩＶ）。用ＮＯＥＳＹ谱确定化合物Ｉ的立体构型。以上４个化合物均为首次从该植物中分离到，化合物ＩＶ为新化合物。     

Continuous infusion cisplatin and etoposide chemotherapy for cancer of unknown primary site (CUPS) in Taiwan, a region with a high prevalence of endemic viral infections

BACKGROUND: To evaluate the efficacy and toxicity of cisplatin/etoposide continuous infusion chemotherapy for cancer of unknown primary site in Taiwan, a region with a high prevalence of endemic viral infections. METHOD: Between April 1994 and February 1996, 20 patients with a diagnosis of CUPS were treated, including 15 males and five females, of average age 63.3 years (range 41-83 years). Continuous intravenous infusion of etoposide 80 mg/m2 and cisplatin 25 mg/m2 was given for 3 days every 3 weeks. Pretreatment tumor marker and viral serology studies were performed for baseline evaluation. Nearly two-thirds of the patients had poorly differentiated carcinoma. The average number of metastatic sites was 2.65 (range 1-4), with liver and lymph node involvement predominating. RESULTS: The overall response rate was 25% (95% CI 17.7-32.3%); 30.7% for poorly differentiated cancers and 25% for well differentiated cancers. Median survival was 4 months (range 1-12 months), 4.8 months for patients attaining partial response. Toxicity was moderate, grade 3 and 4 neutropenia occurred in 55% and grade 3 and 4 thrombocytopenia in 40%; other toxicities were mild. CA125 and CA199 were elevated in more than 50% of patients. Viral serology studies were not significantly different from those of the indigenous population. CONCLUSION: Etoposide and cisplatin combination chemotherapy has modest activity in patients with extensive CUPS and, at the schedule and dosage given, it is associated with moderate toxicity.      

Different risk profiles for hip fractures and distal forearm fractures: A prospective study

In a prospective cohort of elderly persons, aged 70 years and over, we examined risk indicators for which data could be easily obtained, to construct risk profiles for hip fractures and distal forearm fractures. Participants lived independently, in apartment houses for the elderly or in homes for the elderly. At baseline, information was obtained in 2578 subjects on age, gender, residence, mobility and the frequency of going outdoors. Mobility was measured using a walking score ranging from 1 (not able to walk independently) to 3 (able to walk independently for a fair distance). During the study period (median duration 3.5 years, maximum 4 years) 106 participants sustained a hip fracture and 60 participants suffered a distal forearm fracture. Women compared with men, adjusted for age, had a higher risk of hip fracture (adjusted relative risk (RR)=2.4, 95% confidence interval (CI) 1.3–4.3) and distal forearm fracture (RR=3.7, 95% CI 1.5–9.2). Age, adjusted for gender, was related to hip fractures only: the relative risk of fracture for those in the highest age category (>85 years) was 9.5 (95% CI 4.3–21.2) compared with those in the lowest age category (70–75 years). Moderately impaired walking ability compared with normal walking ability, adjusted for age and gender, was associated with a higher risk of hip fracture (RR=1.8, 95% CI 1.2–2.7) but with a lower risk of distal forearm fracture (RR=0.4, 95% CI 0.2–0.8). The outdoor score, adjusted for age and gender, was associated with distal forearm fractures only: going outdoors less than once a week, compared with three times or more, was associated with a lower risk of fractures (RR=0.3, 95% CI 0.1–0.9). In those living in homes for the elderly the risk of hip fracture was higher compared with those living independently (RR=2.4, 95% CI 1.4–4.2), adjusted for age and gender. Risk profiles were constructed using stepwise Cox's proportional-hazards regression. The risk profile predicted probabilites of sustaining a hip fracture in a 4-year period ranging from 0.4% to 25.9%, and of distal forearm fractures ranging from 0.2% to 4.5%, depending on the subject's characteristics as defined by the risk indicators. We conclude that easily obtainable risk indicators can be used in the prediction of fractures and can discriminate among fracture types.