Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.     

骨质疏松性椎体骨折模型的建立

目的:建立大鼠的骨质疏松性椎体骨折模型,探讨骨折愈合程度与X射线、骨结构和力学性能的相互关系,以期能为临床治疗提供科学的指导和理论依据。方法:实验于2005-07/2006-07在解放军兰州军区总医院骨研所完成。实验动物:选择雌性SPF级8个月龄SD大鼠54只。实验分组:采用随机数字法将大鼠分为2组:骨质疏松组和对照组,每组27只。实验干预:骨质疏松组经双背侧手术切除卵巢,对照组行伪手术。术后3个月,所有动物麻醉下,采用L5椎体手术开窗刮除术区内松质骨方法建立人工椎体骨折模型。实验评估:于术后1,2,4,6,8,12周观察两组大鼠腰椎影像学、骨组织切片组织学与受累椎体力学性能。结果:54只SD大鼠全部进入结果分析。①影像学观察:术后两组X射线片示L5椎体有一骨折缺损透光区。对照组在术后6周时原透光区与周围骨质无明显差别,而骨质疏松组原透光区仍清晰可见,于8周时无明显差别。②组织学观察:两组软骨细胞在骨愈合1周时出现,形成软骨岛,但骨质疏松组软骨细胞每高倍视野数量明显少于对照组,另外,软骨细胞改建成成熟骨细胞,骨小梁形成数量,胶原纤维排列与对照组比较有显著性差异。③力学性能:在骨质愈合6～12周,L5椎体的最大载荷、弹性模量、最大应力明显低于同期对照组,差异有显著性意义(P<0.05)。结论:骨质疏松性椎体骨折SD大鼠模型,符合动物模型标准,可用于研究新骨形成与正常骨质结构关系,观察骨质疏松性椎体骨折愈合机制,并证明骨质疏松性松质骨骨折修复过程中,骨折愈合质量降低。     

参三七皂苷Rg1预适应对5-氮胞苷诱导大鼠骨髓间充质干细胞向心肌细胞转化的干预

目的:用药物预适应方法进行干细胞诱导已有报道,本实验观察中药参三七皂苷Rg1对5-氮胞苷诱导大鼠骨髓间充质干细胞向心肌细胞转化中的作用。方法:实验于2003-01/05在南京医科大学药理教研室完成。①实验材料:清洁级SD大鼠8只。参三七皂苷Rg18mg,批号20021017,由云南省长春花生物制剂公司提供,加入不含胎牛血清的IMDM培养液10mL,调配成10-4mol/L溶液,4℃保存。5-氮胞苷(Sigma公司,批号021209)。②实验方法:贴壁法体外培养大鼠骨髓间充质干细胞。设立4组:空白对照组常规培养后进行无血清处理,每3d换液1次;5-氮胞苷单用组单纯以10μmol/L的5-氮胞苷进行处理,其终浓度为1×10-8moL/L,连续诱导15d;5-氮胞苷 参三七皂苷Rg1预适应组分别加入0.1,1μmol/L参三七皂苷Rg1培养液处理24h,再各以10μmol/L的5-氮胞苷进行诱导,其终浓度为1×10-8moL/L,连续诱导15d。③实验评估:取第2代骨髓间充质干细胞,绘制生长曲线并计算群体倍增时间。观察诱导后骨髓间充质干细胞的生长形态学特征和细胞超微结构变化。激光共聚焦显微镜测定细胞表面积变化和细胞内钙离子浓度。结果:①5-氮胞苷诱导后骨髓间充质干细胞的生长形态学特征和细胞超微结构变化:骨髓间质干细胞胞体逐渐增大并伸出细长突起,在突起末端出现分支,部分相邻细胞的突起连接成网,形态学上表现出向心肌细胞方向转化的特征。其超微结构呈梭形,有明显的肌丝,细胞核呈单椭圆形,位于细胞中央,间质干细胞形似心肌细胞。②参三七皂苷Rg1预适应对5-氮胞苷诱导的骨髓间充质干细胞增殖特性的影响:与5-氮胞苷单用组比较,5-氮胞苷 参三七皂苷Rg1预适应组从第3天开始细胞数明显增加,细胞生长曲线均无明显的生长平台期,达到高峰后细胞数开始减少。③参三七皂苷Rg1预适应对5-氮胞苷诱导的骨髓间充质干细胞表面积的影响:与空白对照组骨髓间充质干细胞表面积比较,5-氮胞苷单用组明显降低,0.1,1μmol/L参三七皂苷Rg1预适应则能显著升高5-氮胞苷诱导的骨髓间充质干细胞表面积(P<0.01)。④参三七皂苷Rg1对5-氮胞苷诱导的骨髓间充质干细胞内游离钙水平的影响:与空白对照组比较,5-氮胞苷诱导4周后骨髓间充质干细胞内游离Ca2 相对荧光强度均明显升高(t=6.72,P<0.01),且5-氮胞苷 1μmol/L参三七皂苷Rg1预适应组升高幅度大于5-氮胞苷单用组(t=3.13,P<0.05)。结论:①参三七皂苷Rg1预适应在体外可显著刺激5-氮胞苷诱导的鼠骨髓间充质干细胞向心肌细胞转化和增殖,改善细胞形态,刺激细胞内钙离子增加。②参三七皂苷Rg1与5-氮胞苷对骨髓间充质干细胞向心肌细胞定向分化产生协同效应。     

健康人群隐神经传导中远端和近端方法的比较

隐神经传导对隐、股神经损伤,腰丛病变和L3、L4神经根损伤具有诊断价值.在通过表面电极评估隐神经逆向传导中,有远端和近端两种方法.[收稿日期 2005-09-20     

The chasm of care: Where does the mental health nursing responsibility lie for the physical health care of people with severe mental illness?

The poor physical health of people with a severe mental illness is well documented and health professionals' attitudes, knowledge and skills are identified factors that impact on clients' access to care for their physical health needs. An evaluation was conducted to determine: (i) mental health nurses' attitudes and beliefs about providing physical health care; and, (ii) the effect that participant demographics may have on attitudes to providing physical health care. It was hypothesized that workplace culture would have the largest effect on attitudes. Nurses at three health services completed the “Mental health nurses' attitude towards the physical health care of people with severe and enduring mental illness survey” developed by Robson and Haddad (2012). The 28‐item survey measured: nurses' attitudes, confidence, identified barriers to providing care and attitudes towards clients smoking cigarettes. The findings demonstrated that workplace culture did influence the level of physical health care provided to clients. However, at the individual level, nurses remain divided and uncertain where their responsibilities lie. Nursing leadership can have a significant impact on improving clients' physical health outcomes. Education is required to raise awareness of the need to reduce cigarette smoking in this client population.     

Analysis of serious non‐AIDS events among HIV‐infected adults at Latin American sites

Objective

Acquired immune deficiency appears to be associated with serious non‐AIDS (SNA)‐defining conditions such as cardiovascular disease, liver and renal insufficiency and non‐AIDS‐related malignancies. We analysed the incidence of, and factors associated with, several SNA events in the LATINA retrospective cohort.

Materials and methods

Cases of SNA events were recorded among cohort patients. Three controls were selected for each case from cohort members at risk. Conditional logistic models were fitted to estimate the effect of traditional risk factors as well as HIV‐associated factors on non‐AIDS‐defining conditions.

Results

Among 6007 patients in follow‐up, 130 had an SNA event (0.86 events/100 person‐years of follow‐up) and were defined as cases (40 with cardiovascular events, 54 with serious liver failure, 35 with non‐AIDS‐defining malignancies and two with renal insufficiency). Risk factors such as diabetes, hepatitis B and C virus coinfections and alcohol abuse showed an association with events, as expected. The last recorded CD4 T‐cell count prior to index date (P=0.0056, with an average difference of more than 100 cells/μL) and area under the CD4 cell curve in the year previous to index date (P=0.0081) were significantly lower in cases than in controls. CD4 cell count at index date was significantly associated with the outcome after adjusting for risk factors.

Conclusions

The incidence and type of SNA events found in this Latin American cohort are similar to those reported in other regions. We found a significant association between immune deficiency and the risk of SNA events, even in patients under antiretroviral treatment.     

急性缺血性卒中影像学检查的建议——美国心脏协会的科学声明（上）

卒中是一种常见、严重的疾病,仅美国每年的新发病例就高达795000例,并已成为全世界人类死亡和残疾的主要病因。10年前,重组型组织纤溶酶原激活剂（recombinant tissue plasminogen activator,rt—PA）被批准用于治疗急性缺血性卒中。rt—PA应用指南建议,应在卒中发病后3h内静脉给予rt—PA,给药前应行头部CT检查,排除颅内出血。     

急性缺血性卒中影像学检查的建议——美国心脏协会的科学声明（中）

2．3核磁共振血管成像（MRA） 2．3．1背景和方法：在头部MRI检查中常结合MRA,用于急性卒中患者病情评估以指导治疗决策的制定^[19]。日前,有几种不同的MRA技术用于脑血管成像,包括二维时间飞跃（timeof-flight,TOF）序列、三维TOF序列、