免疫磁珠分选骨髓衍生肝干细胞亚群c-Kit+lin-

目的:利用免疫磁性细胞分选系统分离纯化骨髓衍生肝干细胞亚群c-Kit lin-。方法:实验于2006-07/08在南方医科大学实验动物中心完成。6～8周龄的SPF级纯系BALB/C雄性小鼠10只,体质量18～20g。收集小鼠股骨骨髓细胞,利用免疫磁性细胞分选系统,通过两步法分选纯化c-Kit lin-:将获取的lin-细胞悬液8℃条件下1500r/min离心10min,弃上清,按80μL/107加入Buffer重悬细胞。按20μL/107加生物素抗体磁珠,混匀,4℃冰箱孵育15min,按1mL/107加入Buffer洗细胞1次,8℃条件下1500r/min离心10min,弃上清,按500μL/108加入Buffer重悬细胞。Buffer500μL润MS柱,悬液过柱后,Buffer500μL/次洗柱3次,柱子脱离磁场,加1mLBuffer,用配套柱塞推出柱中的c-Kit lin-细胞,收集到c-kit lin-细胞,细胞计数。取2.0×106个细胞分成10等份,流式细胞仪分析c-Kit lin-细胞纯度,计算回收率,评估纯化效率,苔盼兰染色检测纯化前后的细胞活力。计算活细胞的百分率。细胞纯度和细胞回收率的计算:细胞纯度=分离产物中的阳性细胞数/分离细胞的总细胞数×100%,细胞回收率=分离产物中的阳性细胞数/起始标本阳性细胞总数×100%。结果:10只小鼠均进入结果分析。利用免疫磁性细胞分选系统分选出的骨髓衍生肝干细胞亚群c-Kit lin-细胞纯度和回收率分别为(77.98±2.34)%,75.40%,纯化前后细胞活力不受影响。结论:免疫磁性细胞分选系统能有效分选骨髓衍生肝干细胞亚群c-Kit lin-,纯度和回收率高,且不影响细胞活力。     

冻存复苏条件对髓性白血病细胞株生物学特性的影响

目的:采用不同方法对白血病HL60细胞株进行冻存和复苏,观察生物学特性改变,筛选最佳冻存复苏方案。方法:实验于2006-04/06在吉林医药学院临床检验实验室(国家三级标准)进行。①HL60细胞株由中国科学院上海生物细胞研究所提供。将欲冷冻保存的HL60细胞调整到良好的生长状态(对数生长期),随机数字表法分成4组,离心收集后在含体积分数为0.1小牛血清的RPMI-1640培养基中分别加入二甲基亚砜,使其终浓度依次为50,100,150,200g/L。冻存15d后,每组取1支冻存管复苏并接种于细胞培养板,培养12h后用锥虫蓝拒染法计算细胞复苏率。②选择冻存条件和冻存细胞密度相同的两支冻存管,按不同方法进行HL60细胞复苏。37℃水浴传统复苏法:立即将冻存管置于37℃水浴中,待融化后800r/min离心5min,吸去上清液,加入含体积分数为0.1小牛血清的RPMI-1640培养基,混匀后接种于细胞培养板,1mL/孔,9孔/组,置于体积分数为0.05的CO2培养箱37℃继续培养。40℃溶解后再37℃恒温改良复苏法:将冻存细胞于40℃水浴中迅速溶解,转入37℃水浴箱,融化后离心、加培养基等操作同传统复苏法。复苏细胞培养12h后采用锥虫蓝染色计算细胞存活率。③用无血清RPMI-1640培养基制备HL60细胞悬液,按3×107L-1密度接种于细胞培养板中,1mL/孔,然后分别加入体积分数为0.05,0.1,0.12,0.15,0.2的灭活小牛血清,每种血清浓度设置8个试验孔,并分别于培养12,24,36,48,60,72,84,96h后计数每孔细胞数量,并绘制细胞生长曲线。结果:①不同浓度二甲基亚砜冻存后HL60细胞复苏率的比较:二甲基亚砜200g/L组的细胞复苏率明显低于二甲基亚砜50,100,150g/L组[(64.6±2.8)%,(87.0±1.4)%,(86.4±2.1)%,(85.7±2.8)%;t=25.44,P<0.01],二甲基亚砜50,100,150g/L组间差异无显著性意义(t=0.82～1.44,P>0.05)。②不同复苏方法HL60细胞存活率的比较:与37℃水浴传统复苏法比较,40℃溶解后再37℃恒温改良复苏法的细胞存活率显著升高[(69.5±1.5)%,(87.4±1.8)%,t=23.24,P<0.01]。③RPMI-1640培养基不同血清含量对HL60细胞生长情况的影响:在体积分数为0.05的血清含量培养基中,HL60细胞基本不生长,且有逐渐死亡的趋势;在体积分数为0.1的血清含量培养基中,HL60细胞生长趋势明显,但生长速度相对较慢;在体积分数为0.12,0.15,0.2的血清含量培养基中,HL60细胞生长迅速,3种血清浓度间细胞生长趋势无明显差异。结论:以50g/L二甲基亚砜作为HL60细胞冻存保护剂、联合40℃溶解后再37℃恒温改良复苏法可使细胞保持最佳生物学特性,体积分数为0.12的血清含量为HL60细胞常规培养的最适浓度。     

Moderate and severe reactions in blood donors

During the period April 1985 to March 1986, 217 blood donors were found to have moderate (syncopal) to severe (convulsive) reactions. This population was compared to 5630 randomly selected donors who did not have reactions. An examination of demographic, physical, and societal/emotional factors was conducted to determine if any were predictive of reactions in donors. The results of the research supported the hypothesis that first-time donors have a higher frequency of reactions (1.7%) than do repeat donors (0.19%). A review of the above predictive factors documented that, with regard to demographic factors, 1) the number of prior donations was inversely proportional to the risk of reaction; 2) the gender of the donor was not predictive; and 3) youth was a predictor of reactions. An analysis of the physical factors revealed that donors who reacted were of lower weight (mean, 153.7 lb) than those who did not (mean, 166.4 lb) and that systolic blood pressure was slightly lower in the group with reactions. Although the difference was significant (3 torr), it was not thought to be significant clinically. In a comparison of a group with systolic blood pressure ranging from 80 to 100 torr and a group with systolic blood pressure ranging from 120 to 140 torr, the first group had a 70-percent higher risk of reaction. Finally, with regard to the last category of societal or emotional factors, the research demonstrates 1) that the ingestion of caffeinated beverages was associated with a reduced risk of reactions; 2) that the food intake of donors who reacred was significantly different from that of those who had no reaction, but this difference was not thought to be clinically significant; and 3) that the duration between registration and the onset of phlebotomy was directly predictive of reaction status. The research indicates that first-time donor status and several specific demographic, physical, and societal or emotional factors are predictors of donor reactions.     

颈动脉内中膜厚度与初诊2型糖尿病视网膜病变的关系

目的:研究发现,糖尿病视网膜病变和动脉粥样硬化终点事件相关。试验拟验证颈动脉内中膜厚度与初诊汉族2型糖尿病患者糖尿病视网膜病变相关危险因素的关系。方法:①试验对象:选择2006-06/2007-06本院住院的初诊2型糖尿病患者187例,男114例,女73例;平均年龄(51±14)岁;平均体质量指数(24.7±4.7)kg/m2。均符合1997年美国糖尿病协会的2型糖尿病诊断标准,排除既往已存在心血管疾病者。患者对治疗及试验均知情同意。根据眼底照相检查结果,将所有受检者分为糖尿病视网膜病变组及非糖尿病视网膜病变组进行统计分析。②试验方法及评估:所有患者询问一般情况,测量颈动脉内中膜厚度以及相关生化指标,对糖尿病视网膜病变相关因素进行单因素及多因素Logistic回归分析。结果:纳入2型糖尿病患者187例,均进入结果分析。单因素Logistic回归分析显示,高血压、糖尿病家族史、颈动脉内中膜厚度、尿白蛋白、低密度脂蛋白胆固醇与糖尿病视网膜病变发生呈显著正相关,多因素Logistic回归分析未见显效因素。结论:单因素回归分析中颈动脉内中膜厚度及其他4项指标与糖尿病视网膜病变相关,而多因素回归分析这些因素未进入主效基因模型。     

Determinants of third dose of diphtheria–tetanus–pertussis (DTP) completion among children who received DTP1 at rural immunization centres in Pakistan: a cohort study

Objective  In Pakistan, a high proportion of children fail to complete third dose of diphtheria-tetanus-pertussis (DTP3) after having received the first dose (DTP1). A cohort study was conducted to identify the factors predicting three doses of diphtheria–tetanus–pertussis (DTP3) completion among children who have received DTP1 at six centres of Expanded Programme on Immunization (EPI) in rural Pakistan.
Method  We analyzed a cohort of mother–child pairs enrolled at DTP1 between November 2005 and May 2006 in the standard care group of a larger randomized controlled trial. Data were collected from mothers on a structured questionnaire at enrolment, and each child was followed up at clinic visits for 90 days to record dates of DTP2 and DTP3. Multivariable log-binomial regression analysis was performed to identify the independent predictors of DTP3 completion.
Results  Only 39% (149/378) of enrolled children completed DTP3 during the follow-up period. After adjusting for the centre of enrolment in multivariable analysis, DTP3 completion was higher among children who were ≤60 days old at enrolment [adjusted risk ratio (Adj. RR) 1.39, 95% confidence interval (CI): 1.06–1.82], who were living in a household with monthly household income >Rs. 3000 (US$ 50) (Adj. RR 1.76, 95% CI: 1.16–2.65), and who were living ≤10 min away from EPI centre (Adj. RR 1.31, 95% CI: 1.04–1.66).
Conclusions  Interventions targeting childhood immunization dropouts should focus on bringing more children to EPI centres on-time for initial immunization. Relocation of existing EPI centres and creation of new EPI centres at appropriate locations may decrease the travel time to the EPI centres and result in fewer immunization dropouts.     

Molecular mechanisms of traumatic brain injury: the missing link in management

Head injury is common, sometimes requires intensive care unit admission, and is associated with significant mortality and morbidity. A gap still remains in the understanding of the molecular mechanism of this condition. This review is aimed at providing a general overview of the molecular mechanisms involved in traumatic brain injury to a busy clinician. It will encompass the pathophysiology in traumatic brain injury including apoptosis, the role of molecules and genes, and a brief mention of possible pharmacological therapies.     

Effect of menstrual cycle phase on dopamine D2 receptor availability in female cynomolgus monkeys

Sex differences have been reported in a variety of affective and neurodegenerative disorders that involve dysfunctional dopamine (DA) neurotransmission. In addition, there is evidence for differences in sensitivity to the abuse-related effects of psychostimulants across the menstrual cycle which may result from effects of ovarian hormones on DA function. The goal of the present study was to extend previous work examining menstrual cycle-related changes in DA D2 receptor availability in humans to drug-naive female cynomolgus monkeys (n=7) using the selective D2-like receptor ligand [(18)F]fluoroclebopride (FCP) and a high-resolution microPET P4 scanner. Menstrual cycle phase was characterized by daily vaginal swabs and measurements of serum progesterone levels. PET studies were conducted once during the luteal phase and once during the follicular phase. Regions of interest in the caudate nucleus, putamen, and cerebellum were defined on coregistered MRIs. Distribution volumes were calculated for FCP in each structure and the distribution volume ratio (DVR) for both brain regions relative to the cerebellum was used as a measure of D2 receptor availability. FCP DVRs were significantly higher in the luteal phase compared to the follicular phase in both the caudate nucleus (11.7% difference, p=0.02) and putamen (11.6% difference, p=0.03). These findings extend earlier work in humans and suggest that changes in DA receptor availability may be involved in the variation in symptoms of various neuropsychiatric disorders across the menstrual cycle, including differences in sensitivity to the abuse-related effects of stimulants.     

The impact of cytokines on the expression of drug transporters,cytochrome P450 enzymes and chemokine receptors in human PBMC

Background and purpose:

The function of transporters in peripheral blood mononuclear cells (PBMC) has been characterized, but less is known about cytochrome P450 (CYP) enzyme function in these cells. Given that cytokines are dysregulated in many diseases, the purpose of this work was to assess the impact of cytokines on the expression of CYPs, transporters and chemokine receptors in PBMC.

Experimental approach:

Human PBMC were incubated with cytokines for 48 h. ATP-binding cassette (ABC)B1, ABCC1, ABCC2, CYP2B6, CYP3A4, CXCR4 and CCR5 expression were measured by quantitative polymerase chain reaction and flow cytometry at 0, 4, 8, 24 and 48 h. Enzyme activity was assessed using fluorescent probes.

Key results:

We show here functional activity of CYP3A4 and CYP2B6 in PBMC. Furthermore, cytokines had a significant impact on the mRNA and protein expression of all proteins. For example, interleukin-2 (IL-2) had a marked impact on ABCB1 mRNA (% control 4745 ± 11961) and protein (% control 200 ± 57). Increases in drug efflux transporter expression, in response to cytokines, resulted in reduced cellular accumulation of digoxin [decrease of 17% and 26% for IL-2 and interferon-γ (IFNγ) respectively] and saquinavir (decrease of 28% and 30% for IL-2 and IFNγ respectively). The degree to which drug transporter and chemokine receptor expression changed in response to cytokines was positively correlated (e.g. ABCB1 and CXCR4, r² = 0.545).

Conclusions and implications:

These data have important implications for diseases in which cytokines are dysregulated and for which pharmacological intervention targets immune cells.     

应用BED．CEIA检测干血斑样本监测HIV-1发病率的可行性研究

目的 研究干血斑样本应用于现场HIV-1 BED-CEIA新近感染的检测以及监测HIV-1发病率的可行性.方法 收集27个自愿咨询检测(VCT)哨点的10 313名咨询者的血浆和干血斑样本,进行HIV抗体检测,对经免疫印迹方法确认的349例HIV感染者的血浆和干血斑样本同时进行BED-CEIA检测,检验干血斑样本BED-CEIA新近感染检测结果的稳定性和重复性,比较两种样本检测结果的差异及对HIV发病率估计的一致性.结果 应用干血斑样本检测具有较好的稳定性和重复性,R2值分别达到0.9551和0.95.349对样本的检测结果显示,294对样本被同时判定为长期感染,53对样本被同时判定为新近感染,两种样本对HIV-1是否为新近感染判断的一致性达到99.43%.而2对样本得到不同的结果,其An值均处于临界值附近.在人群总体水平上,两种样本计算得到的HIV-1发病率完全一致.结论 在HIV-1 BED-CEIA新近感染检测中,尽管个别样本检测结果存在差异,但在人群水平上千血斑样本的检测结果对HIV-1发病率的估计与血浆样本没有差异,因此在中国可以应用于血斑样本进行HIV-1 BED-CEIA新近感染检测.