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。     

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。     

The brain‐derived neutrophic factor val66met polymorphism and sudden unexpected infant death

Aim: Findings of hypoxia prior to death and involvement of a dysregulation of the serotonergic network in sudden infant death syndrome (SIDS) may indicate that brain‐derived neutrophic factor (BDNF) also is of importance with regard to sudden unexpected infant death. Based on this, the purpose of this study was to investigate the BDNF val66met polymorphism in SIDS cases, cases of infectious death and controls. Methods: The polymorphism was investigated in 163 SIDS cases, 34 cases of infectious death and 121 controls, using real‐time PCR and fluorescence melting curve analysis. Results: There were no differences in val66met genotype distribution between neither the SIDS cases nor the cases of infectious death and controls (p = 0.95 and p = 0.52, respectively). Conclusion: The study indicates that the val66met polymorphism is not important for sudden unexpected infant death. However, several other SNPs in the BDNF gene, as well as in other genes involved in this pathway, including G‐protein, have to be investigated to fully exclude any involvement of BDNF in SIDS.     

Outcomes of permanent pacemaker implantation for carotid sinus hypersensitivity in a district general hospital with a Falls Fits Faints and Funny Turns Clinic

Permanent pacemaker implantation is considered for carotid sinus hypersensitivity with asystolic pause of > 3 seconds during carotid sinus massage, with or without associated systolic blood pressure drop of > 50 mmHg. AIMS: To assess the outcome of treatment with dual chamber permanent pacemaker implantation for carotid sinus hypersensitivity in a district general hospital and to compare our practice with available national data. METHODS: Patients presenting with syncope, dizziness or unexplained falls were initially assessed as outpatient and investigations, commonly cardiovascular were performed including tilt table test with carotid sinus massage to look for carotid sinus hypersensitivity, vasovagal syncope and postural hypotension. A retrospective analysis was performed on patients who had pacemaker implantations for carotid sinus hypersensitivity. RESULTS: Fifty pacemaker implantations were performed, 14 (28%) were for carotid sinus hypersensitivity. Mean follow-up period was 10 months. Age range was 56-88 (mean = 71.9) years. In the eighteen months prior to pacemaker implantation, 71.4% of patients had syncope, 64% dizziness and 50% had unexplained falls. Following pacemaker implantations, only 2 (14.3%) had symptoms. Scottish national figures show 13.6% of all new pacemaker implants were for carotid sinus hypersensitivity, and in district general hospitals this was 16%. CONCLUSIONS: Health benefits for patients presenting with syncope dizziness and falls can be achieved by cardiovascular investigations including tilt table testing. We have shown carotid sinus hypersensitivity is successfully treated with pacemaker implantation in a district general hospital setting and this type of clinic has an impact on the rate and type of new pacemaker implantation.     

金龙胶囊配合辨证用药治疗中晚期恶性肿瘤40例

1临床资料2003-12/2004-10采用北京建生药业生产的金龙胶囊配合辨证用药治疗中晚期癌症40例.Karnofsky评分≥40分,预计生存期>2 mo.     

Parents' perceptions towards their child's use of inhaled medications for asthma therapy

Objective : Understanding patients' and their parents' perceptions towards asthma medication is important in developing strategies to ensure patient compliance. In this study, parents' perceptions towards their children's use of inhaled medication for asthma treatment were evaluated.
Methodology : A questionnaire was administered by interviewing parents of 210 asthmatic children attending the Department of Paediatrics, National University Hospital.
Results : Our results showed that 76/210 (36%) of parents either felt opposed to inhaler therapy and/or preferred oral medications. The main reasons for their reluctance to use inhalers were related to fear of dependence, side effects and overdosage, and the child's dislike for inhalers. A third of these parents felt that inhalers were only indicated for very severe asthma.
Conclusions : We conclude that a significant proportion of parents have reservations regarding the use of inhalers for the treatment of asthma. These factors should be taken into consideration when planning an effective asthma education programme.     

Neonatal neutropenia and thrombocytopenia following maternal hypertension

Objective : To assess the relationship between the subtypes of hypertension in pregnancy and subsequent neonatal haematology.
Methodology : Retrospective review of the haematology of newborns of hypertensive mothers at a tertiary neonatal unit
Results : Over a 2 year period. 249 infants had full blood examinations. Nineteen (7.6%) were neutropenic and 35 (14.1%) thrombocytopenic, including 11 (4.4%) who were both neutropenic and thrombocytopenic. Neutropenia occurred only in infants whose mothers had severe pre-eclampsia and eclampsia or pre-eclampsia with pre-existing hypertension, whereas thrombocytopenia complicated all maternal hypertension subtypes. Two (10%) of the neutropenic infants developed nosocomial infection while seven (20%) of the thrombocytopenic infants bled. Thirteen (68%) of the neutropenic infants compared with 15 (43%) of the thrombocytopenic infants developed their haematological abnormality within 24 h of birth. All but two infants developed the haematological abnormality by the 5th day of life.
Conclusions : Although haematological abnormalities in infants born to hypertensive mothers are uncommon, serious neonatal complications can occur and therefore early haematological screening of these infants is recommended.     

Fetal programming of fat and collagen in porcine skeletal muscles

Connective tissue plays a key role in the scaffolding and development of skeletal muscle. Pilot studies carried out in our laboratory have shown that the smallest porcine littermate has a higher content of connective tissue within skeletal muscle compared with its largest littermate. The present study investigated the prenatal development of intralitter variation in terms of collagen content within connective tissue and intramuscular fat of the M. semitendinosus. Twenty-three pairs of porcine fetuses from a Large White-Landrace origin were used aged from 36 to 86 days of gestation. The largest and smallest littermates were chosen by weight and the M. semitendinosus was removed from each. Complete transverse muscle sections were stained with Oil Red O (detection of lipids) and immunocytochemistry was performed using an antibody to collagen I. Slides were analysed and paired t-Tests revealed the smallest littermate contained a significantly higher proportion of fat deposits and collagen I content compared with the largest littermate. Recent postnatal studies showing elevated levels of intramuscular lipids and low scores for meat tenderness in the smallest littermate corroborate our investigations. It can be concluded that the differences seen in connective tissue elements have a fetal origin that may continue postnatally.     

Giant Asian honeybee or Bambara stings causing myocardial infarction,bowel gangrene and fatal anaphylaxis in Sri Lanka: a case series

The sting of Giant Asian honeybee (Apis dorsata) or Bambara in Sinhala and Karunge Kulavi in Tamil is a common environmental hazard in Sri Lanka known to cause immediate allergic reactions, which could be fatal in sensitized individuals. We reported myocardial infarction, bowel gangrene and fatal anaphylaxis in a prospectively proven case series and the association of these uncommon complications with delayed removal of stingers from the patients' skin.