脐血造血干细胞短期冻存效果的评价

为了探讨脐血造血干细胞在液氮中短期冻存复苏后的效果，分别将各自为8例冻存6个月、1年、2年的脐血千细胞进行复苏，观察造血干细胞活性。有核细胞(NC)计数采用自动血细胞分析仪测定，CD34^ 细胞采用流式细胞技术测定，用体外造血细胞培养技术分析粒一巨噬细胞集落生成单位(CFU-GM)产率，台盼蓝染色法判断造血细胞存活率。结果表明：脐血造血干细胞在液氮中冻存6个月、1年、2年后解冻，其有核细胞、CD34^ 细胞、细胞活率、CFU-GM产率在3个不同冻存时间的差异无显著性。结论：脐血干细胞于液氯中短期冻存，其干细胞数量和细胞活性没有明显的变化，冻存效果较好。     

大鼠骨髓间充质干细胞的分离培养纯化及冻存

目的:建立大鼠骨髓间充质干细胞(MSCs)体外分离培养及冻存的方法。方法:用密度梯度离心法及贴壁培养法分离纯化大鼠骨髓MSCs,并对其形态学特征进行观察,比较两种分离培养法的细胞生长增殖情况。大鼠MSCs在12.5%二甲亚砜(DMSO)保护下放入液氮冻存,6个月后复苏,测其成活率及观察生长增殖情况。结果:用密度梯度离心法及贴壁培养法均能有效地分离大鼠骨髓MSCs,细胞活力好,增殖能力强。大鼠MSCs可用液氮保存,且复苏后细胞生长增殖旺盛,形态无明显的变化。结论:采用密度梯度离心法及贴壁培养法成功地建立了大鼠骨髓MSCs体外分离和培养体系;采用液氮冻存大鼠MSCs的方法十分有效可行。     

分离培养方法及冻存技术对犬骨髓间充质干细胞生长和增殖的影响

背景：骨髓间充质干细胞在体外长期培养易发生自发分化，而失去多分化潜能，因此必须对培养的细胞及时冻存，需要时再进行复苏。目的：建立犬骨髓间充质干细胞体外分离、培养、增殖、冻存的方法。设计、时间及地点：对照观察细胞学实验，于2005-06／2008—06在浙江中医药大学附属第一医院骨伤研究所完成。材料：健康成年雄性Beagle犬用于骨髓间充质干细胞的提取。方法：采用密度梯度离心法结合贴壁培养法分离纯化骨髓间充质干细胞，并进行传代扩增培养。将第2代骨髓间充质干细胞与12．5％DMSO混匀置于冻存管中，采用慢冻快融法进行冻存和复苏：将冻存管置于-4℃冰箱2h，然后移入-30℃冰箱2h，再置于厚壁塑料泡沫盒中，扎紧密封，置-80℃冰箱过夜后，取出冻存管直接放入液氮中保存，或放入-80℃冰箱保存。复苏时将冻存管从液氮或80℃冰箱中取出，立即置37℃水浴并轻轻摇动，1-2min内迅速解冻。主要观察指标：复苏前后细胞成生长活性及形态变化。结果：分离的骨髓间充质干细胞为以均一的梭形的成纤维细胞样贴壁生长，贴壁及增殖能力强，传代细胞贴壁较快。经过液氮长期冻存后，将复苏后的骨髓间充质干细胞再培养，细胞形态及生长状态与冻存前无差异，均呈长梭形均匀分布生长，细胞生长增殖旺盛。结论：采用密度梯度离心法结合贴壁培养法町获得犬骨髓间充质干细胞，应用慢冻快融技术进行冻存和复苏，较好的保持了冻存细胞的活力和功能。     

人脐带间充质干细胞低温冻存后生物学鉴定

目的观察人脐带间充质干细胞(HUC-MSCs)经低温冻存复苏后的生物学特性,验证其是否仍具有间充质干细胞的基本特征。方法用胶原酶和胰蛋白酶消化脐带,分离诱导获得HUC-MSCs,传代培养,取第3代细胞加入冷冻保护剂(10%DMSO+40%FBS+50%DMEM/F12)后将其冻存于-80℃冰箱过夜,再转入液氮气相中保存1个月后复苏。采用倒置显微镜观察复苏后贴壁生长细胞的形态;台盼蓝拒染法比较冻存前后细胞活力;MTT法测定HUC-MSCs的增殖活性,比较冻存前后HUC-MSCs增殖活性的差异;流式细胞仪检测HUC-MSCs经冻存复苏后表面抗原的表达情况;复苏后HUC-MSCs进行成骨诱导分化培养,检验其是否仍具有多向分化潜能。结果 HUC-MSCs经低温保存复苏后,细胞仍保持贴壁生长等外形特征,复苏后HUC-MSCs存活率为91.17%;增殖活性与未冻存细胞比较,差异无统计学意义;细胞表面高表达CD105、CD73、CD71、CD90、CD29、CD44、CD13抗原,低表达或不表达CD34、CD133、CD45、CD14,HLA-DR、CD86、CD83、CD80、CD1α;复苏后HUC-MSCs具有向成骨细胞诱导分化能力,细胞经茜素红、ALP和Von-Kossa′s染色呈阳性反应。结论经冻存复苏后的HUC-MSCs保持了间充质干细胞的基本形态,其表型满足间充质干细胞的基本要求,仍具有向成骨细胞分化的潜能。     

不同冻存时间与温度对兔骨髓基质干细胞存活率的影响

目的:以二甲基亚砜作为保护剂,采用慢冻快融法观察不同温度及不同时间冻存的兔骨髓基质干细胞复苏后存活率的变化。方法:实验于2003-09/2005-06在浙江省医学科学院生物工程所完成。①选取清洁级3月龄新西兰大白兔5只,抽取兔髂骨及胫骨的骨髓液,密度梯度离心法结合贴壁培养法分离纯化出兔骨髓基质干细胞,并进行增殖。②取第3代骨髓基质干细胞,经消化后离心重悬,用完全培养液调整细胞浓度为6×109L-1。将质量浓度为250g/L的二甲基亚砜缓慢滴入同体积含骨髓基质干细胞的上述培养液中,混匀,二甲基亚砜的终浓度为125g/L,骨髓基质干细胞的终密度为3×109L-1,1mL/安瓿冻存。③4℃冻存直接将安瓿置4℃冰箱中;-20℃及-60℃冻存,则将安瓿置于有脱脂棉的聚丙烯泡沫盒中,壁厚1.5cm,扎紧密封,各置-20℃及-60℃冰箱过夜,取出后分别直接放于-20℃及-60℃冰箱保存;-196℃液氮冻存,则将置有安瓿的泡沫盒于-60℃冰箱过夜后,取出冻存安瓿直接放于液氮中保存。骨髓基质干细胞分别于4℃、-20℃、-60℃、-196℃条件下进行3d,10d,1,3,8个月以及1年的冻存。④经不同温度及不同时间冻存后,给予复苏。复苏时将冻存安瓿从液氮或冰箱中取出,立即置37℃水浴并轻轻摇动,约1min迅速解冻。含骨髓基质干细胞的冻存液经5倍于冻存液量的完全培养液稀释后,离心重悬,进行骨髓基质干细胞存活率测定。复苏后的骨髓基质干细胞以1×104/cm2用完全培养液再培养,观察骨髓基质干细胞的形态变化及生长活性情况。结果:实验选取3月龄新西兰大白兔5只,全部进入结果分析。①兔骨髓基质干细胞原代培养情况:培养3d时,骨髓基质干细胞数量较少,散在分布,呈短梭形形态。培养1周时细胞形成大量的小集落,细胞变长。此后集落细胞迅速增多,逐渐汇合成片,形态为均一的长梭形,呈旋涡状排列,2周时细胞长成单层。②兔骨髓基质干细胞传代培养情况:刚传代的骨髓基质干细胞呈圆形,数小时后迅速贴壁,重新成为梭形。之后细胞呈长梭形均匀分布生长,形态比原代培养更均一,细胞生长旺盛、增殖迅速。连续传代22代无明显变化。但随传代次数增多,细胞形态出现多样性,逐渐呈老化现象。③不同温度不同冻存时间下复苏后的兔骨髓基质干细胞存活率的比较:兔骨髓基质干细胞液氮保存1年,存活率为79%;-60℃保存1,3,8个月的存活率分别为67%,38%和0%;不宜于4℃及-20℃保存。④复苏后的骨髓基质干细胞再培养情况:复苏后的骨髓基质干细胞细胞形态及生长状况与冻存前无明显差异,呈长梭形生长,增殖旺盛。结论:分离纯化的兔骨髓基质干细胞生长增殖能力强,短期保存可用-60℃冻存,长期需液氮保存,为择期进行骨髓基质干细胞实验和应用提供实验依据。     

人骨髓及脐血间充质干细胞体外大量扩增及建库的质量控制

目的:研究人骨髓和脐血间充质干细胞(MSC)的体外培养特性及建立细胞库的可行性。方法:Ficoll离心剂法分离人骨髓及脐血来源单个核细胞,进行体外培养并大量扩增,观察细胞生长特性,流式细胞术检测MSC表面分子标志物表达情况。结果:骨髓样本比脐血标本的MSC更易于体外短期培养扩增,骨髓样本(5×106)体外培养2周约获得MSC3×107～1.2×108,成功率达100%,而脐血样本的MSC较难培养,成功率低(12.9%),细胞数扩增有限。对冻存复苏前后的细胞进行流式细胞术检测提示MSC表面标志物CD29、CD44、CD59、CD90、CD105、CD166等均阳性表达,造血细胞标志物CD34、CD14和CD45为阴性表达,提示MSC对本室的冻存复苏程序耐受能力较好,可获得复苏后扩增培养。结论:本研究表明对成人骨髓MSC建库的条件较成熟,获得成功培养的MSC分子表型在冻存复苏前后无明显改变。而脐血来源的MSC培养条件有待改善方能提高MSC培养成功率。     

大鼠胚胎神经干细胞的低温保存

目的建立大鼠胚胎神经干细胞(neural stem cells,NSCs)的低温保存方法,探讨经冻存后NSCs的存活及生物学特性变化。方法使用二甲基亚砜(DMSO)做NSCs的冷冻保护剂于液氮中冻存。复苏后用间接免疫荧光染色方法对细胞的存活、形态及分化能力做鉴定。结果不同冻存时间及代数的NSCs复苏后存活率无明显差异(P>0.05),且仍能在体外多次传代,并可分化为神经元、星形胶质细胞和少突胶质细胞。结论大鼠NSCs低温冻存、复苏并不影响其原有的形态、增殖及多向分化等生物学特性。     

低温保存对人外周血单个核细胞免疫活性的影响

目的:探讨外周血单个核细胞的分离、冻存及复苏工艺,实现外周血单个核细胞的长久保存。方法:采集50岁以上健康志愿者外周血50-100 ml,应用Ficoll密度梯度离心法分离单个核细胞;采用程控降温法逐级降温,将细胞保存于-196℃液氮中。根据冻存时间进行细胞复苏,分析其冻存前后活性、回收率与表型变化,最后与对照组(直接分离的未经冻存的外周血单个核细胞)一起,经体外激活扩增培养,对比其表型与扩增效率,分析冻存对细胞的影响。结果:Ficoll密度梯度离心后获得的单个核细胞活力为99.6%±0.4%,回收率58.4%±6.52%。冻存24个月后,复苏细胞回收率89.7%±3.82%。细胞经体外激活扩增培养,得到能够杀伤肿瘤细胞的细胞群扩增活化的自体淋巴细胞(AIL)(CD3~+CD8~+细胞毒性T细胞比例≥45%,CD3~+CD56~+NKT细胞比例≥10%,CD4~+CD25~+NKT细胞比例≤10%),其与对照组细胞在扩增能力、细胞纯度、细胞毒活性等方面无显著性差异。结论:体外人工分离的外周血单个核细胞经过长时间冷冻保存后仍可维持其高活性,并可诱导生成具有抗肿瘤活性的细胞群,该结果对单个核细胞保存、扩增活化的自体淋巴细胞的应用具有重要意义。     

兔骨髓间充质干细胞体外分离培养及冻存研究

目的:寻求体外培养、冻存、复苏兔骨髓间充质干细胞(mensechymalstemcells,MSCs)的方法,为兔MSCs进一步的实验研究打下基础。方法:取新西兰白兔胫骨穿刺获取骨髓液,以贴壁筛选法分离、纯化、培养和扩增获得MSCs,并行液氮冻存,通过倒置显微镜观察其生物学表现。结果:MSCs为贴壁生长,形态为均匀成纤维细胞样,增殖能力强,传代及冻存后细胞仍然保持其生物学特性。结论:MSCs可采取贴壁筛选法进行较好的纯化和扩增,并可长期保存于液氮中,在一定的条件下可复苏存活。     

液氮冻存时间对605份脐血造血干细胞质量和临床移植效果的影响

目的 探讨液氮冻存时间对脐血造血干细胞质量和临床移植效果的影响.方法 对上海脐血库已经应用于临床移植的605份脐血进行研究,脐血平均冻存时间为820(88～ 2651)d,测定脐血复苏之后的有核细胞、CD34+细胞数量、细胞回收率、细胞活力,粒-单核细胞集落形成单位(CFU-GM),结合脐血冻存时间、临床移植效果进行统计分析.结果 复苏后细胞回收率、细胞活力与冻存时间无显著相关性,CFU-GM平均(109.6～ 105.7)个/1×105,随冻存时间延长逐渐降低(P=0.011).临床移植患者的造血重建时间、植入失败率、急性移植物抗宿主病、总体生存率与脐血冻存时间长短无显著相关性.结论 深低温冻存时间长短对脐血复苏后细胞回收率、细胞活力、临床移植效果无显著影响,冻存时间较长的脐血复苏后CFU-GM在正常标准范围内略有下降,保存3～7年的脐血仍能维持正常细胞活性和功能、达到良好的临床移植效果.     

现代残疾康复理念、政策与社区康复体系研究

本研究分析了当代残疾康复的理念和理论架构,构建了以国际公约、决议和政策、国内法律和政策以及操作性工具3个层次的康复理论体系;运用包容性发展,分析探讨了当代社区康复的体系和特点;就发展康复事业、为残疾人提供全面系统的康复服务提出相关的政策建议。     

国内外唇腭裂专科护理发展现状的文献研究及分析

目的了解和分析国内唇腭裂专科护理工作发展的现状。方法检索并分析万方医学网、中国知网、Springer Link及PubMed英文数据库近6年有关唇腭裂手术的围术期护理、患儿的喂养、手术安全管理、围术期疼痛研究以及心理护理的相关文献。结果共检索出国内文献89篇(核心期刊20篇)、国外文献22篇,其中围术期护理38篇、患儿的喂养16篇、手术安全管理10篇、围术期疼痛研究10篇、心理护理15篇。结论与国外文献相比,国内论文总结和回顾性居多,应增加研究探讨性论文,从而促进唇腭裂临床护理工作专科化的发展。     

城乡学童个性行为特征调查

目的探讨农村与城市儿童的个性行为特征。方法将97名农村学龄儿童设为农村组,97名城市学龄儿童设为城市组,采用艾森克个性问卷一儿童版和Achenbach’s 行为量表一儿童版进行评定分析。结果两组学龄儿童艾森克个性问卷各维度评分均无显著性差异（P均〉0．05）,且与全国常模相一致（P均〉0．05）;农村组男学童Achenbach’s行为量表违纪行为、攻击行为及外向型因子分均显著高于城市组,女学童退缩、社交问题因子分均显著高于城市组（P〈0．05或0．01）,其他因子分均无显著性差异（P均〉0．05）。结论农村与城市儿童个性无显著差异,而农村儿童存在较多的行为问题,可能与其所受教养和生活方式有关。     

我院文职护士管理和培训的实践与体会

目的构建文职护士培训、使用及管理的科学方法。方法通过规范管理、科学施训、作为骨干大胆任用等多种管理方式,使文职护士得到充分的发展。结果文职护士群体成长为医院优秀骨干,首批满3年的文职护士顺利通过续聘考核。结论科学的管理培训是文职护士成长的重要基石,对医院护理管理有着重要作用。     

Comparison of short-term and long-term protocols for stabilization and preservation of RNA and DNA of Leishmania, Trypanosoma, and Plasmodium

Molecular tools continue to be important in the prevention and control of parasitic diseases. However, using these techniques directly in the field remains a major challenge. Therefore, the preservation of clinical samples collected from endemic field areas for later analysis remains an important preanalytical process. This study aimed at identifying a suitable protocol for stabilization and preservation of RNA and DNA in bioclinical specimens for Trypanosoma, Leishmania, and Plasmodium research. Both spiked and unspiked blood samples were preserved in 7 protocols (different media; storage temperatures). Samples were evaluated for possible degradation of DNA and RNA along the storage duration up to the 10th week. Nucleic acid targets were assessed as follows: (i) Trypanosoma and Plasmodium RNA analysis was done using real-time nucleic acid sequence-based amplification (RT-NASBA) for 18S rRNA and for stage-specific Pfs25 mRNA, respectively; (ii) Trypanosoma DNA assessment analysis was conducted by using a conventional PCR for 18S rDNA; (iii) Leishmania RNA analysis was performed with a quantitative NASBA for 18S rRNA and Leishmania DNA assessment with an RT-PCR for 18S rDNA. Findings suggested that a newly developed L3™ buffer proved to be reliable and suitable for both short- and long-term preservation of parasite nucleic acid material. This buffer is envisaged to be suitable for utilization in field situations where resources are limited.     

History of violence and subjective health of mother and child

Objective: To study the self-reported prevalence of experienced violence among a cohort of women about two years after giving birth, their health during pregnancy, pregnancy outcomes and their experience of their child’s health.

Setting and subjects: In 2011, a total of 657 women participated in phase III of the Childbirth and Health Cohort Study in Icelandic Primary Health Care, 18 to 24 months after delivery. The women had previously participated in phase I around pregnancy week 16 and phase II 5–6 months after delivery. Data were collected by postal questionnaires.

Main outcome measures: Women’s reported history of experienced violence, sociodemographic and obstetric background, self-perceived health, the use of medications and their child’s perceived health.

Results: In phase III, 16% of women reported experiencing violence. These women felt less support from their current partner (p?p?p?p?p?p?=?0.008).

Conclusions: Our study confirms that a history of violence is common among women. A history of violence is associated with various maternal health problems during and after pregnancy, a higher rate of caesarean sections and maternal reports of health problems in their child 18–24 months after birth.

• KEY POINTS

• Violence is a major concern worldwide. Understanding the impact of violence on human health and developing effective preventive measures are important elements of any public health agenda.

• ??The reported prevalence of experiencing violence was 16% among women attending antenatal care in the primary health care setting in Iceland.

• ??Women with a history of violence reported worse health in general during pregnancy and delivered more often by caesarean section, compared to women with no such history.

• ??Mothers with a history of violence also evaluated the general health of their child as worse than women with no such history.

• ??The findings of this study support the importance of recognizing and addressing experienced violence among women in primary care.

     

Rates and risks of transmission of smallpox and mechanisms of prevention

In 1980, the World Health Organization declared smallpox eradicated from the world; the last known natural case had occurred in Somalia in 1977, and the United States had stopped routinely vaccinating its citizens in 1972. However, with increasing concerns regarding domestic and international terrorism, smallpox has resurfaced as a potential threat to global health. We review the direct and indirect modes of smallpox transmission and how patterns of transmission vary substantially, depending on the severity of circulating disease, vaccination status, environmental and socioeconomic factors, and the setting of an outbreak. We examine mechanisms for controlling outbreaks of disease and preventing further transmission in the event of an outbreak, with an emphasis on smallpox vaccination.     

通用设计原则与产品研发策略

本文介绍了通用设计的概念和设计原则,并阐述了根据此原则实施通用设计的产品研发策略。通用设计惠及的不仅是残疾人,也惠及了包括普通公众在内的广泛人群,以及企业。应将通用设计的原理应融入政府相关政策和未来发展的规划中,为残疾人以及更多人群提供合理便利的通用设计产品。     

Prevalence and severity of nausea and vomiting of pregnancy and effect of vitamin supplementation.

OBJECTIVE: Although nausea and vomiting of pregnancy is the most common medical condition during pregnancy, there are many unanswered questions regarding its cause, epidemiologic features and optimal management. The objectives of this study were to ascertain the prevalence of nausea and vomiting in a sample of Canadian women, to characterize the distribution of their severity and to investigate the role of vitamin B6 deficiency in their etiology. DESIGN: Prospective study. SETTING: Antenatal counselling service for pregnant women. PATIENTS: Three cohorts of women: a prospective, population-based cohort of 193 women, to estimate the rate and severity of nausea and vomiting (cohort A); a cohort of 555 women who sought advice for nausea with or without vomiting, to study the correlation between the maximal daily number of episodes of vomiting and maximal weight loss (cohort B); and a prospective cohort of 301 women who reported vomiting, to correlate vitamin supplementation with vomiting (cohort C). INTERVENTIONS: All 3 cohorts were interviewed during the counselling session, and cohort B was followed up prospectively. OUTCOME MEASURES: Frequency of nausea and vomiting, weight loss, maximal number of daily episodes of vomiting, rate of multivitamin supplementation. RESULTS: Overall, 67% of the women in cohort A reported experiencing nausea or vomiting, or both; 22% reported vomiting, and 9% experienced weight loss. In cohort B there was a significant correlation between the maximal number of daily episodes of vomiting and maximal weight loss, although there was wide variation (r2 = 0.25, p < 0.001). There was a highly significant correlation between the number of daily vomiting episodes and mean weight loss (r2 = 0.99). In cohort C, vomiting was significantly associated with lack of supplementation with multivitamins before 6 weeks' gestation (p = 0.002). CONCLUSIONS: The relation between number of daily vomiting episodes and mean weight loss may serve as a clinical tool to assess the severity of nausea and vomiting in pregnancy and the success of anti-emetics and rehydration regimens. Further study is needed to elucidate the biologic basis of the observed association between vomiting and lack of multivitamin supplementation in early pregnancy.     

胃癌DNA含量与E-cad、EGFR表达的关系及其意义

目的　探讨胃癌组织DNA含量与E cad、EGFR表达的关系 ,评价其在浸润、转移潜能及预后判断中的作用。方法　应用图像分析仪及SP免疫组化法检测 30例正常胃黏膜上皮、30例不典型增生和 5 0例胃癌组织DNA含量与E cad、EGFR蛋白表达情况。结果　①正常胃黏膜上皮、Ⅰ、Ⅱ、Ⅲ级不典型增生及胃癌组的DI、PI、S %、>5c %逐渐增高 ,与胃癌分期、淋巴结转移显著相关 (P <0 0 1)。②E cad在正常胃黏膜上皮阳性表达率 10 0 % ,EGFR呈阴性表达 ;Ⅰ、Ⅱ、Ⅲ级不典型增生及胃癌E cad阳性表达率逐渐降低 ,EGFR的阳性表达率逐渐升高 ,且差异显著 (P <0 0 5 ) ;E cad表达减弱、EGFR表达升高与胃癌分化差、淋巴结转移显著相关 (P <0 0 1)。③E cad表达阴性的胃癌DNA指数及EGFR表达水平显著高于E cad阳性 (P <0 0 1)。结论　检测DNA含量及E cad、EGFR蛋白表达有助于胃癌的早期诊断及转移潜能和预后的判断。