-80℃低温冻存保护剂对不同来源的造血干细胞体外冻存效果分析

目的分析-80℃体外冻存保护体系对不同来源的造血干细胞的冻存效果。方法以国产或进口药用二甲基亚砜(DMSO)、羟乙基淀粉(HES)、人血白蛋白(HSA)配制细胞冷冻保护剂,分别对人外周血、纯化的CD34+、骨髓及脐血造血干细胞进行-80℃低温保存,冻融前、后计数MNC、CD34+细胞、CFU-GM数量、回收率及台盼蓝拒染率,了解其生理活性。结果 4种来源的造血干细胞各30例,以120g/L HES加10%(V/V)DMSO和64g/L HSA为保护剂-80℃冻存360d,观察组与对照组在冻融前、后MNC、CD34+细胞、CFU-GM数量、回收率及台盼蓝拒染率无统计学意义(P>0.05)。应用国产试剂对118例外周血干细胞、39例纯化CD34+造血干细胞、3例脐血干细胞进行-80℃低温冻存1～9个月,进行冻存的造血干细胞移植,移植后7～19d内均成功恢复造血重建。结论以120g/L HES加10%(V/V)DMSO和64g/L HSA为细胞冷冻保护剂,能够良好保持干细胞数量及生理活性,且国产试剂与进口试剂无显著差别,具有价格低廉,容易购买,低毒安全,操作简便等特点,可临床应用于造血干细胞-80℃低温短期保存。     

二甲基亚砜与羟乙基淀粉联合冷冻保护外周造血干细胞的效果

目的研究并总结二甲基亚砜与羟乙基淀粉联合冷冻保护外周造血干细胞的效果。方法使用二甲基亚砜和羟乙基淀粉作为外周造血干细胞冷冻保护剂,将外周造血干细胞直接降温至-80℃存放。存放两周后,将外周造血干细胞解冻,植入患者体内。统计单个核细胞回收率以及台盼蓝拒染率。结果回收到细胞(4.00±0.69)×10~9/kg,解冻后细胞回收率89.23%,解冻后台盼蓝拒染细胞比例为89.45%。解冻后外周造血干细胞输入患者体内后(10.83±1.55)d,所有研究对象体内中性粒细胞绝对值升至0.5×10~9/L及以上。结论外周造血干细胞直接冷冻应用二甲基亚砜和羟乙基淀粉联合作为冷冻保护剂,细胞回收率高、细胞存活率高,体内能够短时间内恢复活性。     

志苓胶囊通过激活caspase-3抑制K562细胞增殖和诱导细胞凋亡

目的研究志苓胶囊(ZLJN,抗癌复方Ⅱ号)对人慢性髓系白血病K562细胞株增殖及凋亡的影响。方法将志苓胶囊按其不同中西药成分比例配制成中药、西药和复方组,与K562细胞共培养后,采用MTT法、集落形成实验分别检测细胞存活率和集落形成率;Annexin V-FITC/PI标记法、DNA倍体分析及DNA片段化分析检测细胞凋亡;流式细胞仪检测caspase-3活性;Western blot法检测caspase-3酶原(pro-caspase-3)表达。结果不同药物组与K562细胞共培养后,细胞生长受抑制,集落形成率降低。Annexin V-FITC/PI法检测到早期凋亡细胞;DNA倍体分析可见亚二倍体峰(凋亡峰);琼脂糖电泳见典型的DNA梯状带。流式细胞检测caspase-3活性增强,Western blot检测pro-caspase-3表达减弱。结论志苓胶囊可有效抑制K562细胞增殖,诱导其凋亡,其作用机制可能与caspase-3活性增强有关。     

PI3K/Akt信号通路参与大鼠骨髓间充质干细胞凋亡的发生

为研究体外大鼠骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)在缺血缺氧条件下发生凋亡的作用机制。采取大鼠骨髓,以密度梯度离心分离出单个核细胞(MNCs),于体外培养并由牛垂体提取物(PEX)诱导扩增传代培养出MSCs。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,BMSCs在缺血缺氧条件下培养,通过Annexin V/PI双染细胞凋亡检测比较不同组别细胞的凋亡率和蛋白印迹法(western blot)来观察细胞中蛋白的变化。结果表明,①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示BMSCs培养成功。②对照组(无缺血缺氧)与缺血缺氧组比较,缺血缺氧组的凋亡率显著性增加,而通过磷酸化Akt的表达量显著性增加提示PI3K(phosphoinositide-3kinase)/Akt(proteinkinaseB,PKB)信号通路被激活(P<0.05);同时缺血缺氧组与缺血缺氧+PI3K/Akt抑制剂(LY294002)组比较,缺血缺氧+PI3K/Akt抑制剂组的凋亡率显著降低,而通过磷酸化Akt的表达量显著减少(P<0.05),提示...     

细胞凋亡技术应用于电离辐射遗传损伤的研究

目的了解不同剂量的辐射及不同时间段对小鼠骨髓细胞凋亡率的影响,比较PI/FITC-Annexin V双染和PI单染2种荧光剂检测细胞凋亡方法的差异。方法以60Co射线照射ICR小鼠,制备不同时间段的骨髓单个核细胞的单细胞悬液,用PI/FITC-Annexin V双染和PI单染法,通过流式细胞仪检测样品的凋亡率。结果PI/FITC-Annexin V方法检测出的骨髓单个核细胞早期凋亡率于6~24 h达到最高且与辐射剂量之间无正相关性;较低剂量辐射组凋亡率的下降速度明显高于大剂量辐射。PI单染法只能检测样本的总凋亡率。在各时间点,骨髓细胞的总凋亡率随辐射剂量增大而增高。结论小剂量辐射相对于大剂量辐射单位剂量下的细胞凋亡率更高,因此能更加有效地清除受损细胞,保护机体。只有PI/FITC-Annexin V双染法才能检测出早期细胞凋亡率,有效鉴别细胞凋亡与死亡。     

柠檬醛抑制NB4细胞生长并诱导凋亡机制的研究

目的研究柠檬醛（Citral）对急性早幼粒细胞白血病NB4细胞株生长抑制和诱导凋亡作用;研究柠檬醛诱导NB4细胞凋亡可能的机制。方法采用台盼蓝拒染法测定细胞活力;采用CCK-8比色法检测柠檬醛对细胞增殖的影响;形态学观察和流式细胞仪检测细胞凋亡;流式细胞仪检测线粒体膜电位（MMP）改变情况。结果 2～20 mg·L^-1柠檬醛具有时间和剂量依赖方式抑制NB4细胞增殖,诱导细胞凋亡,明显降低细胞线粒体膜电位。结论柠檬醛诱导NB4细胞内线粒体膜电位崩溃可能是引起细胞凋亡的机制之一。     

用四甲基偶氮唑盐比色法测定5种冷冻保存液对大鼠垂体细胞的保存效果

采用5种低温保存液冷冻保存大鼠垂体细胞，首次运用改良的MTT方法，结合台盼兰拒染法对冻存效果进行评价。研究发现：台盼兰拒染法并不能正确反映细胞活性，MTT方法能较客观地从功能上反映细胞的活性。结果显示：采用DMSO－PVP联合保存液，以1℃／分的速率降温至—80℃，再浸于液氮中保存垂体细胞的方式获得最佳的保存效果。     

力达霉素诱导人胃癌BGC823细胞凋亡和抑制裸鼠移植瘤生长

观察力达霉素(LDM)对人胃癌BGC823细胞的诱导凋亡作用及体内抗肿瘤活性。采用MTT法观察LDM对人胃癌BGC823细胞增殖的抑制作用。利用Annexin V-FITC/PI双染结合流式细胞仪和脱氧核糖核酸末端转移酶介导的缺口末端标记技术检测细胞凋亡的改变。采用Western blotting法检测细胞中VEGF蛋白的表达情况。建立裸鼠胃癌皮下移植瘤模型，观察LDM的体内抗肿瘤活性。LDM能够明显抑制BGC823细胞增殖，诱导细胞凋亡，降低细胞VEGF蛋白的表达，抑制胃癌裸鼠移植瘤的生长。LDM剂量0.02和0.04 mg·kg^-1的抑瘤率分别为57%和72%(P<0.01)。LDM可诱导胃癌细胞凋亡并抑制裸鼠移植肿瘤的生长。     

三氧化二砷诱导Hela细胞凋亡的机制研究

目的:了解三氧化二砷(As2O3)诱导的细胞凋亡是否与线粒体跨膜电位(Δψm)改变有关。方法:以Hela细胞为体外模型,应用碘化丙啶(PI)/Rhodamine123(Rh123)双重染色检测Δψm,通过测定细胞活力、流式细胞仪、HE染色及DNA电泳检测细胞凋亡。结果:As2O3处理Hela细胞后。台盼蓝拒染法测定细胞活力降低,流式细胞仪检测发现凋亡细胞明显增多,HE染色可见明显的细胞凋亡形态特征,琼脂糖凝胶电泳出现DNA梯形条带;As2O3使线粒体膜电位降低(P<0.01)。结论:As2O3在体外诱导Hela细胞凋亡的机制可能与降低线粒体膜电位有关。     

负载MAGE-A3抗原的树突状细胞与细胞因子诱导杀伤细胞共培养对子宫内膜癌肿瘤干细胞及恶性进展的影响

目的:探究负载黑色素瘤相关抗原基因A3(MAGE-A3)的树突状细胞(DC)与细胞因子诱导杀伤细胞(CIK)对子宫内膜癌肿瘤干细胞及恶性进展的影响。方法:采集人外周血分离单个核细胞，利用细胞因子分别诱导生成DC和CIK;MAGE-A3孵育DC后与CIK共培养，流式细胞仪检测DC-CIK、MAGE-A3-DC-CIK的表型；流式细胞仪分选子宫内膜癌细胞系Ishikawa的CD133⁺干细胞，以子宫内膜癌干细胞作为靶细胞，分别以CIK、DC-CIK及MAGE-A3-DC-CIK作为效应细胞，MTT法检测效靶比为10∶1、20∶1、40∶1的细胞杀伤活性，ELISA法检测联合培养细胞上清液中IFN-γ、IL-2、IL-12、IL-17水平，Annexin V-FITC/PI双染法检测子宫内膜癌干细胞凋亡；建立子宫内膜癌干细胞移植瘤裸鼠模型，尾静脉注射DC-CIK或MAGE-A3-DC-CIK,观察裸鼠肿瘤生长情况，每隔2 d测量瘤体大小，21 d后取肿瘤组织，电子天平称重，HE染色观察肿瘤组织病理形态学变化，免疫组织化学染色检测肿瘤组织内Ki-67表达。结果:分离获得细...     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Lung disease and PKCs

The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified.     

Gender-related symptoms and correlates of alcohol dependence among men and women with a lifetime diagnosis of alcohol use disorders

This study explored gender-related symptoms and correlates of alcohol dependence in a crosssectional study of 150 men and 150 women with a lifetime diagnosis of alcohol use disorders (AUD). Participants were recruited in equal numbers from treatment settings, correctional centres and the general community. Standardized measures were used to determine participants' use of substances, history of psychiatric disorders and psychosocial stress, their sensation seeking and family history of substance use and mental health disorders. Multivariate analyses were used to detect patterns of variables associated with gender and the lifetime severity of AUD. Men had a longer history of severe AUD than women. Women had similar levels of alcohol dependence and medical and psychological sequelae as men, despite 6 fewer years of AUD. More women than men had a history of severe psychosocial stress, severe dependence on other substances and antecedent mental health problems, especially mood and anxiety disorders. There were differences in family history of alcohol-related problems approximating same-gender aggregation. The severity of a lifetime AUD was predicted by its earlier age at onset and the occurrence of other disorders, especially anxiety, among both men and women. The limitations in the generalizability of these findings due to sample idiosyncrasies are discussed.     

Biochemical and molecular characterisation of cubozoan protein toxins

Class Cubozoa includes several species of box jellyfish that are harmful to humans. The venoms of box jellyfish are stored and discharged by nematocysts and contain a variety of bioactive proteins that are cytolytic, cytotoxic, inflammatory or lethal. Although cubozoan venoms generally share similar biological activities, the diverse range and severity of effects caused by different species indicate that their venoms vary in protein composition, activity and potency. To date, few individual venom proteins have been thoroughly characterised, however, accumulating evidence suggests that cubozoan jellyfish produce at least one group of homologous bioactive proteins that are labile, basic, haemolytic and similar in molecular mass (42-46 kDa). The novel box jellyfish toxins are also potentially lethal and the cause of cutaneous pain, inflammation and necrosis, similar to that observed in envenomed humans. Secondary structure analysis and remote protein homology predictions suggest that the box jellyfish toxins may act as α-pore-forming toxins. However, more research is required to elucidate their structures and investigate their mechanism(s) of action. The biological, biochemical and molecular characteristics of cubozoan venoms and their bioactive protein components are reviewed, with particular focus on cubozoan cytolysins and the newly emerging family of box jellyfish toxins.     

Dose tolerability of chronically inhaled voriconazole solution in rodents

Invasive pulmonary aspergillosis (IPA) is a fungal disease of the lung associated with high mortality rates in immunosuppressed patients despite treatment. Targeted drug delivery of aqueous voriconazole solutions has been shown in previous studies to produce high tissue and plasma drug concentrations as well as improved survival in a murine model of IPA. In the present study, rats were exposed to 20 min nebulizations of normal saline (control group) or aerosolized aqueous solutions of voriconazole at 15.625 mg (low dose group) or 31.25 mg (high dose group). Peak voriconazole concentrations in rat lung tissue and plasma after 3 days of twice daily dosing in the high dose group were 0.85 ± 0.63 μg/g wet lung weight and 0.58 ± 0.30 μg/mL, with low dose group lung and plasma concentrations of 0.38 ± 0.01 μg/g wet lung weight and 0.09 ± 0.06 μg/mL, respectively. Trough plasma concentrations were low but demonstrated some drug accumulation over 21 days of inhaled voriconazole administered twice daily. Following multiple inhaled doses, statistically significant but clinically irrelevant abnormalities in laboratory values were observed. Histopathology also revealed an increase in the number of alveolar macrophages but without inflammation or ulceration of the airway, interstitial changes, or edema. Inhaled voriconazole was well tolerated in a rat model of drug inhalation.