热休克蛋白90对大鼠缺氧心肌细胞丝氨酸苏氨酸蛋白激酶表达的影响

目的探讨内源性热休克蛋白90（HSP90）在缺氧心肌细胞丝氨酸苏氨酸蛋白激酶（AKT）相关信号通路中的作用。方法建立新生Wistar大鼠心肌细胞缺氧模型，将细胞分为正常组、缺氧组、加入HSP90特异性阻断剂格尔德霉素后再缺氧组（格尔德霉素＋缺氧组）。于缺氧后1、3、6、12、24、48h用噻唑蓝法检测心肌细胞的活力；缺氧24h，原位缺口末端标记法检测心肌细胞凋亡指数（AI）；缺氧1、3、6、12、24h，蛋白质印迹法检测大鼠心肌细胞中内源性HSP90及AKT表达水平。结果（1）缺氧24、48h，缺氧组、格尔德霉素＋缺氧组细胞活力均较正常组明显下降（P〈0．05）；格尔德霉素＋缺氧组细胞活力缺氧12h即开始明显下降，缺氧48h时明显低于缺氧组（P〈0．05）。（2）缺氧24h，缺氧组细胞AI为（10．7±1．2）％，明显高于正常组[（1．9±0．3）％．P〈0．05]；格尔德霉素＋缺氧组细胞AI为（26、3±5．3）％，明显高于缺氧组（P〈0．01）。（3）缺氧12h，缺氧组心肌细胞内源性HSP90及AKT表达水平高于正常组与格尔德霉素＋缺氧组；缺氧24h，缺氧组有所下降．格尔德霉素＋缺氧组则下降更明显。结论内源性HSP90对维持心肌细胞的活力有重要作用．缺氧心肌细胞AKT表达水平可受内源性HSP90表达水平的影响。     

Simultaneous versus sequential optimal design for pharmacokinetic-pharmacodynamic models with FO and FOCE considerations

We consider nested multiple response models which are used extensively in the area of pharmacometrics. Given the conditional nature of such models, differences in predicted responses are a consequence of different assumptions about how the models interact. As such, sequential versus simultaneous and First Order (FO) versus First Order Conditional Estimation (FOCE) techniques have been explored in the literature where it was found that the sequential and FO approaches can produce biased results. It is therefore of interest to determine any design consequences between the various methods and approximations. As optimal design for nonlinear mixed effects models is dependent upon initial parameter estimates and an approximation to the expected Fisher information matrix, it is necessary to incorporate any influence of nonlinearity (or parameter-effects curvature) into our exploration. Hence, sequential versus simultaneous design with FO and FOCE considerations are compared under low, typical and high degrees of nonlinearity. Additionally, predicted standard errors of parameters are also compared to empirical estimates formed via a simulation/estimation study in NONMEM. Initially, design theory for nested multiple response models is developed and approaches mentioned above are investigated by considering a pharmacokinetic–pharmacodynamic model found in the literature. We consider design for situations where all responses are continuous and extend this methodology to the case where a response may be a discrete random variable. In particular, for a binary response pharmacodynamic model, it is conjectured that such responses will offer little information about all parameters and hence a sequential optimization, in the form of product design optimality, may yield near optimal designs.     

重组人骨形态发生蛋白4基因腺相关病毒载体对兔骨髓基质干细胞生物学行为的影响

目的应用重组人骨形态发生蛋白4基因腺相关病毒载体(AAV-hBMP4)转染兔骨髓基质干细胞(BMSCs),观察其对BMSCs生物学行为的影响,从而为骨组织工程寻找理想的病毒载体及种子细胞。方法全骨髓法培养兔BMSCs,按感染复数(MOI)值不同设定为四组,分别转染兔BMSCs,观察病毒量对细胞形态的影响。选取影响最小的MOI值,进行后续实验。转染兔BMSCs,MTT法描记细胞生长曲线,观察AAV对细胞增殖活性的影响。以重组增强型绿色荧光蛋白基因的腺相关病毒载体(AAV-EGFP)为参照,行流式细胞仪检测,计算转染效率。AAV-hBMP4与对照病毒AAV-EGFP分别转染细胞,观察细胞形态,行碱性磷酸酶(ALP)染色、Von Kossa染色及ALP含量测定,观察成骨活性。兔肌袋实验观察异位成骨情况。结果MOI值为5×10~4 vg/cell时,AAV对细胞形态影响最小,以此值进行后续实验。AAV转染后,细胞增殖活性良好,转染效率为55％～65％。AAV-hBMP4转染后,细胞形态呈现典型的成骨改变,ALP染色及Von Kossa染色均出现成骨的特征性改变,而AAV-EGFP组无上述改变。细胞上清ALP含量测定显示,实验组ALP含量显著增高,与对照组比较差异有统计学意义(t=218．65,P＜0．01)。兔肌袋实验术后4周组织学检测可见大量钙盐沉积,矿化结节形成。结论AAV-hBMP4转染效率高,对BMSCs的增殖活性影响小,AAV-hBMP4转染的BMSCs可望成为组织工程化骨的理想种子细胞。     

Mathematical modelling of the spatio-temporal response of cytotoxic T-lymphocytes to a solid tumour.

In this paper a mathematical model describing the growth of a solid tumour in the presence of an immune system response is presented. In particular, attention is focused upon the attack of tumour cells by so-called tumour-infiltrating cytotoxic lymphocytes (TICLs), in a small, multicellular tumour, without necrosis and at some stage prior to (tumour-induced) angiogenesis. At this stage the immune cells and the tumour cells are considered to be in a state of dynamic equilibrium--cancer dormancy--a phenomenon which has been observed in primary tumours, micrometastases and residual disease after ablation of the primary tumour. Nonetheless, the precise biochemical and cellular mechanisms by which TICLs control cancer dormancy are still poorly understood from a biological and immunological point of view. Therefore we focus on the analysis of the spatio-temporal dynamics of tumour cells, immune cells and chemokines in an immunogenic tumour. The lymphocytes are assumed to migrate into the growing solid tumour and interact with the tumour cells in such a way that lymphocyte-tumour cell complexes are formed. These complexes result in either the death of the tumour cells (the normal situation) or the inactivation (sometimes even the death) of the lymphocytes. The migration of the TICLs is determined by a combination of random motility and chemotaxis in response to the presence of chemokines. The resulting system of four nonlinear partial differential equations (TICLs, tumour cells, complexes and chemokines) is analysed and numerical simulations are presented. We consider two different tumour geometries--multi-layered cell growth and multi-cellular spheroid growth. The numerical simulations demonstrate the existence of cell distributions that are quasi-stationary in time and heterogeneous in space. A linear stability analysis of the underlying (spatially homogeneous) ordinary differential equation (ODE) kinetics coupled with a numerical investigation of the ODE system reveals the existence of a stable limit cycle. This is verified further when a subsequent bifurcation analysis is undertaken using a numerical continuation package. These results then explain the complex heterogeneous spatio-temporal dynamics observed in the partial differential equation (PDE) system. Our approach may lead to a deeper understanding of the phenomenon of cancer dormancy and may be helpful in the future development of more effective anti-cancer vaccines.     

兔肢体爆炸伤合并海水浸泡后在不同复温速率及维持浅低体温下机体炎症反应的特征

Objective To investigate effects of different rewarming rates and maintenance of light hypothermia on inflammatory response in rabbits after limb blast injury, coupled with seawater immersion. Methods First, the model of limb blast injury coupled with seawater immersion was reproduced [the animals were immersed to low body temperature of (31.0±0.5℃)]. Then, 24 adult rabbits were randomly divided into group Ⅰ [the rapid rewarming group, n=6, rewarmed to (38±0.5)℃ at a rate of (8.94±0.93)℃/h], group Ⅱ [the slow rewarming group, n=6, rewarmed to (38±0.5)℃ at a rate of (3.88±0.22)℃/h], group Ⅲ [another slow rewarming group, n=6, rewarmed to (38±0.5)℃ at a rate of (2.18±0.12)℃/h], and the H group [the hypothermia group, n =6, rewarmed to (34 - 35)℃ at a rate of (4.49±0.66)℃/h and kept at that temperature till termination of the experiment]. Regulation of ambient temperature and warm transfusion were used to restore body temperature to target levels and maintained there for 6 hours. Blood samples were taken at 5 different times, I.e. Pre-injury time(T0), post-immersion time (T1), the time when rewarming started (T2), 3 h after rewarming (T3), and 6 h after rewarming (T4). Tissue samples from heart, liver, intestinum, lung and kidney were also collected. Levels of TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β) and IL-6 (interleukin-6) in plasma and MPO (myeloperoxidase) in homogenate were detected. Results Following rewarming, TNF-α, IL-1β, IL-6 concentrations in the plasma of the animals in group Ⅰ and group H were significantly higher when compared with those of the animals in group Ⅱ and group Ⅲ (P<0.05, P<0.01), and MPO activity in homogenate was significantly higher when compared with that of the animals in group Ⅱ and group Ⅲ(P<0.01, P<0.05), and no statistical difference could be seen between group Ⅱ and Ⅲ (P>0.05). Conclusions Rapid rewarming and maintenance of light hypothermia could obviously elevate TNF-α, IL-1β, IL-6 concentrations in plasma and MPO activity in homogenate, following limb blast injury coupled with hypothermia induced by seawater immersion, while slow rewarming (with a rewarming rate of 2-4℃/h) could significantly inhibit TNF-α, IL-1β, IL-6 levels and PMN activity.     

多器官功能障碍小鼠高迁移率族蛋白1释放对单个核细胞免疫相关指标的影响

目的 分析多器官功能障碍综合征(MODS)小鼠血清高迁移率族蛋白B1(HMGB1)含量的变化与外周血中单个核细胞免疫相关指标变化的关系,观察MODS发生、发展中HMGB1释放的规律及其对细胞免疫功能的影响.方法 腹腔注射酵母多糖复制小鼠MODS模型,用Westernblot法检测病程不同阶段血清HMGB1含量、流式细胞术测定外周血单核细胞表面组织相容性复合体-Ⅱ类分子(MHC-Ⅱ类分子,IAb)的表达量及T淋巴细胞亚群的比值(CD4+/CD8+).结果 在酵母多糖所致小鼠MODS模型中,当血清HMGB1含量升高时,外周血单核细胞IAb表达量及CD4+/CD8+比值下降;当血清HMGB1含量回复接近正常时,单核细胞IAb表达量及CIM4+/CD8+比值也趋于恢复正常.结论 在MODS的发生、发展过程中,HMGB1可能通过影响血中单个核细胞MHC-Ⅱ类分子(IAb)表达及T淋巴细胞的活性参与免疫调节过程,导致免疫失衡或免疫抑制.     

Characteristics of antibody responses in tick-borne encephalitis vaccination breakthroughs

Tick-borne encephalitis (TBE) virus is an important human pathogenic flavivirus that is endemic in Europe and Asia. The disease can be effectively prevented by inactivated vaccines and vaccination breakthroughs (VBTs) are rare. We investigated the characteristics of antibody responses in such VBTs in comparison to those in unvaccinated TBE patients. In contrast to the unvaccinated controls, most of the VBTs displayed a delayed IgM antibody response and had high avidity and strongly neutralizing antibodies already in the first sample taken upon hospitalization. The antibody profile of these patients therefore had the characteristics of an anamnestic immune response. In the VBTs analyzed, immunological priming and memory were apparently not sufficient or fast enough to prevent the disease.     

17 β雌二醇通过丝裂原活化蛋白激酶抑制前列腺癌细胞系PC3增殖

目的探讨丝裂原活化蛋白激酶在17β雌二醇(E2)抑制前列腺癌PC3细胞生长中的作用。方法检测不同浓度E2作用不同时间后PC3细胞生长抑制率。流式细胞仪分析细胞周期分布,TUNEL染色检测凋亡。Western blot检测ERK1/2,JNK和p38活性。RT-PCR法检测雌激素受体(ER)α、ERβ、P21WAF1和cyclinD1的表达。结果E2抑制PC3细胞增殖,并且可以激活ERK1/2、JNK和p38。处理因素作用48h后,对照组、E2、E2 PD98059、E2 SP600125、E2 SB203580组细胞的凋亡率分别为(0.9±0.1)%;(23.0±1.4)%;(30.0±1.2)%;(10.6±0.8)%和(14.6±0.7)%,(P<0.05)。E2使细胞阻滞在G1期,PD98059、SP600125、SB203580分别预处理1h后细胞分别进一步阻滞在G1期;轻度阻滞在G1期和进入S期。RT-PCR发现PC3细胞表达ERα和ERβ,E2、E2 PD98059、E2 SP600125、E2 SB203580组中cyclinD1、P21WAF1基因表达分别为对照组的(0.42±0.03)、(3.13±0.02)倍;(0.21±0.03)、(3.08±0.05)倍;(0.43±0.01)、(1.31±0.04)倍;(2.81±0.02)、(3.14±0.02)倍(P<0.05)。结论E2激活JNK增加P21WAF1基因表达,并激活p38抑制cyclinD1表达,使细胞阻滞在G1期,JNK和p38通路还介导E2引起PC3细胞凋亡。同时E2又可激活ERK1/2,轻度拮抗上述作用。     

HBV转录激活蛋白HBx、MHBst78MHBst155真核重组载体构建

目的分别构建人乙型肝炎病毒(HBV)X蛋白、羧基端截断的中分子表面蛋白MHBst78、MHBst155编码基因的真核重组表达载体,以便进一步研究其转录激活功能及对宿主细胞信号传导通路的影响。方法设计合成3对寡核苷酸引物,以adr亚型HBV质粒pHBVDNA为模板,采用PCR法分别扩增HBVX基因、MHBst78与MHBst155编码基因片段;用HindⅢ,KpnⅠ双酶切HBVⅩ基因;用HindⅢ和BamHⅠ双酶切MHBst78与MHBst155编码基因片段后,分别定向插入到真核表达载体pcDNA3.1相应酶切位点,转化宿主菌JM109,提取质粒,分别用上述内切酶酶切及DNA测序鉴定重组质粒。结果酶切重组体显示所切下的片段大小均与预计相符,测序结果与文献报道序列及预计结果一致。结论成功构建了HBVX基因、羧基端截断的HBV中分子表面蛋白MHBst78、MHBst155编码基因的真核重组表达载体,为进一步研究HBV转录激活蛋白HBx、MHBst78MHBst155对宿主细胞信号转导通路的影响奠定基础。     

Therapeutic effect of 5-FC on YCD modified murine P388 leukemia in vivo

Cytosine deaminases (CDs) in bacteria andfungi are found to deaminate prodrug 5 - FC intocytotoxic agent 5 - FU .Yeast CD (YCD) is clonedfrom Saccharomyces cerevisiae.It has been shownpreviously that YCD was more efficient inconversing5 - FC than bacterial CD(b CD) [1-3 ] .As anovel suicide gene therapy system,YCD/ 5 - Fcsystem may be promising in cancer therapy andprevention of graft versus host disease.In thepresent study,we established a P388/ DBA murineleukemia model by infusin…