扶芳藤合剂对兔病态窦房结综合征Cx45、Cx43及Cx40蛋白表达的影响

目的观察复方扶芳藤液对兔病态窦房结综合征模型心率（HR） 及窦房结电生理、及心脏窦房结组织Cx45、Cx43及Cx40蛋白表达的影响,并探讨其作用机制.方法将40只清洁级健康日本大耳白兔采用随机、平行对照的方法,按随机数字表分为空白对照组、模型组、心宝丸组及复方扶芳藤液组（中药组）高、低剂量组,每组各 8 只.造模成功后空白对照组、模型组分别予生理盐水15 mL/d,心宝丸组予心宝丸悬浮液,中药组高、低剂量组分别予复方扶芳藤液连续灌胃14 d.取各组兔上下腔静脉之间的窦房结组织,常规切片,免疫组化检测窦房结组织Cx45、Cx43及Cx40蛋白表达.结果大剂量组窦房结组织Cx45、Cx43及Cx40蛋白表达明显高于模型组（P＜0.05）;小剂量组干预后窦房结组织Cx45、Cx43及Cx40蛋白表达较模型组增高,其中,Cx40较模型明显增高（P＜0.05）.结论复方扶芳藤可上调损伤兔窦房结组织Cx45、Cx43及Cx40蛋白的表达,可能是治疗病态窦房结综合征的机制之一.     

Combination anti-CD137 and anti-CD40 antibody therapy in murine myc-driven hematological cancers

In order to stimulate antigen presentation and T cell activity against cancer, we treated three different tumor models in mice with the monoclonal antibodies anti-CD40 plus anti-CD137 (BiMab). In a subcutaneous transplantable MC38 colon cancer model, there was significant enhancement in the survival of mice following BiMab treatment. Anti-CD40 has shown considerable success against lymphoma in previous studies by other investigators, and we also showed in this study that, in a model of Eμ-Myc lymphoma, there was a statistically significant enhancement of survival of mice following BiMab treatment. Following the success of the BiMab treatment in the previous two models, we wished to determine if it would be successful in a mouse model of multiple myeloma. Firstly, we tested a transplantable model of disease in which multiple myeloma cells derived from Vk*MYC mice were injected intravenously. A minor proportion of anti-CD137 and BiMab treated mice experienced prolongation of life beyond 250 days. Then we tested the therapy in a spontaneously occurring multiple myeloma model, in Vk*MYC transgenic mice. The majority of mice treated survived longer than control mice, although statistical significance was not demonstrated.     

脂必泰对高脂血症患者CD40/sCD40L的调节作用

目的 探讨脂必泰对高脂血症患者CD40/sCD40L水平的影响.方法 66例原发性高脂血症患者应用脂必泰治疗8周,分别测定治疗前（0周）、治疗4周和8周后CD40和sCD40L的浓度进行对比,同时入选44例健康者作为正常对照组.结果 高脂血症组与正常对照组相比CD40/sCD40L治疗前表达明显上调,而应用脂必泰治疗4周后sCD40L即有显著下降（P〈0.05）;CD40治疗后亦有下降,但未达到统计学意义.结论 高脂血症刺激细胞CD40/sCD40L表达显著增加,脂必泰具有抑制CD40/sCD40L信号转导通路的作用,这可能是其抗炎、抗动脉硬化的重要机制.     

野生型p53基因的表达及其对子宫颈癌细胞株的生长抑制作用

构建了可在哺乳动物细胞中表达人ｐ５３蛋白的重组质粒ｐ５３─ｐＳＶ２ｎｅｏ，分别转染猴肾细胞株ＣＶ─１及人子宫颈癌细胞株ＳｉＨａ。用ＰＣＲ法检测出转染细胞中的ｐ５３ｃＤＮＡ；用免疫组化ＡＢＣ法检测出ｐ５３蛋白的表达定位于细胞核内，用Ｗｅｓｔｅｒｎｂｌｏｔ检测出转染细胞中存在ｐ５３蛋白。结果表明，外源性野生型ｐ５３基因的表达可以使ＳｉＨａ细胞的细胞克隆形成效减少５０％，使细胞生长速度降低，但对ＳｉＨａ细胞的软琼脂克隆形成效无明显影响。提示ｐ５３基因的肿瘤抑制作用与野生型ｐ５３蛋白的量有关，并且是细胞类型特异性的。     

心脏性猝死病人心肌组织CX43和CX40的表达

目的观察心脏性猝死病人左、右心室肌及室间隔肌缝隙连接蛋白43(CX43)及缝隙连接蛋白40(CX40)的表达,探讨CX43、CX40表达与心脏性猝死的关系。方法应用免疫组化及Simple PCI图像分析系统,分析比较心脏性猝死与非心脏性猝死病人心室肌及室间隔肌CX43、CX40的表达,并用SPSS 13.0统计软件对数据进行统计分析。结果非心脏性猝死病人CX43、CX40主要表达于心肌细胞闰盘处,少数表达于细胞侧面连接及胞浆中。心脏性猝死病人CX43、CX40多数弥散于胞浆及细胞侧面连接处,表达于闰盘的数量减少。心脏性猝死病人CX43在左心室、室间隔及右心室的表达明显低于对照组(t=2.09~8.01,P均<0.05);心脏性猝死病人CX40在左心室、室间隔及右心室的表达也明显低于对照组(t=3.24~9.40,P均<0.05)。结论CX43、CX40表达部位的改变与表达数量的减少,可能与心脏性猝死有一定关系。     

Umbilical cord stem cell transplantation for primary immunodeficiencies

Primary immunodeficiencies (PIDs) are a rare but important cause of mortality and morbidity in childhood: the most severe – known as severe combined immunodeficiency (SCID) – are fatal within the first year of life; other PIDs are less immediately life-threatening, but have a poor long-term outlook. Haematopoietic stem cell transplantation (HSCT) is the best treatment for SCID and is increasingly offered for other PIDs. The best results are achieved with an HLA-matched family donor. Umbilical cord stem cells (UCSCs) are an alternative stem cell source. Results using UCSCs in the treatment of haematological disorders and malignancy are as good as those for which marrow is the stem cell source. Although PIDs make up a small proportion of disorders amenable to treatment by HSCT, UCSCs are an ideal source of haematopoietic stem cells for many of these patients. Of the 52 patients with SCID or other PIDs for whom detailed information on outcome is available, results of engraftment, immune reconstitution, incidence of graft-versus-host disease and survival are comparable with other stem cell sources. Small stem cell dose and prolonged time to viral immunity limit the patients for whom UCSCs can be used. Newer methods of achieving better engraftment, ex vivo expansion of stem cells and generation of antigen-specific cytotoxic T cells are being developed at present, and will widen the application of UCSCs as a viable source for more patients.     

Dendritic cell maturation in active immunotherapy strategies

Dendritic cells (DCs) loaded with tumour antigen have become the centrepiece of clinical trials testing active immunotherapy strategies. Important variables include the source of DCs, the choice of antigens, the method of antigen loading and the route and timing of administration. Recently, the requirement for and the method of, DC maturation have been receiving particular attention. This is due to observations from in vitro studies and animal models demonstrating that mature DCs induce more potent antigen-specific T-cells responses than immature DCs. Furthermore, preliminary observations in human studies suggest that immature DCs might actually downregulate antigen-specific T-cell responses but mature DCs may augment them. Current studies are addressing how to define DC maturation, whether the variety of methods for maturation result in DCs with similar T-cell stimulatory capacity, how to maintain the maturational status and whether maturation in vitro before immunisation, or in vivo, after immunisation, results in better DC function.     

清热祛瘀颗粒对稳定性冠心病患者颈动脉易损斑块的影响

目的 观察清热祛瘀颗粒对冠心病伴颈动脉易损斑块患者的治疗作用。方法 82例稳定性劳力型心绞痛伴颈动脉易损斑块患者中医辨证属痰热血瘀型者,随机分为清热祛瘀颗粒加西医常规治疗的试验组（41例）和采用西医常规治疗加安慰剂治疗的对照组（41例）,共观察25周。采用高频超声检测颈动脉复杂斑块的数目、简单斑块的数目、斑块的Crouse积分及颈动脉内中膜的厚度,观察清热祛瘀颗粒对颈动脉斑块及血浆高敏C反应蛋白(hs-CRP)、CD40L变化及肝肾功能。结果 试验组治疗后颈动脉复杂斑块的数目、斑块的Crouse积分、颈动脉内中膜的厚度较治疗前为低,差异均有统计学意义（P<0.05）;颈动脉斑块的Crouse积分、颈动脉内中膜的厚度也较对照组治疗后为低,差异亦有统计学意义（P<0.05）, 试验组治疗后hs-CRP及CD40L与治疗前及对照组治疗后比较,差异均有统计学意义（P<0.05,P<0.01）。治疗过程中未见明显不良反应。结论 清热祛瘀颗粒对冠心病伴颈动脉易损斑块患者的颈动脉斑块具有一定的稳定作用。     

免疫分选软骨前体细胞并诱导永生化的研究

目的建立永生化大鼠软骨前体细胞株,为细胞移植和转基因治疗提供稳定的细胞来源。方法利用免疫磁珠技术分离纯化具有特异性表面标志成纤维生长因子受体-3(FGFR-3)的软骨前体细胞,用基因转染技术将含有猿肾病毒40大T抗原基因(SV40Tag)的重组质粒pEGFP- IRES2-SV40Tag转染原代培养的新生大鼠软骨前体细胞,经G418筛选,抗性克隆扩大培养。应用FGFR-3、Ⅱ型胶原和X型胶原抗体进行细胞鉴定,检测其分化能力,观察细胞的形态及其生长状况,绘制生长曲线。用逆转录-聚合酶链反应(RT-PCR)、Southern blot和免疫细胞化学法鉴定SV40Tag在转染细胞中的表达。结果获得1个阳性细胞克隆,免疫细胞化学证实为FGFR-3阳性的具有较强增值能力和多分化潜能的软骨前体细胞。经Southern印迹杂交证实,SV40Tag已稳定转染入软骨前体细胞,表达mRNA及其蛋白。贴壁培养的永生化软骨前体细胞株(IPSC),群体倍增时间为23.62 h,传代、冻存和复苏对细胞形态及生长无明显影响。结论SV40Tag导入可诱导软骨前体细胞永生化,为软骨前体细胞的实验研究及其介导的细胞移植治疗提供了稳定的细胞来源。     

创伤性急性肺损伤时兔肺微血管内皮细胞Cx40调控内皮细胞通透性的研究

目的 观察创伤性急性肺损伤(ALI)兔血清对肺微血管内皮细胞连接蛋白40(Cx40)表达和间隙连接通道(GJC)功能的影响,及后者与内皮细胞单层通透性的关系.方法 组织块贴壁法分离、培养兔肺微血管内皮细胞.细胞分为3组,对照组、创伤血清处理组和阻滞剂组.在阻滞剂组,细胞GJC阻滞剂1-庚醇(0.5 mmol/L)加入到培养液中.1 h后,阻滞剂组和创伤血清组的20%胎牛血清的培养液均被更换为含有20%创伤血清的培养液.对照组则只更换培养液.3组细胞继续孵育6 h后,进入实验观察.分别行划痕实验、Cx40蛋白免疫印迹、单层细胞通透性和细胞内Ca2+钙浓度检测.结果 Cx40蛋白印迹见创伤血清组与阻滞剂组蛋白表达水平较对照组有明显降低,且2组之间亦不同,阻滞剂组蛋白水平较创伤血清组低,吸光度和值减少(对照组726293.2±83 684.1,创伤血清组397 798.1±87 145.7,阻滞剂组316977.6±76 325.9,n=4,P＜0.05).3组细胞划痕边缘的细胞均被染料荧光黄(LY)染色,LY在对照组细胞扩散得最远,显色的周边细胞的数量最多,创伤血清组次之,阻滞剂组显色的细胞数量最少,相邻细胞与边缘细胞比值的差异有统计学意义(23±5、11±3比7±2,n=4,P＜0.05).3组细胞伊文思蓝清除率均随着时间的延长而增加,且3组细胞间亦有不同,阻滞剂组清除率高于创伤血清组和对照组.组间差异和时间点差异均有统计学意义(P＜0.05).通过共聚焦显微镜的观察,3组细胞荧光强度不相同,阻滞剂组荧光最强,对照组最弱.创伤血清组和阻滞剂组细胞内Ca2+浓度均显著高于对照组[(494.80±41.94)、(569.80±6.70)nmol/L比(158.80±13.09)nmol/L,P＜0.05].结论 创伤性ALI动物血清抑制肺微血管内皮细胞Cx40表达,进而引起GJC功能下降,导致肺血管内皮细胞单层通透性增加,该机制可能参与了胸部创伤后肺血管通透性增加的形成,促进了伤后急性肺损伤的发病.

Abstract：

Objective To investigate the influence of traumatic acute lung injury (ALI) rabbit serum on gap junction channel ( GJC ) function and connexin40 ( Cx40) expression, and the correlation of GJC and Cx40 with rabbit pulmonary microvascular endothelial cells permeability. Methods Cultured pulmonary microvessel endothelial cells were divided into three groups, control ( Gcontrol) , injured serum (Gserum) , and blocker agent ( Gblocker). GJC function was assessed by scrape-loading and dye transfer techniques. Pulmonary microvessel endothelial cells permeability was measured by Evans blue-labeled albumin transfer, and the expression of Cx40 was measured by Western blotting. Intracellular free calcium concentration was measured by fluo-3am. Results Injured serum decreased GJC function and the level of Cx40,which was aggravated by the GJC blocker (control 726 293. 2 ± 83 684. 1, serum 397 798. 1 ± 87 145. 7, blocker 316 977. 6 ±76 325. 9,n=4,P ＜0. 05). Similarly, pulmonary microvascular endothelial cells permeability was increased significantly in Gserum and Gblocker as compared with Gcontrol (P ＜0. 05). After treatment with injured serum in combination with gap junction blocker in vitro, intracellular free calcium concentration was increased [control (494. 80 ±41.94) nmol/L, serum (569. 80 ±6. 70) nmol/L, blocker (158. 80 ±13. 09) nmol/L, P＜ 0.05]. Conclusion Traumatic ALI rabbit serum inhibits the Cx40 expression and down-regulates GJC function, which contributes to the increase of pulmonary microvascular endothelial cells permeability after ALI This mechanism may participate in the increase of pulmonary vascular permeability and promote the morbidity of ALI after trauma.