次声刺激诱导原代培养大鼠星形胶质细胞释放谷氨酸的研究

摘要 目的：观察16Hz, 130dB次声刺激对原代培养的大鼠星形胶质细胞谷氨酸释放的影响,并探讨其释放机制。 方法：原代培养大鼠海马区星形胶质细胞,将其分为次声刺激组（IE group,n=6）,Gap26+次声刺激组（Gap26+IE group）,对照组（Ctrl group,n=6）。次声刺激组细胞暴露于次声舱中,分别给予15min, 30min, 60min, 90min, 120min和240min的次声刺激;对于Gap26+IE组,则在次声刺激前,选用Cx43半通道阻断剂Gap26预处理星形胶质细胞;对照组也置于次声舱,但不给予次声刺激。次声刺激频率为16Hz,强度为130dB。为了排除Gap26对谷氨酸释放的影响,还选用乱序Gap26肽段(Scrb Gap26)来预处理星形胶质细胞（Scrb Gap26+IE group,n=6）。采用免疫荧光染色测定细胞Cx43的表达情况,采用高效液相色谱（HPLC）来测定细胞外液谷氨酸浓度。 结果：次声刺激后,IE组细胞外液谷氨酸的浓度显著升高,并在刺激90min后,细胞外液谷氨酸浓度达峰值,为（4.6±0.3）nmol/ml,显著高于对照组（2.3±0.2）nmol/ml（P<0.05）,这说明次声刺激可以显著增高星形胶质细胞外液的谷氨酸含量。此外,次声刺激后,IE组细胞Cx43的表达也显著升高,刺激60mins时,Cx43平均荧光强度达到峰值,为（198±33）(AUC),显著高于对照组（P<0.05）。而对于Gap26+IE组,次声刺激后细胞外液谷氨酸浓度的升高受到抑制,90min后,细胞外液谷氨酸浓度为（3.58±0.17）nmol/ml,显著低于次声刺激组（P<0.05）。 结论：次声刺激可以诱导星形胶质细胞释放谷氨酸;Cx43半通道阻断剂Gap26抑制了谷氨酸释放,次声刺激诱导的谷氨酸释放可能是通过Cx43半通道来完成的。     

哈萨克斯坦农业耕作改变引起的棘球蚴病流行病学变化

Shaikenov等为研究棘球蚴病流行病学变化的原因 ,对哈萨克斯坦南方阿拉木图、江布尔和南哈萨克斯坦等 3个州的犬和羊进行了相关检查。该研究检查了 2 4 0 0只羊的肝和肺以明确其是否有细粒棘球蚴寄生 ;对 2 0 71条犬采用槟榔碱法导泻 ,并计数其粪便中的细粒棘球绦虫 ;用漂浮法检查了阿拉木图市 35 0份城市犬粪便中细粒棘球绦虫卵的情况。此外 ,该研究对分别采集自 175个农家小院和 6 5个牧场庭院的 6 2 6份和 36 2份土壤样品进行了虫卵情况的分析 ;收集了从国家流行病办公室获得的关于棘球蚴病感染者的数据和来自阿拉木图市医院的档案 ,还…     

胰岛素对烧伤血清诱导血管内皮细胞核因子κB核移位的抑制作用

Objective To study the inhibitory effects of insulin on nuclear factor-kappa B (NF-κB) nuclear translocation of vascular endothelial cells induced by burn serum and its correlative mechanism. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and divided into 5 groups: blank control group (BC, ordinary culture without any stimulation) , normal serum control group (NS, cultured with nutrient solution containing 20% healthy human serum) , burn serum stimulation group (BS, cultured with nutrient solution containing 20% burn human serum) , burn serum + insulin treatment group (BI, cultured with nutrient solution containing 20% burn human serum and 1 × 10-7 mol/L insulin) ,inhibitor pretreatment group [IP, pretreated with 50 μmol/L protein kinase B (Akt) specific inhibitor LY-294002, then cultured with the same medium as used in BI group 30 minutes later] according to the random number table. Six hours later, the injury and apoptosis of HUVECs was respectively observed by the scanning electron microscope and determined by the flow cytometry. Meanwhile, the phosphorylation of inhibitor kappa B-α (p-IκB-α) and Akt (p-Akt) in cytoplasm, and the content of NF-κB-p65 in nucleus were determined with Western blot. Results (1) Compared with those in BC group, HUVECs in BS group shrank obviously with irregular nuclear structure, and intercellular links jagged or vanished. Slight change was observed in HUVECs structure in NS and BI groups, with the cell ductility and nuclear structure much better than those in BS group. (2) The apoptosis rates of HUVECs in BS group [(28. 5 ± 2. 3) %] , BI group [(22. 3 ±1.8)%], and IP group [(29. 7 ± 2. 4) %] were all obviously higher than that in BC group [(15.7 ±2.2)% , F =14.288, P <0.05otP <0.01]. There was no significant statistical difference between NS group [(17. 0 ± 2. 5) %] and BC group in apoptosis rate (F = 14. 288 , P > 0. 05). The apoptosis rate of HUVECs in BI group was obviously lower than that in BS group (F = 14. 288 , P <0.05). (3)Compared with those in BC group, the protein expressions of p-IκB-α in cytoplasm and NF-κB-p65 in nucleus were up-regulated, and the protein expression of p-Akt in cytoplasm was down-regulated in BS and IP groups. The expression levels of the three proteins in NS and BI groups were close to those in BC group. Conclusions Insulin could inhibit the IκB phosphorylation, and then restrict NF-κB nuclear translocation and improve the vascular endothelial cells function accordingly through regulating phosphatidylinositol 3 ki-nase/Akt pathway.     

胰岛素对烧伤血清诱导血管内皮细胞核因子κB核移位的抑制作用

Objective To study the inhibitory effects of insulin on nuclear factor-kappa B (NF-κB) nuclear translocation of vascular endothelial cells induced by burn serum and its correlative mechanism. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and divided into 5 groups: blank control group (BC, ordinary culture without any stimulation) , normal serum control group (NS, cultured with nutrient solution containing 20% healthy human serum) , burn serum stimulation group (BS, cultured with nutrient solution containing 20% burn human serum) , burn serum + insulin treatment group (BI, cultured with nutrient solution containing 20% burn human serum and 1 × 10-7 mol/L insulin) ,inhibitor pretreatment group [IP, pretreated with 50 μmol/L protein kinase B (Akt) specific inhibitor LY-294002, then cultured with the same medium as used in BI group 30 minutes later] according to the random number table. Six hours later, the injury and apoptosis of HUVECs was respectively observed by the scanning electron microscope and determined by the flow cytometry. Meanwhile, the phosphorylation of inhibitor kappa B-α (p-IκB-α) and Akt (p-Akt) in cytoplasm, and the content of NF-κB-p65 in nucleus were determined with Western blot. Results (1) Compared with those in BC group, HUVECs in BS group shrank obviously with irregular nuclear structure, and intercellular links jagged or vanished. Slight change was observed in HUVECs structure in NS and BI groups, with the cell ductility and nuclear structure much better than those in BS group. (2) The apoptosis rates of HUVECs in BS group [(28. 5 ± 2. 3) %] , BI group [(22. 3 ±1.8)%], and IP group [(29. 7 ± 2. 4) %] were all obviously higher than that in BC group [(15.7 ±2.2)% , F =14.288, P <0.05otP <0.01]. There was no significant statistical difference between NS group [(17. 0 ± 2. 5) %] and BC group in apoptosis rate (F = 14. 288 , P > 0. 05). The apoptosis rate of HUVECs in BI group was obviously lower than that in BS group (F = 14. 288 , P <0.05). (3)Compared with those in BC group, the protein expressions of p-IκB-α in cytoplasm and NF-κB-p65 in nucleus were up-regulated, and the protein expression of p-Akt in cytoplasm was down-regulated in BS and IP groups. The expression levels of the three proteins in NS and BI groups were close to those in BC group. Conclusions Insulin could inhibit the IκB phosphorylation, and then restrict NF-κB nuclear translocation and improve the vascular endothelial cells function accordingly through regulating phosphatidylinositol 3 ki-nase/Akt pathway.     

以护士为主导的快速反应小组抢救住院病人的重症护理新策略

为给病人提供早期干预和降低病人的死亡率,美国佛罗里达州的Tallahassee Memorial医院(TMH)最近采用了一种新的策略,即组建抢救病人的快速反应小组,也称为医疗急救小组(MET)。现正在全州医院运行。MET可以帮助护理人员在急救过程中更早的进行干预,从而避免一些并发症的发生或由     

胰岛素对烧伤血清诱导血管内皮细胞核因子κB核移位的抑制作用

目的 了解胰岛素对烧伤血清诱导的血管内皮细胞NF-κB核移位的抑制作用及其相关机制.方法 体外培养人脐静脉内皮细胞(HUVEC).按照随机数字表法将细胞分为5组:空白对照组,不加任何刺激因素常规培养;正常血清对照组和烧伤血清刺激组,分别用含体积分数20%健康人血清、体积分数20%烧伤患者血清的培养液培养;烧伤血清+胰岛素处理组,在烧伤血清刺激组培养液成分的基础上添加胰岛素(终浓度1×10-7mol/L)进行培养;抑制剂预处理组,预先加入蛋白激酶B(PKB或Akt)特异性抑制剂LY294002(50μmol/L)孵育细胞,30 min后改用培养液(成分同烧伤血清+胰岛素处理组)培养.6 h后,采用扫描电镜观察各组HUVEC损伤情况;用流式细胞仪检测细胞凋亡率;蛋白质印迹法检测细胞胞质磷酸化κB-α抑制蛋白(p-IκB-α)和磷酸化Akt(p-Akt)水平,以及胞核NF-κB-p65的蛋白表达变化.结果 (1)扫描电镜观察:与空白对照组HUVEC比较,烧伤血清刺激组、抑制剂预处理组细胞收缩明显,细胞间呈锯齿状连接或连接消失,胞核结构不规整.正常血清对照组及烧伤血清+胰岛素处理组细胞结构有轻微改变,但细胞延展性及胞核结构明显好于烧伤血清刺激组.(2)细胞凋亡率:空白对照组为(15.7±2.2)%.烧伤血清刺激组为(28.5±2.3)%,烧伤血清+胰岛素处理组为(22.3±1.8)%,抑制剂预处理组为(29.7±2.4)%,均明显高于空白对照组(F=14.288,P<0.05或P<0.01);正常血清对照组细胞凋亡率为(17.0±2.5)%,与空白对照组比较差异无统计学意义(F=14.288,P>0.05).与烧伤血清刺激组相比,烧伤血清+胰岛素处理组细胞凋亡率明显降低(F=14.288,P<0.05).(3)蛋白表达水平:与空白对照组相比,烧伤血清刺激组和抑制剂预处理组细胞胞质p-IκB-α与胞核NF-κB-p65的蛋白表达水平明显升高,p-Akt表达水平明显下降;正常血清对照组和烧伤血清+胰岛素处理组3种蛋白水平均与空白对照组接近.结论 胰岛素通过调节磷脂酰肌醇3激酶/Akt信号通路抑制IκB-α磷酸化,继而限制NF-κB核移位,最终发挥改善内皮细胞功能的作用.     

兔骨髓间充质干细胞体外分离培养及多向诱导分化*

背景：间充质干细胞不仅自身免疫原性弱,还可以调节细胞免疫功能,减轻移植物排斥反应,在组织工程中具有良好的应用前景。但骨髓中间充质干细胞含量稀少,约占单个核细胞的十万分之一到百万分之一。 目的：建立一种分离、培养扩增兔骨髓间充质干细胞的方法,观察体外培养骨髓间充质干细胞的生长特性,及其潜在的诱导分化能力。 方法：采用灌流法获取兔胫骨骨髓,密度梯度离心法联合贴壁培养法体外纯化扩增,相差显微镜观察其形态学特点,MTT法测定绘制传代骨髓间充质干细胞生长曲线,经成骨诱导液(L-DMEN/F12,体积分数为10%胎牛血清,0.1 μmol/L地塞米松,200 μmol/L抗坏血酸,10 mmol/L β-甘油磷酸钠)、成脂诱导液(L-DMEN/F12,体积分数为10%胎牛血清,1 μmol/L地塞米松,200 μmol/L吲哚美辛,0.5 mmol/L IBMX,10 mg/L胰岛素)、成软骨诱导液(L-DMEN/F12,体积分数为10%胎牛血清,10 μg/L转化生长因子β1,0.1 μmol/L地塞米松,50 μmol/L抗坏血酸,6.25 mg/L胰岛素)体外诱导骨髓间充质干细胞向成骨细胞、脂肪细胞及软骨细胞分化,并分别经碱性磷酸酶染色、油红O染色和甲苯胺蓝染色法鉴定。 结果与结论：通过密度梯度离心法联合贴壁培养法可在体外大量扩增、纯化骨髓间充质干细胞,所获细胞具有高度自我更新能力和多向分化潜能,原代及传代骨髓间充质干细胞为梭形。经成骨细胞诱导,细胞碱性磷酸酶染色阳性;经成脂肪细胞诱导,细胞内出现红色脂滴,经成软骨细胞诱导,甲苯胺蓝染色阳性。     

兔骨髓间充质干细胞体外分离培养及多向诱导分化

背景:间充质干细胞不仅自身免疫原性弱,还可以调节细胞免疫功能,减轻移植物排斥反应,在组织工程中具有良好的应用前景.但骨髓中间充质干细胞含量稀少,约占单个核细胞的十万分之一到百万分之一.目的:建立一种分离、培养扩增兔骨髓间充质干细胞的方法,观察体外培养骨髓间充质干细胞的生长特性,及其潜在的诱导分化能力.方法:采用灌流法获取兔胫骨骨髓,密度梯度离心法联合贴壁培养法体外纯化扩增,相差显微镜观察其形态学特点,MTT法测定绘制传代骨髓间充质干细胞生长曲线,经成骨诱导液(L-DMEN/F12,体积分数为10%胎牛血清,0.1 μmol/L地塞米松,200 μmol/L抗坏血酸,10 mmol/L β-甘油磷酸钠)、成脂诱导液(L-DMEN/F12,体积分数为10%胎牛血清,1 μmol/L地塞米松,200 μmol/L吲哚美辛,0.5 mmol/L IBMX,10 mg/L胰岛素)、成软骨诱导液(L-DMEN/F12,体积分数为10%胎牛血清,10 μg/L转化生长因子β1,0.1 μmol/L地塞米松,50 μmol/L抗坏血酸,6.25 mg/L胰岛素)体外诱导骨髓间充质干细胞向成骨细胞、脂肪细胞及软骨细胞分化,并分别经碱性磷酸酶染色、油红O染色和甲苯胺蓝染色法鉴定.结果与结论:通过密度梯度离心法联合贴壁培养法可在体外大量扩增、纯化骨髓间充质干细胞,所获细胞具有高度自我更新能力和多向分化潜能,原代及传代骨髓间充质干细胞为梭形.经成骨细胞诱导,细胞碱性磷酸酶染色阳性;经成脂肪细胞诱导,细胞内出现红色脂滴,经成软骨细胞诱导,甲苯胺蓝染色阳性.     

人脂肪间充质干细胞的分离培养及生物学特性研究

目的：探索高效分离和扩增人脂肪间充质干细胞（adipose derived mesenchymal stem cells,ADSCs）的方法,观察其生物学特性,并通过形态学、免疫表型及多向分化能力进行鉴定。方法：以手术中剩余的人皮下脂肪组织为材料来源,利用胶原酶消化法分离ADSCs,体外扩增后传代,倒置显微镜观察,MTT比色法测定细胞生长曲线,流式细胞仪检测细胞表面标记CD29、CD31、CD34、CD45、CD90、CD105的表达。在DMEM及胎牛血清培养基、地塞米松、IBMX、吲哚美辛的诱导下向脂肪细胞定向分化;在DMEM及胎牛血清培养基、地塞米松、抗坏血酸、β-磷酸甘油、胰岛素的诱导下向成骨细胞定向分化。结果：原代和传代细胞呈梭形外观,生长增殖能力良好。细胞传代后2天内处于潜伏期,第3天进入生长期,5天后进入平台期。流式细胞仪检测CD29、CD31、CD34、CD45、CD90、CD105阳性率分别为73.4%、3.6%、4.5%、2.0%、97.3%、80.4%。经定向诱导分化后,细胞分别呈现脂肪细胞、骨细胞的表型特征。结论：酶消化法能有效分离纯化人ADSCs,细胞生长稳定,增殖能力活跃,具有ADSCs的一般生物学特性,为其成为组织工程理想的种子细胞提供了进一步的支持。     

研究型医院科技创新体系构建

研究型医院是以医学知识、医疗技术为基础,坚持临床与科研教学并举,不断推动临床诊疗水平、社会卫生事业,以及人类健康持续稳定发展的新型的一流现代化医院。构建符合其发展要求的科技创新管理体系,是保证研究型医院自主创新、高新技术研发、科技成果转化,以及高层次人才培养等内涵要素持续提高的关键环节。