目的:β淀粉样蛋白(β-amyloid protein,Aβ)聚集形成的老年斑是阿尔茨海默病(Alzheimer's disease,AD)的重要病理特征之一,由淀粉样前体蛋白(amyloid precusor protein,APP)经成淀粉途径多次水解形成.人APP基因表达受可变剪接调控,人脑内有三种APP可变剪接异构体产物.根据这些APP可变剪接异构体是否表达外显子7,可分为APP-KPI+和APP-KPI-两种类型.据报道,APP-KPI+在脑内的表达水平与Aβ的产生呈正相关.GSK3β是脑内和AD相关的最主要的激酶之一,它的活性在AD脑内增加,是AD治疗中的一个潜在的重要靶点.我们的前期研究发现,二苯乙烯苷(stibene glucoside,TSG)能够减少APP转基因小鼠脑内Aβ含量和淀粉样斑块数量,改善学习记忆功能,但TSG对APP可变剪接和GSK3β的影响尚不清楚.方法:构建包含APP外显子7、8可变剪接的微型基因(mini-gene).人胚胎肾细胞(HEK-293FT)、小鼠神经母细胞瘤细胞(Neuro-2a,N2a)内转染APP mini-gene来模拟APP外显子7、8在体内的剪接.在神经细胞N2a内过表达GSK3β或下调其表达,用RT-PCR和qPCR观察GSK3β对APP可变剪接的影响.在HEK-293FT细胞内共表达剪接因子ASF和磷酸激酶GSK3β,用HA抗体免疫共沉淀ASF,GSK3β抗体检测ASF与GSK3β之间是否存在相互作用.将ASF、GSK3β单独或者共转染至HeLa细胞中,用荧光二抗进行染色,观察ASF和GSK3β的细胞定位.APP/PS1转基因鼠TSG(50 mg·kg-1·day-1)灌胃处理12个月,用Western blot或qPCR观察鼠脑内相关蛋白在mRNA和蛋白水平发生的改变.结果:APP mini-gene在HEK-293FT和N2a细胞系内进行表达,RT-PCR结果可见三个条带,经测序分别是APP770,APP751和APP695.过表达GSK-3β促进APP微型基因产物APP-KPI+在N2a细胞内的表达,siGSK3β则抑制APP-KPI+表达量;不同浓度LiCl处理SH-SY5Y细胞,内源性APP-KPI表达量随LiCl浓度增高而降低.SR蛋白ASF对APP-KPI+的表达影响最大,是最为重要的APP可变剪接调控因子.GSK3β可以被ASF免疫共沉淀,存在生理上的相互作用,并且免疫共定位实验显示ASF与GSK3β有很好的细胞内共定位.TSG抑制细胞内APP-KPI+的表达量,同时发现细胞内AKT-GSK3β信号通路可被TSG激活.在体内实验中,5月龄APP/PS1转基因小鼠灌胃给药TSG12个月后,脑内AKT和GSK3β磷酸化水平增加,APP-KPI+表达水平降低.结论:成功构建了研究APP-KPI表达的微型基因.TSG可以激活神经细胞和APP/PS1转基因鼠脑内的AKT-GSK3β通路;GSK3β通过与剪接因子ASF相互作用,从而抑制其促进APP-KPI+生成的能力;在体内长期喂食TSG可降低APP-KPI+表达.

Objective To explore the effects of tetrahydroxystilbene glucoside (TSG) on the expression of amyloid precursor protein (APP) and its mechanism in cultured nerve cells.Methods The expression of APP and cAMP responsive element-binding protein (CREB) was detected by Western blotting assay.The CRE cis-elements of APP promoter were analyzed by Matinspector software.The plasmids expressing serial deletion mutants of APP promoter were constructed.The luciferase activity in cells was measured by luciferase double reporter gene system.Results Activation of CREB by Forskolin down-regulated APP expression in SH-SY5Y cells.The luciferase expression plasmids of APP promoter containing CRE elements were successfully constructed.TSG treatment decreased APP level in SH-SY5Y cells,and reduced luciferase activity of APP reporter gene containing CRE1 and CRE2 cis-elements in APP promoter plasmidstransfected cells.Conclusion TSG down-regulated APP expression in nerve cells,and its mechanism may be related to affecting the binding of CREB with CRE eis-elements.

目的 探讨重组人脑利钠肽(rhBNP)对重症心力衰竭患者血清氨基末端脑利钠肽前体(NT-proBNP)和心肌钙蛋白T(cTnT)的影响.方法 21例重症心力衰竭患者行rhBNP治疗,观察治疗前后心率、血压、24-h尿量、血清NT-proBNP和cTnT含量的变化.结果 与治疗前相比,治疗后24-h尿量增多(P＜0.01),心率、收缩压及血清cTnT水平下降(P＜0.01或P＜0.05),NT-proBNP水平无明显改变(P＞0.05).血清NT-proBNP与eTnT水平呈正相关(r=0.735,P＜0.01).结论 rhBNP对重症心力衰竭患者有治疗作用,可在一定程度上改善心肌损伤程度.

目的：：评价全心舒张末期容积(global end diastolic volume index, GEDVi)以及胸腔内血容积(intrathoracic blood volume index, ITBVi)在急性心力衰竭患者治疗过程中的价值和意义。方法：对23例急性心力衰竭患者的脑利钠肽(brain natriuretic peptide, BNP)、GEDVi以及ITBVi进行回顾性分析，观察其治疗前后的变化，同时对其变化进行相关性分析。结果：患者入院治疗1周后BNP、GEDVi以及ITBVi均明显下降(P<0.001)，且BNP的变化与GEDVi及ITBVI两者的变化值呈正相关。结论：监测GEDVi和ITBVi对急性心力衰竭患者的治疗效果及评价预后有一定的指导意义。