人肾间质纤维母细胞培养及血管紧张素Ⅱ2型受体转染表达

目的：体外培养人肾间质纤维母细胞（hRIF)并用腺病毒介导转染表达血管紧张素Ⅱ（AngⅡ）2型受体（AT2R）。方法：用肾穿刺活检法取慢性肾小球肾炎病人肾组织,以常规组织块贴壁法培养hRIF;构建带AT2R基因的重组复制缺陷型腺病毒载体（AdCMV-AT2R),体外转染hRIF;用RT-PCR方法检测AT2RmRNA表达,免疫组织化学法及蛋白免疫印迹法检测AT2R蛋白表达,流式细胞仪检测AT2R表达0率,同时检测AT1R的表达变化。结果：用肾活检组织成功培养出hRIF,构建的AdCMV-AT2R体外转染培养hRIF表达率为90.57%,以免疫组化、免疫印迹和RT-PCR检测AT2R表明,转染后AT2R表达明显增强。并随表达时间延长而增加,48h为表达峰值。AT2R转染表达不影响AT1R表达,结论：腺病毒载体可较高效率介导AT2R在hRIF的转染表达,ATIR表达不受其影响。转染表达AT2R的hRIF可作为研究AngⅡ在肾间质纤维化中作用的良好细胞模型。     

IRX1真核表达载体的构建及在胃癌细胞SGC-7901中的定位

目的 构建pEGFP-IRX1真核表达载体,体外转导至SGC-7901胃癌细胞株,观察IRX1基因转染后蛋白表达与细胞内定位.方法 PCR扩增IRX1全长1443 bp编码序列,将PCR扩增产物连接人pGEM-Teasy载体,经测序确认序列无误后,亚克隆入pEGFP-N1载体,构建pEG-FP-IRX1真核表达载体.采用Lipofectamine 2000转染SGC-7901胃癌细胞株,在荧光显微镜下观察外源性IRX1基因导入后在胃癌细胞内的表达与亚细胞定位.结果 成功构建有绿色荧光蛋白融合的IRX1真核表达载体,外源性IRX1基因在胃癌SGC-7901细胞中表达成功,在荧光显微镜下IRX1表达蛋白定位于细胞核内.结论 pEGFP-IRX1真核表达载体构建成功,并可在细胞内表达,这将为IRX1在胃癌内的功能研究奠定基础.     

携带ROCK2基因siRNA有效片段的慢病毒载体的筛选

目的:筛选能够显著抑制自发性高血压大鼠(SHR)阴茎海绵体平滑肌细胞内ROCK2基因表达的携带ROCK2基因siRNA的慢病毒载体。方法:设计并合成4个靶向ROCK2的siRNA片段,构建并包装成慢病毒载体。随机将5只SHR阴茎海绵体平滑肌细胞分为6组,每组每个样本3×104个细胞,每组5个样本,分别为:A组(未转染对照组)、B组(携带慢病毒转染组)、C~F组(分别携带靶向ROCK2基因siRNA 1~4号靶点的慢病毒转染组),以感染复(MOI)=80转染SHR阴茎海绵体平滑肌细胞,转染后48 h荧光显微镜下观察细胞GFP表达情况,并用RT-PCR检测各组被转染细胞ROCK2 mRNA的表达。结果:荧光显微镜下观察各组细胞转染效率均50%。与A组相比,B组ROCK2 mRNA的表达无明显改变(P﹥0.05);C、D、F组SHR阴茎海绵体平滑肌细胞ROCK2基因mRNA的表达较A组极显著下降(P0.01),抑制效率分别达到(43.91±8.19)%、(47.15±6.64)%、(25.7±6.03)%;E组SHR阴茎海绵体平滑肌细胞ROCK2基因mRNA的表达较A组显著下降(P0.05),抑制效率为(16.81±5.94)%。结论:本研究构建的4种携带ROCK2基因的siRNA慢病毒载体均能够显著抑制SHR阴茎海绵体平滑肌细胞内ROCK2基因的表达,其中有1种慢病毒载体抑制作用最强。     

Source–receptor probability of atmospheric long‐distance dispersal of viruses to Israel from the eastern Mediterranean area

Viruses that affect the health of humans and farm animals can spread over long distances via atmospheric mechanisms. The phenomenon of atmospheric long‐distance dispersal (LDD ) is associated with severe consequences because it may introduce pathogens into new areas. The introduction of new pathogens to Israel was attributed to LDD events numerous times. This provided the motivation for this study which is aimed to identify all the locations in the eastern Mediterranean that may serve as sources for pathogen incursion into Israel via LDD . This aim was achieved by calculating source–receptor relationship probability maps. These maps describe the probability that an infected vector or viral aerosol, once airborne, will have an atmospheric route that can transport it to a distant location. The resultant probability maps demonstrate a seasonal tendency in the probability of specific areas to serve as sources for pathogen LDD into Israel. Specifically, Cyprus’ season is the summer; southern Turkey and the Greek islands of Crete, Karpathos and Rhodes are associated with spring and summer; lower Egypt and Jordan may serve as sources all year round, except the summer months. The method used in this study can easily be implemented to any other geographic region. The importance of this study is the ability to provide a climatologically valid and accurate risk assessment tool to support long‐term decisions regarding preparatory actions for future outbreaks long before a specific outbreak occurs.     

广西环江县传疟按蚊种群密度变化

目的 分析原以嗜人按蚊为主要传疟媒介的广西环江县在消除当地疟疾后的传疟按蚊种类及密度监测结果。方法 收集整理该县历年人房和牛房传疟按蚊资料及近2年按蚊监测数据,采用Excel 2003版软件进行统计分析和图表制作。结果 该县历史上存在按蚊种类20种,传疟按蚊种群密度依次为嗜人按蚊、中华按蚊、微小按蚊、日月潭按蚊。嗜人按蚊的人房密度高于其它传疟按蚊,季节消长与当地疟疾发病高峰密切相关,嗜人按蚊的唾液腺子孢子自然感染率为0.3%（5/1 585）。1982—1983年采用通宵人工捕蚊的方法观察结果显示,人房嗜人按蚊密度占55.68%,中华按蚊占21.77%,日月潭按蚊占19.81%,微小按蚊占0.05%。牛房嗜人按蚊密度占6.21%,中华按蚊占67.55%,日月潭按蚊占13.89%,微小按蚊占0.07%。2015—2016年在该县大安乡采用通宵灯诱方法捕蚊观察,2015年结果显示,牛房、室外和人房共捕获按蚊604只,经鉴定全部为中华按蚊,其中牛房占35.43%,室外占34.27%,人房占30.30%。2016年结果显示,牛房、室外和人房共捕获按蚊811只,经鉴定99.63%为中华按蚊,其中牛房占54.25%,室外占36.37%,人房占9.00%,在5月份的牛房和室外分别捕获嵌斑按蚊1只和日月潭按蚊2只。结论 该县目前的传疟媒介是以单一的中华按蚊为优势蚊种。     

慢病毒介导FGFR1 沉默对肺癌细胞 增殖和凋亡的影响

目的 探讨慢病毒介导成纤维生长因子受体1（FGFR1）沉默对肺癌细胞增殖和凋亡的影响。 方法 构建慢病毒表达载体LV-shFGFR1,对其进行包装和病毒颗粒制备。将A549 肺癌细胞分为干扰组、 空载体组及对照组。采用实时荧光定量聚合酶链反应和Western blotting 检测各组细胞FGFR1 mRNA 和蛋白 相对表达量,MTT 比色法检测细胞增殖情况,流式细胞术和Hoechest 染色检测细胞凋亡情况。结果 ① 3 株 肺癌细胞中,A549 细胞的FGFR1 mRNA 相对表达量高于H3255 和A-427 细胞（P <0.05）。② 3 组A549 细 胞中FGFR1 mRNA 和蛋白的相对表达量比较,差异有统计学意义（P <0.05）;空载体组与对照组比较,差异 无统计学意义（P >0.05）;干扰组低于对照组（P <0.05）。③干扰组、空载体组、对照组0、12、24、48 和 72 h 的A549 细胞增殖情况比较,不同时间点、各组间、随时间变化趋势均有差异（P <0.05）。④ 3 组A549 细胞凋亡率比较,差异有统计学意义（P <0.05）;空载体组与对照组比较,差异无统计学意义（P >0.05）;干 扰组高于对照组（P <0.05）。结论 慢病毒介导FGFR1 沉默抑制A549 肺癌细胞增殖,同时促进肺癌细胞凋亡, 提示FGFR1 可能参与肺癌细胞的发生、发展过程。     

miR-186海绵载体的构建及其在EA.hy926细胞株中的表达

目的：构建微小RNA-186（miR-186）基因的海绵载体,建立稳定敲减miR-186的EA.hy926细胞株。方法：化学合成miR-186海绵体序列,并克隆到慢病毒FV040表达载体上;采用lipofectamine 2000共转染FV040-miR-186-sponge载体和慢病毒辅助包装质粒到HEK293T细胞,包装慢病毒,测定病毒滴度;FV040-control慢病毒作为对照组,FV040-miR-186-sponge作为实验组,分别感染EA.hy926细胞,筛选稳转细胞株;采用荧光定量PCR （qPCR）法检测空白对照组、FV040-control对照组及FV040-miR-186-sponge实验组中EA.hy926细胞miR-186的相对表达水平。结果：测序结果显示克隆的目的基因序列与设计的miR-186海绵体序列完全一致。在感染的EA.hy926细胞中观察到绿色荧光蛋白（GFP）的表达。FV040-control包装的慢病毒滴度为2×10⁸ TU·mL^-1,FV040-miR-186-sponge组慢病毒滴度为6×10⁸ TU·mL^-1;成功建立了稳定表达miR-186-sponge的EA.hy926细胞株,感染效率达95%;qPCR检测,与空白对照组和FV040-control组比较,FV040-miR-186-sponge组EA.hy926细胞中miR-186相对表达水平降低（P<0.01）。结论：成功构建了miR-186基因的海绵载体,建立了稳定下调miR-186表达的EA.hy926-miR-186-sponge细胞株。     

绿色荧光蛋白基因逆转录病毒载体转染人骨髓间充质干细胞及表达的研究

目的　绿色荧光蛋白 (EGFP)基因的逆转录病毒载体转染人骨髓间充质干细胞(MSCs)及其表达情况。方法　pEGFP与逆转录病毒载体经过酶切、连接等构建重组的EGFP 逆转录病毒载体 ,转染PA3 17细胞 ,用G418筛选出抗性克隆 ,获病毒上清 (滴度达到 8.5× 10 5cfu/ml)并感染人MSCs。流式细胞仪测定转染效率后加入成骨细胞分化培养夜 (地塞米松 (10 -7mol/L)、β 甘油磷酸钠 (10mmol/L)、维生素C(5 0mg/L ) ) ,2周后观察转染细胞的分化情况。 结果　 48h后细胞转染效率为 7% ,G418筛选 2周后约 97%细胞出现抗性克隆 ,表达维持 4周。基因转染细胞能够分化为成骨细胞。结论　重组EGFP逆转录病毒载体能转染MSCs ,且不影响细胞的功能。     

A divide-and-combine method for large scale nonparallel support vector machines

Nonparallel Support Vector Machine (NPSVM) which is more flexible and has better generalization than typical SVM is widely used for classification. Although some methods and toolboxes like SMO and libsvm for NPSVM are used, NPSVM is hard to scale up when facing millions of samples. In this paper, we propose a divide-and-combine method for large scale nonparallel support vector machine (DCNPSVM). In the division step, DCNPSVM divide samples into smaller sub-samples aiming at solving smaller subproblems independently. We theoretically and experimentally prove that the objective function value, solutions, and support vectors solved by DCNPSVM are close to the objective function value, solutions, and support vectors of the whole NPSVM problem. In the combination step, the sub-solutions combined as initial iteration points are used to solve the whole problem by global coordinate descent which converges quickly. In order to balance the accuracy and efficiency, we adopt a multi-level structure which outperforms state-of-the-art methods. Moreover, our DCNPSVM can tackle unbalance problems efficiently by tuning the parameters. Experimental results on lots of large data sets show the effectiveness of our method in memory usage, classification accuracy and time consuming.     

Noninvasive recording of His bundle electrograms using vectorial leads system

His bundle electrograms were recorded in 8 rats by a signal averaging technique using the Takayasu vectorial lead system. Animals were anesthetized and placed in the prone position. The polarity of three orthogonal leads were: the X axis, from right to left; the Y axis, from top to bottom; the Z axis, from back to front. Potentials from the X, Y, and Z leads were amplified by 20,000 with high-pass 12 dB/octave filtering at 80 Hz, and signal averaging of 2000 beats was performed at a sampling interval of 100 microseconds. The His bundle potential could be clearly defined in all 8 rats. The mean amplitude of the His potential was larger in the X-axis (23.6 +/- 9.2 mu V) or the Y-axis (28.4 +/- 14.5 mu V) than the Z-axis lead (11.5 +/- 8.6 mu V). Directions of the His potential vectors were to the left in 5 of 8 rats (62.5%), to the caudal in 4 of 6 rats (66.7%), and to the dorsal site in 6 of 8 rats (75.0%). This vectorial lead system, devised in accordance with McFee and Johnston's theory on lead field, was useful for recording His bundle electrograms.