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Establishment of a recombinant adeno-associated virus expressing hVEGF_(165)

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Neural Regeneration Research 2008年第6期3卷 610-613页

作者： Xianghui Huang Zhibin Shi Xiaoqian Dang Chen Zhang pengbo yu Kunzheng Wang Department of Orthopedics Second Affiliated Hospital of Xi＇an Jiaotong University Xi＇an 710004 Shaanxi Province China Laboratory of Shaanxi Provincial Center for Disease Control and Prevention Xi＇an 710054 Shaanxi Province China

BACKGROUND： Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE： To construct a non-pathogenic, recombinant adeno-associated virus （AAV） simultaneously expressing human vascular endothelial growth factor 165 （hVEGF165） and green fluorescent protein （GFP）. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS： AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS： The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC （the rep/cap-gene containing plasmid）, and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES： Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter （FACS） upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS： The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. Th

关键词： vascular endothelial factor adeno-associated virus nerve regeneration

2020—2022年陕西省手足口病病原的监测及主要型别的流行特征分析

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天津医药 2024年第4期52卷 432-437页

作者：杜燕华关路媛冯瑄郑媛余鹏博陕西省疾病预防控制中心 710054

目的了解2020—2022年陕西省手足口病主要病原构成情况和流行规律。方法收集陕西省各省辖市手足口监测病例的核酸检测结果和病例资料,分离肠道病毒核酸检测阳性样本,并对流行优势病原的肠道病毒VP1区进行PCR扩增、序列测定和毒株基因... 详细信息

目的了解2020—2022年陕西省手足口病主要病原构成情况和流行规律。方法收集陕西省各省辖市手足口监测病例的核酸检测结果和病例资料,分离肠道病毒核酸检测阳性样本,并对流行优势病原的肠道病毒VP1区进行PCR扩增、序列测定和毒株基因亚型鉴定。结果 2020—2022年陕西省共采集6 352例手足口病例样本,肠道病毒核酸检测阳性4 417例(69.54%)。2020年和2021年,以除外EV-A71和EV-A16的其他肠道病毒为主要流行型别,构成比分别为95.43%和74.35%;2022年,以CV-A16型为主要流行型别,构成比52.88%。对8个省辖市4 145例手足口实验室确诊病例开展其他肠道病毒中CV-A6型和CV-A10型检测,2020年—2022年CV-A6型在其他肠道病毒的占比分别是75.31%、62.56%和61.15%,是陕西省其他肠道病毒中的优势流行型别。2020—2022年陕西省分离测序毒株239株,CV-A16型151株,CV-A6型72株,CV-A10型6株,CV-A4型2株,CV-B3型8株。CV-A16型和CV-A6型是主要流行型别,经系统进化分析,CV-A6型以D3亚型为主要流行亚型,CV-A16型以B1a和B1b共存,2021年和2022年B1a亚型占比逐年升高,并取代B1b成为主要流行亚型。结论 2020—2022年陕西省手足口病较往年处于低流行水平,主要病原以其他肠道病毒、CVA16交替出现。

关键词：手足口病肠道病毒病原构成系统进化树分析陕西

经皮耳甲迷走神经刺激治疗慢性意识障碍临床观察

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中国实用神经疾病杂志 2024年第5期27卷 552-556页

作者：雷彪王晓峰杨若琳于蓬勃李加龙邵威乔育陕西中医药大学陕西咸阳712046 宝鸡市中医医院陕西宝鸡721001 宝鸡第三医院陕西宝鸡721004

目的观察经皮耳甲迷走神经刺激(taVNS)治疗慢性意识障碍(pDoC)的临床效果。方法选取宝鸡第三医院2020-06—2022-12收治的68例pDoC患者,随机分成对照组与治疗组各34例。对照组接受常规促醒治疗,治疗组在对照组的基础上接受taVNS治疗,2... 详细信息

目的观察经皮耳甲迷走神经刺激(taVNS)治疗慢性意识障碍(pDoC)的临床效果。方法选取宝鸡第三医院2020-06—2022-12收治的68例pDoC患者,随机分成对照组与治疗组各34例。对照组接受常规促醒治疗,治疗组在对照组的基础上接受taVNS治疗,2组患者均治疗4周。对比2组患者格拉斯哥昏迷量表(GCS)评分、昏迷恢复量表(CRS-R)评分、全身无反应性量表(FOUR)评分、安全性及临床疗效。结果治疗后,2组患者的GCS单项评分(睁眼、言语和运动)及总分、CRS-R单项评分(听觉、视觉、运动、言语、交流和唤醒)及总分和FOUR单项评分(眼部、运动、脑干和呼吸)及总分均升高(均P0.05)。2组患者均未发生不良事件。治疗组总有效率(79.41%)显著高于对照组(61.76%)(P<0.05)。结论 taVNS治疗pDoC有一定的疗效,可改善患者意识障碍程度,且安全可靠。

关键词：经皮耳甲迷走神经刺激慢性意识障碍重型颅脑损伤

Mild Breakthrough Infection in a Healthcare Professional Working in the Isolation Area of a Hospital Designated for Treating COVID-19 Patients—Shaanxi Province,China,March,2021

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China CDC weekly 2021年第19期3卷 397-400页

作者： Chaofeng Ma Songtao Xu Yecheng Yao pengbo yu You Xu Rui Wu Hailong Chen Xiaoping Dong Xi’an Center for Diseases Control and Prevention Xi’anShaanxiChina State Key Laboratory for Infectious Disease Prevention and Control Collaborative Innovation Center for Diagnosis and Treatment of Infectious DiseasesNational Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijingChina National Institute for Nutrition and Health Chinese Center for Disease Control and PreventionBeijingChina Shaanxi Center for Diseases Control and Prevention Xi’anShaanxiChina Xi’an No.8 Hospital Xi’anShaanxiChina Center for Global Public Health Chinese Center for Disease Control and PreventionBeijingChina Center for Biosafety Mega-Science Chinese Academy of SciencesWuhanHubeiChina China Academy of Chinese Medical Sciences BeijingChina.

Summary What is already known about this topic?Healthcare workers are at high risk of acquiring COVID-19 from occupational exposure to COVID-19 virus during their daily medical service *** infection prevention and control measures and adequate personal protective equipment(PPE)are essential to reduce the risk of hospital-acquired COVID-19.

关键词： prevention protective COV

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一株丁酸梭菌的分离鉴定及生物学特性研究

家禽科学 2024年第7期46卷 9-17,I0007页

作者：翟凯旋李金香冷敦鹏臧学运于蓬勃苗越齐慧东青岛中仁动物药品有限公司山东青岛266300 威海市文登区米山畜牧兽医工作站山东威海264200

本试验通过厌氧培养法从健康鸡的新鲜粪便中分离筛选得到1株菌株,从形态学观察、生理生化试验和16S rDNA基因序列分析等方面对菌株进行鉴定,确定该菌株为丁酸梭菌。对其生物学特性进一步研究,发现该菌株能够产生乙酸、丁酸等挥发性脂肪... 详细信息

本试验通过厌氧培养法从健康鸡的新鲜粪便中分离筛选得到1株菌株,从形态学观察、生理生化试验和16S rDNA基因序列分析等方面对菌株进行鉴定,确定该菌株为丁酸梭菌。对其生物学特性进一步研究,发现该菌株能够产生乙酸、丁酸等挥发性脂肪酸,对产气荚膜梭菌、金黄色葡萄球菌、沙门氏菌、大肠杆菌等多种肠道致病菌均有一定的抑菌能力。综上所述,该菌株在生长、抗逆、产酸、抑菌方面表现较好,具有很好的应用价值。

关键词：丁酸梭菌分离鉴定生物学特性

Construction of recombinant adeno-associated virus vector co-expressing hVEGF_(165) and hBMP_7 gene

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Journal of Nanjing Medical University 2008年第4期22卷 205-210页

作者： Zhibin Shi Xianghui Huang Kunzheng Wang Xiaoqian Dang Pei Yang pengbo yu Department of Orthopedics the Second Affiliated Hospital of Xi＇ an Jiaotong University Xi＇ an 710004 Shaanxi Province China Laboratory of Virus Shaanxi Provincial Center for Disease Control and Prevention Xi＇ an 710054 Shaanxi Province China

Objective： To construct recombinant adeno-associated virus co-expressing human vascular epithelial growth factor 165（hVEGF165） and bone morphogenetic protein 7（hBMPT）, measure the virus titer and verify the recombination. Methods：The AAV helper-free system was used as basis to generate recombinant AAV. The IRES sequence of plasmid plRES was cut down and subcloned into ITR/ MCS containing vector pAAV-MCS to construct recombinant plasmid pAAV-MCSa-IRES-MCSb. The hVEGF165 and hBMP7 gene was amplified by PCR and inserted into upstream MCSa and downstream MCSb respectively. Then, recombinant plasmid pAAV- hVEGF165-IRES-hBMP7, pAAV-RC and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hBMP7 packaging. The GFP labeled rAAV-IRES-GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-hrGFP. The efficiency of AAV packaging was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells. The virus titer was measured by infecting AAV-HT1080 cells, and the recombinant AAV-hVEGF165-IRES-hBMP7 was verified by PCR of the exogenous interest genes. Results：Recombinant pAAV-hVEGF165-IRES-hBMP7 was verified by double digestion. GFP expression in AAV-293 could be observed under fluorescent microscope 72 h after transfection and the system provided a high packing ratio of 95%. The recombinant adeno-associated virus has a high titer of 5.5 ×10^11vp/ml, and AAV-HT 1080 was infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP7 and hVEGF165 gene. Conclusion：Recombinant rAAV-hVEGF165-IRES-hBMP7 was successfully constructed with a high virus titer, which may offer foundation for in vitro and in vivo experiments of hVEGF165 and hBMP7 co-expression and provide a new method for gene therapy of bone regeneration.

关键词： adeno-associated virus human vascular endothelial factor human bone morphogenetic protein intemal ribosome entry site

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洼38块蒸汽驱开发技术研究

油气藏评价与开发 2015年第2期5卷 42-44页

作者：于蓬勃中国石油辽河油田金马油田开发公司辽宁盘锦124010

以小洼油田洼38块东三段油层蒸汽驱先导试验为例,分析了深层特稠油油藏蒸汽驱采用常规反九点井网开发存在的技术瓶颈,提出高干度注汽、"以采定注"、"回字型"井网设计等改善深层特稠油蒸汽驱开发效果的新思路,对于同类油藏开发有参考价值。

关键词：蒸汽驱深层特稠油油藏井网采注比高干度

低含油饱和度油藏成因研究与开发实践——以辽河油田W38块东二段为例

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油气藏评价与开发 2015年第4期5卷 38-41页

作者：于蓬勃中国石油辽河金马油田开发公司辽宁盘锦124010

辽河油田W38块东二段直井开发存在油井低产液、普遍高含水等问题,断块长期低速开采。利用测井解释、岩心分析、数值模拟及动态分析等技术手段,揭示了区块为含有可动水的低含油饱和度油藏。从油气运移及成藏机理出发,研究了油藏低含油饱... 详细信息

辽河油田W38块东二段直井开发存在油井低产液、普遍高含水等问题,断块长期低速开采。利用测井解释、岩心分析、数值模拟及动态分析等技术手段,揭示了区块为含有可动水的低含油饱和度油藏。从油气运移及成藏机理出发,研究了油藏低含油饱和度的成因。按照分层开发的技术思路,利用水平井技术提高单井产量,累计实施水平井20口,区块产量由3.2万吨上升至6.7万吨,采收率提高7.8%,大幅度改善了W38块东二段低含油饱和度油藏的开发效果。

关键词：低含油饱和度油藏水平井分层开发提高采收率

利用非溶剂诱导相分离法制备低介电常数聚酰亚胺微孔薄膜

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高分子材料科学与工程 2018年第3期34卷 132-137页

作者：吴鹏李忠伦余智刘鹏波高分子材料工程国家重点实验室四川大学高分子研究所四川成都610065

利用非溶剂诱导相分离法,制备了一种低介电常数聚酰亚胺(PI)微孔薄膜。扫描电镜观察表明,聚酰亚胺微孔薄膜中泡孔均匀,其平均孔径在6~28μm之间。随着聚酰胺酸(PAA)浓度的增大及凝固浴温度的升高,所制得的聚酰亚胺微孔薄膜的孔隙率和平... 详细信息

利用非溶剂诱导相分离法,制备了一种低介电常数聚酰亚胺(PI)微孔薄膜。扫描电镜观察表明,聚酰亚胺微孔薄膜中泡孔均匀,其平均孔径在6~28μm之间。随着聚酰胺酸(PAA)浓度的增大及凝固浴温度的升高,所制得的聚酰亚胺微孔薄膜的孔隙率和平均孔径均增大。介电性能分析表明,聚酰亚胺微孔薄膜的介电常数明显下降,当孔隙率为80%时,聚酰亚胺微孔膜的介电常数(1 MHz)为1.81。拉伸性能测试表明,随着孔隙率增加,聚酰亚胺微孔膜的拉伸强度和拉伸模量均逐渐下降,但断裂伸长率增大。

关键词：非溶剂诱导相分离聚酰亚胺低介电常数微孔薄膜