抑瘤细胞运动和侵袭的鼠FRNK重组腺病毒的构建

目的应用AdEasy复制缺陷型腺病毒载体系统构建小鼠FRNK基因重组腺病毒，并在293HEK细胞中扩增制备重组病毒。方法把小鼠FRNK基因克隆入腺病毒穿梭载体pAdTrack—CMV中，构建腺病毒穿梭质粒pAdTrack—CMV-FRNK，经PmeⅠ内切酶酶切成线性化后，采用电穿孔转化将其转化到含有腺病毒骨架载体pAdEasy-1的感受态大肠杆菌BJ5183中，通过卡那霉素筛选获得阳性腺病毒重组质粒。再将获得的腺病毒重组质粒转染到293HEK细胞进行包装，获得FRNK重组腺病毒pAdFRNK。荧光显微镜下观察绿荧光的表达。结果经酶切和绿荧光表达证明了小鼠FRNK基因的重组腺病毒载体pAdFRNK构建成功，并制备出高滴度的重组病毒。结论成功构建携带小鼠FRNK基因的重组腺病毒pAdFRNK，为研究FRNK的基因治疗奠定了基础。     

双片段survivin短发夹RNA腺病毒载体的构建与鉴定

目的:构建负载双片段survivin短发夹RNA(shRNA)的腺病毒载体,为PCa的基因治疗提供实验基础。方法:分别设计2条靶向survivin基因的shRNA,克隆至穿梭质粒载体中,特异性酶切后亚克隆至腺病毒骨架,构建负载2条survivin shRNA的RNA干扰重组腺病毒载体。提取环状重组病毒质粒进行PCR、酶切双重鉴定后,大规模提取纯化正确的克隆。最后将线性化的重组腺病毒基因组转染至HEK293A包装细胞包装成病毒,并扩增到所需滴度。结果:负载双片段survivin shRNA的穿梭质粒载体pShuttle2经MluⅠ酶切鉴定,证实双片段survivinshRNA插入正确;完成目的片段至腺病毒骨架的亚克隆后,PCR及PI-SceⅠ/I-CeuⅠ双酶切鉴定均证实双片段sur-vivin shRNA插入正确。结论:负载双片段survivin shRNA腺病毒载体构建成功。     

应用AdEasy腺病毒载体系统构建血管内皮生长因子165重组腺病毒

目的利用AdEasy腺病毒载体系统构建血管内皮生长因子165（VEGF165）重组腺病毒，并在293T细胞中扩增。方法将PCR获取的VEGF165基因酶切并插入到pAdTrack-CMV中，构建成腺病毒穿梭质粒pAdTrack-VEGF165，经PmeI酶切线性化后，采用电穿孔转化到事先电转化腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌感受态细胞中，挑选同源重组菌落。提取质粒并用Pac I酶切鉴定，线性化重组质粒pAdEasy-VEGF165转染293T细胞，包装成重组腺病毒颗粒，荧光显微镜下观察绿色荧光表达，并扩增收集重组腺病毒，测定病毒滴度。结果经限制性内切酶检测、基因测序及和绿色荧光观察证实成功构建了携带VEGF165基因的重组腺病毒，并扩增出10^9pfu／mL的高滴度重组腺病毒。结论成功构建了携带VEGF165基因的重组腺病毒载体，为研究骨组织工程血管化局部基因治疗奠定了基础。     

大鼠PnNOS基因修饰脂肪源性干细胞的构建

目的 构建过表达大鼠阴茎神经源性一氧化氮合酶（PnNOS）基因大鼠脂肪源性干细胞（ADSCs）,为基因修饰ADSCs移植治疗勃起功能障碍（ED）大鼠模型提供种子细胞.方法 获取大鼠PnNOS基因,并与线性化的腺病毒真核表达载体pDC315-EGFP定向克隆连接.构建正确的pDC315-PnNOS-EGFP穿梭质粒转染293 T细胞,采用Western Blot检测PnNOS基因在293 T细胞中的表达情况.利用AdMax腺病毒包装系统包装产生重组腺病毒,包装后扩增纯化并测定病毒滴度.PnNOS基因重组腺病毒转染大鼠ADSCs,观察GFP表达情况和Western Blot检测PnNOS基因表达情况.结果 经PCR鉴定、限制性酶切分析、测序鉴定和目的质粒Western Blot表达检测鉴定,证实pDC315-PnNOS-EGFP重组腺病毒载体构建成功.重组腺病毒包装成功且病毒滴度为5.0×109 PFU/ml.Western Blot于大鼠ADSCs中检测到约161 KDa大小条带,其与PnNOS蛋白分子量大小基本相一致.结论 大鼠PnNOS基因修饰的ADSCs构建成功,从而为基因修饰ADSCs移植治疗ED提供了良好的基因工程细胞.     

肝细胞肝癌靶向性r-Caspase-3重组腺病毒的构建与表达

目的 构建肝细胞肝癌特异性表达反向半胱氨酸蛋白酶3(r-Caspase-3)重组腺病毒,为肝细胞肝癌的基因治疗提供新策略.方法 构建甲胎蛋白(AFP)增强子和白蛋白 (ALB) 启动子腺病毒载体(pAdTrack-EAFP-PALB),然后将目的 基因r-Caspase-3亚克隆到载体pAdTrack-EAFP-PALB上, 获得重组腺病毒穿梭载体pAdTrack-EAFP-PALB /r-Caspase-3, 经PmeⅠ酶切线性化后与pAdEasy-1同源重组,获得重组腺病毒骨架pAdEasy-EAFP-PALB /r-Caspase-3; 鉴定正确的pAdEasy-EAFP-PALB /r-Caspase-3 经PacⅠ酶切线性化后脂质体转染AD293 细胞进行包装、扩增,获得病毒.绿色荧光蛋白(green fluorescent protein,GFP)监测病毒滴度和感染效率; RT-PCR和Western blot 法检测r-Caspase-3在HepG2细胞中的表达; SRB染色法评估重组腺病毒对HepG2细胞的抑制作用,初步观察HepG2细胞凋亡状况.结果 穿梭载体pAdTrack-EAFP-PALB/r-Caspase-3酶切、测序正确.穿梭载体、pAdEasy-1载体同源重组后PCR 及PacⅠ酶切鉴定结果表明pAdEasy-EAFP-PALB/r-Caspase-3 重组成功; 经PacⅠ酶切线性化后,pAdEasy-EAFP-PALB/r-Caspase-3 转染AD293 细胞即可观察到GFP 的表达; 回收病毒可重复感染AD293 细胞,RT-PCR和Western blot 均可检测到r-Caspase-3的表达,证实Ad-EAFP-PALB/r-Caspase-3病毒颗粒包装成功; SRB染色检测发现Ad-EAFP-PALB/r-Caspase-3具有凋亡诱导特异性.结论 靶向性Ad-EAFP-PALB/r-Caspase-3重组腺病毒构建成功,并具有凋亡诱导靶向性,为进一步研究靶向性r-Caspase-3基因治疗肝细胞肝癌及其生物学功能奠定了基础.     

构建携带EGFP标志人白细胞介素10基因的重组腺病毒载体

目的 构建携带EGFP标志人白细胞介素10(hIL-10)基因腺病毒载体.方法 以pSNAV2.0-hIL-10重组质粒为模板.PCR扩增hIL-10基因,获得hIL-10 cDNA片段,并酶切连接到带有含强绿色荧光蛋白基因(EGFP)标记的pDC316-IRES-EGFP-lacZalpha载体质粒上,Pmel线性化重组质粒pDC316-hIL-10-IRES-EGFP,与腺病毒包装系统AdMax转染293包装细胞,包装产生复制缺陷型重组腺病毒,经反复感染293细胞扩增病毒后,进行离子交换法纯化病毒,并测定病毒颗粒数、滴度.结果 经PCR鉴定、限制性酶切分析及序列测定,证明已正确构建重组穿梭质粒pDC316-hIL-10-IRES-EGFP和重组腺病毒质粒Ad-hIL-10-IRES-EGFP.扩增纯化后.测得重组腺病毒颗粒数为3.2×1011VP/ml,OD260/OD280值约为2.0,滴度为1.1×1010TCID50/ml.结论 成功构建了重组腺病毒质粒Ad-hIL-10-IRES-EGFP,为进一步研究hIL-10基因奠定实验基础.     

应用AdEasy腺病毒载体系统构建人骨形成蛋白2基因重组腺病毒

目的利用AdEasy腺病毒载体系统构建人骨形成蛋白2(BMP2)基因重组腺病毒并在293E细胞中扩增制备重组病毒。方法自人骨形成蛋白2真核表达载体pcDNA3-hBMP2中酶切出hBMP2基因，插入pAdtrackCMV中构建成腺病毒穿梭质粒pAdtrackCMV—hBMP2，经酶切线性化后，采用电穿孔转化到事先电转化腺病毒骨架质粒pAdEasy的BJ5183大肠杆菌电感受态细菌中，挑选同源重组质粒，酶切线性化重组质粒并转染293E细胞包装成重组病毒颗粒，荧光显微镜观察绿色荧光表达。重组病毒上清感染293细胞，荧光显微镜观察绿色荧光表达。结果经限制性内切酶检测和GFP表达证实成功地构建了携带hBMP2基因的重组腺病毒载体并制备出高滴度重组病毒。结论成功地构建了携带hBMP2基因的重组腺病毒载体，为进一步研究rBMP2基因治疗奠定了基础。     

急性早幼粒性白血病基因重组逆转录病毒载体构建及表达

目的构建重组人早幼粒白血病基因(PML)的逆转录病毒载体及稳定表达PML的膀胱肿瘤细胞株。方法应用DNA重组技术,将PML基因亚克隆后连接在逆转录病毒载体pLXSN上,经酶切鉴定正确后,磷酸钙沉淀法转染到PA317包装细胞包装病毒,并计算重组病毒的滴度。聚合酶链反应(PCR)鉴定重组PML逆转录病毒能够成功感染靶细胞。激光共聚焦和Westernblot鉴定PML蛋白的表达。结果经酶切分析证实有大约2.1kb大小的片段插入到逆转录病毒载体中。PCR结果显示感染重组PML逆转录病毒的细胞可以扩增出304bp大小的片段。激光共聚焦显微镜显示,感染重组PML病毒的膀胱肿瘤细胞胞核内有散在的斑点样的绿色荧光亮点。Westernblot也发现感染PML组有大约90×103的特异性的蛋白条带。结论我们成功构建重组人PML基因的逆转录病毒载体并且建立了稳定表达PML的膀胱肿瘤细胞株。     

负载PCA3启动子及CD-TK自杀基因腺病毒载体的构建及包装

目的 构建负载PCA3启动子联合CD-TK基因的腺病毒载体,并对其进行包装及滴度测定.方法 将PCA3启动子无缝克隆到pHBAd-U6-GFP上,取代原有U6启动子形成pHBAd-PCA3-GFP,AgeI单酶切该重组载体,将CD-TK基因片段无缝克隆至线性化pH-BAd-PCA3-GFP上,抽提质粒抗性筛选后经PCR及测序鉴定为pHBAd-PCA3-CD-TK载体.取上述重组载体质粒及骨架质粒pHBAd-BHG,用LipofiterTM转染试剂进行转染HEK293细胞包装成病毒,大量扩增并测定病毒感染性滴度.结果 经PCR及测序验证证实成功构建负载PCA3启动子及CD-TK自杀基因的重组腺病毒pHBAd-PCA3-CD-TK并包装扩增,病毒滴度为1×1010PFU/mL.结论 构建负载PCA3启动子及CD-TK自杀基因的重组腺病毒,为进一步体内外对前列腺癌细胞的杀伤效应研究奠定了基础.     

可调控表达人骨形态发生蛋白2基因的腺病毒载体系统的构建

目的构建可调控人骨形态发生蛋白2（hBMP2）基因表达的重组腺病毒载体系统,为骨缺损的修复提供新思路。方法基因测序验证pcDNA3．1－hBMP2质粒的准确性,将酶切得到的hBMP2基因片段亚克隆到穿梭载体质粒pTRE—Shuttle2的多克隆位点,将pTRE—Shuttle2-hBMP2线性化并连接至腺病毒骨架质粒Adeno—XSystem 1 Viral DNA上,构建重组的Adeno—X－pTRE—hBMP2载体;用PacI酶将其线性化后脂质体法（Lipofectamine 2000）转染HEK293细胞,获得有感染能力的重组腺病毒颗粒,收集病毒上清进行hBMP2基因PCR鉴定;将包含rtTA激活因子的Adeno—X Tet—Onvirus Stock感染HEK293细胞包装扩增病毒。结果hBMP2基因被成功定向克隆到腺病毒骨架质粒Adeno—X System 1 Viral DNA上,转染HEK293细胞后获得有感染能力的重组腺病毒颗粒。结论本研究成功构建了hBMP2基因的腺病毒Tet—On表达系统,为实现hBMP2基因在靶细胞水平上的调控表达提供前期实验基础。     

Human genetics and gender

Human genetics is the study of genetics and biological variation in Homo sapiens and medical genetics is the science of such variation as it relates to health and disease. The scope of medical genetics includes teaching, basic research, genetic counseling, as well as genetic testing services. These services offer genetic testing for confirmation of clinical diagnoses, predictive genetic testing and prenatal genetic testing. There are clear gender-specific differences in the importance and consequences of the specific application of such genetic tests (e.g. genetic aspects of infertility, hereditary cancer syndromes). Accordingly, gender-specific genetic counseling must be part of the management of every prenatal, postnatal or predictive genetic testing.     

Human and animal bites

Mammalian bites are potentially dangerous injuries. Despite the severity and frequency of the bite-related complications, there are still many controversies regarding the approach to these injuries. The generally accepted view that human bites are worse than animal bites cannot be substantiated by the existing data. Bites on the hands are associated with a high incidence of sepsis and they should be managed more aggressively than other bites. Most clean bite wounds can safely be sutured primarily after meticulous irrigation and debridement. The microbiology and management of mammalian bites are described in detail.     

Therapeutic Targets of Human AKI: Harmonizing Human and Animal AKI

The opportunity to make advances in the prevention and treatment of AKI has never been greater than it is today. Major advances have been made in the understanding of the biology of AKI, the design of clinical trials, and the use of diagnostic and prognostic biomarkers. These advances have been supplemented by the coordinated effort of societies, federal agencies, and industry, such that we are poised in the ensuing years to positively address the unrelenting harm that this disorder has created. Over the past decade, major advances have been made in understanding the pathophysiology of AKI, mainly through the study of small animal models. However, translating these findings to human AKI remains a barrier, which is typified by the absence of effective therapeutic agents. The purpose of the Acute Dialysis Quality Initiative (ADQI) XIII was to harmonize human and animal studies and determine what is known about potential therapeutic targets and what gaps in knowledge remain. A series of invited reviews will distill key concepts from this initiative that focus on different pathogenic features of AKI, including hemodynamics, immunity and inflammation, cellular and molecular pathways, progression, and regeneration and repair. This series will convey the status of our knowledge of the pathophysiology of human AKI and propose therapeutic targets for further investigation.     

Calcitonin in Human Seminal Plasma and its Localization on Human Spermatozoa

We determined the calcitonin (CT) levels in peripheral plasma and in seminal fluid of 15 normal human subjects: the concentration of the hormone in seminal fluid was about 30 times higher than the concentration found in peripheral plasma. We also studied the localization of calcitonin on human spermatozoa by means of an indirect immunofluorescent technique, using an anti-human CT rabbit serum and a fluorescein-isothiocyanate conjugated goat anti-rabbit immunoglobulins serum. A bright fluorescence was observed at the middle piece and neck, while the tail's principal piece was weakly stained. With an anti-human CT rabbit serum pre-absorbed with human CT no fluorescent staining was detectable. These findings demonstrate that calcitonin is localized onto spermatozoa and suggest a potential role for calcitonin in the calcium dependent-mechanisms of spermatozoa.     

人绒毛膜促性腺激素与胚胎种植

人绒毛膜促性腺激素(HCG)是一种由早期胚胎滋养球细胞和胎盘滋养层合体细胞分泌的、两个亚基以非共价健结合的糖蛋白激素.目前认为它是滋养层细胞和众多胚胎源性物质中最早出现的标志物.β-HCG通过内分泌和旁、自分泌途径与靶细胞表面HCG/LH受体(HCG/LH-R)结合在胚胎发育、妊娠早期黄体功能维持和胚胎种植与胎盘形成过程中发挥着不可缺少的生物学作用.     

Human performance testing and simulators

Virtual-reality (VR) simulation offers the opportunity to practice surgical techniques and gain experience outside the operating room. Using VR simulators and tools recently developed by human performance researchers, different aspects of human performance (innate ability) can be measured objectively. These novel tools, General Systems Performance Theory, and application of nonlinear causal resource analysis (NCRA) may allow us to utilize VR simulators, not only to train physicians, but possibly to predict their performance prior to training. By analyzing objective measures of basic performance resources (BPRs) with performance models developed with NCRA, we showed that BPRs can predict a subject's ability to perform high-level tasks. Two pilot studies suggested that this approach may objectively identify limitations of the surgeons with the most worrisome performance scores. Thus, VR simulation and performance testing may help with identification and remediation of residents with poor endoscopic skills and with optimization of surgical training protocols. Although the initial observations provide encouraging results for objective prediction of surgical performance and identification of performance-limiting resources, further investigation in larger cohorts of surgical trainees appears warranted.