重组腺病毒载体Ad5-hBDNF-EGFP的构建与鉴定*☆

背景：基因治疗是目前脊髓损伤治疗的方向，目的基因和载体是基因治疗的关键。 目的：构建携带增强型绿色荧光蛋白基因(Enhanced green fluorescence protein, EGFP)标志人脑源性神经营养因子(human brain-derived neurotrophic factor，hBDNF)基因重组腺病毒载体。 设计、时间及地点：单一样本观察，实验于2007-09/2008-06在福建医科大学附属第一医院完成。 材料：感受态大肠杆菌DH-5α购自美国Stratagene公司；pDC316-hBDNF、载体质粒pDC316-mCMV-EGFP、腺病毒骨架质粒pBHGlox_E1,3Cre、腺病毒包装系统AdMax和包装细胞株293购自加拿大Mixcrobix-Biosystems公司。 方法：以pDC316-hBDNF为模板，聚合酶链反应扩增酶切获得hBDNF基因片段，连接到带有EGFP标记基因的载体质粒pDC316-mCMV-EGFP上，构建穿梭质粒pDC316-hBDNF-mCMV-EGFP。利用AdMax包装系统，穿梭质粒与骨架质粒pBHGlox_E1，3Cre共转染293包装细胞, 同源重组产生复制缺陷型重组腺病毒载体Ad5-hBDNF-EGFP，反复感染293细胞扩增病毒后，离子交换法纯化病毒，并测定病毒颗粒数及滴度。 主要观察指标：①hBDNF基因原始质粒聚合酶链反应鉴定。②穿梭质粒pDC316-hBDNF-mCMV-EGFP的构建及鉴定。③重组腺病毒Ad5-hBDNF-EGFP的包装、扩增及纯化。④毒种目的基因的聚合酶链反应鉴定。⑤纯化病毒的滴度测定结果。 结果：经聚合酶链反应鉴定、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-hBDNF-mCMV-EGFP和重组腺病毒载体Ad5-hBDNF-EGFP；扩增纯化后，测得重组腺病毒颗粒数为2.4×1011VP / mL，A260/A280值约为2.0，滴度为0.8×1010 CCID50/ mL。 结论：已成功构建重组腺病毒载体Ad5-hBDNF-EGFP，为hBDNF基因功能及基因治疗的进一步研究奠定实验基础。     

甲胎蛋白特异性小干扰RNA重组腺病毒载体的构建

背景：RNA干扰技术的应用，关键在于能够采用1个有效的基因转移系统将小干扰RNA转入至靶细胞，目前广泛应用的是构建小干扰RNA表达载体。 目的：利用AdMax腺病毒载体系统构建表达人甲胎蛋白-小干扰RNA的腺病毒载体。 设计、时间及地点：开放性实验，于2007-03/10在山东大学附属济南市中心医院中心实验室完成。 材料：穿梭质粒pDC316-EGFP-U6为本元正阳基因技术公司产品；AdMax KitD试剂盒与低代数HEK293细胞为Microbix Biosystems Inc.（Canada）公司产品。 方法：选择针对甲胎蛋白 mRNA的特异性小干扰RNA靶序列，设计合成为相应的双链DNA，并将其与酶切线性化的pDC316-EGFP-U6载体片段连接，构建好的穿梭质粒pDC316-EGFP-U6-AFP-siRNA和腺病毒骨架质粒pBHGlox_E1,3Cre共转染HEK293细胞，同源重组产生重组腺病毒。 主要观察指标：对重组腺病毒进行聚合酶链反应鉴定及扩增、纯化、滴度测定。 结果：构建的穿梭质粒载体经聚合酶链反应鉴定和测序分析，证实与设计一致。重组腺病毒Ad-AFP-siRNA经聚合酶链反应和绿色荧光蛋白表达检测证实构建成功，测定滴度为1.4×109 nfu/L。 结论：实验成功构建了Ad-EGFP-U6-AFP-siRNA重组腺病毒。     

腺病毒介导嗜铬瘤细胞中p75神经营养因子的RNA干扰

课题前期研究发现,在急性马尾神经受压模型大鼠中,马尾神经元华勒氏变性和腰骶脊髓前角运动神经元的凋亡及马尾相应阶段脊髓中有p75NTR的高表达存在正向变化关系。本文设想用腺病毒携带shRNA的方式,进行针对p75NTR基因沉默病毒的包装及体外验证重组腺病毒的基因沉默效果。实验按照Reynolds siRNA模板寡核苷酸的设计原则,设计合成3条p75 siRNA模板寡核苷酸片段（shRNA）,随后分别克隆进Shuttle vector 1.0 CMV,与表达加强绿色荧光蛋白的腺病毒载体骨架共转染HEK293细胞成功包装出具有p75shRNA的腺病毒,所得病毒分别命名为Ad.shRNA1、Ad.shRNA2、Ad.shRNA3,重组腺病毒分别感染体外培养的PC12细胞。48h后收集PC12细胞中p75NTRmRNA及总蛋白,发现相对于阴性对照,在mRNA水平,Ad.shRNA1,Ad.shRNA2,Ad.shRNA3组RNA干扰率为分别为（98.49±0.68）％,（95.08±1.79）％,（96.60±1.14）％;在蛋白水平,其RNA干扰率分别为（72.89±2.17）％,（58.83±1.15）％,（59.88±0.44）％。由此得出重组腺病毒成功的抑制了p75NTR的表达,其中以Ad.shRNA1基因沉默效果最为显著。     

重组腺病毒Ad5-mHes1的构建及其在小鼠海马组织中的表达

目的 构建含小鼠Hes1基因的重组腺病毒(rAd)并观察其在小鼠海马组织中的表达情况,为进一步研究Hes1基因在成年小鼠神经再生中的作用奠定基础。 方法 利用限制酶对pEGFP-mHes1质粒和pDC316质粒进行酶切,将获得的mHes1目的基因片段和pDC316质粒酶切产物回收,在T4 DNA连接酶作用下连接,形成pDC316-mHes1穿梭质粒,并用PCR法及EcoR Ⅰ+HindⅢ双酶切法对pDC316-mH-Hes1进行鉴定。利用AdMax包装系统,穿梭质粒pDC316-mHes1与骨架质粒pBHGlox_E1,3Cre共转染293细胞,同源重组产生复制缺陷型腺病毒Ad5-mHes1,其报告病毒是包含高强度绿色荧光蛋白(EGFP)的腺病毒Ad5 -EGFP。向成年C57BL/6小鼠海马中立体定向注射Ad5 -mHes1及Ad5-EGFP,Western blotting检测注射后7 d Hes1蛋白在海马中的表达情况,荧光显微镜观察EGFP在海马中的表达情况。 结果 用PCR法及EcoR Ⅰ+HindⅢ双酶切法对pDC316-mHes1鉴定的实验结果与预期结果相符,经测序后其序列与mHes1 CDS序列一致。注射后7d,Hes1蛋白在PBS注射组和Ad5-mHes1注射组海马组织中均有表达,其Hes1蛋白与GAPDH的比值分别为0.363±0.053和0.705±0.128,差异有统计学意义(P＜.05)。荧光显微镜下在海马齿状回颗粒细胞层中观察到Ad5-EGFP表达的绿色荧光。 结论 本研究成功构建了Ad5-mHes1表达载体系统,并在C57BL/6成年小鼠海马组织中表达了Hes1基因。     

白细胞介素12基因修饰大鼠胶质瘤9L/rIL-12细胞的建立

目的 构建大鼠白细胞介素12（rIL-12）基因真核表达载体质粒,建立rIL-12基因修饰并稳定表达的大鼠胶质瘤9L/rIL-12细胞.方法 用Trizol提取大鼠腹腔巨噬细胞总RNA,RT-PCR方法分别获取rp40和rp35 cDNA,依次克隆人pcDNA3.1（-）/mycHis A质粒中,以IRES连接双亚基,构建成双顺反子真核表达载体质粒pcDNA3.1/rIL-12.将构建的重组质粒转染大鼠胶质瘤9L细胞,G418筛选单克隆细胞株,ELISA法检测其培养上清rIL-12（p70）蛋白含量,同时抽提细胞RNA,RT-pCR方法检测rp40、rp35基因在9L/rIL-12细胞中的表达.结果 所得rp40cDNA序列与Gene Bank NM022611及AF133197、rp35cDNA序列与Gene Bank NM053390及AF177031均一致,构建的质粒经PCR、酶切及测序鉴定正确.质粒转染9L细胞后,获得2个rIL-12基因稳定、高效表达的9L/rIL-12单克隆细胞株,其培养上清rIL-12（p70）蛋白含量分别为139.0、162.1 pg/ml,RT-PCR结果显示细胞中rIL-12基因表达呈阳性.结论 成功构建了rIL-12真核表达质粒pcDNA3.1/rIL-12,并建立了9L/rIL-12细胞株,为rIL-12基因修饰的胶质瘤疫苗研究打下了基础.     

携带人血红素加氧酶-1基因重组腺病毒的构建及其在大脑皮层神经元中的表达

目的构建人血红素加氧合酶-1（hHO-1）基因的重组腺病毒,并研究其感染大脑皮层神经元的效率。方法采用基因克隆技术,将hHO-1基因克隆到腺病毒穿梭质粒pShuttle—IRES—hrGFP中,构建pShuttle-hHO-1-IRES—hrGFP质粒,利用细菌BJ5183将穿梭质粒与pAdEasy-1进行重组,获得重组的腺病毒质粒。线性化的腺病毒质粒经脂质体转染AD293细胞,进行重组腺病毒的包装和扩增。采用CsCl梯度离心进行病毒纯化。获得的腺病毒感染大脑皮层神经元,观察其感染效率和hHO-1表达水平。结果通过酶切鉴定证明腺病毒载体构建成功,包装出携带hHO-1基因的腺病毒,病毒滴度为2．0×10^10pfu／ml,获得的腺病毒对大脑皮层神经元的感染效率高于95％以上。结论成功地利用细菌内同源重组方法构建了携带hHO-1基因的腺病毒,并能够在大脑皮层神经元中高效地表达,为利用HO—1进行基因治疗提供了材料。     

力生长因子重组腺病毒载体构建及其在成骨细胞中的表达

背景：研究表明,力生长因子(mechano-growth factor,MGF)能激活卫星细胞,促进成肌细胞增殖,在治疗肌损失、预防心肌损伤和修复神经损伤等方面有重要的作用。机械拉伸可使成骨细胞表达MGF,但是MGF对骨组织生理生化行为的影响机制尚不清楚。 目的：应用携带MGF基因的重组腺病毒载体转染成骨细胞,观察MGF在成骨细胞中的表达。 方法：将MGF基因插入到腺病毒穿梭质粒pAdTrack-CMV中,构建pAdTrack-MGF重组体。pAdTrack-MGF与腺病毒骨架质粒pAdEasy-1在BJ5183菌中进行同源重组,生成重组腺病毒质粒pAdEasy-MGF。脂质体介导pAdEasy-MGF转染293A细胞,包装成整的重组腺病毒Ad/MGF。用Ad/MGF感染原代培养的大鼠成骨细胞,荧光示踪计数法测定感染效率,RT-PCR法对重组腺病毒感染结果进行鉴定。 结果与结论：实验构建的重组腺病毒载体Ad/MGF滴度可达8.5×108 pfu/mL。Ad/MGF能高效感染体外培养的Wistar大鼠成骨细胞并表达目的基因,当感染复数为100时,感染效率最高。说明实验构建的Ad/MGF重组腺病毒可在大鼠成骨细胞中有效表达。     

大鼠GLUT3基因重组腺病毒载体的构建及鉴定

目的构建携带大鼠葡萄糖转运体3（GLUT3）基因的复制缺陷型重组腺病毒载体，为研究GLUT3的生物学功能提供工具。方法采用逆转录-聚合酶链反应（RT-PCR）的方法获取大鼠GLUT3基因全长cDNA片段，将其克隆至穿梭质粒pShuttle中构建穿梭质粒pShuttle-GLUT3，经酶切后与线性化的腺病毒骨架质粒pAdeno-X体外连接并转化大肠杆菌DH5α，构建重组腺病毒质粒pAd-GLUT3，酶切线性化重组腺病毒质粒后转染293细胞包装成重组病毒颗粒，重组腺病毒在293细胞中反复扩增数代后，分别用聚合酶链反应（PCR）及免疫印记法（western blot）的方法从基因和蛋白表达水平鉴定重组的腺病毒。结果经DNA测序和PCR分析显示GLUT3 cDNA序列正确。成功筛选出重组腺病毒质粒pAd-GLUT3后在HEK293细胞中成功包装出重组病毒，包装后冻融细胞行PCR及western blot检测表明重组腺病毒包装成功。结论本研究成功构建了携带大鼠GLUT3基因的复制缺陷型重组腺病毒载体。     

应用AdEasy腺病毒载体系统构建鼠白细胞介素10基因重组腺病毒★

目的：AdEasy系统可在原核细胞E.coli BJ5183中完成穿梭质粒与骨架质粒之间的高效同源重组，经抗生素培养板筛选重组体，然后转染293细胞获得重组病毒，极大简化了载体的构建过程。实验利用AdEasy腺病毒载体系统构建鼠白细胞介素10基因重组腺病毒。 方法：实验于2006-08/2007-05在青岛大学医学院病毒学实验室完成。①实验材料：清洁级SD大鼠5只，实验过程中对动物的处置符合动物伦理学标准。腺病毒穿梭质粒pAdTrack-CMV，骨架质粒pAdeasy-1，大肠杆菌BJ5183和DH10B，人胚肾293细胞均由青岛大学医学院微生物教研室罗兵教授惠赠。②实验方法：从脂多糖刺激培养的大鼠脾细胞中提取细胞总RNA，应用反转录多聚酶链反应技术扩增白细胞介素10 cDNA，定向亚克隆至pAdtrack-CMV中构建腺病毒穿梭质粒pAdtrack-CMV-IL-10，酶切线性化后转化到含腺病毒骨架质粒pAdEasy的BJ5183大肠杆菌中，获得重组腺病毒质粒pAd-IL-10，Lipofectamin包装转染293细胞，获得重组腺病毒vAd-IL-10，荧光显微镜下观察绿色荧光蛋白的表达。将293细胞接种于96孔板中，每孔1×104个细胞，浓缩后的病毒贮存液按不同比例稀释加至培养板中，测定重组腺病毒滴度。用重组腺病毒vAd-IL-10感染293细胞3 d后，以TRIzol一步法提取细胞总RNA，所获RNA加入DNaseⅠ消化处理可能污染的DNA后，PCR产物行琼脂糖电泳检测白细胞介素10 mRNA的表达。 结果：①重组腺病毒的包装及滴度测定：经限制性内切酶转染293细胞16 h后，荧光显微镜观察到细胞中有GFP荧光，可间接反映目的基因的表达。将病毒上清反复感染293细胞扩增重组腺病毒，通常传至第4~5代于接种病毒后24~48 h，几乎可观察到所有细胞出现荧光，此时收获病毒，重组病毒命名为vAd-IL-10。vAd-IL-10滴度为5.5×108 pfu/mL，vAd-GFP滴度为9.0×108 pfu/mL。②目的基因RT-PCR产物的鉴定：提取重组腺病毒感染293细胞总RNA，RT-PCR扩增后琼脂糖电泳显示特异性580 bp预计大小的片段，表明该重组腺病毒可在293细胞中有效转录。 结论：利用AdEasy腺病毒载体系统成功构建了携带白细胞介素10基因的重组腺病毒载体，并制备出高滴度的重组腺病毒vAd-IL-10，可在293细胞中有效转录。     

大鼠蛋白酪氨酸磷酸酶1B基因靶向RNA干扰重组腺病毒的构建

背景：研究者们早已发现蛋白酪氨酸磷酸酶1B（protein tyrosine phosphatase，PTP1B）可负性调节胰岛素信号转导，但目前制约PTP1B功能研究的重要环节是缺乏有效的工具。 目的：拟构建PTP1B基因靶向RNA干扰重组腺病毒。 设计、时间及地点：体外细胞学基因转染实验，于2007-05/12在解放军第二军医大学长海医院中心实验室完成。 材料：293细胞由中科院上海细胞研究所提供，真核表达质粒psiRNA-PTP1B由上海吉凯生物公司合成，AdEasy-1菌、穿梭载体pAdTrack、大肠杆菌DH5α由本室保存，重组腺病毒PCR鉴定引物由上海生工生物公司设计合成。 方法：由生物公司合成针对大鼠PTP1B mRNA的特异性siRNA真核表达质粒psiRNA-PTP1B，双酶切得到siRNA-PTP1B表达片段，插入pAdTrack载体上，获得转移质粒pAdTrack-siRNA-PTP1B。后者经线性化后，转化含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183进行同源重组，PacⅠ酶切鉴定，筛选出正确重组腺病毒质粒pAdEasy-siRNA-PTP1B，酶切线性化后转染293细胞包装成重组病毒颗粒。通过反复感染扩增病毒，采用氯化铯密度梯度离心法纯化重组腺病毒。 主要观察指标：荧光显微镜观察绿色荧光蛋白的表达，鉴定重组腺病毒穿梭载体及重组腺病毒是否构建成功。 结果：PacⅠ酶切鉴定证实重组腺病毒质粒pAd-siRNA-PTP1B构建成功，转染293细胞后有绿色荧光蛋白表达。PCR鉴定表明重组腺病毒中含有siRNA-PTP1B片断，成功构建了携带PTP1B干扰RNA的重组腺病毒Ad-siRNA-PTP1B，293细胞扩增纯化后，获得约3.6×109 efu/mL滴度的重组腺病毒。 结论：应用AdEasy-1系统可以高效快速制备表达PTP1B干扰RNA的重组腺病毒。     

Type 1 human poliovirus binds to human synaptosomes

Poliovirus is a neurotropic virus that selectively infects human motoneurons in vivo. The basis for the specificity of this infection is not fully understood. It has been suggested that this tropism occurs because motoneurons are the only neurons to express poliovirus receptors. We have examined this hypothesis by measuring the binding of 125I-labeled poliovirus type 1 to neural tissues. With this assay we have detected highly specific binding sites in human but not rodent neural tissue. Regional assays of binding in human central nervous system homogenates demonstrate that binding sites are not confined to motoneurons. Rather, they are widely distributed throughout the human neuraxis. Particularly in the forebrain, binding is more abundant in gray than white matter. For this reason, we performed tissue fractionation studies which indicate that poliovirus binding sites are enriched in synaptosomes, the subfraction of central nervous system gray matter tissue rich in synaptic endings. The preferential expression of poliovirus binding sites in synaptic endings may be an important factor in the motor tropism of this virus, inasmuch as the major category of neurons with synaptic endings outside the central nervous system are motoneurons; thus, particles of virus may preferentially bind to this cell type during poliovirus viremia.     

Symptomatic human neurocysticercosis

Abstract. Objective: To evaluate the relevance of exposure and host biological factors in the heterogeneity of the clinical, radiological and inflammatory picture of neurocysticercosis (NCC). Methods: 105 Mexican symptomatic NCC patients confirmed by imaging were studied before they received any specific treatment. The relationships studied were those between a) the patients characteristics (gender, age and level of exposure), b) the type of clinical picture and c) the radiological and inflammatory characteristics of the disease (number, aspect, localization of the parasites, and CSF leukocytecounts). Results: Results Seizures were the most frequent symptom and multiple subarachnoid cysticerci the most frequent localization. Symptomatology related to the developmental stage, number and localization of the parasites as well as the CSF leukocyte-counts. The total number of cysticercal lesions and of vesicular cysticerci increased with age,whereas the number of colloidal cysticerci decreased. CSF leukocyte-counts were higher in women than in men. Levels of exposure did not correlate with the clinical and radiological pictures. Conclusions: The variability found in the number, stage, localization and inflammation in the parasite lesions is strongly associated with the heterogeneity of NCC symptoms. The increased number of vesicular cysticerci and the decreased number of degenerating cysticerci with aging, as well as the prominence of inflammation in women suggest that immuno-endocrinological factors may play a role in susceptibility and pathogenesis. The data also show that with increasing age and exposure there is no increment in severity, a suggestion that there might be ways of regulating pathogenicity.     

Disseminated human neurocysticercosis

Summary This study was based on two cases of disseminated human neurocysticercosis from India. The material availabel was examined grossly, and by light microscopy, histochemistry, immunomorphology and electron microscopy. The results showed that the parasites commonly embolized to the anatomically discernable gray-white matter junction of the brain and were located in cavities, the walls of which were dilated vascular channels. The parasite-nutrition process was through endocytosis and microtrichal activity. To camouflage themselves from the host-defense mechanisms, the parasites apparently covered themselves with host-tissue-like material. Host reactivity to the parasite was heralded morphologically by the physical anchoring of the parasite by activated endothelial cells, loss of the host-tissue-like cover and an acute polymorphonuclear leucocytic response.     

Age-related reduction of human growth hormone-binding sites in the human brain

Previous studies have shown that alterations in various neuroendocrine functions occur with increasing age. We here report a study of growth hormone (GH)-binding sites in different areas of post-mortem human brains collected from individual males and females of different age. The results indicate that there exists a significant negative correlation between the density of GH-binding sites and increasing age. This phenomenom was observed in both sexes in brain areas such choroid plexus, hippocampus, hypothalamus, pituitary and putamen but not in e.g. thalamus, In all tissues (expect for choroid plexus), the GH binding was significantly higher in those originating from females than those from males. This discrepancy was found likely to be associated with the affinity of GH to lactogenic rather than to somatogenic sites as no pronounced sex difference in binding was observed in the presence of excessive amounts of human prolactin. Data also indicate that the putative GH receptors in the various brain regions differ with regard to binding constants and to the estimated molecular size of the hormone-binding units. The loss of GH receptors in brain of elderly people may have consequences in several physiological courses. The decrease in GH binding at hypothalamic and pituitary levels may be of importance for the mechanisms behind the release or secretion of the hormone.     

Enhanced DNA synthesis of human glial cells exposed to human leukocyte products

DNA synthesis was studied in primary glial cell cultures derived from adult human non-neoplastic and neoplastic brain tissues. Enhanced DNA synthesis occurred in 5/5 non-neoplastic astrocyte, one oligodendroglioma, and 2/5 astrocytoma cultures after exposure to medium containing 1.25-12.5% supernatant fluid (SF) from insoluble concanavalin A (Con A) stimulated unseparated or T lymphocyte-enriched human mononuclear leukocytes (MNL). Analyses of SF indicated that the presence of platelet-derived growth factor (PDGF) could not account for glial cell stimulation, and exposure to semi-purified interleukin-2 (IL-2) in amounts comparable to those in SF from Con A-stimulated MNL had no effect on glial cells. These data indicate that non-neoplastic astrocytes and other human glial cells are stimulated by products of human MNL.