重组外源性人热休克蛋白70基因真核表达载体的构建及鉴定

背景：热休克蛋白70的肿瘤免疫作用近年来受到国内外学者的广泛关注,然而目前常用的诱导内源性热休克蛋白70表达的方法,如热应激、缺血预处理等均存在一些弊端。 目的：拟构建携带并正确表达外源性人热休克蛋白70基因的重组真核表达载体。 方法：外源性热休克蛋白70基因连接入真核表达载体pDC315-EGFP中,转化大肠杆菌感受态细胞,重组真核表达载体外源基因测序及PCR检测鉴定阳性克隆后转染人胚肾293细胞,荧光显微镜及western blot鉴定外源基因在细胞中的表达。 结果与结论：经PCR和测序证实外源性人热休克蛋白70基因正确重组入真核表达载体pDC315-EGFP,转染293细胞后荧光显微镜观察到绿色荧光蛋白GFP的表达,Western blot检测到热休克蛋白70在293细胞中有效表达。     

大鼠热休克蛋白72基因真核表达载体构建及在COS7细胞中的表达

背景：体外克隆、表达热休克蛋白,尤其正常时少量或不表达,而应激时大量表达的热休克蛋白72对研究其缺血再灌注损伤中的作用尤为重要。 目的：构建热休克蛋白72基因真核表达载体并于COS7细胞内表达,为HSP72蛋白免疫调节功能的研究奠定基础。 方法：采用RT-PCR技术从BABL/C大鼠肝细胞中扩增出热休克蛋白72 cDNA,经限制性核酸内切酶消化后,插入真核表达载体pcDNA3.1(+)的相应酶切位点,并将重组质粒转染COS7细胞,进行基因表达鉴定。 结果与结论：重组质粒插入基因序列检测证实为热休克蛋白72 cDNA,并能在COS7细胞稳定表达。成功构建热休克蛋白72真核表达载体,并于COS7细胞中成功转录与表达。     

构建人热休克蛋白70基因重组腺病毒载体及鉴定

背景：腺病毒载体作为低毒高效的基因载体已被广泛应用,但是人热休克蛋白70基因腺病毒载体较为少见。 目的：构建重组人热休克蛋白70基因的腺病毒载体,鉴定外源基因在真核细胞中的良好表达。 方法：采用AdMax腺病毒系统将外源基因人热休克蛋白70基因重组入腺病毒载体中,转染人胚肾293细胞并重组包装出毒,检测外源基因的表达和病毒滴度。 结果与结论：观察转染后的人胚肾293细胞出现明显细胞病变效应后,收获并纯化重组病毒;荧光显微镜观察绿色荧光蛋白表达情况良好,Western blot检测人热休克蛋白70蛋白表达良好,收获病毒的滴度为1×1011 efu/mL,证明实验已成功构建携带人热休克蛋白70基因的重组腺病毒载体。     

人类Munc18-1重组质粒的构建及蛋白表达和定位

目的构建人类Munc18-1(hMunc18-1)真核表达质粒pEGFP-hMunc18-1并检测其融合蛋白GFPhMunc18-1在细胞内表达及定位。方法以人胎脑cDNA文库中的cDNA为模板,聚合酶链反应PCR扩增hMunc18-1全长编码基因,亚克隆至绿色荧光蛋白真核表达载体pEGFP-C1中。将构建的重组质粒pEGFPhMunc18-1送到Invitrogen公司进行基因测序,根据NCBI数据库中的hMunc18-1基因序列判断所得重组质粒的基因序列是否正确。将测序正确的质粒pEGFP-hMunc18-1转染到非洲绿猴肾细胞COS-7细胞中,提取细胞蛋白以Western blot法检测融合蛋白GFP-hMunc18-1表达,利用共聚焦激光扫描显微镜观察其在非洲绿猴肾细胞COS-7内的定位。结果 hMunc18-1全长基因序列被成功克隆到绿色荧光蛋白真核表达载体pEGFP-C1中,经酶切后鉴定其片段大小为1785bp。Western blot检测到融合蛋白GFP-hMunc18-1相对分子质量约为94 000,主要分布在细胞质,尤其是细胞膜附近。结论成功构建了hMunc18-1全长基因真核表达质粒pEGFPhMunc18-1,其融合蛋白GFP-hMunc18-1主要定位于COS-7细胞质内,尤其是细胞膜附近。     

Beclin1 基因慢病毒表达载体的构建和鉴定*☆

背景：Beclin1基因是哺乳动物的自噬调控基因。 目的：实验拟构建Beclin1 基因慢病毒过表达载体。 方法：聚合酶链反应扩增目的基因Beclin1 后插入慢病毒表达载体pLenex中,构建重组载体pLenex-Beclin1。使用聚合酶链反应、双酶切和DNA的测序方法对其进行鉴定,并与辅助包装质粒共感染293T细胞。慢病毒颗粒转染非小细胞肺癌A549细胞后,用蛋白质印迹法检测Beclin1 基因的过表达效率。 结果与结论：聚合酶链反应鉴定结果显示扩增的阳性片段已插入pLenex载体,聚合酶链反应、双酶切和DNA测序结果表明,重组慢病毒载体pLenex-Beclin1 的插入序列完全正确,重组慢病毒载体感染A549细胞后,细胞内Beclin1蛋白高效表达。结果证实,实验成功构建了Beclin1 基因慢病毒过表达载体。     

pEGFPN1-GDNF真核表达载体的构建及其表达

目的 构建人胶质细胞源性神经营养因子(GDNF)基因真核表达载体并观察其在COS-7细胞内的表达.方法 应用RT-PCR从U251细胞总RNA中扩增GDNF cDNA,将其克隆至真核表达载体pEGFPN1,经酶切鉴定及序列分析后,以Fugene HD介导转染COS-7细胞,应用免疫细胞化学和Western blot鉴定其在细胞内的表达.结果 RT-PCR产物为650bp的特异片段,重组质粒pEGFPN1-GDNF经双酶切产生650bp和4.7kb的片段,测序分析结果与文献报道结果完全一致.将其转染COS-7细胞后,免疫细胞化学、Western blot结果表明GDNF蛋白能在COS-7细胞中正确表达.结论 成功构建了pEGFPN1-GDNF真核表达载体,为进一步开展帕金森病的基因治疗研究奠定了基础.     

小鼠E1A激活基因阻遏子RNA干扰表达载体的构建

背景：血管平滑肌的增殖是动脉粥样硬化及支架内再狭窄发生的重要机制，小鼠E1A激活基因阻遏子（Cellular repressor of E1A-stimulated genes，CREG）可以抑制血管平滑肌的增殖，成为心血管领域新的治疗靶点。 目的：构建小鼠CREG小分子RNA干扰真核表达载体，下调小鼠细胞中CREG基因的表达。 设计、时间及地点：观察性实验，于2007-04/10在解放军沈阳军区总医院心血管研究所完成。 材料：小鼠成纤维母细胞系（NIH3T3）由美国新泽西州Robert Wood Johnson医学院病理实验科李少华教授馈赠。 方法：应用RNA干扰在线设计工具设计4对可与小鼠CREG基因cDNA结合的短发夹RNA。通过化学合成法合成并克隆至pEN_mH1c载体中，与目的载体pDS_hpEY进行LR重组得到4种表达不同shCREG片段的表达载体pDS_shCREGs。应用LipofectamineTM 2000将pDS_shCREGs转染至小鼠NIH3T3细胞，G418筛选获得稳定转染的细胞克隆。 主要观察指标：应用反转录-聚合酶链反应和蛋白免疫印迹分别检测CREG的沉默效率。 结果：经酶切和DNA测序证实，4种重组pDS_shCREG载体中插入片段的序列正确。反转录-聚合酶链反应和蛋白免疫印迹证实，4种稳定转染细胞中的CREG表达水平均有不同程度的下降，稳定转染pDS_shCREG1的细胞克隆中CREG基因mRNA和蛋白表达分别降低了87%和70%。 结论：成功构建小鼠CREG基因真核干扰表达载体，使体外培养NIH3T3细胞中CREG蛋白表达明显降低。     

人骨形成蛋白2真核表达载体的构建和体外表达

背景：骨形态发生蛋白2(bone morphogenetic protein 2,BMP-2) 是已知的所有生长因子中对骨的形成作用最强的生长因子，被认为是最具有前途的骨诱导物质。 目的：构建人骨形成蛋白2真核表达载体并观察其体外表达情况。 设计、时间及地点：自身对照实验，于2005-07/2006-05在华中科技大学同济医学院分子生物中心实验室完成。 材料：pcDNA3.1(+)载体由华中科技大学同济医学院左石博士惠赠；成骨肉瘤组织由华中科技大学同济医学院附属协和医院骨科提供。 方法：从人成骨肉瘤细胞中提取细胞总RNA，利用反转录-聚合酶链反应方法扩增获得人BMP-2基因cDNA，将基因片断重组到pGEM-T质粒中构建pGEM-T- hBMP-2重组质粒载体，转化到大肠杆菌DH5α后筛选阳性克隆，利用限制性酶切和DNA序列分析鉴定重组质粒。分别用RcoRI和NotI双酶切pGEM-T- hBMP-2质粒和pcDNA3.1真核表达载体，将克隆载体中人骨形成蛋白2基因重组到pcDNA3.1真核表达载体，提取质粒作酶切电泳、聚合酶链反应鉴定及DNA测序后，用脂质体体外转染小鼠骨髓基质细胞，反转录-聚合酶链反应检测BMP-2的表达。 主要观察指标：①人骨肉瘤细胞总RNA 反转录-聚合酶链反应结果。②重组质粒pGEM-T-hBMP-2 和pcDNA3.1-hBMP-2的构建和酶切鉴定。③BMP-2在小鼠骨髓基质细胞内的表达。 结果：人骨肉瘤细胞总RNA经反转录-聚合酶链反应扩增后，获得1.2 kb条带。经酶切电泳、聚合酶链反应鉴定及DNA测序证实实验成功克隆BMP-2基因，重组质粒pcDNA3.1- hBMP-2构建正确;该重组质粒能在体外培养的小鼠骨髓基质细胞中有效表达BMP-2。 结论：实验成功克隆人骨形成蛋白2基因并构建了此基因的真核表达载体。     

人隔蛋白7的真核表达载体的构建及表达

目的 构建人隔蛋白7(SEPT7,hCDC10/SEPT7)的表达载体并对U251人脑恶性胶质瘤细胞系进行转染且检测其表达.方法 RT-PCR方法扩增SEPT7 cDNA片段,并将扩增的片段插入pCDNA3真核表达载体,构建成含SEPT7cDNA的重组载体pCDNA3/SEPT7,以脂质体介导该质粒转染人脑恶性胶质瘤细胞系U251,应用RT-PCR和免疫荧光染色鉴定转染细胞中hCDC10/SEPT7的表达.用流式细胞术对转染前后的U251细胞进行细胞周期分析.结果 成功构建含SEPT7cDNA的重组载体pCDNA3/SEPT7,并使其稳定转染U251细胞,在转染的细胞中,证实有SEPT7mRNA表达上调.细胞周期检测结果G0/G1期细胞增多,SPF降低.结论 成功构建SEPT7表达载体,并在人脑恶性胶质瘤细胞系获得表达,延迟细胞周期进展.     

真核细胞中人神经生长因子cDNA的表达

目的 构建人神经生长因子cDNA的表达载体pcDNA3．1-NGF,并在哺乳动物细胞中进行一过性表达。为以后人神经生长因子的基因治疗打下基础。方法 在本研究室已克隆人神经生长因子cDNA的基础上。构建表达载体pcDNA3．1-NGF。用DEAE-葡聚糖转染技术将pcDNA3．1-NGF导入COS-7细胞，将人神经生长因子cDNA进行一过性表达。并采用Westernblot方法检测表达情况。以鸡胚背根神经节的生长检测表达蛋白的生物活性。结果 成功构建了表达载体pcDNA3．1-NGF，Western blot方法检测出COS-7细胞表达有目的蛋白，并具有一定的生物活性。结论 用哺乳动物细胞一过性表达了人神经生长因子，并具有一定的生物活性。     

Denervation study of synapses of organ of corti of old world monkeys

Fine structural characteristics of synapses in the spiral organ of Corti were examined, with reference to differences between inner and outer haircell systems, and to location of neurons of origin of efferent axons. Surgical interruption of crossed olivocochlear bundle, of vestibular nerve, of facial nerve, and excision of superior cervical ganglia were used to determine the pathways of efferent axons. Interruption of the vestibular nerve near the brainstem results in degeneration of all efferent terminals on outer hair cells. Mid-line lesions at, and caudal to, the facial colliculus result in degeneration of about half of these efferent terminals. Efferent synaptic bulbs to the inner hair-cell system are small, of the order of one micron, and form type 2 junctions with afferent dendrites. They tend to have more large dense-core vesicles (about 80 nm) than the large efferent terminals of the outer hair-cell system, and appear to be the terminals of axons in the habenula perforata, which exhibit varicosities laden with large dense core vesicles. The varicosities are unaffected by excision of the superior cervical ganglia. So far as our material can reveal, it appears that the varicosities in the habenula perforata do not survive vestibular root interruption, nor do the efferent processes in the internal spiral bundle or at the base of inner hair cells. Most interestingly, the afferent processes of the inner hair-cell system, as identified for example by their relation to pre-synaptic bodies in the inner hair cells, are subject to a trans-synaptic reaction after severance of the vestibular root. They undergo a dramatic cytological transformation, characterized by increase of volume, engorgement with microtubules, microfilaments, microvesicles of various sizes, and clusters of lysosomes. Thus, both the efferent and afferent terminals of the inner hair-cell system show marked cytological differences from the corresponding terminals of the outer hair cell system.     

Mechanism of action of tubocurarine on nicotinic cholinoreceptors of neurons of the sympathetic ganglia of rats

Tubocurarine (Tc) effect on membrane currents elicited by acetylcholine (ACh) was studied in isolated superior cervical ganglion neurons of rat using patch-clamp method in the whole-cell recording mode. The "use-dependent" block of ACh current by Tc was revealed in the experiments with ACh applications, indicating that Tc blocked the channels opened by ACh. Mean lifetime of Tc-open channel complex, tau, was found to be 9.8 +/- 0.5 s (n = 7) at -50 mV and 20-24 degrees C. tau exponentially increased with membrane hyperpolarization (e-fold change in tau corresponded to the membrane potential shift by 61 mV). Inhibition of the ACh-induced current by Tc (3-30 microM/1) was completely abolished by membrane depolarization to the level of 80-100 mV. Inhibition of ACh-induced current was augmented at increased ACh doses. It is concluded that the open channel block produced by Tc is likely to be the only mechanism for Tc action on nicotinic acetylcholine receptors in superior cervical ganglion neurons of rat.     

The effect of age of onset of PD on risk of dementia

Background Dementia occurs in the majority of patients with Parkinson’s disease (PD). Late onset of PD has been reported to be associated with a higher risk for dementia. However, age at onset (AAO) and age at baseline assessment are often correlated. The aim of this study was to explore whether AAO of PD symptoms is a risk factor for dementia independent of the general effect of age. Methods Two community-based studies of PD in New York (n = 281) and Rogaland county, Norway (n = 227) and two population-based groups of healthy elderly from New York (n = 180) and Odense, Denmark (n = 2414) were followed prospectively for 3–4 years and assessed for dementia according to DSM-IIIR. All PD and control cases underwent neurological examination and were followed with neurological and neuropsychological assessments. We used Cox proportional hazards regression based on three different time scales to explore the effect of AAO of PD on risk of dementia, adjusting for age at baseline and other demographic and clinical variables. Findings In both PD groups and in the pooled analyses, there was a significant effect of age at baseline assessment on the time to develop dementia, but there was no effect of AAO independent of age itself. Consistent with these results, there was no increased relative effect of age on the time to develop dementia in PD cases compared with controls. Interpretation This study shows that it is the general effect of age, rather than AAO that is associated with incident dementia in subjects with PD. Received in revised form: 22 December 2005     

Limits of deinstitutionalization--perspectives of relatives of psychiatric patients

After a hopeful beginning, the social process of the reintegration of those with severe mental illness has come to a standstill. I am led to wonder whether "the community" really wants to live together with people suffering from severe mental illness, and if so, how closely? As long as the medical treatment of mental illness provided by the general practitioners is fundamentally deficient, as they are not able to prescribe the necessary interventions--such as out-patient psychiatric nursing, and service providers in the out-patient sector are content with offering increasingly intensive forms of care for the less seriously ill at the cost of the Social Welfare System--the reintegration of those with serious mental illness remains an illusion--which is mainly to the benefit of providers of residential care in homes and hostels.