慢病毒过表达Cdh1蛋白对星形胶质细胞反应性增殖作用的初步研究

目的探讨慢病毒过表达Cdh1蛋白对星形胶质细胞反应性增殖的影响。方法体外纯化培养大鼠星形胶质细胞:(1)随机分为Cdh1慢病毒组及空病毒组,荧光显微镜观察绿色荧光蛋白的表达,Western blot检测Cdh1及Skp2蛋白表达的变化;(2)随机分为单纯氧糖剥夺/复氧组、Cdh1慢病毒干预组、空病毒干预组,氧糖剥夺1h复氧48h后,CCK-8法检测细胞增殖的变化。结果与空病毒组相比,Cdh1慢病毒组Cdh1蛋白表达总量增加,Skp2蛋白表达下降(P0.05);与单纯氧糖剥夺/复氧组相比,Cdh1慢病毒干预组细胞反应性增殖受到抑制(P0.05),空病毒组没有变化(P0.05)。结论慢病毒过表达Cdh1蛋白对氧糖剥夺再复氧后星形胶质细胞的反应性增殖有一定的抑制作用。     

bFGF基因对兔颈内动脉瘤壁的修复研究

目的 通过注射携带bFGF的重组慢病毒(lentivirus)制动脉瘤模型,观察bFGF对颈内动脉瘤壁的修复作用.方法 模型建立后每天从兔耳缘静脉注射携带bFGF的慢病毒滴液,每隔1周处死1组兔并收集标本,RT - PCR检测瘤壁平滑肌细胞表型基因SM22α mRNA、HRG-1 mRNA表达,免疫组织化学染色观察瘤壁PDGF的表达.结果 慢病毒滴液干预的动脉瘤比对照组体积小,弹性纤维断裂程度减轻,平滑肌细胞数目较多,SM22α mRNA、HRG-1 mRNA表达呈下降趋势,瘤壁PDGF表达呈递增趋势.结论 携带bFGF的重组慢病毒能修复动脉瘤壁细胞的破坏,有抑制动脉瘤动态生长的作用.     

脂肪干细胞中慢病毒-血管内皮生长因子165过表达的研究

【摘要】背景与目的：目前,越来越多的研究探讨干细胞成为基因治疗载体的最可能性,但是有关于脂肪干细胞的研究还很少,因此我们拟构建慢病毒-VEGF165的过表达载体并转染脂肪组织来源的干细胞以产生能用于基因治疗的种子细胞。 方法：将克隆EHS1001-6895048 5313912通过PCR方法突变成VEGF165的转录本片段(NM_001025368)。采用三质粒系统（pGC-FU、pHelp1.0和pHelp2.0）包装过表达慢病毒载体。载体构建成功并行滴度测定后转染ADSCs。使用免疫荧光、ELISA和western blot分析鉴定VEGF165在ADSCs中的表达。 结果：使用DNA测序及将质粒转染293T细胞后的表达检测证明所构建的载体为VEGF165与GFP融合表达载体。RT-qPCR检测后计算得到的病毒滴度达到2×108TU/ml。免疫荧光证实大约95%的ADSCs能表达绿色荧光蛋白,ELISA检测出在转染VEGF165后的ADSCs培养基里VEGF浓度为850.86～1202.13pg/ml(平均 923±31.22pg/ml)而在只转染GFP的ADSCs培养基里并不能检测到(p<0.001),并且这一结果也得到western blot检测结果的证实。 结论：本研究通过慢病毒- VEGF165转染ADSCs获得稳定表达VEGF165的脂肪组织来源干细胞为广泛地进行血管再生研究,特别是糖尿病性勃起障碍奠定基础。     

Expression of exogenetic enhanced green fluorescent protein in rat endocranium through lentivirus infection

The study aims to investigate whether exogenetic green fluorescent protein is able to express in the endocranium of rats, and to establish a method for further study in exogenetic gene knock-in or gene overexpression. Forty female Sprague Dawley (SD) rats were randomly divided into 4 groups with 10 in each: low and high dose groups, treated with 10% and 100% EGFP-lentivirus, respectively; negative control group, treated with virus enhancer; sham group, treated with normal saline. Seven days later, half rats’ brain tissues were perfusion fixed and fresh brain tissues were obtained from the rest after euthanasia in each group. Immunohistochemical analysis, Western blotting and RT-PCR were respectively performed to detect the site where EGFP expressed and its levels. Immunohistochemical analysis demonstrated that EGFP was successfully expressed in brain tissue of those rats infected with EGFP-lentivirus. Both Western blotting and RT-PCR showed that EGFP was expressed after treatment with EGFP-lentivirus, and the expression level increased with the dosage of the vector. Exogenetic EGFP gene can express in brain tissue of the rat, which laid a solid foundation for future studies in exogenetic gene knock in or gene overexpression.     

Evaluation of high functional avidity CTL to Gag epitope clusters in EIAV carrier horses

Cytotoxic T lymphocytes (CTL) are critical for lentivirus control including EIAV. Since CTL from most EIAV carrier horses recognize Gag epitope clusters (EC), the hypothesis that carrier horses would have high functional avidity CTL to optimal epitopes in Gag EC was tested. Twenty-two optimal EC epitopes were identified; two in EC1, six in EC2, and seven each in EC3 and 4. However, only five of nine horses had high functional avidity CTL (相似文献   

Lack of trans-activation function for Maedi Visna virus and Caprine arthritis encephalitis virus Tat proteins

All lentiviruses contain an open reading frame located shortly upstream or inside of the env gene and encoding a small protein which has been designated Tat. This designation was mainly with respect to the positional analogy with the first exon of the trans-activator protein of the well studied human immunodeficiency virus type 1 (HIV-1). In this work we comparatively studied the trans- activation activity induced by Tat proteins of the small ruminant Maedi Visna virus (MVV) of sheep and Caprine arthritis encephalitis virus (CAEV) of goats on MVV and CAEV LTRs with that induced by the human lentivirus HIV-1 on its own LTR. The HIV-1 LTR alone weakly expresses the reporter GFP gene except when the HIV-1 Tat protein is coexpressed, the GFP expression is increased 60-fold. In similar conditions only minimal trans-activation increasing two- to three-fold the MVV and CAEV LTR activity was found with MVV Tat protein, and no trans-activation activity was detected in any used cell type or with any virus strain when CAEV Tat was tested. These results indicate that the small ruminant lentiviruses (SRLV) differ from the primate lentiviruses in their control of expression from the viral LTRs and put into question the biological role of the encoded protein named "Tat."     

中药淫羊藿苷对TDP-43介导的骨关节炎软骨细胞病变的作用机制

目的:研究分析中药淫羊藿苷对TDP-43介导的骨关节炎软骨细胞病变的作用机制,从而为临床有效治疗骨关节炎软骨细胞病变提供参考依据。方法:选取并构建TDP-43慢病毒转染人软骨细胞,并予以不同浓度的中药淫羊藿苷进行干预。采用荧光定量PCR检测不同组别人软骨细胞的TDP-43基因表达情况,采用流式细胞仪检测TDP-43慢病毒转染的人软骨细胞加入中药淫羊藿苷前后细胞凋亡情况,通过连续细胞计数观察细胞增殖情况。分别比较3组人软骨细胞中靶基因TDP-43与Ⅰ型胶原蛋白α1表达情况,TDP-43慢病毒转染的人软骨细胞加入中药淫羊藿苷前后细胞凋亡情况,加入不同浓度中药淫羊藿苷培养后的软骨细胞计数水平。结果:TDP-43慢病毒转染的人软骨细胞中TDP-43基因表达水平为(16. 02±1. 45),比正常人软骨细胞、空白慢病毒转染的人软骨细胞的(1. 86±0. 13)、(1. 88±0. 12)高,差异有统计学意义(P 0. 05);而3组Ⅰ型胶原蛋白α1基因表达差异无统计学意义(P 0. 05)。加入不同浓度的中药淫羊藿苷后TDP-43慢病毒转染的人软骨细胞凋亡率相比加入中药淫羊藿苷前均降低,差异有统计学意义(P 0. 05)。培养3 d后、6 d后、12d后高浓度中药淫羊藿苷培养的软骨细胞计数比中浓度高,而中浓度中药淫羊藿苷培养的软骨细胞计数水平比低浓度高,差异有统计学意义(P 0. 05)。结论:TDP-43可能在骨关节炎软骨细胞病变过程中发挥负调控作用,而中药淫羊藿苷可通过改善上述过程,改善TDP-43介导的骨关节炎软骨细胞病变。     

长链非编码 RNA Linc00461干扰慢病毒的构建与鉴定

目的:制备shRNA干扰人长链非编码RNA Linc00461的慢病毒载体（shRNA- Linc00461）,为深入研究长链非编码RNA功能奠定基础。方法：利用在线干扰设计网页进行干扰引物设计,根据小干扰RNA的设计原则,选取三对针对Linc00461特异性干扰引物送由公司合成。合成的片段连接到慢病毒载体质粒pSIH1-H1,形成pSIH1-H1-shRNA- Linc00461质粒。用PCR及测序对其进行鉴定,之后与慢病毒包装质粒混匀,由脂质体转染至HEK293T细胞进行包装,产生慢病毒shRNA- Linc00461,并收集上清液使其感染乳腺癌MDA-MB-231细胞,通过RT-PCR鉴定重组慢病毒干扰效果。并选取干扰效果最好的Linc00461感染MDA-MB-231,MTT和流式细胞术检测细胞增殖和周期改变。结果： SIH1-H1-shRNA Linc00461质粒经PCR验证和测序后,证实其构建成功。shRNA- Linc00461在HEK293T细胞中重组成慢病毒,并成功感染MDA-MB-231,感染效率约为70%。shRNA-Linc00461感染乳腺癌MDA-MB-231细胞后Linc00461的表达下调65.32%; 感染shRNA-Linc00461第3天MTT 结果显示：实验组吸光度值（0.232±0.032）明显低于实验对照组（0.398±0.037）。流式结果提示实验组G2M 期细胞（47.55±5.063）％高于实验对照组（8.876±3.565）％。结论：成功构建了shRNA- Linc00461慢病毒载体,并能有效抑制Linc00461表达,为深入研究人Linc00461基因功能奠定了基础。     

沉默VEGF对人鼻咽癌放射抗拒细胞株CNE-2R侵袭迁移的影响

目的 探讨沉默血管内皮生长因子（vascular endothelial growth factor,VEGF）对人鼻咽癌放射抗拒细胞株CNE-2R迁移和侵袭能力的影响。方法 制备含VEGF-shRNA的慢病毒载体（VEGF-LV1、VEGF-LV2、VEGF-LV3）和对照shRNA重组慢病毒（VEGF-NC）,并转染人鼻咽癌放射抗拒细胞株CNE-2R,建立稳定沉默VEGF细胞（CNE-LV1、CNE-LV2和CNE-LV3）和阴性对照细胞（CNE-NC）。采用RT-qPCR和Western blot法检测细胞转染效果,划痕实验和Transwell小室实验检测VEGF沉默后细胞的迁移和侵袭能力。结果 成功构建VEGF-shRNA重组慢病毒并建立了稳定沉默VEGF细胞CNE-LV3。划痕实验和Transwell小室迁移实验显示,与空白对照组和CNE-NC组相比,CNE-LV3组细胞迁移率明显降低［（50.17±1.76）% vs （37.97±1.56）%,P＝0.001;（50.63±1.52）% vs （37.97±1.56）%,P＝0.001］,穿膜细胞数亦明显下降［（95.3±3.8） 个 vs （41.3±1.5） 个,P＜0.001;（94.3±4.0） 个 vs （41.3±1.5） 个,P＜0.001］;Transwell小室侵袭实验结果亦显示,CNE-LV3组细胞穿膜细胞数较空白对照组和CNE-NC组明显下降［（46.3±2.1） 个 vs（105.3±1.5） 个,P＜1.001;（46.3±2.1） 个 vs （93.3±2.5） 个,P＜0.001］。结论 沉默VEGF表达可抑制人鼻咽癌放射抗拒细胞株CNE-2R的迁移和侵袭能力。