Vegfr2-luc转基因子代小鼠的鉴定

背景与目的 Vegfr2-luc转基因小鼠体内血管内皮生长因子受体2(vascular endothelial growth factor recep- tor 2,VEGFR2)的表达可以驱动荧光素酶报告基因(luciferase,luc)的表达,是活体动物水平实时监测血管生成情况的有利工具。本研究旨在对子代Vegfr2-luc转基因小鼠进行鉴定,以确定能否用于血管生成研究。方法 PCR检测新生小鼠基因组内luc基因;利用活体成像技术观察新生Vegfr2-luc转基因小鼠生长发育过程中以及皮肤伤口修复过程中 luc基因表达水平的变化情况;荧光素酶报告基因检测试剂盒检测成年(8周龄)转基因小鼠各脏器荧光素酶的活性和Real-time PCR检测各器官VEGFR2 mRNA的表达水平。结果 PCR结果显示50%(56/112)的新生小鼠携带luc基因。活体成像结果显示随着Vegfr2-luc转基因小鼠发育成熟,luc表达量逐渐降低(P<0.001);在皮肤伤口修复过程中, 伤口处luc表达水平先增强后降低(P<0.001)。雌性成年转基因小鼠各脏器VEGFR2 mRNA的表达水平与荧光素酶活性呈正相关(r=0.948,P<0.001)。将睾丸组织除外,雄性成年转基因小鼠各脏器VEGFR2mRNA的表达水平与荧光素酶活性同样呈正相关(r=0.836,P<0.001)。结论 Vegfr2-luc转基因子代小鼠体内luc表达水平的变化可以反映VEGFR2 的表达情况。     

人多巴胺D2受体基因内含子区51103 T/C多态性对基因转录活性的影响

目的：探讨人多巴胺D2受体(DRD2)基因第3内含子区51103 bp位点单核苷酸多态性(rs2075652)对DRD2基因转录活性的影响,进一步揭示DRD2基因第3内含子区的单核苷酸多态性影响创伤后应激障碍发病风险的分子机制。方法以人全血基因组DNA为模板,扩增DRD2基因第3内含子区序列(399 bp),51103 bp处分别为T或C,与报告基因载体pmirGlo相连接,构建重组荧光素酶表达载体,经限制性内切酶酶切及测序得以鉴定,然后分别与pRL-CMV共转染人胚肾癌细胞株HEK293,检测细胞中荧光素酶的相对表达量。结果双酶切及测序结果证实成功构建重组质粒pmirGlo-T/pmirGlo-C,荧光素酶检测结果显示,当第3内含子区51103 bp位点为C时荧光素酶活性显著高于为T时(P<0．01)。结论成功构建了pmirGlo-T/pmirGlo-C报告基因重组质粒,DRD2基因第3内含子区rs2075652位点由T突变为C后,可能增强基因转录活性,该结果为进一步研究DRD2基因内含子区的功能奠定了基础。     

黄芪中7种活性成分对缺氧诱导因子(HIF-1)表达的影响

目的:研究黄芪中的7个主要活性成分芒柄花素、芒柄花苷、毛蕊异黄酮葡萄糖苷、毛蕊异黄酮、乙酰黄芪皂苷Ⅰ、黄芪皂苷Ⅱ、黄芪甲苷对缺氧诱导因子(HIF-1)表达的影响。方法:将包含有高度保留的HIF-1结合位点的缺氧应答原件(HRE)插入到含有荧光素酶报告基因的pBI-GL质粒中,磷酸钙沉降法将构建的质粒转染到人胚肾(HEK)293T纤维原细胞。用不同浓度的药物处理48h后测定荧光素酶的活性,通过荧光素酶报告基因系统评估HIF-1的表达。结果:10μmol·L-1芒柄花素可促进HIF-1的表达。芒柄花苷与HIF-1的表达呈浓度-效应依赖关系,低浓度促进其表达,高浓度抑制其表达。其它5种单体与对照比较无明显作用趋势。结论:本试验可为黄芪的抗肿瘤作用及其他疾病的治疗提供一定依据。     

Application of bioluminescence imaging to therapeutic intervention of herpes simplex virus type I – Thymidine kinase/ganciclovir in glioma

Lentiviral vector containing the HSV1-tk and firefly luciferase (fLuc) gene was infected into C6 and C6-TL expressing HSV1-tk and fLuc gene was generated. C6-TL showed higher [¹²⁵I]IVDU uptake than C6. The survival rate of C6-TL decreased more rapidly with increasing GCV dose and was well correlated with fLuc activity. The images of microPET clearly demonstrated higher uptake of [¹⁸F]FHBG into the C6-TL tumor. Inhibition of tumor growth was observed in C6-TL tumor-bearing mice treated with GCV through tumor size measurement and bioluminescence imaging. The therapeutic effect of HSV1-tk/GCV system can be monitored using bioluminescent imaging and tumor size measurement.     

Rhythmic expression of per1 in the dentate gyrus is suppressed by corticosterone: Implications for neurogenesis

The stimulating action of anti-depressant drugs on the number of mitotic cells on the dentate gyrus on the adult rat depends on an intact diurnal rhythm of corticosterone. This suggests that there may be a clock mechanism in the dentate gyrus which is sensitive to corticoids. This paper reports the diurnal expression the ‘clock’ gene per1 in the dentate gyrus, and how it is altered by clamping the diurnal rhythm in corticosterone. We show that there is a diurnal rhythm in the number of mitotic progenitor cells in the dentate gyrus of the hippocampus in adult male per1-luciferase rats, approximately 6 h out of phase with the plasma corticosterone rhythm. This is suppressed by clamping the daily corticosterone levels by a subcutaneous implant of corticosterone (100 mg). There was also a daily rhythm of per1 expression in both suprachiasmatic nucleus (SCN) and the dentate gyrus, which were in phase with one another. The per1 rhythm in the dentate gyrus, but not the SCN, was suppressed by clamping the plasma corticosterone rhythm. These results are related to the previous finding that clamping the corticosterone rhythm also prevents the stimulating action of fluoxetine and other controlling agents on the mitotic activity of the progenitor cells.     

报告基因研究及其应用进展

报告基因是现代分子生物学研究领域中用于分析结构基因旁侧区域潜在的顺式元件(如启动子、增强子和沉默子等)和反式作用因子相互作用关系的一种重要工具,通过它的表达产物便捷、可靠、灵敏地解析基因时空表达调控的内在规律.报告基因技术广泛应用于基因表达调控、信号转导、转基因、启动子分析、受体功能鉴定、基因治疗以及药物筛选等领域,该文将对报告基因的主要类型、特点及其应用研究进展进行综述.     

PLGA/chitosan composites from a combination of spray drying and supercritical fluid foaming techniques: New carriers for DNA delivery

The use of poly(_{D, L}-lactic-co-glycolic acid) for DNA delivery application is limited by its negative surface charge and acidic degradation products. The motivation of the present work was to study the effects of chitosan incorporation into PLGA foams on DNA delivery. PLGA/chitosan composite foams loaded with luciferase plasmid were fabricated by a combination of spray drying and supercritical CO₂ foaming techniques. The resultant composite foams showed good morphology and chitosan was found to be poorly crystallized in the PLGA matrixes. The composite foams exhibited a sustained release of DNA (5–9weeks) with decreasing release rate upon increasing content of chitosan. With this encapsulation technique, it was also observed that the integrity of plasmid was well maintained. Moreover, cell culture results proved that the bioactivity of plasmid released from all foams was well maintained and the incorporation of chitosan in foams helps increase cell adhesion and maintain high cell viability. Therefore, it can be concluded that PLGA/chitosan composite foams fabricated by combining spray drying and supercritical CO₂ foaming are promising in DNA delivery applications.     

碱性成纤维细胞生长因子和环磷酸腺苷对神经前体细胞内源性端粒酶反转录酶基因转录水平的调控

目的 探讨人类神经前体细胞中内源性人端粒酶反转录酶(hTERT)基因的转录活性及增殖与分化相关调控机制.方法 采用系列hTERT基因启动子的荧光素酶报告载体和β-半乳苷酶表达载体,瞬时共转染HeLa细胞和hNPCs-G3细胞,检测不同长度启动子的活性.分析碱性成纤维细胞生长因子(bFGF)促进增殖过程中hTERT基因启动子活性,分析cAMP诱导分化过程中hTERT基因启动子活性.结果 hNPCs-G3神经前体细胞内源性hTERT基因的转录活性较低,主要依赖近端启动子区.bFGF上调hNPCs-G3神经前体细胞内源性hTERT基因的表达,其调控主要作用在-2 098～-1 099bp区域和-3 216bp～-2 098bp区域内;cAMP 抑制hNPCs-G3神经前体细胞hTERT基因的转录,其调控主要作用在近端启动子区.结论 神经前体细胞内源性hTERT基因的转录活性较低,bFGF可上调神经前体细胞内源性hTERT基因的表达,cAMP则可抑制hTERT基因的转录.