大鼠弹力蛋白原基因重组腺病毒载体的构建及在血管平滑肌细胞中的表达

目的构建携载大鼠弹力蛋白原（tropoelastin）基因的重组腺病毒载体，并在体外培养大鼠血管平滑肌细胞（VSMC）中表达。方法利用基因重组技术将腺病毒骨架质粒pAdEasy-1以及线性化的重组穿梭质粒pShuttleTropoelastin-GFP进行同源重组，筛选出重组黏粒pAdTropoelas-tin-GFP，经过包装、扩增、纯化后获得重组腺病毒AdTropoelasfin-GFP，测定病毒滴度。腺病毒体外感染大鼠主动脉平滑肌细胞，一步法提取细胞总RNA，逆转录后，采用实时荧光定量聚合酶链反应（real-timePCR）检测tropoelastin的mRNA。结果得到了携载tropoelastin基因的重组腺病毒，纯化后滴度为5×10^11pfu／ml，腺病毒转染VSMC后1、3dtropoelastinmRNA表达较空病毒明显增高（P〈0．01），并超出空病毒转染组2倍以上。结论成功构建了携带tropoelastin的重组腺病毒载体，体外转染的VSMC有效表达目的基因。     

大鼠VEGF腺病毒基因转染系统的构建及鉴定

目的:探讨构建携带大鼠血管内皮细胞生长因子(VEGF)的重组腺病毒载体方法，为后续的基因转染研究作准备。方法:采用RT-PCR扩增的方法获取鼠源性的VEGF,并克隆入穿梭质粒pDC316。构建的质粒pDC316-VEGF经酶切及测序鉴定正确后，通过lipofectamine2000的介导与腺病毒包装质粒pBHGE3共转染至人胚肾细胞HEK293，经同源重组后获得携带鼠VEGF的重组腺病毒VDC316-VEGF。应用PCR鉴定重组腺病毒，空斑传代纯化病毒并反复冻融扩增病毒。以50%组织培养感染剂量法(TCID50)测定病毒滴度。结果:PCR鉴定证实重组腺病毒含有鼠VEGF,病毒滴度为3×109pfu/mL。结论:成功构建的携带大鼠VEGF的重组腺病毒载体能在HEK293细胞内扩增获得足够高的病毒滴度,可作为后续基因治疗研究工作中可靠的基因转染工具。     

人骨保护素基因重组腺病毒的构建及鉴定

目的构建人骨保护素(hOPG)基因的重组腺病毒载体并检测其转染能力。方法采用基因工程技术,将hOPG基因片段插入到腺病毒载体质粒pAdTrack-CMV上,利用PadEasy系统与骨架质粒在大肠杆菌BJ5183胞内进行同源重组,经293细胞包装、扩增,得到携带hOPG基因的重组腺病毒AdEasy-PAdtrack-CMV-hOPG,将重组腺病毒AdhOPG对大鼠骨髓基质细胞(rMSCs) 进行感染。采用PCR方法对重组腺病毒载体进行鉴定,利用绿色荧光报告基因转染结果,以荧光计数法检测重组腺病毒的滴度。结果酶切鉴定及PCR结果证明hOPG基因重组腺病毒载体成功, 病毒滴度可达2．5×108pfu／ml,具有很强感染能力,结论应用细菌内同源重组法,成功构建了含 hOPG重组腺病毒载体。     

携带融合基因CTLA4-Ig重组腺病毒载体的构建及表达

目的　为进行基因转移阻断T细胞共刺激通路诱导移植免疫耐受的研究 ,构建携带融合基因CTLA4 Ig的重组腺病毒载体。方法　构建含人CTLA4 Ig的重组腺病毒载体质粒pShuttle Tracker CMV CTLA4 Ig ,与腺病毒骨架质粒 pAdEasy 1 △E1、△E3共转化大肠杆菌BJ5 183 ,经细菌内同源重组 ,获得重组腺病毒质粒pAd Tracker CTLA4 Ig ,转染 2 93细胞 ,制备携带CTLA4 Ig的重组腺病毒 ,体外感染L O2细胞 72h后ELISA检测其外分泌性表达。 结果　获得携带CTLA4 Ig的重组腺病毒 ,滴度为 6× 10 1 3 PFU L ;在其感染的L O2细胞培养上清中能检测到可溶性重组蛋白CTLA4 Ig的表达 (P N =4.6 ,阳性 )。结论　制备的重组腺病毒在体外能有效感染L O2细胞 ,受感染细胞能表达、分泌可溶性的融合蛋白CTLA4 Ig ;为进行体内移植免疫耐受的基因治疗研究奠定基础。     

重组Akt腺病毒的构建及其在肝硬化大鼠肝脏中的表达

目的:探讨持续活化Akt且带有HA标签(myr-HA-Akt)基因的重组腺病毒在肝硬化大鼠肝脏中的表达特性。方法:酶切正向插入目的基因的真核表达载体pcDNA3.1-myr-HA-Akt,获得myr-HA-Akt cDNA后，将其定向克隆到穿梭质粒pDC316中,然后将重组质粒与病毒骨架质粒pBHGloxΔE1,3Cre共转染293 细胞,获得复制缺陷型重组腺病毒Ad-myr-HA-Akt,并进行扩增、纯化。通过观察腺病毒感染293细胞后是否出现细胞病变效应;PCR和基因测序方法对所构建病毒进行鉴定，并采用TCID50法检测病毒滴度。自尾静脉注射重组腺病毒Ad-myr-HA-Akt感染肝硬化模型大鼠。免疫印迹法检测大鼠肝组织内Akt 和p-Akt蛋白的表达。结果:感染的293 细胞出现明显的细胞病变效应，PCR产物电泳证实重组腺病毒的存在，基因测序证实myr-HA-Akt的cDNA正确插入穿梭质粒且与pBHGloxΔE1,3Cre正确同源重组;病毒滴度为5.5×1011 vp/mL。从蛋白水平证实感染病毒的肝硬化模型大鼠有外源性Akt基因的表达。结论:构建的重组腺病毒Ad-myr-HA-Akt能有效地感染肝硬化模型大鼠,并可在模型大鼠中正确转录和翻译，为进一步研究腺病毒介导的Akt基因对肝硬化的治疗奠定了实验基础。     

腺病毒介导野生型PTEN基因对乳腺癌MDA MB 468细胞周期的影响

摘要：目的 探讨腺病毒介导野生型PTEN基因对乳腺癌MDA MB 468细胞周期时相的影响。方法 构建含PTEN目的基因的腺病毒穿梭质粒pAdTrack CMV PTEN与骨架质粒pAdEasy 1，将重组腺病毒pAd PTEN制备成纯化高效的含绿色荧光蛋白（GFP）的PTEN腺病毒，体外转染人乳腺癌MDA MB 468后，检测PTEN表达后对乳腺癌MDA MB 468细胞周期的影响。结果 携带PTEN基因重组腺病毒载体成功构建，检测病毒滴度为2.5×1010pfu/mL。表明重组腺病毒已含有PTEN蛋白。流式细胞术显示对照组处于G1期的细胞占41.18%，PTEN蛋白表达后处于G1期的细胞占56.47%。结论 利用腺病毒载体系统AdEasy 1可快速构建表达PTEN基因的腺病毒载体，可高效制备均一的高滴度重组病毒；野生型PTEN基因转染使乳腺癌细胞周期停滞于G1期。     

携带白蛋白启动子和IDO基因重组腺病毒的构建

目的 构建携带小鼠白蛋白启动子和IDO基因的重组腺病毒载体,研究肝脏Hepa l石细胞的IDO基因mRNA及蛋白表达情况.方法 酶切含有小鼠全长IDO cDNA的IDO质粒,亚克隆至穿梭载体pAdTrack.ALB上,在BJ5183细菌中和AdEasy-1进行同源重组,生成并筛选阳性克隆,测序、鉴定正确后,转染AD-293细胞进行包装、扩增,检测病毒滴度,RT-PCR和荧光显微镜鉴定重组腺病毒转染AD-293细胞后IDO的表达.重组腺病毒进一步感染Hepa 1-6细胞,RT-PCR和WesternBlot法分别检测IDO基因在细胞内表达情况.结果 经酶切及测序证实携带白蛋白启动子和IDO基因重组腺病毒载体构建成功,RT-PCR检测到转染后AD-293细胞内IDO的表达,病毒感染滴度为2.9×10~6pfu/ml.感染Hepa 1-6细胞后,RT-PCR和Western Blot可以检测到IDO mRNA水平和蛋白水平表达.结论 构建了携带白蛋白启动子和IDO基因的重组腺病毒载体.     

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。     

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。     

人血管内皮细胞生长因子重组腺病毒载体的构建、鉴定及在骨髓间充质干细胞的表达

目的构建人血管内皮细胞生长因子(VEGF)165腺病毒表达载体,体外转染大鼠骨髓间充质干细胞研究其相关特性。方法利用细菌内同源重组技术快速构建Ad-VEGF165腺病毒重组质粒,经酶切及测序鉴定正确后转染人胚肾细胞HEK293包装成为重组Ad-VEGF165腺病毒,并进行电镜观察及滴度测定。感染大鼠骨髓间充质干细胞观察VEGF165基因的转录和表达。结果酶切鉴定及基因测序证实重组腺病毒质粒含有hVEGF165cDNA,电镜显示包装细胞中有大量病毒颗粒存在,测定包装的病毒滴度为6.3×1010TCID50/ml。逆转录-聚合酶链反应(RT-PCR)、免疫组织化学及免疫印迹检测骨髓间充质干细胞内有VEGF165的转录和表达。结论构建的VEGF腺病毒表达载体可有效感染骨髓间充质干细胞,并在体外高效表达,为将表达VEGF基因的骨髓间充质干细胞用于基因治疗提供了实验依据。     

二溴海因/二氧化硅复合粒子的制备及其杀菌性能评价

通过实验室制备，合成了二溴海因／二氧化硅复合粒子，运用扫描电镜（SEM）与红外光谱（IR）进行了表征，SEM结果显示，在SiO2的表面覆合了二溴海因，红外光谱图表明，复合粒子中二溴海因和二氧化硅之间以物理作用方式结合，将单纯二溴海因与二溴海因／二氧化硅复合粒子溶解于水，测定其在水中的释放速度，结果表明：二溴海因／二氧化硅复合粒子中的二溴海因在水中释放的速度明显慢于纯二溴海因，且能维持较长的作用时间，比较纯二溴海因与含等量二溴海因的复合粒子对大肠杆菌、金黄色葡萄球菌、白葡萄球菌和枯草芽孢杆菌的抑制效果，发现在二溴海因含量高于有效抑茵浓度时，复合粒子对茵体的抑制效果高于纯二溴海因。     

The effect of pH and PCO2 on epidural analgesia with 2% 2-chloroprocaine

Increasing the pH of local anesthetics with sodium bicarbonate has been reported to hasten their onset of action. The purpose of this study was to compare the onset and duration of epidural analgesia with the use of sodium bicarbonate and tromethamine to increase the pH of 2% chloroprocaine (2CP). Five groups of patients were studied: Group I received 2CP; Group II received 2CP buffered to a pH of 7.1 with tromethamine; Group III received 2CP buffered to a pH of 7.1 with sodium bicarbonate; Group IV received 2CP buffered to a pH of 7.7 with tromethamine; and Group V received 2CP buffered to a pH of 7.7 with sodium bicarbonate. The final pH and PCO2 of each solution were measured. Time to onset of analgesia was significantly delayed with either of the tromethamine buffered groups (II [5.6 +/- 1.0 minutes] and IV [5.4 +/- 0.4 minutes]) when compared with data from the unbuffered control (I [4.4 +/- 0.1 minutes]) and the sodium bicarbonate buffered (III [4.5 +/- 0.8 minutes] groups and Group V [2.7 +/- 0.9 minutes]). Only when sodium bicarbonate buffer adjusted to pH 7.7 (Group IV) was onset significantly more rapid than the unbuffered 2CP (I) and tromethamine buffered 2CP (II and IV). Multiple regression analysis revealed that onset times were significantly related to both pH and PCO2. The coefficient of determination for this model was 0.5156.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the association of IGF2BP2 variants with type 2 diabetes in French Caucasians

OBJECTIVE—We performed a comprehensive genetic association study of common variation spanning the IGF2BP2 locus in order to replicate the association of the “confirmed” type 2 diabetes susceptibility variants rs4402960 and rs1470579 in the French Caucasian population and to further characterize the susceptibility variants at this novel locus.RESEARCH DESIGN AND METHODS—We genotyped a total of 21 tagging single nucleotide polymorphisms spanning the IGF2BP2 locus in our type 2 diabetes case-control cohort comprising 3,093 French Caucasian subjects.RESULTS—IGF2BP2 variants rs4402960 and rs1470579 were not associated with type 2 diabetes in the present study (P = 0.632 and P = 0.896, respectively). Meta-analysis of genotype data from over 34,000 subjects demonstrated that our inability to replicate rs4402960/rs1470579 was consistent with the findings from several previous genome-wide association study (GWAS) datasets that were underpowered to detect this modest association signal (odds ratio [OR] 1.14). We obtained novel evidence that rs9826022, a borderline rare variant (5% minor allele frequency) in the 3′ downstream region, was associated with type 2 diabetes (P = 0.0002; OR 1.53 [95% CI 1.22–1.91]). This result was corroborated by the meta-analysis of 10,542 genotypes from the current study and GWAS datasets using both fixed (P = 9.47 × 10⁻⁶; 1.30 [1.16–1.46]) and random effects (P = 0.001; 1.30 [1.11–1.52)] calculations.CONCLUSIONS—We were unable to replicate the confirmed rs4402960/rs1470579 susceptibility variants but found novel evidence for a rare variant in the 3′ downstream region of IGF2BP2. Further genetic and functional studies are required to identify the etiological IGF2BP2 variants.The insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) gene on chromosome 3q27 is a paralog of IGF2BP1, a known regulator of IGF2 gene expression. Genome-wide association studies (GWASs) carried out by the Finland-U.S. Investigation of NIDDM Genetics (FUSION) (1), the Wellcome Trust Case Control Consortium (WTCCC) (2), and the Diabetes Genetics Initiative (DGI) (3) groups each found modest evidence that single nucleotide polymorphisms (SNPs) in the IGF2BP2 region are associated with type 2 diabetes. The subsequent meta-analysis of primary and replication datasets from these GWASs corroborated these findings and identified two strongly correlated IGF2BP2 variants, rs1470579 and rs4402960, as “confirmed” type 2 diabetes susceptibility variants (1–3). By contrast, the French/Canadian GWAS (4) typed 10 SNPs across the IGF2BP2 locus, including rs1470579, in 1,363 subjects, but found no nominal (P < 0.05) association signals at IGF2BP2. In an attempt to replicate the IGF2BP2 association findings in the French Caucasian population in a larger study and to further characterize the susceptibility variants at this novel locus, we performed an association study of HapMap Phase II tag SNPs spanning the IGF2BP2 locus in 3,093 French Caucasian subjects.