抗凋亡基因bcl-2转染哺乳动物细胞的研究

目的：构建抗凋亡基因bcl-2的哺乳动物细胞表达型质粒并转染哺乳动物细胞293T细胞。方法：利用基因重组技术，将bcl-2全编码cDNA序列，导人pCMV-Tag1表达型质粒；应用磷酸钙共沉淀技术，将其转染293T细胞：并利用免疫沉淀和Westernblotting技术检测Bcl-2融合蛋白的表达。结果：bcl-2全编码cDNA序列成功导人pCMV-Tag1表达型质粒；在转染后的293T细胞成功地检测出bcl-2融合蛋白的表达。融合蛋白大小与预期完全一致。结论：该表达型质粒可以成功转染哺乳动物细胞，为对胰岛细胞的转染奠定了基础。     

SYBR-Green实时定量PCR用于Vero宿主细胞DNA残留量的检测

目的:建立基于SYBR-Green荧光染料实时定量PCR检测Vero宿主细胞DNA残留量的方法。方法:提取Vero细胞基因组DNA,设计细胞基因组高度重复顺序AGMr(HindIII)-1基因片段的特异性引物,通过PCR扩增AGMr(HindIII)-1序列中一段特异性cDNA片段,克隆至pGEM-T Vector,重组质粒经酶切及测序鉴定合格后命名为pGEM-T/AGMr(HindIII)-1-S。结果:使用重组质粒和细胞基因组DNA分别作为检测标准品,均取得了良好的结果,其检测灵敏度分别达到了0.03 fg.μL-1和0.03 pg.μL-1。结论:SYBR-Green实时定量PCR可用于Vero宿主细胞DNA残留量的准确定量。     

细胞分裂素产生菌质粒DNA的分离和鉴定

本文首次报道从细胞分裂素产生菌泾阳链霉菌A_3中分离得到两种质粒,并经电镜观察得到了证实。琼脂糖凝胶电泳分析,表明质粒DNA的分子量分别为4.8×10~6道尔顿(7.3kb)和3.4×10~6道尔顿(5.7kb),并命名为pSJ_1、和pSJ_2。快中子消除质粒处理,得到的无质粒菌株(B_(11))与原株(A_3)比较,两者在培养特征方面没有差异,但在产生细胞分裂素及硝酸盐还原酶方面存在差异。菌株B_(11)的细胞分裂素产量和酶活性高于和显著高于原株A_3。结果表明,质粒pSJ_1和pSJ_2可能携带调控细胞分裂素生物合成及硝酸盐还原酶的某些基因。     

质粒DNA类基因治疗药物的研究现状及发展趋势

基因治疗是将外源基因导入人体以纠正基因缺陷的方法,通过转录和翻译实现对细胞基因表达的调控,从而合成特异性蛋白治疗相关疾病。质粒DNA是经基因工程改造过的环状DNA分子,其结构简单,具有自主复制能力,可以携带治疗基因导入人体细胞,被广泛用于基因治疗的研究中。目前已上市及正处于临床研究的基于质粒DNA的基因药物包括cambiogenplasmid、Tavo等。对遗传病、恶性肿瘤等适应证,质粒DNA的基因治疗比传统的治疗方案有明显优势。CRISPR-Cas9等相关的质粒基因编辑技术成为质粒DNA类基因治疗的一大研发趋势,基因枪、聚合物纳米载体等递送系统以透皮给药、静脉注射等方式为质粒DNA导入体内提供了更多可能。基于质粒DNA类的基因治疗已成为基因治疗领域的一种理想技术手段。     

利用哺乳动物细胞双杂交体系研究仙灵骨葆对雌激素受体的作用

目的:利用哺乳动物细胞双杂交体系研究仙灵骨葆对雌激素受体(ERs)活性的影响。方法:通过MTT法检测仙灵骨葆对Hela细胞活力的影响,并构建含有ERs配体结合域(LBD)的质粒pCMV-BD及含有报告基因Lusferase的质粒pFR-Luci,将二者与内标质粒pFRTlaczeo共转染到Hela细胞中,形成Gal4DBD-ERa和ERb LBD细胞杂交报告系统。以染料木苷为阳性对照验证本杂交系统,并利用本系统考察仙灵骨葆对ERs活性的影响。结果:仙灵骨葆在25～2 500 ng·mL-1浓度范围内可以促进HeLa细胞的增殖。10μmol．L-1染料木苷可以显著提高Gal4DBD-ERα和ERβLBD细胞杂交报告系统中ERa和ERb的活性,证明本系统构建成功。仙灵骨葆对ERa具有浓度依赖性的激活作用,2500 ng·mL-1浓度时ERa的相对荧光活性是DMSO对照组的18倍;仙灵骨葆对ERb也具有一定的激活作用,2500 ng·mL-1浓度时ERb的相对荧光活性是DMSO对照组的4倍。结论:本研究成功构建了含有雌激素受体配体结合域的细胞杂交系统,仙灵骨葆对该系统的ERs具有激活作用。     

人DNA聚合酶β高表达细胞HLFβ的细胞学分析

目的获取人HLF细胞内稳定高表达人DNA聚合酶beta(pol-β)，并对pol-β稳定高表达细胞的细胞学特征进行分析。方法使用构建的人pol-β全长cDNA的绿色荧光蛋白GFP表达载体pEGFP-Cl-β，转染人HLF细胞，经G418筛选，获得单克隆的pol-β稳定高表达的细胞HLFβ，并对HLFβ细胞的细胞增殖、细胞周期、自发突变率、恶性增殖能力等特征进行分析。结果Western印迹显示，HLFβ细胞的pol-β蛋白表达稳定，较空载体转染对照细胞高60％以上，且在不同代间表达稳定。HLFβ细胞增殖能力较对照细胞增强。HLFβ细胞自发突变率略有增加，但与对照细胞的差异无显著性，细胞周期分布及细胞恶性程度亦无明显改变。结论该研究成功获得人pol-β稳定高表达细胞HLFβ，在正常生理状况下，pol-β的高表达对细胞周期、自发突变和恶性增殖能力未见明显影响。     

细胞凋亡调控及其在生物技术中的应用

对表示细胞凋亡控制特性的分子相互作用的大量知识,正用于确定一些阻止细胞死亡和产生较大活细胞生物量的新方法。这些方法用于重组哺乳动物细胞可提高工业规模蛋白表达的成本－效益。     

生长抑素DNA疫苗在COS细胞中的瞬时表达

构建了生长抑素DNA疫苗制裁粒pCHSS(pCDNA/HBsAg/SS)，并在COS细胞中的获得较高水平的瞬时表达，用HBsAgElisa检测试剂盒跟踪发现，约有27%的融合蛋白分泌到细胞外，说明SS的插入没有影响HBsAg的免疫反应性和分泌表达，免疫电镜观察，HBsAg可以组装成22nm-30nm的颗粒，说明SS的插入没有影响HBsAg的组装，该实验为动物实验打下基础。     

凝血因子Ⅷ(FⅧ)在哺乳动物细胞中的表达研究

对FⅧ cDNA进行分子改建，并分别克隆到表达载体中，在多种哺乳动物细胞表达体系（CHO、BHK等）成功地获得了表达。结果证实：基因的改建可以有效的提高FⅧ在细胞中的表达。FⅧ B结构域的缺失比全长FⅧ的表达量提高了3倍左右，而重链和轻链的共表达及vWF与FⅧ的共同转染使得其表达量进一步提高。与CHO表达系统相比较，FⅧ在BHK表达系统中的表达量提高了近10倍。     

外源性rIL-10基因质粒静脉注射及其在大鼠体内表达

目的建立大鼠体内质粒DNA尾静脉快速大容量注射法,使外源性rIL-10在肝组织中高表达,为探索肝脏疾病的基因治疗提供动物试验手段。方法以rIL-10为报告基因,将含不同体积及不同质粒浓度的质粒DNA溶液经大鼠尾静脉快速注射,在注射后的不同时间内分别收集血浆及肝、肾、肺、脾和心组织,使用RT-PCR、ELISA和免疫组化法检测rIL-10在体内的表达情况。结果尾静脉快速大容量注射rIL-10DNA溶液可使rIL-10基因在大鼠肝脏有较明显转录及表达。血浆中rIL-10浓度可通过质粒的重复注射使其保持在一个稳态。结论尾静脉快速大容量注射法是一种简单、方便和有效的大鼠体内基因转移及基因表达的方法,为进一步探讨rIL-10基因在肝脏疾病的基因治疗打下基础。     

Development of the Liver- and Lobe-Selective Nonviral Gene Transfer Following the Instillation of Naked Plasmid DNA Using Catheter on the Liver Surface in Mice

Purpose. The present study has undertaken the liver- and lobe-selective nonviral gene transfer following the instillation of naked plasmid DNA (pDNA) using catheter on the liver surface in mice. Methods. The polyethernylon catheter was inserted intraperitoneally through the abdominal wall and was retained on the surface of the liver right and left medial lobes. pDNA was administered through the catheter to the liver right and left medial lobes. Results. The luciferase levels produced in the applied liver lobes at 6 h after liver surface instillation of pDNA were significantly higher than those produced in other liver lobes and other tissues assayed, and ranged from approximately 5 folds higher in other lobes to 20-30 folds higher in other tissues. Following liver surface instillation of pDNA at a time from 2 to 24 h or at a volume from 15 to 60 l, the gene expressions of the applied liver lobes were always significantly higher than those of other liver lobes and other tissues. Conclusion. We have demonstrated the liver- and lobe-selective gene transfection following the instillation of naked pDNA using catheter on the liver surface in mice.     

Synergistic cytotoxicity and DNA strand breaks in cells and plasmid DNA exposed to uranyl acetate and ultraviolet radiation

Depleted uranium (DU) has a chemical toxicity that is independent of its radioactivity. The purpose of this study was to explore the photoactivation of uranyl ion by ultraviolet (UV) radiation as a chemical mechanism of uranium genotoxicity. The ability of UVB (302 nm) and UVA (368 nm) radiation to photoactivate uranyl ion to produce single strand breaks was measured in pBR322 plasmid DNA, and the presence of adducts and apurinic/apyrimidinic sites that could be converted to single strand breaks by heat and piperidine was analyzed. Results showed that DNA lesions in plasmid DNA exposed to UVB‐ or UVA‐activated DU were only slightly heat reactive, but were piperidine sensitive. The cytotoxicity of UVB‐activated uranyl ion was measured in repair‐proficient and repair‐deficient Chinese hamster ovary cells and human keratinocyte HaCaT cells. The cytotoxicity of co‐exposures of uranyl ion and UVB radiation was dependent on the order of exposure and was greater than co‐exposures of arsenite and UVB radiation. Uranyl ion and UVB radiation were synergistically cytotoxic in cells, and cells exposed to photoactivated DU required different DNA repair pathways than cells exposed to non‐photoactivated DU. This study contributes to our understanding of the DNA lesions formed by DU, as well as their repair. Results suggest that excitation of uranyl ion by UV radiation can provide a pathway for uranyl ion to be chemically genotoxic in populations with dermal exposures to uranium and UV radiation, which would make skin an overlooked target organ for uranium exposures. Copyright © 2014 John Wiley & Sons, Ltd.     

中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域

目的：采用中心组合设计法优化载基因壳聚糖纳米粒的最佳转染制备区域。方法采用复凝聚法制备载质粒基因的壳聚糖纳米粒,选择壳聚糖浓度和质粒基因浓度作为实验考察因素,应用两因素五水平中心组合设计优化最佳转染制备区域,优化指标选择平均粒径和基因转染率。通过透射电镜观察纳米粒的形态;通过动态光散射和电泳光散射技术分别测量纳米粒的粒径和Zeta电位;通过凝胶电泳分析考察质粒在纳米粒制备过程中的稳定性;通过倒置荧光显微镜观察质粒基因在细胞内的表达;通过流式细胞技术测定纳米粒的转染效率。结果成功优化了载基因壳聚糖纳米粒的最佳转染制备区域。优选条件下制备的纳米粒大多呈球形,纳米粒平均粒径为217．6 nm,粒径多分散系数为0．241,表明粒径分布较窄。纳米粒zeta电位为＋22．4 mV,表明纳米粒表面带有正电荷,可以增加纳米粒混悬液的稳定性。凝胶电泳分析结果表明质粒基因在纳米粒制备过程中没有遭到破坏。纳米粒的细胞转染效率比较高,能够高效地将绿色荧光蛋白质粒基因递送到细胞内,并且基因表达产生绿色荧光蛋白。结论本研究建立的数学模型具有良好的预测性。在优化的制备区域内制备的载基因壳聚糖纳米粒的转染性能比较理想。     

In vitro transfection of plasmid DNA by amine derivatives of gelatin accompanied with ultrasound irradiation

Purpose. The purpose of this study is to examine the ultrasound (US)-enhanced gene expression by the complexes of a plasmid DNA with gelatin derivatives of aminization. Methods. Gelatin derivatives with different introduced extents of ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) were prepared with a water-soluble carbodiimide. The molecular size and zeta potential of the gelatin derivatives before and after complexation with the plasmid DNA were examined. After incubation with the complexes with or without US exposure, the DNA expression of rat gastric mucosal cells was measured to evaluate the effect of the type of gelatin derivatives on their gene expression. The cell uptake of the complexes, the cell viability, and the buffering effect of gelatin derivatives were examined. Results. The apparent molecular size and zeta potential of gelatin derivatives became larger as their aminization extent increased although the Sm gelatin derivative of higher aminization showed a larger value than other corresponding derivatives. Irrespective of the type of gelatin derivatives, the apparent molecular size of plasmid DNA was reduced by increasing the gelatin-DNA mixing ratio to attain a saturated value of about 150 nm. The condensed gelatin-DNA complexes showed the zeta potential of 10-15 mV. The cells incubated with the complex exhibited significantly stronger luciferase activities than free plasmid DNA, and the activity was further enhanced by US irradiation. The enhancement was significant for the Sm derivative compared with the corresponding Ed and Sd derivatives. The amount of plasmid DNA internalized into the cells was significantly increased by the complexation with every gelatin derivative, whereas US irradiation did not significantly increase the DNA internalization. US irradiation had no effect on the viability of cells incubated with every gelatin derivative-plasmid DNA complex, although the viability was decreased by the complex incubation. The buffering capacity of Sm derivative was higher than that of Ed and Sd derivatives and comparable with that of polyethylene amine. Conclusion. Among amine derivatives of gelatin, the Sm derivative enabled the plasmid DNA to induce the US-enhanced gene expression of cells in vitro most effectively because of the superior buffering effect.     

蚓激酶重组逆转录病毒载体pLN-BCP-LK~R的构建及其在山羊乳腺组织中的表达

目的在山羊奶中表达蚓激酶。方法将含有蚓激酶基因的表达盒插入到逆转录病毒载体pLNCL中获得了蚓激酶重组逆转录病毒载体,转染泌乳期奶山羊乳腺小叶并获得暂时表达。利用脂质体转染PA317包装细胞,G418进行筛选得到阳性细胞克隆,克隆扩大培养后病毒上清感染妊娠期奶山羊乳腺小叶组织,山羊产后采奶纤维蛋白平板溶圈法检测蚓激酶活性。结果产后奶样经纤维蛋白平板溶圈法检测羊奶具有明显纤溶活性。结论蚓激酶基因已经整合到奶山羊乳腺组织并能实现相对稳定的表达。     

弓形体SAG1基因DNA诱导小鼠体液免疫应答及保护性研究

目的 :用重组真核表达质粒 pc DNA3- SAG1直接免疫 BAL B/ c小鼠 ,观察其 DNA免疫所诱导的小鼠体液免疫反应和保护性作用。方法 :大量制备重组质粒 pc DNA3- SAG1,然后将其导入 BAL B/ c小鼠体内。抗体滴度用 EL ISA法进行测定 :采用 PCR方法对小鼠的血液组织外源基因进行检测 ;对试验组及对照组进行 RH株攻击感染 ,并对其进行分析。结果 :用 EL ISA法检测的抗体 Ig G滴度为 1∶ 2 5 6 0 ;免疫后三周、六个月从免疫鼠的血液组织中用 PCR仍可检测到特异的SAG1基因的存在 :体外弓形虫攻击感染实验组和对照组 ,可见实验组的存活时间较对照组时间延长 (P<0 .0 5 )。结论 :重组质粒 pc DNA3- SAG1免疫 BAL B/ c小鼠可诱导一定的体液免疫应答和一定的保护性作用     

Enhanced In Vitro Oligonucleotide and Plasmid DNA Transport by VP1 Virus-like Particles

Purpose. We developed a prokaryotic expression system to express the major capsid protein of Polyomavirus, VP1. Furthermore, we investigated the transport of single stranded (ss) and double stranded (ds) DNA, mediated through VP1 as drug delivery system into mouse fibroblasts. Methods. To study DNA delivery we used two kinds of DNA, a ssDNA fragment (19mer) and dsDNA (plasmid pEGFPN1, 4.7 kb or a FITC-labelled dsDNA fragment, 1.8 kb). Results. The uptake of VP1 capsoids loaded with FITC-labelled oligodeoxynucleotides (FODNs) was observed. VP1 pentamers loaded with condensates of dendrimer/dsDNA fragments (FITC-labelled) resulted in significantly higher fluorescence signal in the cytoplasm of NIH 3T3 cells in comparison to control experiments without VP1. Additionally, VP1 capsoids loaded with plasmid pEGFPN1 without dendrimers resulted in an approximately 10 fold higher transfection rate in comparison to blank DNA controls. Conclusions. Our results demonstrated the potential of VP1 capsoids as DNA delivery system. EGFP expression was significantly enhanced when plasmid DNA was delivered via VP1 capsoids, compared to control experiments with naked DNA.