带HSV－tk基因的重组腺病毒杀伤离体肺癌细胞的研究

带有单纯疱疹病毒脱氧胸苷激酶基因（ＨＳＶ－ｔｋ）的腺病毒结合ｇａｎｃｉｃｌｏｖｉｒ（ＧＣＶ）对分裂细胞有很强的杀伤作用．在感染复数（Ｍ．Ｏ．Ｉ,ＭｕｌｔｉＰｌｉｃｉｔｙｏｆＩｎｆｅｃｔｉｏｎ）达到１０００时对人体肺腺癌细胞Ａ５４９的杀伤几乎达到１００％．四种人体肺癌细胞株（Ａ５４９,ＬＡＸ,ＳＰＣ,ＳＫＹ）对带ＨＳＶ－ｔｋ的腺病毒（ＡＤＶ／ＲＳＶ－ｔｋ）的杀伤作用表现出不同的敏感性．另Ａｃｙｃｌｏｖｉｒ（ＡＣＶ）和ＧＣＶ对感染了重组腺病毒ＡＤＶ／ＲＳＶ－ｔｋ的细胞都有一定杀伤作用,但杀伤效果有很大差别;就Ａ５４９而言,ＧＣＶ的杀伤作用比ＡＣＶ高７～８倍．此外ＡＤＶ／ＲＳＶ－ｔｋ结合ＧＣＶ杀伤肿瘤细胞时有“旁观者效应”,即感染了ＡＤＶ／ＲＳＶ－ｔｋ的细胞与未感染细胞混合后,后者也明显地遭到杀伤．     

转录靶向性KDR启动子调控双自杀基因治疗肺癌的实验研究

从人肺癌细胞株中克隆KDR基因的启动子(kinasedomainreceptorpromotor,KDRp),构建KDR基因启动子调控的双自杀基因(CDglyTK)真核表达质粒pcDNA3-KDRp-CdglyTK,将其导入ECV304、L9981和NL9980细胞,建立相应的转基因细胞系,并应用不同的前药处理。体内、外实验结果显示:KDR启动子调控的双自杀基因在KDR高表达人肺癌细胞和人脐静脉内皮细胞靶向表达,而在KDR不表达的正常细胞或正常血管内皮细胞中未检测到双自杀基因表达;联合应用5-FC和GCV处理,对转双自杀基因细胞的杀伤作用显著高于单独应用5-FC或GCV,且二者显示了良好的药物协同作用。     

IL-24联合大蒜素体外抗肺癌细胞生长的实验研究

目的探讨抑癌基因IL-24联合大蒜素对肺癌A549细胞凋亡的影响,并初步探讨其作用机制.方法提取前期构建的p DC316-h IL-24-EGFP质粒,转染肺癌A549细胞,分为A组(空白对照组)、B组(单独大蒜素组(40μL/m L))、C组(脂质体+IL-24组)、D组(大蒜素(40μL/m L)+脂质体+IL-24).荧光显微镜下观察质粒的转染情况;流式细胞仪检测细胞凋亡;Western blot法检测p DC316-h IL-24-EGFP、大蒜素和联合用药作用A5 4 9细胞4 8 h后IL-24,Bax,Bcl-2和Caspase-3蛋白表达水平.结果大蒜素(40μL/m L)和p DC316-h IL-24-EGFP单用48 h对A549细胞均有明显抑制作用;大蒜素(40μL/m L)联合p DC3 1 6-h IL-24-EGFP比单用p DC3 1 6-h IL-24-EGFP作用更强(P0.05);大蒜素(40μL/m L)、p DC316-h IL-24-EGFP和两药联用48 h后,A549细胞凋亡率分别为32.7%,39.4%和68.8%;大蒜素(40μL/m L)和/或IL-24作用于A549细胞48 h后,Bax/Bcl-2的比值及Caspase-3表达均上调,联合用药组效果尤其明显.结论p DC316-h IL-24-EGFP联合大蒜素可协同抑制人肺癌A549细胞生长,机制可能是通过调节基因Bax/Bcl-2的表达比率,并上调Caspase-3的表达,进而诱导A549细胞的凋亡.     

Construction and<Emphasis Type="Italic">in vitro</Emphasis> study of an E1B-defective adenovirus

An E1B-defective adenovirus named rl/Ad was constructed by homologous recombination.The construction,selection and propagation of recombinant virus was done in the human embryonic kidney 293 cells (HEK293).The in vitro study demonstrated that the recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as the human glioblastoma tumor cells (U251) and human bladder tumer cells (EJ) but not in the normal cells with functional p53 such as the human fibroblast cells (MRC-5).Also,based on the cytopathic effect (CPE),it was demonstrated that the U251 cells were more sensitive to the infection of rl/Ad than that of EJ cells under identical conditions.In this paper,it was found that rl/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus.This may help to determine the safety of using any E1B-defective adenoviruses in cancer gene therapy.     

hPNAS-4腺病毒载体的构建及其体外抗肿瘤作用

[摘要] 目的：构建携带人的凋亡相关新基因PNAS-4(hPNAS-4)的重组腺病毒,并观察其感染人肺癌A549细胞所引起的hPNAS-4过表达对体外肿瘤细胞凋亡的影响。方法：用RT-PCR从293A细胞中克隆hPNAS-4编码区cDNA,将其酶切后连接至pENTR11载体上,再通过pENTR11与腺病毒骨架载体pAd/CMV/V5-DEST之间的同源重组作用将hPNAS-4基因片段重组至pAd/CMV/V5-DEST上,最后经293细胞的包装扩增后得到携带hPNAS-4基因的重组腺病毒;将重组腺病毒体外感染人肺癌A549细胞;用RT-PCR检测感染细胞中hPNAS-4的过表达情况;通过MTT、流式细胞术及DNA Ladder分别检测感染细胞的增殖与凋亡情况。结果：从293A细胞中中克隆到hPNAS-4全长cDNA并成功构建腺病毒表达载体Ad-hPNAS-4,经测定其滴度为：2.4×108 pfu/ml,感染人肺癌A549细胞后：经RT-PCR测得其mRNA表达明显上调,MTT检测结果为细胞增殖受到明显抑制,流式细胞术测得细胞凋亡率明显升高,琼脂糖凝胶电泳显示其基因组DNA有明显的梯状条带（DNA ladder）;结论：hPNAS-4腺病毒载体感染人肺癌A549细胞后,发现hPNAS-4过表达对肿瘤细胞的生长有明显的抑制和诱导凋亡作用,为今后研究其分子机制以及hPNAS-4腺病毒载体应用于肿瘤动物实验和肿瘤基因治疗提供实验资料。     

基于报告基因能筛选下调p66shc基因转录的药物普筛系统的建立

将p66shc基因的启动子序列克隆到含有荧光素酶报告基因和新霉素抗性基因的pGL3B-neo载体中,然后将重组质粒pGL3B-neo-p66shc转染入人的肺癌细胞A549中,通过单集落分离法得到含有重组质粒的稳定细胞系A549/p66shc。利用这个以报告基因为基础的药物普筛系统,筛选了数百种中药提取物及单体化合物,发现抑制率达到50%以上的2种中药提取物和8种单体化合物。     

hSOD基因在大白鼠肺上皮细胞中的表达

本文以哺乳动物细胞表达载体ｐＲｃ／ＣＭＶ为运载体,构建了受控于ＣＭＶ启动子的ｈＳＯＤ基因的表达载体ｐＲｃ／ＣＭＶ＿ＳＯＤ,用脂质体转染法转染大白鼠肺上皮细胞Ｌ２细胞后,经抗生素Ｇ４１８选择性培养,获得与细胞染色体ＤＮＡ稳定整合的ｈＳＯＤｃＤＮＡ导入细胞,经ＳｏｕｔｈｅｒｎＢｌｏｔｓ和ＷｅｓｔｅｒｎＢｌｏｔｓ分析,结果表明,ｈＳＯＤ在细胞中得到稳定表达,同时测定细胞ＳＯＤ酶活性,ｈＳＯＤｃＤＮＡ导入细胞的ＳＯＤ酶活性是空载体导入细胞和父本细胞的约２倍,并且在选择细胞克隆之后的６０天内是持续的．     

Enhancement of transfection efficiency for HeLa cells via incorporating arsinine moiety into chitosan

Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan （Arg-CS）. Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and ^13C NMR. Arg-CS/DNA polyelectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering （DLS） and atomic force microscopy （AFM）. The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2：1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candidate as a safe and efficient vector for gene delivery and transfection.     

Optimization of hypocrellin B derivative amphiphilicity and biological activity

To satisfy the dual requirements of the fluent transportation in blood and the affinity to the target tissues of vascular diseases, hypocrellin derivatives with optimized amphiphilicity are expected. In this work, 3-amino-l-propanesulfonic acid and 4-amino-l-butanesulfonic acid substituted hypocrellin B, named compounds 1 and 2, were designed, synthesized in high yields and characterized. Besides greatly strengthened red absorption, the maximum solubility of compound 2 in phosphate buffered saline （PBS） is 4.2 mg/mL which is just enough to prepare an aqueous solution for intravenous injection in clinically acceptable concentration, while the partition coefficient between n-octanol and PBS, 5.6, benefits the cell-uptake and biological activity as well. Furthermore, EPR measurements reveal that the photosensitization activities of the two compounds to generate semiquinone anion radicals, superoxide anion radicals and singlet oxygen are a little bit higher than those of taurine substituted hypocrellin B （THB）, but the photodynamic activities to human lung cancer A549 cells are several times that of THB, mainly due to increases in lipophilicity and cell-uptake.     

Expression of green fluorescent protein gene with baculovirus vector in insect cells

The green fluorescence of bioluminescent jellyfishAequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10³ dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus-insect cell expression system. Supported by the National Natural Science Foundation of China Hu Jianhong: born in July, 1972, Master graduate student     

X连锁凋亡抑制蛋白对肺癌细胞生长和细胞周期的影响

为了研究靶向X连锁凋亡抑制蛋白的发夹状RNA对肺癌细胞中抗肿瘤作用,构建XIAP基因的shRNA表达载体,并设计阴性对照(psiRNA-Con)质粒,分别转染A549细胞;实时定量PCR和免疫印记法分别检测XIAP的mR-NA和蛋白的表达;四甲基偶氮唑盐试验(MTT)检测A549细胞的增殖;流式细胞术检测细胞周期分布。实验结果表明:转染psiRNA-XIAP组细胞XIAP mRNA和蛋白水平均低于正常对照组,生长慢于正常组(t=16.82和t=12.13,均P<0.01),G2/M期细胞所占比例增高(t=3.78,P<0.05),并出现亚G1峰。而阴性对照质粒psiRNA-Con未能下调XIAP的表达水平,对A549细胞增殖和细胞周期亦无明显影响。结论:针对XIAP的RNA干扰质粒特异性地抑制了其RNA和蛋白水平,使肺癌细胞A549生长减慢,诱导凋亡,并使其发生G2/M期阻滞,有望发展为新的抗肿瘤药物。     

乙肝病毒基因上一个新的维甲素受体反应元件的发现

在对人乙肝病毒ＡＤＲ－１进行计算机序列检索时，发现在Ｂａｍ ＨＩ和ＨｉｎｃⅡ酶切位点内存在一保守的维甲素受体反应元件序列。用构建好的包含这一片段的乙肝病毒基因亚克隆ｐＢａｍＨＩ－ＨｉｎｃⅡ－ＣＡＴ２与人维甲素Ａ受体ｈＲＡＲα或人维甲素Ｘ受体ｈＲＸＲα共转染人特异性肝癌细胞ＨｅｐＧ２，通过相应配体ＡＴＲＡ或９－ｃｉｓＲＡ的刺激，使细胞内氯霉素乙酰基转移酶活力较不加配体刺激的对照细胞明显增强。为了排除     

bFGF真核表达载体构建及表达

旨在构建可在哺乳细胞中表达碱性成纤维细胞生长因子（basic Fibroblast Growth Factor,简称bFGF）的真核表达载体，以便用于研究bFGF基因治疗在骨组织工程中的应用，应用基因重组技术，将已经克隆的bFGF基因从PBR322－bFGF载体上亚克隆到真核表达载体pcDNA3．1上，构建的重组质粒经脂质体介导转染3T3细胞，36h后观察瞬时表达情况，酶切，PCR和DNA测序结果均证实了插入片段的正确性，免疫组织化学检测bFGF的表达分泌情况，结果显示部分经转染的细胞呈阳性（棕色颗粒）。结果表明已成功构建了bFGF真核表达载体pcDNA3.1-bFGF，并且bFGF能够在3T3细胞表达。     

乙型肝炎病毒(HBV)DNA免疫的初步研究

用聚合酶链反应（ＰＣＲ）扩增了编码ＨＢＶｓＡｇ的基因序列,将其插入到ｐｃＤＮＡ３载体中,位于人巨细胞病毒（ＣＭＶ）早期启动子下游．重组质粒ｐｃＤＮＡ３－ｓＡｇ转染细胞后检测到ＨＢｓＡｇ表达．用纯化后的重组质粒直接注射到ＢＡＬＢ／Ｃ小鼠骨骼肌内,诱发实验小鼠产生了抗ＨＢｓＡｇ特异性抗体．ＰＣＲ扩增未检测到注射部位及肝脏细胞染色体中有外源ＨＢＶＤＮＡ整合     

RFLP analysis of nov gene in tumor and normal cells

Using human nov (nephroblastoma-overexpressed, nov) DNA as probe, hybridization to total cellular DNAs of tumor and normal cells digested by restriction enzymes BamHI or EcoRI respectively was carried out through Southern blot. It was observed that nov gene in these cells is not only highly conserved, but also certains RFLP characteristic. The correlation between RFLP characteristic of nov gene and its function was analyzed. Supported by the Natural Science Fundation of China and Doctor Station Foundation Zeng Xianchun: born in June, 1966, Ph. D. graduate student     

人Endostatin基因的克隆及其在Hela细胞中的表达

利用RT-PCR克隆人Endostatin基因,分别构建到双顺反子表达载体pIRES2-EGFP和融合表达载体pEGFP-C1中.两个重组表达载体利用脂质体介导转染真核细胞Hela,48 h后可在荧光显微镜下观察到被转染细胞发出绿色荧光.传代10次后,绿色荧光仍持续表达,说明是稳定转染.以转染细胞的总DNA为模板PCR检测Endostatin基因已整合到细胞染色体上.利用兔抗人Endostatin抗体对已转染细胞进行免疫组化检测,显微镜下观察,转染的细胞显棕红色,表明Endostatin在转染细胞内稳定表达.本试验通过报告基因检测,DNA检测,免疫细胞化学检测三个水平证明Endostatin已整合到细胞的染色体上,为基因治疗以及联合治疗打下基础.     

TRAIL与kallistatin联合表达载体的构建及抗肿瘤活性分析

为研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)与组织激肽释放酶结合蛋白(kallistatin)联合用药的抗肿瘤作用,构建TRAIL与kallistatin双表达的重组质粒pAM-CAG-Kal-IRES-TRAIL,将重组质粒转染A549,LO-2,NCI-H446和Hela细胞,考察其抗肿瘤活性.实验结果表明:构建的双表达载体能同时表达TRAIL与kallistatin,且均能分泌至培养基中;TRAIL与kallistatin联合表达对肿瘤细胞活力的抑制作用明显增强,诱导肿瘤凋亡的作用也明显增强,说明联合表达TRAIL与kallistatin能够增强抗肿瘤活性.     

Inhibiting mechanism of baculovirus p35 gene to apoptosis

We transfected Sf9 cells with an expressing vector p35IE1Neo containing antiapoptotic p35 gene and neomycin-resistant gene (as a selection marker). By G418 screening, we got transformed cells that appeared resistant to G418 and picked one clone named Sf9-35. By hybridization in situ, it was found that p35 gene had integrated into the chromosome of Sf9-35 cells; By using actinomycin D treatment and cellular DNA electrophoresis, Sf9-35 cells were found to resist apoptosis induced by infection of vAcΔp35 deleting p35 gene and actinomycin D treatment; And it was also found that apoptosis induced by viral infection and actinomycin D treatment can only be delayed, but can not be stopped in Sf9-35. Supported by the National Natural Science Fundation of China Li Xiaofeng: born in 1965, Lecturer     

新型Bola两亲分子作为基因治疗药物输递载体的研究

为检测新型Bola两亲分子(Orn-C16-G)用作基因载体的性能,制备了Bola与siRNA的复合物(Bolaplexes),用原子力显微镜观察其在不同复合比下的形貌;并转染Hela细胞,用CCK-8检测Bolaplexes的细胞毒性,用荧光显微镜、流式细胞仪表征Bolaplexes的细胞内吞效果。实验结果显示,复合比1∶1条件下,Bola分子可以有效地压缩siRNA分子形成较小尺寸的复合物(100～200 nm)。Bolaplexes可高效地进入Hela细胞,并且毒性低于商售转染试剂,有较好的应用前景。本研究为下一步Bola两亲分子用于基因治疗的临床研究奠定了基础。     

人annexin A2基因真核表达的构建及活性

为了构建人Annexin A2基因真核表达载体。通过TRIzol法提取Hela宫颈癌细胞中总RNA,采用RT-PCR技术(逆转录-聚合酶链反应)扩增Annexin A2的基因片段,并成功构建成VR1012-Annexin A2-HA重组质粒。经酶切、测序检测其构建的正确性,将质粒瞬时转染到293T人胚肾细胞,使用免疫荧光和蛋白印迹(Western blot)法检测基因表达,使用流式细胞术检测转染后细胞凋亡率。结果表明实验成功构建了具有表达活性的Annexin A2真核表达质粒,为进一步研究Annexin A2的抗凋亡作用奠定了基础。