婴儿双歧杆菌/胞嘧啶脱氨酶肿瘤靶向性基因治疗系统的构建

Objective： To construct Bifidobacterium Infantis/CD targeting gene therapy system. Methods： CD gene was amplified from E. Coli K12λ using PCR method, pGEX-1LamdaT plasmid and CD gene were digested with dual restriction endonucleas of EcoR Ⅰ and BamH Ⅰ and two segments of 4.9 kb and 1.3 kb were obtained. T4 DNA ligase was added to these two segments to make a recombinant CD/pGEX-1LamdaT plasmid. Then the recombinant plasmid was transfected into Bifidobacterium Infantis by electroporation. The recombinant plasmid was extracted from the positively transfected Bifidobacterium Infantis and digested with dual restriction endonucleases. Then the size of digested fragments was detected and sequencing of the gene segment inserted in extracted recombinant plasmid was performed according to the method of Sanger dideoxynucleotide triphosphate chain termination. Results： 6.2 kb recombinant plasmid was obtained from the positively transfected bacterial colony of Bifidobacterium Infantis. After being digested with dual restriction endonucleases, two segments of approximate 4.9 kb and 1.3 kb were gained from the extracted recombinant plasmid, which were equal to the size of pGEX-1LamdaT plasmid and CD gene, respectively. The full length and sequence of nucleotide acid of the inserted gene in extracted recombinant plasmid was completely identical to the CD gene. Conclusion： The foreign gene, CD gene was correctly inserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium Infantis. Bifidobacterium Infantis/CD targeting gene therapy system was successfully constructed.     

婴儿双歧杆菌/胞嘧啶脱氨酶肿瘤靶向性基因治疗系统的构建

目的 构建婴儿双歧杆菌/胞嘧啶脱氨酶肿瘤靶向性基因治疗系统。方法 PCR扩增胞嘧啶脱氨酶(CD)基因，TSS法在大肠杆菌，M109中扩增pGEX-1LambdaT质粒。EcoRⅠ和BamHⅠ对CD基因和pGEX-1LambdaT质粒分别进行双酶酶切。琼脂糖电泳凝胶分离并切取其中4．9kb和1．3kb两个片段长度的凝胶。凝胶DNA提取试剂盒提取其中所含的DNA片段，T4DNA连接酶连接这两个片段，最后用电穿孔法将重组片段转染婴儿双歧杆菌，并挑选阳性菌落，提取质粒双酶切后进行琼脂糖凝胶电泳检测。结果 应用电穿孔法转染婴儿双歧杆菌，获得含6．2kbp重组质粒的阳性转染婴儿双歧杆菌。结论 成功构建婴儿双歧杆菌/胞嘧啶脱氨酶肿瘤靶向性基因治疗系统。     

含胞嘧啶脱氨酶基因的靶向腺病毒载体的构建及应用

目的　建立癌胚抗原 (CEA )启动子驱动的含胞嘧啶脱氨酶基因 (CD )自杀基因的腺病毒载体AdCEACD ,使CD基因只能在CEA阳性的细胞中表达。方法　采用同源重组法在人胚肾细胞株 2 93细胞中重组腺病毒 ,用点杂交、PCR法进行鉴定 ,并进行纯化和滴度测定 ,分别感染CEA阳性和CEA阴性的细胞 ,采用MTT法检测感染细胞对 5 Fc的敏感性。结果　重组腺病毒中含有CEA基因及CD基因 ,纯化后滴度可以达到 5 .0× 10 11pfu/ml ,CEA阴性的Hela细胞感染AdCMVCD后对 5 Fc很敏感 ,而感染AdCEACD后不能被 5 Fc杀伤 ,与之相反 ,CEA阳性的Lovo细胞感染AdCMVCD和AdCEACD后有相似的表达活性 ,均对5 Fc敏感。AdCEACD/ 5 Fc系统有明显的“旁观者”效应。结论　本实验建立的CEA启动子驱动的含CD自杀基因的腺病毒载体AdCEACD ,能使CD基因专一性地在CEA阳性细胞中表达 ,有助于对CEA阳性的肿瘤细胞靶向性自杀基因治疗。     

肿瘤厌氧靶向双自杀基因治疗系统pTRKH2-PsT/CD、pTRKH2-PsT/UPRT的构建

目的 利用婴儿双歧杆菌对实体瘤低氧区的靶向效应,构建肿瘤厌氧靶向双自杀基因治疗系统pTRKH2-PsT /CD和pTRKH2-PsT /UPRT。方法 用PCR的方法从质粒pGEX/CD和pGEX/UPRT中扩增出CD基因和UPRT基因,双酶切CD基因、UPRT基因和质粒pTRKH2-PsT,分别连接后重组于大肠杆菌中。之后用电转化的方法将重组质粒转入婴儿双歧杆菌中。用RT-PCR检测该系统mRNA水平的表达,SDS-PAGE检测该系统在蛋白质水平的表达。在黑色素瘤B16-F10细胞上检测该系统的体外肿瘤细胞杀伤效果。结果 成功地将CD基因和UPRT基因转入了质粒pTRKH2-PsT,CD基因和UPRT基因的测序结果表明序列与Genebank公布的序列一致。RT-PCR检测到CD和UPRT mRNA水平的明显表达。在含有CD基因的婴儿双歧杆菌细胞全蛋白中发现了CD蛋白质的表达,在含有UPRT基因的婴儿双歧杆菌上清液中发现了UPRT蛋白质的表达。黑色素瘤细胞的低存活率证明了pTRKH2-PsT/CD、pTRKH2-PsT/UPRT自杀基因治疗系统对黑色素瘤的显著杀伤作用。结论 肿瘤厌氧靶向双自杀基因治疗系统pTRKH2-PsT/CD、pTRKH2-PsT/UPRT构建成功并显示出杀伤肿瘤细胞的作用。     

婴儿型双歧杆菌对肠癌大鼠化疗性肠黏膜炎的影响

目的 探讨婴儿型双歧杆菌对肠癌大鼠化疗所致的肠道黏膜炎的疗效。方法 用皮下注射二甲基肼联合直肠黏膜种瘤造模方法建立肠癌大鼠模型41只,随机分为对照组（生理盐水）13只、化疗组（5-氟尿嘧啶+奥沙利铂）14只、双歧杆菌组（5-氟尿嘧啶+奥沙利铂+婴儿型双歧杆菌灌胃）14只,记录3组肠癌大鼠的腹泻、体重、绒毛高度、隐窝深度及促炎性细胞因子IL-6、IL-1β、TNF-α的水平。结果 41只大鼠中12只成功致瘤,对照组3只,化疗组5只,双歧杆菌组4只。在肠癌模型大鼠中,治疗72 h后,婴儿型双歧杆菌可以阻止由化疗引起的体重降低（P＜0.05）;减轻化疗后引起的腹泻次数（1.43±0.53 vs. 2.43±0.79,P＜0.050）;减轻由化疗所致的小肠黏膜炎症反应,使绒毛长度升高［379.37±53.80）μm vs. （335.33±89.54）μm,P＜0.05］,隐窝深度变浅［（219.22±38.05）μm vs. （331.24±69.24）μm,P＜0.05］;降低由化疗引起的肠道促炎性细胞因子IL-6、IL-1β水平的升高（P＜0.05）,亦可使TNF-α水平降低,但与化疗组比较,差异无统计学意义（P＞0.05）。而血浆中促炎性细胞因子水平的差异均无统计学意义（P＞0.05）。结论 婴儿型双歧杆菌可有效减轻肠癌大鼠化疗介导的黏膜炎。     

肿瘤双自杀基因治疗系统pTRKH2-PsT/CD,pTRKH2-PsT/UPRT在厌氧婴儿双歧杆菌中的构建

Objective: We recombine the suicide gene CD, UPRT into plasmid pTRKH2 and clone the recombinant dual suicide gene therapy system into tumor-hypoxia-targeting vector Bifidobacterium infantis and characterize its function. Methods: CD gene, UPRT gene and lactic acid bacteria expression plasmid pTRKH2 were digested by restriction endonuclease BamH I and Sal I, and constructed recombinant plasmids pTRKH2/CD and pTRKH2/UPRT in E. coll. The recombinant plasmids were then transfected into Bifidobacterium Infantis by electroporation. Identification of pTRKH2/CD and pTRKH2/UPRT was processed by dual restriction endonuclease digesting and sequencing. RT-PCR and SDS-PAGE were used to examine the expression of CD and UPRT genes at RNA and protein levels. The killing effects on Melanoma B16-F10 cells by pTRKH2/CD and pTRKH2/UPRT suicide gene therapy system with 5-FC were examined by MTT assay. Results: The CD gene and UPRT gene was successfully recombined into lactic acid bacteria expression plasmid pTRKH2. After dual endonuclease digestion of plasmid purified from the positively transfected E. co/i, two fragments of 6.9 Kb and 1.3 Kb were found for CD gene and two fragments of 6.9 Kb and 620 bp were found for UPRT gene. The sequencing of CD gene and UPRT gene proved consistent sequences with Genebank published data. A fragment of 1.3 Kb for CD gene and fragment of 620 bp for UPRT gene was found in recombinant Bifidobac- terium by RT-PCR. A 52 KDa protein for CD gene was identified in whole-cell protein of recombinant Bifidobacterium and a 26 KDa protein for UPRT gene was identified in supernatant fluid of recombinant Bifidobacterium. The survival rate of tumor cells treated by extracts from culture of recombinant Bifidobacterium with 5-FC showed a strong killing effects of pTRKH2/CD and pTRKH2/UPRT dual suicide gene therapy system on Melanoma B16-F10 cells. Conclusion: CD gene and UPRT gene are suc- cessfully inserted into pTRKH2 and transfected into tumor-hypoxia-targeting vector Bifldobacterium Infantis. This dual suicide gene therapy system shows a high efficiency for tumor cells killing.     

肿瘤基因治疗中HSV1-TK的靶向性研究

自杀基因HSV1-TK可选择性杀伤表达HSV1-TK的细胞,在肿瘤的基因治疗中尤为重要.HSV1-TK 除了可以引起细胞自杀作用外,还有独特的旁观者效应,已经被美国FDA批准进入Ⅲ期临床试验研究.近几年人们把HSV1-TK的研究重点放在了肿瘤靶向性治疗上,力求提高HSV1-TK的转移效率以及HSV1-TK基因表达的特异性,使抗肿瘤治疗更有效,更安全.     

靶向人Pokemon基因的shRNA重组质粒的构建(英文)

Objective:The aim of this study was to establish the foundation for studying the role of pokemon gene in tumorigenesis and development by constructing recombinant plasmids that can express small interfering RNA(siRNA)targeting human Pokemon gene.Methods:Hairpin siRNA templates targeting Pokemon gene were synthesized and cloned into plasmid vector psiRNA-H1neo.Three vectors derived siRNAs(psiRNA1,2,3)and one mocking psiRNAc(as control)were con- structed.The recombinant Pokemon siRNA plasmids were constructed...     

肿瘤光动力治疗系统--PHOTOFRIN/DIOMED(R)630PDT介绍

光动力治疗(photodynamic therapy,PDT)又称光敏疗法、光化学疗法.由于它具有有别于以往的手术、放疗和化疗三大传统肿瘤治疗模式的诸多优点而受到广大医务工作者和肿瘤患者的欢迎.近30年来,国内外学者经过大量临床和实验研究取得了一系列成果,尤其在光敏剂和相应配套的激光机的研发上取得巨大成功[1].     

肿瘤选择性增殖腺病毒CNHK300治疗乳腺癌的体外实验研究(英文)

目的观察肿瘤选择性增殖腺病毒 CNHK300对乳腺癌的选择性杀伤作用。方法用 RT-PCR 方法检测各种细胞株的端粒酶活性;CNHK300、ONYX-015(E1B 55 KDa 蛋白缺失的2型和5型嵌合型腺病毒)、wtAd5(野生型腺病毒)分别行病毒增殖实验和细胞生长抑制实验,验证 CNHK300选择性复制和杀伤能力;Western Blot 检测腺病毒E1A 在细胞中的表达。结果乳腺癌细胞株 MCF-7、BT-549和 SK-BR-3端粒酶 hTERT mRNA 均为阳性表达,而正常成纤维细胞株 MRC-5和 BJ 端粒酶hTERT mRNA 为阴性。CNHK300在乳腺癌细胞 MCF-7、BT-549和 SK-BR-3中48 h 复制倍数分别为40 625、1265和20000倍,与wtAd5的增殖能力相似,较 ONYX-015增殖能力强,在 MCF-7和 BT-549细胞中复制能力甚至强于野生型腺病毒。然而,在正常成纤维细胞 MRC-5和 BJ 中 CNHK300病毒增殖能力减弱,48 h 增殖倍数为63～192倍,而 wtAd5增殖仍可高达3160～4846倍CNHK300 MOI 10 PFU/cell 作用7天,可有效杀伤半数乳腺癌细胞,与 ONYX-015相比,CNHK300具有更强的肿瘤杀伤能力CNHK300对正常成纤维细胞的杀伤力较 wtAd5明显减弱,CNHK300在 MOI 100 PFU/cell 时 BJ 细胞存活率50%以上。正常成纤维细胞株中未检测到 CNHK300 E1A 基因表达,在293细胞和感染 CNHK300的乳腺癌细胞株中能够检测到 E1A 基因表达。结论 hTERT 启动子可成功地调控腺病毒 CNHK300选择性在端粒酶阳性的乳腺癌细胞中复制,并产生溶瘤作用。可望成为治疗乳癌的一种新的治疗策略。     

CD/5－FC体系对人乳腺癌基因治疗作用的实验研究

目的：探讨ＣＤ／５－ＦＣ体系对人乳腺癌的实验治疗作用。方法：应用ＭＴＴ法测定人乳腺癌细胞对５－ＦＣ的敏感性。结果：实验显示,５－ＦＣ对导入ＣＤ基因的人乳腺癌细胞有明显的细胞毒作用,而对未导入ＣＤ的人乳腺癌细胞的毒性较低,５－ＦＣ对导入和未导入ＣＤ基因的人乳腺癌细胞的ＩＣ５０分别为４１８μｇ／ｍｌ和１２４９μｇ／ｍｌ。结论：ＣＤ／５－ＦＣ体系对体外转基因的人乳腺癌细胞有一定的抗肿瘤作用。     

p16基因重组质粒的构建及其对人肺癌细胞的抑制作用

表达大肠杆菌胞嘧啶脱胺酶(CD)基因的重组腺病毒AdCD体外转染小鼠黑色素瘤细胞B16F10,结果显示转染了CD基因的B16F10细胞对5-氟胞嘧啶(5FC)的敏感性显著提高.将经AdCD/5FC系统处理的B16F10细胞上清倍比稀释后.加至野生型B16F10细胞中,发现当上清仅占6.25%时即可对野生型B16F10细胞发挥明显的杀伤作用,提示AdCD/5FC介导的旁观者效应可能是通过5FC经CD酶代谢产生的毒性产物扩散而实现的.本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠经注射AdCD并连续10天给予5FC治疗后,与PBS、对照病毒AdLacZ/5FC治疗小鼠比较,小鼠肿瘤生长明显受到抑制,小鼠存活期明显延长.     

hTERT启动子调控的融合自杀基因CD:UPRT载体的构建及其应用

目的 构建hTERT启动子调控的融合自杀基因CD:UPRT表达载体,研究其对人胃癌细胞SGC7901的体外靶向性杀伤作用。方法 PCR扩增hTERT核心启动子片段,克隆入荧光素酶报告基因质粒pGL3 Basic,检测hTERT启动子在人胃癌细胞SGC7901和人成纤维细胞HLF中的转录活性。构建hTERT启动子调控的CD:UPRT基因表达载体hTERT CD:UPRT,将其和CMV启动子调控的CD:UPRT基因表达载体pcDNA3.1 CD:UPRT用脂质体转染法分别转染入SGC7901和HLF细胞,筛选稳定表达细胞系,用RT PCR和Western blot方法检测CD基因的表达,用MTT法检测5 FC对转染细胞的杀伤作用。结果 成功克隆hTERT核心启动子;荧光素酶活性检测显示,hTERT启动子在SGC7901细胞中的转录活性为阳性对照的(21.50±2.15)%,而在HLF细胞中仅有背景活性。成功构建hTERT启动子调控的CD:UPRT基因表达载体,转染pcDNA3.1 CD:UPRT的SGC7901和HLF细胞以及转染hTERT CD:UPRT的SGC7901细胞在mRNA和蛋白质水平均可检测到CD基因的表达,且对5 FC敏感;而转染hTERT CD:UPRT的HLF细胞未检测到CD基因的表达,对5 FC不敏感。结论 构建的hTERT启动子调控的融合自杀基因系统CD:UPRT/5 FC能在体外靶向性杀伤SGC7901细胞。     

大肠杆菌CD自杀基因转染诱导肿细胞凋亡及旁观者效应的研究

以重组腺病毒AdCD为载体将大肠杆菌胞嘧啶脱氨酶(CD)基因体外传染小鼠红白血病细胞FBL3,结果显示,转染了CD基因的FBL3细胞对5-氟胞嘧啶(5-FC)的敏感性显著提高,进一步研究发现,AdCD/5-FC系统可以诱导肿瘤细胞发生凋亡;将经AdCD/5-FC处理过的FBL3细胞上清倍比稀释后,加入到野生型FBL3细胞中,发现当上清仅占6.25％时,即对野生型FBL3细胞发挥明显的杀伤作用,提示旁观者效应在AdCD介导的细胞毒作用中起着重要的作用。本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠局部注射AdCD并连续10天给予5-FC(300mg/kg)治疗后,小鼠皮下肿瘤结节的生长受到明显抑制。     

CD/5-FU联合亚叶酸钙对直肠癌细胞杀伤作用的研究

目的 :探讨亚叶酸钙 (CF)联合 5 氟胞嘧啶 (5 FU)对转染胞嘧啶脱氨酶 (CD)基因的直肠癌细胞的杀伤作用。方法 :构建含CD基因的真核表达载体pCDA。应用四唑盐 (MTT)比色试验检测瘤细胞的存活率 ,观察CF联合 5 氟尿嘧啶 (5 FU)对CD阴性细胞及CF联合 5 FU对CD阳性细胞的杀伤作用 ,同时观察CF对CD/ 5 FU旁观者效应的影响。结果 :CF增强 5 FU对 834 8细胞的杀伤作用。单独CD/ 5 FU和CD/ 5 FU联合CF对 834 8细胞的IC5 0分别为 0 .8mmol/L、0 .3mmol/L ,CF明显降低5 FU的IC5 0 (P <0 .0 1)。CF对CD/ 5 FU系统的旁观者效应也有增强作用。结论 :CF明显增强CD/5 FU系统对直肠癌细胞的杀伤效能 ,可作为一种增效剂应用于CD自杀基因治疗直肠癌     

腺病毒介导的大肠杆菌CD自杀基因体内外对肿瘤杀伤作用的研究

编委会 Editorial Board名誉主编 Honorary Editor-in-chief吴孟超 Wu Meng chao(Second Military Medical University,Shanghai 200433学术顾问 Academic Advisers巴德年 Ba Denian(Chinese Academy of Medical Sciences,Beijing 100730)刘新垣 Liu Xinyuan(Shanghai Institute of Biochemistry,Chinese Academy of Sciences,Shanghai 200031)吴旻 WuMin(Cancer Institute,Chinese Academy of Medical Sciences,Beijing 100021)汤钊猷 Tang Zhaoyou(Shanghai Medical University,Shanghai 200032)主编 Editor-in-chief张友会 Zhang Youhui(Cancer Institute,Chinese Academy of Medical Sciences,Beijing,100021)副主编 Associate Editor-in-chief崔正言 Cul Zhenyan(Department of Immunology,Shandong Academy of Medical Sciences,Jinan 250001)钱振超 Qian Zhenchao(Department of Patho-physiology,Dalian Medical University,Dalian 116027)何球藻 He Qiuzao(Department of Immunology,Shanghai Medical University,Shanghai 200032)董志伟 Dong Zhiwei(Cancer Institute,Chinese Academy of Medical Sciences,Beijing 100021)常务副主编 Managing Editor-in-chief     

癌胚抗原启动子控制胞嘧啶脱氨酶基因体外对结肠癌细胞专一性杀伤

目的：研究癌胚抗原（CEA）启动子是否能控制大肠杆菌胞嘧啶脱氨酶（EC－CD）基因在CEA阳性结肠癌细胞中专一性表达和杀伤结肠癌细胞。方法：构建由CEA启动子驱动的含EC－CD基因的重组腺病毒载体AdCEACD与由巨细胞病毒（CMV）启动子驱动的含EC－CD基因的腺病毒载体AdCMVCD，分别感染CEA阳性的人结肠癌细胞株Lovo细胞和CEA阴性的Hela细胞，用RT－PCR法检测受染细胞中EC－CD基因的表达，并用MTT法检测感染后细胞对5－氟胞嘧啶（5－FC）的敏感性。结果：Lovo细胞在AdCMVCD与AdCEACD感染后均有EC－CDmRNA表达，且对5－FC的敏感性明显增强，Hela细胞在AdCMVCD感染后有EC－CDmRNA表达，对5－FC的敏感性增强，而在AdCEACD感染后则没有EC－CDmRNA表达，5－FC对其亦无杀伤作用。结论：CEA启动子能够控制EC－CD基因专一性地在CEA阳性的结肠癌细胞中表达，从而实现EC－CD基因的靶向性作用。