含血管内皮生长因子启动子驱动CD自杀基因重组腺病毒的制备及其体外杀伤作用观察

OBJECTIVE: To efficiently construct a recombinant adenovirus containing cytosine deaminase (CD) gene driven by vascular endothelial growth factor (VEGF) promoter using an AdEasier-1 system and observe its killing effect on LoVo cells in vitro. METHODS: The CD gene was amplified by PCR, and amplicons were cloned in JM109 bacteria, and then recombined into pREP8 to obtain pREP8-CD, which was then digested by HindIIIand XbaIfor the CD fragment with polyadenylation site (CD-pA) subcloned into the VEGFP -containing shuttle plasmid pAdtrack-VEGFP to generate pAdtrack-VEGFP-CD-pA. After linearization with PmeI, pAdtrack-VEGFP-CD-pA was transformed into AdEasier-1 cells, and the transformants were selected on LB agar plates containing 25 microg/ml kanamycin followed by identification of the positive pAdEasy-VEGFP-CD with electrophoretic analysis and enzymatic digestion. pAdEasy-VEGFP-CD was then digested with PacIand transfected into 293 cells to produce the recombinant adenovirus Ad-VEGFP-CD, which was finally confirmed by PCR. The positive recombinant adenoviruses were transfected into LoVo cells to observe their in vitro anti-tumor effect. RESULTS: pAdEasy-VEGFP-CD was constructed with a success rate of 70%. After being packaged in 293 cells and purified by CsCl banding, the titer of the recombinant adenovirus Ad-VEGFP-CD reached as high as 4.8x10(12) CFU particle/ml, and the adenovirus was further confirmed by PCR analysis. In the presence of the prodrug 5-FC, the recombinant adenoviruses remarkably inhibited the growth of LoVo cells. CONCLUSION: The recombinant adenoviruses containing CD gene under the control of VEGF promoter can be efficiently generated using the AdEasier-1 system, and exhibit potent anti-tumor effect in vitro.     

新法制备含VEGF启动子驱动的CD／TK融合基因重组腺病毒

目的 应用简化的细菌内同源重组法高效制备含血管内皮细胞生长因子(VEGF)启动子驱动的CD／TK融合基因重组腺病毒。方法 先以氯化钙法替代电穿孔法将腺病毒基因组质粒pAdEasy-1导入BJ5183细菌，制备BJ5183pAdEasy-1细菌；再以后者作感受态细菌，与线性化的转移质粒pAdtrack-VEGFP-CDglyTK进行同源重组，获得重组腺病毒质粒pAdEasy-VEGFP-CDglyTK，转染293细胞，制备含VEGF启动子驱动的CD／TK融合基因重组腺病毒。结果 构建重组腺病毒质粒的成功率为60％(6／10)。转染293细胞后7～12d可收获病毒。结论 简化的细菌内同源重组法简便易行，重组效率较高，易于推广应用。     

新法制备含VEGF启动子驱动的CD/TK融合基因重组腺病毒

目的 应用简化的细菌内同源重组法高效制备含血管内皮细胞生长因子(VEGF)启动子驱动的CD/TK融合基因重组腺病毒。方法 先以氯化钙法替代电穿孔法将腺病毒基因组质粒pAdEasy-1导入BJ5183细菌,制备BJ5183pAdEasy-1细菌;再以后者作感受态细菌,与线性化的转移质粒pAdtrack-VEGFP-CDglyTK进行同源重组,获得重组腺病毒质粒pAdEasy-VEGFP-CDglyTK,转染293细胞,制备含VEGF启动子驱动的CD/TK融合基因重组腺病毒。结果 构建重组腺病毒质粒的成功率为60%(6/10)。转染293细胞后7~12d可收获病毒。结论 简化的细菌内同源重组法简便易行,重组效率较高,易于推广应用。     

双自杀基因融合基因重组腺病毒的制备与鉴定

目的：制备含胞嘧部氨酶（CD）基因，胸苷激酶（TK）基因的融合基因重组腺病毒，为恶性肿瘤的基因治疗研究作准备。方法：构建包含有CD，TK融合基因的重组粘粒，将其与腺病毒DNA末端肽复合物混合后以磷酸钙共沉淀法转染人胚肾293细胞株细胞，获取重组腺病毒，并进行鉴定。结果：酶切结果及PCR结果显示，重组粘粒中CD，TK融合基因插入正确，经鉴定所获重组腺病毒带有融合基因，并且无具有复制能力的腺病毒存在。结论：所获重组病毒为E1，E3缺陷型，含有所需的双自杀基因，可进一步用于基因治疗研究。     

双自杀基因重组腺病毒对胃癌细胞及血管内皮细胞的体外特异性杀伤作用

目的研究腺病毒介导的KDR启动子驱动CD／TK融合基因（AdKDR—CDglyTK）对胃癌细胞及血管内皮细胞的选择性杀伤作用。方法选择表达KDR的MGC803细胞、ECV304细胞和不表达KDR的LS174T细胞，用腺病毒重组体AdEasy-KDR—CDglyTK和AdEasy—CMV—CDglyTK感染之，观察其感染效率并以RT—PCR方法检测转基因细胞CDglyTK的表达．然后给予不同浓度的前药5一FC及GCV处理，观察该体系对不同细胞株的杀伤效应。结果两种重组体对各细胞株的感染率相似，其感染率随腺病毒滴度的增高而递增。RT—PCR检测发现：除感染AdKDR—CDglyTK的LS174T细胞外．感染AdCMV—CDglyTK的所有细胞及感染AdKDR—CDglyTK的MGC803细胞、ECV304细胞均有目的基因CDglyTK的表达。以感染复数为100的两种腺病毒分别感染各细胞株，其表现出对前药不同的敏感性：感染AdCMV—CDglyTK的所有细胞株和感染AdKDR—CDglyTK的MGC803细胞、ECV304细胞对前药具有较高的敏感性．且其敏感性差异无显著性意义（P〉0．1）；相比之下，感染AdKDR—CDglyTK的LS174T细胞对前药不敏感（P〈0．001）。双自杀基因的疗效优于任一单自杀基因的疗效（P〈0．001）。将感染腺病毒的细胞与未感染细胞以不同比例混合培养，观察到该体系明显的旁观者效应。结论KDR基因启动子可调控双自杀基因系统选择性杀伤表达KDR胃癌细胞及血管内皮细胞。     

血管内皮生长因子受体启动子驱动重组腺病毒双自杀基因系统选择性杀伤大肠癌细胞

OBJECTIVE: To observe the selective killing effect of adenovirus (Ad)-mediated double suicide gene driven by kinase domain-containing receptor(KDR) promoter on human colorectal cancer LoVo cells and human umbilical vein endothelial ECV304 cells. METHODS: The plasmid pAdEasy-KDR-CDglyTK was transfected into 293 packaging cells for amplification of the infectious Ad and used to infect the KDR-producing cells (ECV304 and LoVo) and the KDR-nonproducing cells (LS174T) respectively. The three cells were treated with the prodrugs 5-flurocytosine (5-FC) and ganciclovir (GCV) at different concentrations after infection. The killing effects of the fusion gene system on the cells were evaluated. The distribution of cell cycle was detected by flow cytometry. RESULTS: The infection rates of the recombinant Ad were similar among the 3 cells, gradually increasing with the increment of multiplicity of infection (MOI) and reaching 100% with the MOI of 200. The LoVo cells and ECV304 cells infected with Ad-KDR-CDglyTK were highly sensitive to both of the prodrugs (P>0.1), whereas the infected LS174T cells failed to exhibit similar sensitivity (P<0.001). The killing effect of CD/TK fusion gene on the target cells was much stronger than that of either suicide gene (P<0.001). The cell cycle of LoVo cells was arrested at G1 phase. CONCLUSION: The CD/TK fusion gene system driven by KDR promoter can selectively kill KDR-expressing human colorectal cancer LoVo cells and endothelial cells.     

双自杀基因重组腺病毒对胃癌细胞及血管内皮细胞的体外特异性杀伤作用

目的 研究腺病毒介导的KDR启动子驱动CD/TK融合基因(AdKDR-CDglyTK)对胃癌细胞及血管内皮细胞的选择性杀伤作用。方法 选择表达KDR的MGC803细胞、ECV304细胞和不表达KDR的LS174T细胞,用腺病毒重组体AdEasy-KDR-CDglyTK和AdEasy-CMV-CDglyTK感染之,观察其感染效率并以RT-PCR方法检测转基因细胞CDglyTK的表达,然后给予不同浓度的前药5-FC及GCV处理,观察该体系对不同细胞株的杀伤效应。结果 两种重组体对各细胞株的感染率相似,其感染率随腺病毒滴度的增高而递增。RT-PCR检测发现:除感染AdKDR-CDglyTK的LS174T细胞外,感染AdCMV-CDglyTK的所有细胞及感染AdKDR-CDglyTK的MGC803细胞、ECV304细胞均有目的基因CDglyTK的表达。以感染复数为100的两种腺病毒分别感染各细胞株,其表现出对前药不同的敏感性:感染AdCMV-CDglyTK的所有细胞株和感染AdKDR-CDglyTK的MGC803细胞、ECV304细胞对前药具有较高的敏感性,且其敏感性差异无显著性意义(P>0.1);相比之下,感染AdKDR-CDglyTK的LS174T细胞对前药不敏感(P<0.001)。双自杀基因的疗效优于任一单自杀基因的疗效(P<0.001)。将感染腺病毒的细胞与未感染细胞以不同比例混合培养,观察到该体系明显的旁观者效应。结论 KDR基因启动子可调控双自杀基因系统选择性杀伤表达KDR胃癌细胞及血管内皮细胞。     

血管内皮生长因子受体启动子驱动重组腺病毒双自杀基因系统选择性杀伤大肠癌细胞

目的 探讨血管内皮生长因子受体(KDR)启动子驱动重组腺病毒CDglyTK融合基因体系对结直肠癌细胞株LOVO及人脐血管内皮细胞株ECV30的选择性杀伤作用.方法 将质粒pAdEasy-KDR-CDglyTK在293细胞内包装、扩增后,体外感染表达KDR的LoVo、ECV30细胞和对照组不表达KDR的LS17T细胞,并给予不同浓度的前药GCV(ganciclovir)和/或5-FC(5-fluorocytosine),观察该体系对细胞株的杀伤效应.结果 制备的病毒滴度为2.0×1012pfu/ml.3种细胞的感染率相似,且感染率随腺病毒滴度的递增而增加,当MOI为200时,所有细胞株均接近100%感染.以MOI为100的重组体分别感染各细胞株,发现其对前药的敏感性不同:表达KDR的LoVo和ECV30细胞对前药具有较高的敏感性,且二者敏感性无显著差异(P>0.1);与前二者相比,LS17T细胞对前药不敏感(P<0.001).同时,CDglyTK双自杀基因的疗效优于任一单自杀基因(P<0.001).流式细胞术检测表明该体系抑制LoVo细胞DNA的合成,表现为S期细胞比率增多及G1期细胞减少(P<0.001).结论 KDR基因启动子调控的CDglyTK融合基因体系可选择性杀伤结直肠癌LoVo细胞和血管内皮细胞.     

人NP9基因蛋白重组腺病毒的制备

目的：制备表达人NP9蛋白的复制缺陷型重组腺病毒。方法：酶切pMD18T-NP9质粒获得NP9基因编码区序列并克隆至芽梭载体构建重组pAcTrack-CMV-NP9载体，线性化后与pAdEasy-1共转化BJ5183，通过同源重组得到重组的pAD-NP9病毒载体将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒。结果：酶切鉴定得到阳性pAD-NP9重组腺病毒载体，该载体能有效转染HEK293细胞并在细胞内成功包装，转染3d后可以观察到绿色荧光蛋白(CFP)表达并逐渐增多、增强：结论：成功构建了NP9基因的重组腺病毒载体并制备重组腺病毒颗粒，为进一步研究NP9基因的功能及应用NP9进行基因治疗奠定基础。     

CD基因对大肠癌杀伤作用的实验研究

目的：探讨ＣＤ基因对大肠癌靶向治疗的作用。方法：构建以ＣＥＡ基因顺式转录调控序列（ＴＲＳ）驱动ＣＤ基因的组织特异性重组逆转录病毒载体Ｇ１ＣＥＡＣＤＮa,脂质体裹 法将重组组织特异性载体pＣＤ２在逆转录病毒包装细胞ＰＡ３１７细胞中进行包装,收获病毒上清后分别转染入高分泌ＣＥＡ的大肠癌细胞ＬoＶo中,经Ｇ４１８筛选阳性克隆扩增后进行体外前药敏感实验及观察旁观者交应,然后再将转基因ＬoＶo细胞接种对裸鼠皮下成     

Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor

Objective To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. ResultsThe newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3×109 pfu/ml. ConclusionThe recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.     

Survivin启动子介导的胞嘧啶脱氨酶(CD)自杀基因的抗肿瘤研究

目的 探讨Survivin启动子介导的CD/5-FC自杀系统的抗肿瘤效果.方法 利用Survivin启动子替换pEGFP-N1载体上的巨细胞病毒(CMV)启动子,构建重组表达载体pSurP-EGFP,通过报告基因绿色荧光蛋白(GFP)的表达确定Survivin启动子的特异性;将酿酒酵母的胞嘧啶脱氨酶(CD)基因克隆在pSurP-EGFP中,构建成重组载体pSurP-CD,转染肿瘤细胞后利用毛细管电泳仪检测前药5-氟胞嘧啶(5-FC)的转化效率,同时分析肿瘤细胞的凋亡.结果 GFP的表达表明Survivin启动子具有较好的肿瘤靶向性;毛细管电泳检测表明CD能够有效地将5-FC转化为5-FU,显微镜观察和流式分析表明了Survivin启动子介导的CD的抗肿瘤效果.结论 Survivin启动子能有效地介导CD自杀基因的表达,具有较好的靶向性抗肿瘤效果.     

人血管内皮细胞生长因子165基因真核表达载体的构建及其在大肠杆菌的表达

目的　获得人血管内皮生长因子 (hVEGF165)cDNA ,构建真核表达载体 ,并研究其在大肠杆菌中的表达情况。方法　采用PCR方法 ,从HL6 0细胞中扩增出hVEGF165cDNA ,将其克隆至 pcDNA3 真核表达载体上 ,构建成为 pCD hVEGF165重组质粒。将重组质粒转染感受态的大肠杆菌BL2 1(DE3)中进行表达 ,用Westernblot检测表达产物。结果　经酶切鉴定及基因测序证实hVEGF165cDNA基因克隆成功 ,并建立了高效表达的 pCD hVEGF165重组质粒。Westernblot检测表明 ,组建了具有高效表达hVEGF165的大肠杆菌菌株。结论　成功地克隆和表达出了hVEGF165基因 ,为进一步建立VEGF转基因动物模型 ,研究其在视网膜新生血管性疾病中的应用奠定了基础     

Construction and Identification of Recombinant Adenovirus Vector Containing the hVEGF165 Gene

Summary To construct the recombinant adenovirus vector containing the cDNA for human vascular endothelial growth factor (hVEGF₁₆₅), the cDNA for hVEGF₁₆₅ was subcloned into pACCMV · pLpA. Subsequently, this recombinant pACCMV · hVEGF was co-transfected into 293 cells together with pJM17 to obtain the replication-deficient recombinant adenovirus containing hVEGF gene — Ad-CMV · hVEGF. The VEGF gene expression was detected by using RT-PCR and Western blot in rabbit aorta vascular smooth muscle cells (VSMC) infected with AdCMV · hVEGF. Cultured human umbilical vein endothelial cells (HUVEC) were incubated with the conditioned medium (CM) from above mentioned VSMC infected with AdCMV · hVEGF to observe the effect of VEGF on proliferation of HUVEC. 48 h after the infection with AdCMV · hVEGF, VSMC demonstrated VEGF expression, and the expressed VEGF could stimulate the proliferation of HUVECin vitro. Successfully prepared AdCMV · hVEGF₁₆₅ could express biologically active VEGF in infected VSMC, and stimulate proliferation of HUVEC.     

酪氨酸酶启动子调控的CD基因治疗恶性黑色素瘤

目的：克隆酪氨酸酶（Ｔｙｒ）启动子并探索其调控细菌胞嘧啶脱氨酶（ＣＤ）在黑色素瘤基因治疗中的应用价值。方法：用ＰＣＲ法从小鼠恶性黑色素瘤细胞Ｂ１６基因组中克隆Ｔｙｒ启动子片段并与正常序列比较，并于逆转录病毒载体ＧｌＮａ中调控ＣＤ基因（ＴｙｒＣＤ）。测体外表达和体内抗肿瘤作用。结果：来源于Ｂ１６基因组的Ｔｙｒ启动子重要反式作用位点未见突变。该Ｔｙｒ启动子可使ＣＤ基因在Ｂ１６中特异表达。转ＴｙｒＣＤ的     

Killing effect of adenoviral mediated cytosine deaminase gene on human pancreatic cancer cell line PaTu8988

Pancreaticadenocarcinomarankssixthofcancer-relateddeathinChina,andittendstobeadvancedatthetimeofpresentation,70%－90%patientsatthetimeofdiagnosishadinoperabledisease.5-Fluorouracil(5FU)iscommonlyusedaloneorwithradiotherapyinthetreatmentofresectablepancreaticcancerandinthepalliationofunresectablepancreaticcancer.Butthecurativeeffectof5FUislimitedbyitssideeffects.Suicidegenetherapyisanewexperimentalformofcancerchemotherapybeingevaluatedinhumantrials.Cytosinedeaminase(CD)isanenzymefoundinava-r…     

The mechanical study of vascular endothelial growth factor on the prevention of restenosis after angioplasty

Restenosis following percutaneous transluminalcoronary angioplasty ( PTCA) is one of the majorre-search subjects of coronary heart disease( CHD) .The mechanism is quite complicated and not yetclear,buttheimpairmentofendothelium isknown asthe primary evocative factor of the progress ofrestenosis.It is supposed to prevent the restenosisfollowing PTCA by accelerating restoration of en-dothelial integrity and function.In the experimentalstudies,it was proved that exogenic vascular en-dothelial…     

含CD基因的腺病毒载体制备及体外抑瘤效果观察

目的：制备含胞嘧啶转氨酶（CD）基因的重组腺病毒（Ad-CD)并进行体外肿瘤的自杀基因治疗。方法：构建含CD或LacZ基因的重组腺病毒载体。在293细胞内重组包装后,体外感染小鼠结肠癌细胞株C26,并给予不同浓度的前药氟胞嘧啶（5-FC）进行肿瘤杀伤效果观察。结果：重组腺病毒载体构建成功。用制备的Ad-CD液感染C26细胞,并予以不同浓度5-FC后,肿瘤细胞生长受不同程度抑制,而对照组细胞和原位转LacZ基因的C26细胞的生长未出现明显的变化。结论：应用腺病毒载体转CD基因进行体外肿瘤治疗效果明显,为自杀基因应用于体内结肠癌的基因治疗研究奠定基础。