百福生胶囊对荷瘤小鼠的抗肿瘤作用研究

目的：探讨百福生胶囊对荷瘤小鼠体内和体外的抗肿瘤机理。方法：体内抑瘤实验，分别对肉瘤S180和H22腹水肝癌小鼠模型灌胃给药后取瘤称重，计算抑瘤率判断百福生胶囊对两种瘤体的抑制作用，生存期实验；荷瘤小鼠接种癌细胞后24hr开始灌胃给药，10天于每3天给药一3次，比较洛组小鼠存活时间；体外抑瘤实验，用体外培养法研究百福生胶囊对人宫颈部Hela细胞生长的抑制作用。结果：百福生胶囊对两种实体瘤均有显的抑制作用，并明显延长荷瘤小鼠的存活天数，同时，该产品可有效抑制Hela细胞的生长。     

肺腺癌组织特异性自杀基因治疗实验研究

目的 探讨肺腺癌组织特异性自杀基因治疗的安全性及有效性。方法 采用病毒感染法，将癌胚抗原(CEA)基因启动子所驱动的CD基因的组织特异性逆转录病毒载体(G1CEACDNa)，导入分泌CEA的肺腺癌细胞系A549细胞．研究裸鼠体内抑瘤效果；应用重组逆转录病毒裸鼠体内治疗A549肿瘤，观察G1CEACDNa／5-氟胞嘧啶(5-FC)对A549细胞致瘤裸鼠的治疗作用及毒副反应。结果 (1)将转基因的A549细胞和未转基因的A549细胞接种至裸鼠皮下．两者成瘤性无明显差异；(2)在转基因细胞致瘤裸鼠实验中，5-FC对转CEA启动子调控自杀基因的肿瘤生长具有明显的抑制作用；(3)将G1CEACDNa重组逆转录病毒上清直接注射到裸鼠成瘤部位．然后腹腔内注射5-FC同样获得明显的抑瘤效果；(4)与直接注射5-FU相比，组织特异性自杀基因治疗对骨髓的抑制明显降低。结论 组织特异性自杀基因治疗可能成为肿瘤治疗个体化的重要方法之一。     

AdTK治疗肺癌的实验研究

目的：构建含HSV—TK基因的重组腺病毒AdTK，在两种肺癌细胞和鼠肺癌模型中对AdTK／GCV系统进行抑瘤买验研究。方法：采用同源重组法构建AdTK，用MTT法测定AdTK／GCV系统对H460、PG49人肺癌细胞的生长抑制率，建立T739鼠肺癌模型，观察AdTK／GCV系统对瘤体大小及鼠存活期的影响。结果：成功构建AdTK，AdTK转染H460、PG49细胞后对细胞抑制率随AdTK、GCV及药物作用时间的增加而增高，有显著性差异，小鼠体内实验中观察到AdTK／GCV系统对鼠肺癌有明显生长抑制作用，且明显延长了小鼠的存活期。结论：本文建立的AdTK／GCV系统在体内外对肺癌细胞均有明显抑制作用，为AdTK用于基因治疗奠定了基础。     

阳离子脂质体介导pE-CEA-cd-tk对CEA阳性肺癌的杀伤作用

目的：增强E.coli cd/HSV-1 tk自杀基因的细胞毒性，实现阳离子脂质体介导pE-CEA-cd-tk/5-FC GCV体系靶向杀伤CEA阳性肺癌，方法：PCR法分别扩增出CMV增强子，CWA启动子，cd-tk，构建真核表达载体pE-CEA-cd-tk;MTT法检测pE-CEA-cd-tk/5-FC GCV体系的体外细胞毒性，体内实验采用肺腺癌细胞SPC－A－1裸鼠皮下移植瘤模型，通过肿瘤局部或鼠尾静脉注射脂质体／pE-CEA-cd-tk复合物，腹腔注射GCV＋5－FC前体药物进行治疗。结果：阳离子脂质体介导pE-CEA-cd-tk/5-FC GCV体系体外可靶向杀伤CEA阳性肺癌细胞，这种杀伤作用存在显著的细胞差异；体内可抑制小鼠皮下肺肿瘤结节的生长，荷瘤鼠存活期延长。结论：阳离子脂质体介导pE-CEA-cd-tk/5-FC GCV体系体内外对CEA阳性肺癌均具有较强的杀伤作用，有望用于CEA阳性肺癌的治疗。     

CD自杀基因联合淋巴细胞趋化因子基因疗法的肿瘤治疗作用及免疫机理研究

本研究以腺病毒作为载体,将大肠杆菌胞嘧啶脱氨酶(CD)基因与小鼠淋巴细胞趋化因子(Ltn)基因体内联合转染,观察了其抗肿瘤效应并分析了免疫机理.小鼠皮下接种结肠腺癌CT26细胞后3天,肿瘤局部注射表达Ltn的重组腺病毒AdLtn和表达CD的重组腺病毒AdCD,然后连续10天给予5一氟胞嘧啶(5-FC)300mg/kg进行治疗,结果表明,联合治疗组荷瘤小鼠皮下肿瘤结节的生长受到明显抑制,小鼠存活期明显长于单用AdLtn治疗组或单用AdCD/5-FC治疗组.经联合治疗后小鼠脾细胞的NK活性和对(37结肠腺癌细胞的CTL杀伤活性明显增强.瘤体细胞FACS分析结果表明,经联合基因治疗后,肿瘤组织CD4~ 、CD8~ 细胞浸润增加,结肠腺癌细胞表达H-2Kd和B7-1分子明显增加.提示经CD自杀基因和Ltn基因联合治疗后,肿瘤细胞免疫原性增加.本研究结果表明联合应用自杀基因和Ltn基因治疗可以提高机体对肿瘤细胞免疫的应答,增加机体的抗肿瘤作用,是肿瘤基因治疗中一条新的途径.     

IL-2基因与自杀基因联合转移对小鼠红白血病的治疗作用

用腺病毒作为载体,将大肠杆菌胞嘧啶脱氨酶(CD)基因体外转染小鼠红白血病FBL-3细胞,结果CD基因转移后该细胞对5-氟胞嘧啶(5-FC)的敏感性提高了近1000倍,FBL-3细胞有明显的凋亡发生,且观察到明显的旁观者效应.小鼠体内接种FBL-3红白血病细胞后3天,肿瘤局部注射小鼠白细胞介素2(IL-2)基因的表达载体(Ad-IL-2)和Ad-CD腺病毒载体,然后连续10天给予5-FC 300mg/kg行治疗.结果FBL-3皮下肿瘤的生长明显受到抑制,部分小鼠肿瘤消失.联合治疗小鼠的存活期明显大于单用Ad-IL-2治疗组和单用自杀基因CD与5-FC治疗组.体内免疫功能检测表明,经小鼠自杀基因与IL-2联合基因治疗后小鼠脾细胞的CTL杀伤     

前药5-FC联合胃泌素拮抗剂CI-988对转CD基因大肠癌SW480细胞的杀伤作用

目的：探讨联合应用前药5-氟胞嘧啶（5-iluorocytosine，5-FC）和胃泌素拮抗剂CI-988对转组织特异性胞嘧啶脱氨基酶（cytosinedeaminase，CD）基因的大肠癌SW480细胞生长的影响。方法：将转CD基因大肠癌SW480细胞接种至4块24孔细胞培养板中，依次分为实验组1、2、3及对照组，分别以含有前药5-FC、胃泌素拮抗剂CI-988、5-FC联合CI-988及正常培养液进行培养。每天胰酶消化法计数各组4孔，共6d，绘制细胞生长曲线并计算生长抑制率。按上述分组方法另接种培养4组细胞，于培养72h行M1Tr法检测490nm处吸光度A值，计算细胞杀伤率。结果：应用5-FC联合CI-988处理的转CD基因大肠癌SW480细胞在6d后生长抑制率达97％；于培养72h时，实验组1、3细胞杀伤率较对照组有显著性差异（P〈0．01）；实验组2与对照组无显著性差异（P〉0．05）；实验组3较实验组1、2有显著性差异（P〈0．01）。结论：CD／5-FC系统和胃泌素拮抗剂CI-988对转基因大肠癌SW480细胞具有杀伤或抑制作用，联合应用胃泌素拮抗剂可以提高CD／5-FC自杀基因系统对大肠癌细胞的杀伤效应。     

前药 5-FC 联合胃泌素拮抗剂 CI-988 对转 CD 基因大肠癌 SW480细胞的杀伤作用

目的：探讨联合应用前药5-氟胞嘧啶（5-iluorocytosine，5-FC）和胃泌素拮抗剂CI-988对转组织特异性胞嘧啶脱氨基酶（cytosinedeaminase，CD）基因的大肠癌SW480细胞生长的影响。方法：将转CD基因大肠癌SW480细胞接种至4块24孔细胞培养板中，依次分为实验组1、2、3及对照组，分别以含有前药5-FC、胃泌素拮抗剂CI-988、5-FC联合CI-988及正常培养液进行培养。每天胰酶消化法计数各组4孔，共6d，绘制细胞生长曲线并计算生长抑制率。按上述分组方法另接种培养4组细胞，于培养72h行M1Tr法检测490nm处吸光度A值，计算细胞杀伤率。结果：应用5-FC联合CI-988处理的转CD基因大肠癌SW480细胞在6d后生长抑制率达97％；于培养72h时，实验组1、3细胞杀伤率较对照组有显著性差异（P〈0．01）；实验组2与对照组无显著性差异（P〉0．05）；实验组3较实验组1、2有显著性差异（P〈0．01）。结论：CD／5-FC系统和胃泌素拮抗剂CI-988对转基因大肠癌SW480细胞具有杀伤或抑制作用，联合应用胃泌素拮抗剂可以提高CD／5-FC自杀基因系统对大肠癌细胞的杀伤效应。     

T细胞及相关免疫分子在淋巴细胞趋化因子增强肿瘤自杀基因疗法中的作用

本室以往的研究表明,用腺病毒作为载体将大肠杆菌胞嘧啶脱氨酶(CD)基因与小鼠淋巴细胞趋化因子(Itn)基因联合体内转染,具有显著的抗肿瘤效应.本文对其免疫机理进行了进一步研究,发现CT26结肠腺癌细胞体外经过CD/Ltn基因转染并给予前体药物5-FC后,CT26结肠腺癌细胞表达CD80和CD54分子明显增加,提示经CD自杀基因和Ltn基因联转移后,肿瘤细胞免疫原性增加.荷瘤小鼠体内经联合治疗后,小鼠脾细胞分泌IL-2和IFN-γ明显增加.体内用抗CD4、CD8抗体阻断实验研究结果的表明,联合应用自杀基因和Ltn基因治疗主要通过诱导CD8~ T细胞发挥抗肿瘤作用.     

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。     

逆转录病毒介导的CD/5-FC对胰腺癌细胞的杀伤作用

目的 :探讨大肠埃希菌胞嘧啶脱氨基酶 (CD) /5 -氟胞嘧啶 ( 5 FC)系统对胰腺癌细胞的体外生长抑制作用。方法 :将含CD基因的重组逆转录病毒载体导入胰腺癌细胞形成转化细胞系 ,对转化细胞进行体外药物敏感实验 ,包括 :1)转化细胞在前体药物 5 FC作用下的细胞生长抑制率 ;2 )MTT法检测旁观者效应。结果 :体外实验 5 FC的有效浓度 >2mmol/L时 ,就表现出杀伤作用 ,当 5 FC的有效浓度 >6mmol/L时 ,几乎未见细胞生长 ,PA3 17/CD与TD2混育比例在 90 %时 ,杀伤作用最大 ,相同药物浓度下 ,混育 96h杀伤作用明显高于 2 4h。结论 :CD/5 FC系统对胰腺癌细胞系细胞具有实验性基因治疗作用     

Nanoparticle-delivered VEGF-silencing cassette and suicide gene expression cassettes inhibit colon carcinoma growth in vitro and in vivo

The strategies for tumor-specific expression of suicide genes and target tumor angiogenesis have been tested in tumors. However, the anti-tumor efficacy of the combination of these two strategies, particularly, delivering suicide gene and anti-angiogenesis agent by nanoparticles, has not yet been evaluated in colon carcinoma. We constructed a cassette to silence VEGF-A expression and express a fused yCDglyTK gene driven by tumor-specific promoter (shVEGF-CDTK). The DNA carrying shVEGF-CDTK was delivered into colon carcinoma cells by calcium phosphate nanoparticles (CPNPs). Cell proliferation was measured by MTT assay, and apoptosis was detected by flow cytometry. The anti-tumor effect of the combined cassette was tested in xenograft animal model. With 5-fluorocytosine (5-FC), CPNP-delivered shVEGF-CDTK DNA (CPNP-shVEGF-CDTK) showed high expression of fused yCDglyTK gene and effectively silenced VEGF-A expression in vitro and in vivo, which significantly inhibited colon carcinoma cell proliferation and induced apoptosis in vitro. With 5-FC, the systemic delivery of CPNP-shVEGF-CDTK significantly inhibited tumor growth in the colon carcinoma xenograft animal model. The combined cassette is obviously effective in inhibiting tumor cell proliferation and inducing apoptosis in vitro and tumor growth in vivo than the CPNP-shVEGF or CPNP-CDTK alone. The combination of VEGF-A-silencing and tumor-specific expression of suicide gene is an effective strategy for colon carcinoma treatment.     

Combined effects of the orally active cisplatin analog, JM216, and radiation in antitumor therapy

Purpose: We evaluated the orally administered platinum agent, JM216, in combination with ionizing radiation both in vivo and in vitro against human tumor cells. Methods: H460 human lung carcinoma cells were used as a subcutaneous xenograft in nude mice. JM216 (30 mg/kg) was administered orally, and radiation treatments (2 Gy) were given 1 h after JM216 delivery for five consecutive days. For in vitro analysis, attached H460 cells were treated with JM216 (15 μM) for 1 h and then irradiated. Cells were rinsed 20 min later, and survival was determined by clonogenic assay. Results: Tumor growth delay measurements showed that the combination of JM216 and radiation was additive in vivo, with an enhancement ratio of 1.24. In vitro clonogenic survival experiments demonstrated a dose enhancement ratio of 1.23. Isobologram analysis showed that this interaction was also additive. Conclusions: These data demonstrate that the combination of JM216 and fractionated radiotherapy is more effective against human lung cancer xenografts than either agent alone, and the in vivo results were supported by those observed using an in vitro system with the same tumor cell line. Received: 20 December 1999 / Accepted: 24 May 2000     

Enhancement of Anti-tumor Activity of Newcastle Disease Virus by the Synergistic Effect of Cytosine Deaminase

This study was conducted to investigate enhancement of anti-tumor effects of the lentogenic Newcastledisease virus Clone30 strain (NDV rClone30) expressing cytosine deaminase (CD) gene against tumor cells andin murine groin tumor-bearing models. Cytotoxic effects of the rClone30-CD/5-FC on the HepG2 cell line wereexamined by an MTT method. Anti-tumor activity of rClone30-CD/5-FC was examined in H22 tumor-bearingmice. Compared to the rClone30-CD virus treatment alone, NDV rClone30-CD/5-FC at 0.1 and 1 MOIs exertedsignificant cytotoxic effects (P<0.05) on HepG2 cells. For treatment of H22 tumor-bearing mice, recombinantNDV was injected together with 5-FC given by either intra-tumor injection or tail vein injection. When 5-FCwas administered by intra-tumor injection, survival for the rClone30-CD/5-FC-treated mice was 4/6 for 80 daysperiod vs 1/6 , 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC and saline alone, respectively. When5-FC was given by tail vein injection, survival for the rClone30-CD/5-FC-treated mice was 3/6 vs 2/6 , 0/6 and0/6 for the mice treated with rClone30-CD, 5-FC or saline alone, respectively. In this study, NDV was used forthe first time to deliver the suicide gene for cancer therapy. Incorporation of the CD gene in the lentogenic NDVgenome together with 5-FC significantly enhances cell death of HepG2 tumor cells in vitro, decreases tumorvolume and increases survival of H22 tumor-bearing mice in vivo.     

低氘水对肺癌细胞增殖的抑制作用及其可能的机制

目的：初步探讨低氘水（deuteriumdepleted water,DDW）在体内外对人肺癌细胞增殖的抑制作用及其可能机制。方法：MTT法检测DDW对肺癌A549细胞和正常人胚肺成纤维细胞HLF1增殖的抑制作用,TUNEL法检测A549细胞凋亡情况,流式细胞仪检测细胞周期的变化。建立BALB/c裸鼠人肺癌H460细胞移植瘤模型,低氘水饮用60 d后观察裸鼠移植瘤的生长情况。结果：与对照组比较,培养10 h时体积分数为0.0025％、0.0050％和0.0105％的DDW对A549 细胞增殖具有显著抑制作用（P<0.05）,随后抑制作用消失,48 h后又逐渐出现抑制现象,72 h抑制作用显著（P<0.05）;相同条件下不同体积分数的DDW对正常人胚肺成纤维细胞HLF1无显著抑制作用。TUNEL染色显示,0.005%DDW作用后A549细胞出现凋亡,凋亡率显著高于对照组细胞\[（25.38±3.90）% vs (10.87±1.11)%), P<0.05\]。流式细胞仪检测显示,DDW作用后A549细胞S期细胞显著增加（P<0.05）。人肺癌细胞H460移植瘤裸鼠饮用DDW后,能够明显提高裸鼠的生活质量,抑瘤率达30.08%。结论：DDW对肺癌细胞增殖的抑制作用仅限于一定的剂量范围,且具有波动性和时段性的特点;其机制可能与诱导肺癌细胞S期阻滞和细胞凋亡有关。     

双自杀基因CD和TK对K562细胞体内外杀伤作用的实验研究

目的:探讨双自杀基因CD和TK对K562细胞的体内外抑制作用及前体药物对肿瘤的杀伤作用。方法:将目的基因转染入K562细胞,MTT法观察细胞在体内外的增殖状况及5-FC/GCV对转染细胞的杀伤作用,电子显微镜观察其超微结构变化。将K562/CDgly TK和K562细胞接种于裸鼠皮下,观察各种肿瘤细胞在体内的成瘤情况及对前体药物治疗的敏感性。结果:单独使用GCV或5-FC对K562及K562/CDgly TK细胞产生明显的杀伤作用,联合应用该2种药物对肿瘤细胞的杀伤作用更强。将K562细胞和K562/CDgly TK细胞接种于小鼠皮下后小鼠成瘤率为100%,GCV或5-FC可明显抑制裸鼠体内的肿瘤形成,联合应用GCV和5-FC治疗K562/CDglyTK细胞在小鼠体内形成的肿瘤,较单独应用GCV和5-FC及对照组小鼠形成的肿瘤体积明显缩小,生存期也明显延长。结论:双自杀基因在体内外对K562细胞均有明显的抑制作用,可增加前体药物GCV和5-FC对瘤细胞的杀伤率。     

Potentiation of platinum antitumor effects in human lung tumor xenografts by the angiogenesis inhibitor squalamine: effects on tumor neovascularization.

Squalamine is a novel anti-angiogenic aminosterol that is postulated to inhibit neovascularization by selectively inhibiting the sodium-hydrogen antiporter exchanger. To determine how to most effectively use this agent in patients with cancer, we examined the antitumor effects of squalamine with or without cytotoxic agents in human lung cancer xenografts and correlated these observations with the degree of tumor neovascularization. No direct cytotoxic effects of squalamine against tumor cells were observed in vitro with or without cisplatin. Squalamine was effective in inhibiting the establishment of H460 human tumors in BALBc nude mice but was ineffective in inhibiting the growth of H460, CALU-6, or NL20T-A human tumor xenografts when administered i.p. to mice bearing established tumors. However, when combined with cisplatin or carboplatin, squalamine increased tumor growth delay by > or =1.5-fold in the three human lung carcinoma cell lines compared with cisplatin or carboplatin alone. No enhancement of antitumor activity was observed when squalamine was combined with paclitaxel, vinorelbine, gemcitabine, or docetaxel. Repeated cycles of squalamine plus cisplatin administration delayed H460 tumor growth >8.6-fold. Squalamine plus cisplatin reduced CD31 vessel formation by 25% compared with controls, squalamine alone, or cisplatin alone; however, no inhibition in CD31 vessel formation was observed when squalamine was combined with vinorelbine. These data demonstrate that the combination of squalamine and a platinum analog has significant preclinical antitumor activity against human lung cancer that is related to the anti-angiogenic effects of squalamine.     

Combination of cytosine deaminase suicide gene expression with DR5 antibody treatment increases cancer cell cytotoxicity

Combined treatment using adenoviral-directed enzyme/prodrug therapy and immunotherapy has the potential to become a powerful alternative method of cancer therapy. We have developed adenoviral vectors encoding the cytosine deaminase gene (Ad-CD) and cytosine deaminase:uracil phosphoribosyltransferase fusion gene (Ad-CD:UPRT). A monoclonal antibody, TRA-8, specifically binds to death receptor 5, one of two death receptors bound by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of Ad-CD:UPRT and TRA-8 against human pancreatic cancer and glioma cell lines. The present study demonstrates that Ad-CD:UPRT infection resulted in increased 5-FC-mediated cell killing, compared with Ad-CD. Furthermore, a significant increase of cytotoxicity following Ad-CD:UPRT/5-FC and TRA-8 treatment of cancer cells in vitro was demonstrated. Animal studies showed significant inhibition of tumor growth of MIA PaCa-2 pancreatic and D54MG glioma xenografts by the combination of Ad-CD:UPRT/5-FC plus TRA-8 as compared with either agent alone or no treatment. The results suggest that the combination of Ad-CD:UPRT/5-FC with TRA-8 produces an additive cytotoxic effect in cancer cells in vitro and in vivo. These data indicate that combined treatment with enzyme/prodrug therapy and TRAIL immunotherapy provides a promising approach for cancer therapy.