瘤内原位转导UPRT基因治疗小鼠胃癌的实验研究

背景与目的:5-FU是临床上重要的化疗药物,但总体疗效有限,如何提高其抗癌疗效一直是临床上亟待解决的难题。本研究采用基因治疗的原理与方法,探讨UPRT(uracilphosphoribosyltransferase)基因对5-FU杀伤效应的增敏作用。方法:采用PCR技术从大肠杆菌K12基因组中扩增UPRT基因,并构建pLXSN逆转录病毒表达载体,进一步转染小鼠胃癌细胞株MFC。采用MTT法检测转导UPRT基因前后MFC对5-FU敏感性的变化。建立615小鼠胃癌皮下移植瘤模型,瘤内反复多次注射携带UPRT基因的浓缩、纯化的逆转录病毒,同时腹腔内给予5-FU,观察抑瘤效果。结果:体外MTT法检测结果显示,转导UPRT基因后可使MFC对5-FU的敏感性提高约17.26倍。以逆转录病毒介导UPRT基因瘤内原位转导,同时联合应用5-FU进行治疗,结果肿瘤生长明显受抑(P<0.005),抑瘤率为87.18%。结论:UPRT基因修饰小鼠胃癌细胞株MFC,能明显提高MFC对5-FU杀伤的敏感性,以逆转录病毒载体介导UPRT基因肿瘤原位基因转导,同时联合应用5-FU,能获得良好的抑瘤效应。     

hTERT启动子调控的CD:UPRT/5-FC自杀基因对人胃癌裸鼠移植瘤的抑制作用

目的:研究人端粒酶逆转录酶(hTERT)启动子调控的胞嘧啶脱氨酶:尿嘧啶磷酸核糖转移酶/5-氟胞嘧啶(CD:UPRT/5-FC)融合自杀基因系统在体内对人胃癌SGC7901细胞的杀伤作用.方法:构建胃癌皮下移植瘤模型,随机分成3组处理.观察30天,测量肿瘤体积,计算抑瘤率.肿瘤组织做常规病理检查,免疫组化检测CD蛋白的表达.结果:成功构建胃癌皮下移植瘤模型.治疗30天后,B组(瘤内注射hTERT-CD:UPRT+腹腔注射PBS )和C组(单纯腹腔注射PBS)的肿瘤生长迅速,肿瘤体积分别为(2.72±0.35)cm3、(2.67±0.30)cm3,A组(瘤内注射hTERT-CD:UPRT+腹腔注射5-FC)的肿瘤生长受到明显抑制,第30天体积为(1.10±0.13)cm3,与前两组相比差异有统计学意义(P＜0.05),其抑瘤率为59.6%.裸鼠移植瘤病理切片镜检,A组可见大量肿瘤细胞排列稀疏,部分肿瘤细胞核固缩、核碎裂;B组和C组则见大量肿瘤细胞排列紧密,细胞形态和细胞核呈多形性,核大深染,核分裂像多见.免疫组化显示,A组和B组可见CD的表达,C组CD表达呈阴性.结论:hTERT启动子调控的CD:UPRT/5-FC融合自杀基因系统对裸鼠胃癌皮下移植瘤有显著的杀伤作用,为胃癌的基因治疗提供了一种可能的方法.     

hTERT启动子调控的CD：UPRT／5-FC自杀基因对人胃癌裸鼠移植瘤的抑制作用

目的：研究人端粒酶逆转录酶（hTERT）启动子调控的胞嘧啶脱氨酶：尿嘧啶磷酸核糖转移酶／5-氟胞嘧啶（CD：UPRT／5-FC）融合自杀基因系统在体内对人胃癌SGC7901细胞的杀伤作用。方法：构建胃癌皮下移植瘤模型,随机分成3组处理。观察30天,测量肿瘤体积,计算抑瘤率。肿瘤组织做常规病理检查,免疫组化检测CD蛋白的表达。结果：成功构建胃癌皮下移植瘤模型。治疗30天后,B组（瘤内注射hTERT—CD：UPRT＋腹腔注射PBS）和c组（单纯腹腔注射PBS）的肿瘤生长迅速,肿瘤体积分别为（2．72±0．35）cm’、（2．67-＇i-0．30）tin’,A组（瘤内注射hTERT—CD：UPRT＋腹腔注射5-Fc）的肿瘤生长受到明显抑制,第30天体积为（1．10±0．13）cm’,与前两组相比差异有统计学意义（P〈0．05）,其抑瘤率为59．6％。裸鼠移植瘤病理切片镜检,A组可见大量肿瘤细胞排列稀疏,部分肿瘤细胞核固缩、核碎裂;B组和C组则见大量肿瘤细胞排列紧密,细胞形态和细胞核呈多形性,核大深染,核分裂像多见。免疫组化显示,A组和B组可见CD的表达,C组cD表达呈阴性。结论：hTERT启动子调控的cD：UPRT／5-FC融合自杀基因系统对裸鼠胃癌皮下移植瘤有显著的杀伤作用,为胃癌的基因治疗提供了-种可能的方法。     

UPRT/5-FU系统对小鼠胃癌细胞体外杀伤效应的实验研究

基因治疗是当今肿瘤治疗领域中的热点,尤以药物敏感基因治疗(自杀基因治疗)倍受瞩目,可通过基因修饰手段,将编码外源蛋自的基因转导入肿瘤细胞,使其获得表达,编码某种特定的酶,可将无毒或低毒的前药转化为细胞毒性药物从而选择性杀伤转染目的基因的靶细胞.尿嘧啶磷酸核糖转移酶(UPRT)是存在于细菌等微生物当中的一种酶,哺乳动物当中不存在,可特异性作用于尿嘧啶及其类似物,催化合成嘧啶核苷酸前体UMP,后者是5-FU在体内发挥抗瘤效应最为重要的中间产物,然后才能进一步转化为细胞毒性的代谢产物发挥抗瘤效应.我们采用分子克隆技术成功构     

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。     

中药R对亚硝基肌氨酸乙酯诱发小鼠前胃癌的预防实验研究

本文应用NSAR诱发NIH小鼠前胃癌的动物模型,研究了中药R阻断癌变的预防作用及其对NK和SOD活性的影响。结果表明,中药R有显著的阻断癌变作用,且呈现明显的剂量效应关系.其中以每公斤体重4g的前期用药组效果较好,其抑制率为77.77％(P<0.01),提示该药主要作用于致癌过程的始发致突变阶段。中药R并可明显地提高NK和SOD活性,具有增强细胞免疫和解毒功能。是一种有苗头的化学预防药物,值得进一步开展人群干予试验和作用机制研究。     

新型阿霉素前体药PDOX对人胃癌原位移植瘤模型的分子靶向治疗研究

  目的   比较PDOX与DOX对裸鼠人胃癌原位移植瘤模型的疗效和毒副作用。   方法  建立MGC-803人胃癌原位移植瘤模型35只, 随机分为PDOX组(n=12)、DOX组(n=12)和Control组(n=11), 接种后第10、17、24天按体质量给药, 每周进行两次详细全面记录。末次给药后1周终止实验, 取血行常规、生化检查; 解剖裸鼠, 详细记录胃部肿瘤及腹腔播散情况, 对肿瘤进行称重, 计算抑瘤率; 进行组织病理学检查, 分析肿瘤细胞增殖、凋亡, 肿瘤血管生成、侵袭转移等指标, 行统计学分析。   结果  至研究终点时,Control组和PDOX组全部存活,DOX组6只死亡。在肿瘤局部控制指标中,PDOX组和DOX组的胃肿瘤质量、体积均显著低于Control组（P<0.05）,PDOX组和DOX组抑瘤率分别为26.4%和24.9%,两组疗效相当（P>0.05）。在血常规指标中,PDOX组血小板计数高于Control组（P<0.05）,淋巴细胞比例低于Control组（P<0.05）,而红细胞计数和血红蛋白在三组间差异无统计学意义（P>0.05）。在心功能指标中,PDOX组CK-MB水平高于Control组（P<0.01）。CK、LDH、肝功能及肾功能指标三组间差异均无统计学意义（P>0.05）。PDOX组和DOX组淋巴管癌栓发生率显著低于Control组（P<0.05）。免疫组织化学结果显示PDOX组Ki-67水平显著低于DOX与Control组,三组间两两比较差异均有统计学意义（P<0.01）;Bcl-2、Bcl-6、Tunel、VEGF差异均无统计学意义（P>0.05）,CD34、D2-40有明显下降趋势。   结论  PDOX治疗人胃癌原位移植瘤模型的整体疗效优于DOX, 毒副作用低于DOX。      

胃癌耐药细胞株SGC-7901/5-FU的建立及其耐药机制的初步探讨

目的构建耐5-FU的胃癌耐药细胞株,初步探讨细胞发生5-FU耐药的可能机制.方法 通过药物耐药间歇冲击并浓度梯度递增法诱导建立胃癌耐药细胞株SGC-7901/5-FU.应用MTT法检测亲代细胞对4种化疗药物的IC50并计算耐药指数(RI).通过MTT法绘制细胞的生长曲线计算细胞倍增时间.流式细胞仪分析细胞周期分布.RT-PCR检测抑癌基因P53、癌基因Bcl-2、促凋亡基因半胱氨酰天冬氨酸特异性蛋白酶-3(caspase-3)、死亡相关蛋白激酶(death associated protein kinase,DAPK)家族成员(DAPK-1、DAPK-2、DAPK-3)、多药耐药蛋白(multidrug resistance proteins,MRP)亚家族中的MRP-6、多药耐药基因1(multidrug resistance 1,MDR1)、切除交叉互补修复基因(excision repair cross-complementing 1,ERCC-1)的表达.Western Blot检测MDR1 编码的P糖蛋白(P-gp)的表达水平.结果SGC-7901/5-FU对5-FU、顺铂、环磷酰胺的耐药指数分别为6.26、3.71、5.25.SGC-7901与SGC-7901/5-FU细胞倍增时间分别为(30.82±1.46)h和(46.01±4.15)h,SGC-7901/5-FU细胞增殖速率显著变缓(P＜0.05).与亲代细胞比较,耐药子株S期细胞显著减少,G0/G1期细胞则显著增高(P＜0.01),而G2/M期细胞无显著差异(P＞0.05).耐药子株SGC-7901/5-FU细胞MDR-1、MRP-6、DAPK-3和P-gp表达显著上调(P＜0.01),而ERCC-1、caspase-3、P53、DAPK-1、DAPK-2、Bcl-2的表达均无显著变化(P＞0.05). 结论成功构建胃癌耐5-FU细胞株SGC-7901/5-FU,其对顺铂、环磷酰胺具有交叉耐药,耐药机制与细胞周期S期细胞比例减少、G0/G1期细胞比例升高有关,可能与MDR-1、MRP-6、DAPK-3基因及P-gp蛋白表达上调有关.     

树突状细胞和基因缺陷腺病毒联合治疗小鼠前胃癌的实验研究

目的观察树突状细胞(den-driticcells,DCs)和基因缺陷腺病毒联合治疗小鼠前胃癌的疗效。方法建立小鼠前胃癌动物模型,在体外分化、诱导DCs;瘤内注射基因缺陷腺病毒,观察肿瘤坏死情况;再在瘤体内注射DCs,观察给药侧瘤体及对侧瘤体体积、小鼠生存情况和特异性细胞毒T淋巴细胞(cytotoxicT-lymphocyte,CTL)活性。结果肿瘤组织局部应用基因缺陷腺病毒后,产生明显坏死;瘤内应用DCs,对侧瘤体体积明显缩小,P=0·000;小鼠的生存率提高,且能产生特异性CTLs,能特异性杀伤小鼠前胃癌细胞,P<0·01。结论DCs和基因缺陷腺病毒联合治疗可抑制小鼠前胃癌生长,诱导机体特异性抗肿瘤免疫反应,二者联合应用,可产生协同效应并加强其抗肿瘤效果。     

Annexin V-targeted enzyme prodrug therapy using cytosine deaminase in combination with 5-fluorocytosine

A fusion protein, consisting of cytosine deaminase (CD) linked to human annexin V, was created for use in an enzyme prodrug therapy targeted to the tumor vasculature and associated cancer cells in the primary tumor and distant metastases. The major finding of this study is that the CD-annexin V fusion protein in combination with the prodrug 5-fluorocytosine has significant cytotoxic activity against endothelial cells and two breast cancer cells lines in vitro that expose phosphatidylserine on their surface. The cytotoxicity experiments verified this novel enzyme prodrug system has the ability to produce therapeutic levels of 5-fluorouracil and thus appears promising.     

106例胃癌术中放射治疗

目的 探讨胃癌术中照射范围、剂量和疗效。方法 对106例胃癌进行术中放射治疗。采用6－16MeV电子线，剂量在10－30Gy之间，靶区范围以瘤床及瘤床及周围淋巴引流区为主。与1975年至1989年期间441例单纯手术进行对照。结果 接受治疗的106例患者无近期并发症，照射范围和剂量均适用于临床。Ⅰ、Ⅳ期胃癌术中放射治疗不能提高5年生存率，Ⅱ、Ⅲ期胃癌术中放射治疗提高5年生态率19.4%、15.3%。结论 胃癌术中放射治疗不增加术后并发症，能提高Ⅱ、Ⅲ期胃癌5年生存率。术中放射治疗是治疗胃癌较安全的重要手段之一。     

Intra-aortic infusion therapy with sequential methotrexate (MTX) and 5-FU in advanced gastric carcinoma

Fifteen patients with advanced gastric carcinoma were treated by intraaortic infusion therapy with sequential MTX and 5-FU. Intraaortic bolus injection with 50-100 mg/body MTX was followed 3 hours later with 500-750 mg/body 5-FU and 24 hours later with 30 mg/body leucovorin. Treatment was repeated weekly. Of these 15 patients who were evaluated, 4 had PR and 3 had MR. Response rate was 27%. Two patients had WBC nadir of less than 3,000 cells/mm and other two had a platelet count nadir of less than 10/mm. Gastrointestinal symptoms such as nausea and vomiting were mild. Three patients had diarrhea and 3 had mucositis. No other toxicity was seen. This regimen has been well tolerated for long periods.     

洛铂联合５-ＦＵ／ＣＦ治疗晚期胃、结直肠癌的剂量爬坡试验

目的　观察国人对不同剂量洛铂联合５-ＦＵ／ＣＦ治疗晚期胃、结直肠癌的耐受性、毒性反应以及与剂量的关系,探讨洛铂在联合化疗中的人体安全耐受剂量。方法　选取洛铂从低剂量逐渐至高剂量,５-ＦＵ和ＣＦ剂量不变,每剂量组至少３例受试者。初始剂量为洛铂２５ｍｇ／ｍ^２ｄ_１,５-ＦＵ４００ｍｇ／ｍ^２ｄ_１～ｄ_５,ＣＦ２００ｍｇ／ｍ^２ｄ_１～ｄ_５,２１ｄ为１周期;如无明显毒性反应则进入下一剂量组,直至最大耐受量（ＭＴＤ）或５０ｍｇ／ｍ^２。结果　１８例晚期胃、结直肠癌患者进入试验,分别进行了６个剂量组的研究,最高剂量组为５０ｍｇ／ｍ２,未观察到ＭＴＤ。主要毒性反应为血红蛋白减少、白细胞减少,其次为恶心、呕吐和腹泻,所有患者未出现３～４级不良反应。结论　洛铂联合５-ＦＵ／ＣＦ毒性反应较轻,患者耐受性较好。推荐使用洛铂４５ｍｇ／ｍ^２联合５-ＦＵ４００ｍｇ／ｍ^２和ＣＦ２００ｍｇ／ｍ^２用于进一步临床研究。     

Treatment outcomes of oxaliplatin, 5-FU, and leucovorin as salvage therapy for patients with advanced or metastatic gastric cancer: a retrospective analysis

Purpose  We performed a single-institution retrospective study to evaluate the efficacy and toxicities of oxaliplatin, 5-fluorouracil (5-FU), leucovorin (LV) combination chemotherapy as salvage treatment in patients with metastatic or advanced gastric cancer. Methods  Sixty-two patients with advanced gastric cancer previously treated were eligible for the study. Patients received oxaliplatin 100 mg/m² and LV 100 mg/m² (2-h intravenous infusion) followed by 5-FU 2,400 mg/m² (46-h continuous infusion) every 2 weeks, and responses were assessed after every three cycles. Results  Fifty-nine out of 62 patients were assessable for response. Among them, 46 patients had previously been treated with cisplatin based chemotherapy. Patients had a median age of 57 years (range 32–76 years), 72.6% had an Eastern Cooperative Oncology Group performance status of 0 or 1. Total 296 courses of chemotherapy were administered as second-line (67.7%) or third-line (27.4%), and the median courses per patient was three cycles. Out of 59 evaluable patients, 14 partial responses were observed (overall response rate, 22.6%). Stable disease was observed in 22 patients (35.5%), and progressive disease in 23 patients (37.1%). The median response duration, time to progression, and overall survival were 2.3, 3.0, and 8.0 months, respectively. The major toxicities were neutropenia, mucositis, and peripheral neuropathy. Grade 3 or 4 hematologic toxicities included neutropenia in nine patients (14.5%) and thrombocytopenia in one patient (1.6%). Other grade 3 or 4 toxicities included mucositis in one patient (1.6%) and vomiting in two patients (3.2%). Grade 1 or 2 peripheral neuropathy were observed in 18 patients (29.0%), however there were no cases of grade 3 or 4 peripheral neuropathy and no treatment-related deaths. Conclusion  The combination of oxaliplatin, 5-FU and LV was effective and safe salvage chemotherapy in advanced gastric cancer patients.     

5-FU节拍化疗策略优化及调节胃癌免疫微环境的体内实验研究

背景与目的：5-氟尿嘧啶（5-fluorouracil,5-FU）是胃癌化疗的骨架药物,传统大剂量5-FU常导致严重不良反应及耐药。低剂量5-FU节拍化疗在不影响疗效的前提下可明显降低药物毒性,但何种给药节拍可达到最佳抗肿瘤作用尚不清楚。本研究旨在探索5-FU的最佳节拍化疗模式,并研究其对胃癌免疫微环境的影响。方法：建立SGC-7901胃癌细胞系的BALB/c裸小鼠皮下移植瘤模型,成瘤后随机分成4组：最大耐受剂量（maximumtolerateddose,MTD）组、每日节拍化疗（daily metronomic chemotherapy,MET-qd）组、隔日节拍化疗（every other day metronomic chemotherapy,MET-qod）组和每周2次节拍化疗（twice-weekly metronomic chemotherapy,MET-biw）组。21 d为1个疗程,共2个疗程。治疗期间观测裸小鼠的一般状况,每周称重并测量瘤体,绘制肿瘤生长曲线。采用流式细胞术检测裸小鼠外周血内皮祖细胞（circulating endothelialprogenitors...     

Sequential methotrexate and 5-fluorouracil therapy for gastric cancer patients with peritoneal dissemination: a retrospective study

Background. Most gastric cancer patients with peritoneal dissemination have been excluded from clinical studies because they usually have no measurable lesions. They also have a high risk of toxicity because of complications such as intestinal obstruction and ascites. We conducted a retrospective analysis to evaluate the efficacy and feasibility of sequential methotrexate (MTX) and 5-flurorouracil (5FU) therapy for this population. Methods. This analysis was based on 56 consecutive chemotherapy-naive patients with confirmed peritoneal dissemination of gastric cancer who were being treated with sequential MTX/5FU. The therapy comprised a weekly schedule of MTX 100 mg/m², given as a bolus infusion 3 h prior to a bolus infusion of 5FU 600 mg/m². Leucovorin 10 mg/m² was administered six times, every 6 h, starting 24 h after MTX administration. Results. Evidence of peritoneal dissemination was confirmed by laparotomy in 16 patients, by cytologic examination of ascites in 11 patients, and by clinical imaging in 29 patients (15 with ascites, 13 with intestinal obstruction; in 10 of the 29 patients, detection was by barium enema or computed tomography [CT] scan). Neutropenia of grade 3 or worse and anemia were observed in 8 (14%) and 10 (18%) of the 56 patients, respectively. There was one treatment-related death due to neutropenic sepsis. Of the 26 patients with measurable lesions, 9 showed a response (36%). The median survival time and median time to treatment failure were 259 days and 167 days, respectively. Objective improvement of ascites was seen in 13 of 26 patients (50%), including 5 with showed complete disappearance of ascites. Seven of the 15 patients (47%) with intestinal obstruction showed resolution, and 8 of the 21 patients (38%) who needed nutritional support before the treatment were free of that support for a median duration of 220 days after the completion of the treatment. Forty-seven of the 56 patients (84%) could be treated at outpatient clinics. Conclusions. This regimen may be of clinical benefit for patients with peritoneal dissemination of gastric cancer. Received: August 24, 2001 / Accepted: October 19, 2001     

A study on the treatment of gastric cancer by CD gene combined with 5-FC in vitro and in vivo

Objective: To study the killing effect of suicide gene CD on mouse gastric cancer. Methods: CD gene was transduced with the retroviral vector. The killing effect and bystander effect of CD gene on mouse gastric cancer cell line MFC were observed. The mouse gastric cancer model was used for in vivo study. The CD gene containing virus was injected into the tumors. The volumes of the tumors in every group were measured in time. Results: Significant killing effect and bystander effect were observed by CD gene in vitro, 70–80% cell death resulting from 20% of CD gene transduction. In vivo, CD/5-Fc caused tumor to diminution. Conclusion: CD/5-Fc system has significant killing effect on mouse gastric cancer Foundation item: This work was supported by the Scientific and Research Foundation of the Ministry of Public health of China(No. 98-1-312) and Shanghai Natural Scientific Foundation (No. 98ZB14028). Biography: GUO Shan-yu(1966–), doctor of medicine, attending physician, Shanghai Ninth People’s Hospital, majors in biological therapy of gastrointestinal cancer.     

OHLF3方案治疗晚期胃癌的临床观察

为了比较奥沙利铂（L-OHP）联合羟基喜树碱（HCPT）、亚叶酸钙（CF）和5-氟尿嘧啶（5-FU）组成OHLF3方案与顺铂（DDP）联合HCPT、CF和5-FU组成的HLFP方案的疗效及不良反应,采用非随机分组方法将56例晚期胃癌患者分为OHLF3组（治疗组）与HLFP组（对照组）.化疗方案：OHLF3方案：L-OHP 130 mg/m^2,静脉滴入,d1;HCPT 6 mg/m^2,静脉滴入,d1～d5;CF 100 mg/m^2,静脉滴入,d2～d6;5-FU 500 mg/m^2,静脉滴入,d2～d6.HLFP方案：DDP 25 mg/m^2,静脉滴入,d1～d3;HCPT 6 mg/m^2,静脉滴入,d1～d5;CF 100 mg/m^2,静脉滴入,d6～d10;5-FU 500 mg/m^2,静脉滴入,d6～d10.28 d为1个周期,完成3个周期评价疗效.治疗组32例有效率56.3%（18/32）,对照组24例有效率41.2%（10/24）,治疗组高于对照组,但两组比较差异无统计学意义,χ^2=1.167,P=0.280.两组不良反应主要是白细胞减少、恶心呕吐、口腔炎、腹泻及周围神经炎.初步研究结果提示,OHLF3方案治疗晚期胃癌疗效好,毒副反应轻,值得临床进一步观察.