Wt—p53／p16基因联合CD／5-FC系统对U251细胞化疗效果的研究

[目的]研究p53、p16基因联合胞嘧啶脱氨酶（CD）／5-氟胞嘧啶（5-FC）治疗神经胶质瘤的可行性。[方法]体外试验以Fe3O4磁性纳米颗粒（Fe3O4 magneticna noparticles,Fe3O4 MNP）为基因载体将质粒pCDNA3．1-p16、pYD5-p53,及pCMVCD转染入U251人胶质瘤细胞,给以不同浓度的5-FC,以MTT法检测细胞生长抑制率。[结果]成功以Fe3O4MNP为载体将wt-p53、wt-p16及CD基因转染U251细胞,并稳定表达。体外转染wt-p53、wt-p16基因都能明显提高CD／5-FC人对胶质瘤细胞的化疗效果。[结论]基于Fe3O4磁性纳米基因载体的p53、p16基因和CD／5-FC系统的联合基因治疗可作为临床上优化自杀基因治疗方案的一种可能选择,可以明显提高治疗效果。     

肿瘤的基因治疗前期研究:反转录病毒介导的肿瘤坏死因子α基因抗C6胶质瘤效应

目的探讨反转录病毒介导的肿瘤坏死因子α(TNF-α)基因对C6胶质瘤的抗瘤效应,从而为肿瘤的基因治疗提供基础研究数据.方法将重组TNF-α反转录病毒载体PLJ+TNF转染大鼠C6胶质瘤细胞,ELISA法检测该细胞中目的基因的表达,MTT法检测体外增殖能力;将实验大鼠随机分为实验组和对照组,每组10只,实验组大鼠右股内侧接种转基因2×106 C6细胞,对照组同法同部位接种未转基因C6细胞,常规饲养5周,每周测量肿瘤大小,绘制肿瘤生长曲线,比较两组皮下肿瘤大小并进行统计学分析.结果C6胶质瘤细胞与C6细胞的体外增殖能力比较,差异无显著性意义(P＞0.05);转基因前后TNF-α分泌情况1×106个C6细胞体外培养24 h每毫升上清液分泌TNF-α(2.42±0.76)u;1 ×106个C6胶质瘤细胞体外培养24 h每毫升上清液分泌TNF-α(17.53±2.31)u,两者比较,差异有显著性意义(P＜0.01);动态TNF-α检测显示转基因C6细胞能稳定地表达高水平的TNF-α;大鼠皮下接种肿瘤细胞5周后,实验组3只未见肿瘤生长,其余肿瘤直径为(1.5±0.3)cm,对照组肿瘤直径为(2.4±0.2)cm,两组比较,差异有显著性意义(P＜0.01).结论TNF-α基因转染大鼠C6胶质瘤细胞后能持续分泌高水平的TNF-α,对大鼠C6胶质瘤皮下肿瘤具有明显的抗瘤效应.     

5-ALA介导的PDT对大鼠体内外C6胶质瘤的杀伤作用研究

目的探讨5-ALA介导的光动力对大鼠C6胶质瘤细胞体内外杀伤作用。方法MTT法探讨培育时间和5-ALA浓度对光动力效应的影响,为动物试验提供时间和剂最参考。取近皮层大鼠胶质瘤模型39只,其中PDT组12只,单纯开骨窗组9只,单用5-ALA＋开骨窗组9只,开骨窗＋激光组9只,各组处理后观察生存状态和生存期,部分大鼠行病理及透射电镜检查。结果5-ALA与C6细胞共同培育5h其介导的PDT作用达到最强;5-ALA浓度为0．8mmol／L以上时对细胞的抑制率可超过50％;电镜示PDT组瘤组织可见较多量凋亡,细胞间连接松散。结论该实验设计的大鼠近皮层模型可以很好地满足脑肿瘤动物实验的要求。5-ALA介导的PDT对体外及颅内种植C6细胞都有杀伤作用。     

肿瘤的基因治疗前期研究：反转录病毒介导的肿瘤坏死因子α基因抗C6胶质瘤效应☆

目的：探讨反转录病毒介导的肿瘤坏死因子α(TNF-α)基因对C6胶质瘤的抗瘤效应，从而为肿瘤的基因治疗提供基础研究数据。方法：将重组TNF-α反转录病毒载体PLJ+TNF转染大鼠C6胶质瘤细胞，ELISA法检测该细胞中目的基因的表达，MTT法检测体外增殖能力；将实验大鼠随机分为实验组和对照组，每组10只，实验组大鼠右股内侧接种转基因2&;#215;10^6C6细胞，对照组同法同部位接种未转基因C6细胞，常规饲养5周，每周测量肿瘤大小，绘制肿瘤生长曲线，比较两组皮下肿瘤大小并进行统计学分析。结果：C6胶质瘤细胞与C6细胞的体外增殖能力比较，差异无显著性意义(P&;gt;0．05)；转基因前后TNF-α分泌情况：1&;#215;10^6个C6细胞体外培养24h每毫升上清液分泌TNF-α(2．42&;#177;0．76)U；1&;#215;10^6个C6胶质瘤细胞体外培养24h每毫升上清液分泌TNF-α(1753&;#177;2．31)U，两者比较，差异有显著性意义(P&;lt;0．01)；动态TNF-α检测显示：转基因C6细胞能稳定地表达高水平的TNF-α；大鼠皮下接种肿瘤细胞5周后，实验组3只未见肿瘤生长，其余肿瘤直径为(15&;#177;0．3)cm，对照组肿瘤直径为(2．4&;#177;0．2)cm，两组比较，差异有显著性意义(P&;lt;0．01)。结论：TNF-α基因转染大鼠C6胶质瘤细胞后能持续分泌高水平的TNF-α，对大鼠C6胶质瘤皮下肿瘤具有明显的抗瘤效应。     

自杀基因联合化疗干预卵巢癌的动物实验研究

目的探讨单纯疱疹病毒胸腺激酶（herpes simplex virus thymidine kinase,HSV-TK）基因/更昔洛韦（ganciclovir,GCV）系统联合化疗药物拓扑替康（Topotecan）对卵巢癌细胞NuTu-19杀伤作用及自杀基因系统与化疗药物不同的给药次序的疗效差异。为临床治疗提供最佳方案。方法常规方法培养PA317/TK细胞、NuTu-19细胞;耐药集落形成法测定病毒滴度;自杀基因转导,PCR方法检测TK基因阳性克隆细胞;测定NuTu-tk细胞对GCV敏感性;建立动物模型：接种NuTu-19细胞及同时接种PA317/TK细胞和NuTu-19细胞的大鼠成瘤后应用化疗药物（Topotecan）,GCV及GCV联合化疗药物,测量肿瘤大小并计算抑瘤率。结果经PCR检测证实PA317/TK已经导入NuTu-19中;GCV浓度在0.01-30时对NuTu–tk细胞均有明显的杀伤作用,GCV对NuTu–tk细胞的杀伤率与其浓度呈明显正相关（r=0.612,t=2.04,P〈0.01）;大鼠接种肿瘤细胞后10-12天局部长出约0.4×0.5cm大小的肿瘤,然后局部逐渐呈膨胀性生长,质硬,活动,与腹壁不粘连,呈球形或椭圆形或多分叶状实体瘤,表面可见丰富皮下血管网;HSV-TK/GCV系统、Topotecan及HSV-TK/GCV联合Topotecan对大鼠皮下移植肿瘤有抑制作用;先给自杀基因组较先给化疗药物组疗效明显,均有统计学意义。结论 HSV-TK/GCV与拓扑替康联合杀伤卵巢癌细胞有协同作用;先用HSV-TK/GCV后用拓扑替康治疗效果最佳。自杀基因联合化疗治疗卵巢癌的动物实验研究为HSV-TK自杀基因系统的临床应用提供了实验基础,也为复发性、难治性卵巢癌患者治疗开辟新的途径。     

逆转录病毒载体介导的耐药基因在造血细胞中基因转导和表达的研究

本文对于逆转录病毒载体介导的基因转移方法中如何提高逆转录病毒滴度进行了研究。以磷酸钙共沉淀的方法转染包装细胞PA17,经氨甲蝶呤(MTX)10~(-7)mol/L筛选,扩增得到产病毒细胞株,经点杂交证实dhfr基因成功导入并产生病毒颗粒。混合细胞克隆PA317/pSPD产生的逆转录病毒滴度为(1.1-2.3)×10~3 cfu/ml。随MTX筛选浓度增加,产病毒细胞PA317/pSPD抗性增加。当培养基中MTX浓度由10~(-7) mol/L增加到10~(-6)mol/L时,逆转录病毒滴度增加了近10倍[(1.02-2.09)×10~4 cfu/ml]。结果显示可以通过增加MTX筛选浓度来提高含dhfr cDNA的逆转录病毒滴度。     

γ-干扰素基因对大鼠脑胶质瘤细胞生长及生存期的影响

背景反转录病毒载体介导细胞因子杀伤恶性肿瘤细胞为目前肿瘤科研领域的热点,逆转录病毒介导γ-干扰素是否抑制大鼠脑胶质瘤细胞的生长及延长其生存期的作用尚无肯定性结论.目的观察反转录病毒载体介导的γ-干扰素对大鼠脑胶质瘤细胞的生长抑制作用,探讨与生存期的关系.设计以动物为研究对象,完全随机设计.单位一所大学附属医院的神经外科.材料实验于2002-07/2004-07在哈尔滨医科大学神经外科研究所完成,选择哈尔滨医科大学附属第一临床医学院动物实验中心提供的60只Wistar雄性大鼠.随机分为C6对照组、C6/PA317neo对照组及C6/PA317γ-干扰素治疗组,每组20只.干预应用反转录病毒载体将鼠γ-干扰素基因转入反转录病毒包装细胞PA317,通过G418抗性筛选,得到高滴度产毒PA317 γ-干扰素细胞株.3组大鼠右侧基底核区接种1×106C6细胞.C6对照组接种后不做其他处理;C6/PA317neo对照组接种4 d后原位注射1×106PA317neo细胞;C6/PA317 γ-干扰素治疗组接种4 d后原位注射1×106 PA317 γ-干扰素细胞.实验观察①各组大鼠生存期≤60 d的情况.②每组动物C6细胞接种后11 d及20 d后随机取5只处死,取出整个脑组织制备冰冻组织切片,采用SABC免疫组化方法检测肿瘤组织CD4和CD8 T淋巴细胞浸润情况.③显微镜下借助图像分析软件测量肿瘤体积.主要观察指标①各组大鼠生存时间.②肿瘤体积测量.③CD4,CD8淋巴细胞浸润情况.结果60只大鼠进入实验分析.C6对照组及C6/PA317neo对照组大鼠平均生存时间分别为(27.10±0.82)d及(26.60±0.93)d,治疗组平均生存时间为(49.30±4.17)d,有5只大鼠存活期超过60 d.两对照组间生存期差异P＞0.05,治疗组较对照组生存期明显延长,P＜0.01.C6对照组及C6/PA317neo对照组肿瘤体积差异P＞0.05,治疗组肿瘤体积显著小于对照组,P＜0.01.计数肿瘤组织CD4及CD8 T淋巴细胞浸润情况发现治疗组CD4+及CD8+T淋巴细胞显著多于对照组,P＜0.01,两对照组之间差异P＞0.05.免疫组化检测Brdu阳性细胞,结果移植后7 d发现有Brdu阳性细胞,移植后16 d未发现Brdu阳性细胞,对照组均未发现阳性细胞.结论PA317 γ-干扰素细胞瘤内注射能够诱导大鼠产生抗肿瘤免疫反应,反转录病毒介导的γ-干扰素抑制肿瘤细胞生长,可明显延长生存期.     

表达融合基因FCU1骨髓间充质干细胞自杀基因系统的建立及其对结肠癌抑制作用的体外验证

目的:构建腺病毒载体介导表达融合自杀基因FCU1的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),验证其对结肠癌CT-26细胞生长的抑制作用。方法:体外分离小鼠BMSCs,构建重组FCU1基因腺病毒表达载体,包装获取病毒上清并感染BMSCs,MTT法检测5-FC对感染重组腺病毒后的BMSCs的抑制作用。感染重组腺病毒后BMSCs与CT-26细胞共培养,MTT法检测5-FC对CT-26细胞增殖的影响。结果:成功构建FCU1融合基因重组腺病毒载体,包装获得病毒滴度为2.6×1010pfu/mL,可有效感染小鼠BMSCs,MOI为200时,可感染95%以上的BMSCs,并且对5-FC敏感。与CT-26细胞共培养后,重组腺病毒感染的BMSCs达到7%时,可对超过50%的CT-26细胞发挥旁观者效应。结论:研究建立的表达融合基因FCU1的骨髓间充质干细胞自杀基因系统,可有效实现对CT-26细胞的抑制作用。     

逆转录病毒介导野生型P^53与反义mdm2融合基因对Mc3细胞凋亡的影响

目的 探讨P53与反义mdm2 cDNA序列真核表达载体诱导人黏液表皮样癌高转移细胞株Mc3细胞凋亡的影响.方法 将P53与反义mdm2 cDNA序列真核表达载体通过脂质体转染Mc3细胞,采用形态学观察凋亡细胞;琼脂糖凝胶电泳检测"梯状条带";末端转移酶标记法(TUNEL)检测凋亡指数;透射电镜检测凋亡小体.结果 转染48 h后形态学观察到细胞体积缩小;核浓染碎裂成大小不等的碎片;核染色质成新月形,琼脂糖凝胶电泳可见180～200 bp典型的凋亡带,TUNEL检测凋亡指数为25.22±1.01(转染后),与对照组2.01±1.10(转染前)比较差异有统计学意义(P<0.05),转染空载体(2.02±1.11)与对照组比较差异无统计学意义(P>0.05),透射电镜可见染色质浓集分布不均,形成由核膜包裹断裂的核碎片的凋亡小体.结论 P53与反义mdm2基因融合体真核表达载体能诱导和促使体外培养的人黏液表皮样癌高转移细胞株Mc3细胞发生凋亡.     

RNA干扰和血液肿瘤融合基因的靶向治疗

RNA干扰(RNAi)是生物进化过程中的一种保守反应,双链小分子干扰RNA所引起的RNAi可序列特异性地使相应mRNA降解。作为一种能使靶基因表达下降的有效工具,RNAi已用于功能基因组学和许多涉及异常基因表达疾病的研究。许多血液肿瘤产生的分子基础是染色体易位所致的融合基因异常表达,运用RNAi技术对融合基因进行靶向治疗已成为一种新的治疗策略。     

骨髓间充质干细胞-胞嘧啶脱氨酶/5-氟胞嘧啶自杀基因治疗系统的构建及效应

背景:体内研究已经证实骨髓间充质干细胞能够像神经干细胞一样在脑内迁移和整合,如果骨髓间充质干细胞用于系统途径移植成为可能,将会显著简化移植的步骤.目的:构建含胞嘧啶脱氨酶(CD)基因的重组表达质粒pEGFP-N3-CD,用脂质体Lipofectamine2000转染大鼠骨髓间充质千细胞,体外观察CD基因的表达及BMSCs-CD/5-氟胞嘧啶自杀基因治疗系统对C6胶质瘤细胞的杀伤作用.方法:构建pEGFP-N3-CD质粒,酶切、DNA测序鉴定.全骨髓贴壁法分离培养骨髓间充质干细胞,脂质体Lipofectamine2000介导重组表达质粒pEGFP-N3-CD转染大鼠骨髓间充质干细胞,G418筛选培养获取阳性克隆(BMSCs-CD细胞),免疫细胞化学染色检测BMSCs-CD细胞的CD基因蛋白表达.Transwell小室共培养BMSCs-CD细胞和C6胶质瘤细胞,24 h后加入前体药物5-氟胞嘧啶,72 h后TUNEL、MTT、流式细胞术检测C6胶质瘤细胞的凋亡情况.结果与结论:构建的重组表达质粒pEGFP-N3-CD含完整的CD基因序列,CD基因转染至骨髓间充质干细胞并在基因及蛋白水平有完整表达.BMSCs-CD和C6胶质瘤细胞体外共培养条件下,C6胶质瘤的凋亡率呈剂量依赖性,与对照组相比差异有显著性意义(P<0.01).结果提示体外共培养条件下,BMSCs-CD/5-氟胞嘧啶自杀基因治疗系统对C6胶质瘤细胞生长有显著的抑制作用.     

In vitro and in vivo activity of 5-fluorocytosine on Acanthamoeba

The results of our studies indicated that the avirulent Neff strain of Acanthamoeba was more susceptible to the activity of the anti-metabolite 5-fluorocytosine (5-FC) than was the virulent A-1 strain or a mouse brain reisolate of this strain, designated A-3. Results of competition experiments in which cultures were exposed simultaneously to 5-FC and either uracil, thymidine, or both uracil and thymidine demonstrated that the drug was directed against both deoxyribonucleic acid and ribonucleic acid in the avirulent strain, whereas ribonucleic acid was mainly affected in the virulent amebas. Concentrations >10 mug of 5-FC per ml were amebicidal to the avirulent strain; lower concentrations of the drug, which only affected growth slightly, significantly impaired the capacity of the cells to spontaneously encyst in stationary-phase cultures. On the other hand, the virulent strains were capable of growing in the presence of 5-FC (40 mug/ml) after an initial period of susceptibility. After a few transfers in growth medium lacking the drug, 5-FC-treated virulent amebas exhibited growth parameters typical of untreated cells. However, after successive subcultures in drug-free medium, 5-FC-treated cells lost their resistance and were again susceptible to the drug. This result suggested that the capacity of the cells to develop resistance resulted from a drug-induced mechanism. Spontaneous encystment, which was normally minimal in stationary-phase A-1 or A-3 cultures, was enhanced in A-3 but not A-1 cultures treated with 5-FC (>30 mug/ml). Results obtained from experiments to determine the effectiveness of 5-FC in protecting mice experimentally infected with either A-1 or A-3 amebas indicated that the clinical usefulness of 5-FC may be limited by the capacity of the amebas to develop resistance.     

The potential of 5-fluorocytosine/cytosine deaminase enzyme prodrug gene therapy in an intrahepatic colon cancer model

Colorectal cancer can metastasize to the liver, but remain liver confined for years. A critical step in developing treatments for intrahepatic cancer involves assessment in an orthotopic intrahepatic model. The purpose of this study was to develop a noninvasive intrahepatic tumor model to study the efficacy of 5-flucytosine/yeast cytosine deaminase (5FC/yCD)-based gene therapy for liver tumors. Luciferase expressing human colorectal carcinoma (HT-29luc) cells were generated by retroviral infection and implanted in the left liver lobe of nude mice. The bioluminescence was measured every week for a period of 1 month, then animals were killed and tumors were measured by calipers. After we found a correlation between photon counts and tumor size, animals were implanted with tumors composed of either 0%, 10%, or 100% yCD/HT-29luc cells, and treated with 5FC. Tumor bioluminescence was measured during treatment and tumor histology examined at the time of death. We found that 5FC caused significant regression of yCD expressing tumors. Furthermore, visible tumors at the time of death, which emitted little bioluminescence, contained little or no viable tumor. We then developed an adenoviral vector for yCD. Intraperitoneal administration of adenovirus containing yCD led to the production of yCD enzyme within intrahepatic tumors. These results suggest that (1) intrahepatic cancer responds to 5FC when cells express yCD; (2) the luciferin-luciferase system permits non-invasive real time imaging of viable intrahepatic cancer; and (3) this system can be used to carry out gene therapy experiments using yCD adenovirus.     

Complementation analysis of resistance to 5-fluorocytosine in Candida albicans.

A complementation test was devised to study allelism among the genetic determinants of resistance to 5-fluorocytosine in Candida albicans. Complementation was demonstrated in control hybrids produced by crossing a resistant strain that was deficient in cytosine deaminase activity with four other resistant strains deficient in UMP pyrophosphorylase activity. This complementation test was used to test allelism of the resistance determinants present in five clinical isolates. All were found to bear recessive alleles of the locus (FCY1) that determined 5-fluorocytosine resistance associated with low levels of UMP pyrophosphorylase activity.     

5-fluorocytosine susceptibility of pathogenic fungi in the presence of allopurinol: potential for improving the therapeutic index of 5-fluorocytosine.

The minimal inhibitory concentration of 5-fluorocytosine in 18 pathogenic fungal isolates was not altered by either allopurinol (100 microM) or oxypurinol (100 microM). Since allopurinol at this level clinically has been demonstrated to interfere with 5-fluorouracil anabolism, thereby reducing toxicity owing to 5-fluorouracil, allopurinol may be useful in counteracting the 5-fluorouracil-induced myelotoxicity observed in patients being treated with 5-fluorocytosine without interfering with the antifungal activity of 5-fluorocytosine.     

Coexpression of the uracil phosphoribosyltransferase gene with a chimeric human nerve growth factor receptor/cytosine deaminase fusion gene, using a single retroviral vector, augments cytotoxicity of transduced human T cells exposed to 5-fluorocytosine

Donor T lymphocytes genetically engineered to express a "suicide gene" to facilitate negative selection represent a promising strategy for the management of graft-versus-host disease occurring after allogeneic hematopoietic cell transplantation (HCT). For this purpose, the herpes simplex virus thymidine kinase (HSV-tk) gene, although well studied, has limitations. Cytosine deaminase (CD), an alternative gene for negative selection, converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). Sensitivity of cells to 5-FU can be further increased by expression of uracil phosphoribosyltransferase (UPRT), which catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate. By using a chimeric gene (NG/CD) expressing the truncated human nerve growth factor receptor (NGFR) for positive selection fused to the Saccharomyces cerevisiae CD gene, we investigated strategies to achieve optimal T cell eradication by CD and UPRT expression, utilizing a single retroviral vector. Three vector strategies were compared on the basis of NGFR expression by flow cytometry, western analysis, and enzymatic activity. A construct (NG/CDiU) expressing UPRT and NG/CD, using a bicistronic message, provided the greatest UPRT activity and killing, reducing the lethal dose of 5-FC sufficient to eradicate 90% of cells from 38.7 microg/ml (300 microM) (NG/CD expression alone) to 0.13 microg/ml (1 microM). This approach provides an effective alternative to the HSV-tk system for eradication of donor T lymphocytes after allogeneic HCT.     

Candida albicans resistance to 5-fluorocytosine: frequency of partially resistant strains among clinical isolates

Resistance to 5-fluorocytosine was studied in 137 independent Candida albicans clinical isolates. Seventy-eight isolates (57%) were susceptible; 51 isolates (37%) were partially resistant; 8 isolates (6%) were highly resistant. All partially resistant isolates gave rise to variants which were highly resistant. Some susceptible isolates gave rise to variants which were highly resistant; two such isolates were shown to be heterozygous for resistance, and these isolates define a new type of heterozygote. A partially resistant isolate gave rise to resistant variants which were auxotrophic for lysine; this result was interpreted as preliminary evidence that the allele which determined resistance was linked to an allele which determined auxotrophy for lysine. It is suggested that heterozygotes constitute a source of preexisting mutant alleles which determine resistance, and that 5-fluorocytosine treatment of infections due to heterozygotes may result in significant selection for resistant variants. A simple screening procedure is described by which partially resistant strains may be recognized.     

Antagonistic effects of fluconazole and 5-fluorocytosine on candidacidal action of amphotericin B in human serum.

This study addressed the effects of fluconazole and 5-fluorocytosine on the candidacidal activity of amphotericin B in the presence of human serum. A Candida albicans isolate that was susceptible to all three agents according to standard testing procedures was employed. Fungicidal activity was estimated by using a flow cytometric procedure that exploited the fact that yeast cells killed by amphotericin B diminish in size and take up propidium iodide. The following findings were made. (i) Fluconazole and 5-fluorocytosine each failed to inhibit pseudohyphal formation and cell aggregation even when applied at 10 and 50 micrograms/ml, respectively, for up to 10 h. Hence, these agents were not fungistatic when tested in the presence of serum. (ii) Simultaneous application of 5-fluorocytosine had neither enhancing nor inhibitory effects on the fungicidal activity of amphotericin B. However, yeasts that were preincubated for 20 h with 5-fluorocytosine became less susceptible to killing by amphotericin B. (iii) Fluconazole exerted a frank antagonistic effect on the fungicidal activity of amphotericin B. Thus, under our in vitro conditions, both fluconazole and 5-fluorocytosine can overtly antagonize the candidacidal action of amphotericin B.     

人参皂甙Rh2对脑胶质细胞瘤侵袭性的影响

目的　探讨人参皂甙Rh2对C6胶质细胞瘤体内侵袭的影响。方法　将携带增强型绿色荧光蛋白 (EGFP)基因的pegfp N3质粒体外转染C6胶质瘤细胞 ,将C6阳性克隆以立体定向法植入SD大鼠脑实质内 ,建立大鼠胶质瘤移植模型。一周后经MRI检测接种成功的大鼠 ,随机分成两组 ,采用阴性对照及腹腔给药的方法 ,用病理检查、荧光显微镜及MRI观察Rh2对胶质瘤侵袭的影响。结果　稳定转染EGFP基因的C6瘤细胞于体内、外均可发出绿色荧光 ,在荧光显微镜下易于区分肿瘤与非肿瘤区。 16 μl/mlRh2可明显抑制肿瘤体内侵袭。 结论　Rh2降低了C6瘤细胞的体内侵袭性 ,抑制肿瘤生长。     

Cytosine deaminase and thymidine kinase gene therapy in a Dunning rat prostate tumour model: absence of bystander effects and characterisation of 5-fluorocytosine metabolism with 19F-NMR spectroscopy

The rat prostate tumour cell line R3327 AT-1 was transfected with a gene coding for a fusion protein comprised of cytosine deaminase (CD from E. coli) and thymidine kinase (TK from Herpes simplex virus, HSV-1). The resulting AT-1/CDglyTK cell line was sensitive to the prodrug 5-fluorocytosine (IC(50) = 78 microM, 96-h incubation) via CD and to ganciclovir (GCV, IC(50) = 1 microM, 96 h) via TK. Subcutaneous tumours generated from 100% CDglyTK(+) cells responded well to 5-FC therapy (500 mg/kg, i.p., 14 daily treatments, four out of seven animals in remission) and to GCV therapy (30 mg/kg, i.p., 14 daily treatments, five of six animals in remission). However, experiments with mixtures of CDglyTK(+) and CDglyTK(-) cells showed low levels of connexins (intercellular gap junctions) and no bystander effect for nontransfected cells using either 5-FC or GCV therapy. Furthermore, (19)F-NMR spectroscopy showed that incubation of cultured CDglyTK(+) cells with 774 microM 5-FC for 16 h resulted in the following intracellular concentrations: 5-FC = 314 microM, 5-FU = 52 microM, cytotoxic fluoronucleotides = 163 microM; extracellular 5-FU reached only 6.4 microM. Thus, in this model system intracellular trapping of 5-FU (slow export) contributes to the failure of the CD/5-FC bystander effect via an extracellular route.