【作者】 贾雪梅；

【导师】 王淑玉； 刘嘉茵；

【作者基本信息】 南京医科大学 ， 妇科肿瘤， 2004， 硕士

【摘要】 目的： 研究融合型自杀基因Fcy∷Fur联合5-FC对卵巢癌细胞株HO-8910的杀伤效果。 方法： 1．根据逆转录病毒表达载体pLXSN和LZRSpBMN-Z的多克隆位点及质粒pORF5-Fcy∷Fur上Fcy∷Fur的基因序列，设计两对PCR引物，在引物的5’端分别引入EcoRⅠ和BamHⅠ酶切位点，采用常规PCR从pORF5-Fcy∷Fur中扩增出Fcy∷Fur融合基因，经核酸序列测定正确后，分别克隆进pLXSN和LZRSpBMN-Z。经双酶切鉴定后，分别转染包装细胞PT67和Pheonix，扩大培养阳性克隆并收集上清，获得重组逆转录病毒。 2．重组病毒感染靶细胞HO-8910，分别以G418和嘌呤霉素筛选得到稳定表达Fcy∷Fur的阳性细胞株(命名为HO-8910-Fcy∷Fur)，并经抽提细胞基因组DNA和RNA分别进行PCR和RT-PCR等鉴定目的基因的整合与表达。 3．在HO-8910-Fcy∷Fur中加入前药5-FC，MTT法测定肿瘤细胞杀伤效应并计算细胞生长抑制率，倒置相差显微镜观察细胞形态变化，流式细胞仪检测细胞凋亡比例以及免疫组化检测凋亡相关蛋白Fas和Fas-L等的表达。 结果： 1．在成功构建逆转录病毒载体的基础上，转染包装细胞获得重组逆转录病毒，感染HO-8910后抗生素筛选获得HO-8910-Fcy∷Fur，南京医科大学硕上学位论文经PCR及RT-PCR鉴定无误。 2.倒置相差显微镜观察细胞形态发现，加入5一FC后，HO一8910一Fcy::Fur从第4天起发生形态改变，第5天部分细胞死亡，而未转染基因的HO一8910经5一FC作用后无明显形态改变。 3.5一FC终浓度为0.1、1、10、100、1000林mol/L的范围内，对正常未转染Fcy::F ur基因的Ho一89 10的生长无明显影响，而在5一FC终浓度大于10“mol/L时，对HO一8910一Fcy::Fur显示明显的生长抑制，5一FC的终浓度为1000林mol几，可使75%的细胞生长受抑制。 4.流式细胞仪检测实验组在正常二倍体细胞的Go/Gl峰前出现一个明显的凋亡峰，细胞凋亡率为33.8%，而正常HO一8910细胞经5一FC作用后，其凋亡细胞所占的百分率为2.86%。 5.免疫组化结果显示:正常的HO一8910细胞FaS和Fas一L染色均为阴性;而HO一8910一Fcy::Fur在加入5一FC4天后，Fas和Fas一L染色均为阳性。结论 融合型自杀基因Fcy::Fur联合5一Fc可对卵巢癌细胞株HO一8910产生明显的杀伤作用;凋亡可能参与了杀伤机制;5一FC加入后，发生了FaS和Fas一L表达的上调，提示杀伤机制可能与FaS和Fas一L诱导的凋亡相关。更多还原

【Abstract】 ObjectiveThe purpose is to study anticancer effects of fusion suicide gene Fcy::Fur/5-fluorocytosine therapies to human ovarian cancer cell line HO-8910.Methods1. Two pairs of PCR primers for Fcy: :Fur genes were designed according to the multiclone site of retroviral expression vector pLXSN, LZRSpBMN-Z and the sequence of Fcy::Fur , in which EcoR I and BamH I sites were introduced at the two 5’ ends of the primers respectively. Fcy::Fur genes were amplified from the pORF5-Fcy::Fur using PCR . After DNA sequencing and analysis , Fcy::Fur genes were further inserted into pLXSN and LZRSpBMN-Z respectively with sense open reading frame. The recombinant expression plasmids, which had been confirmed by enzymes digestion, were transfected into packing cells PT67and Pheonix differently. Supernatant of the cloned packing cells were harvested and tested for the presence of the recombinant retrovivus.2. HO-8910 was infected with two kinds of recombinant retrovivus and selected by neomycin(G418) and puromycin separately ,and the HO-8910 cell lines expressing Fcy::Fur stably were then obtained (named HO-8910-Fcy::Fur ). PCR and RT-PCR were taken to demonstrate if Fcy::Fur gene had been intergrated into the chromosome of resistant HO-8910 and expressed correctly .3. After administration of 5-FC, the changes of these cells were observed through inverted and phase-contrast microscopy .The cytotoxicity efficacy was evaluated by MTT methods, then the growth inhibition rate(GIR) was counted. Immunohistochemical assay (SABC method) was used to detect the expression of Fas and FasL, and the apoptosis was analysed by flow cytometry.Results:1. HO-8910 cells were infected with the recombinant retrovirus acquired from the transfected packing cells, then, after selected by antibiotic we obtained HO-8910-Fcy ::Fur . PCR and RT-PCR demonstrated that Fcy::Fur gene had been intergrated correctly into the chromosome of resistant HO-8910 .2. .With the administration of 5-FC, morphologic variation and cells mortality were observed in HO-8910-Fcy::Fur compared with no obvious changes in control by inverted and phase-contrast microscopy.3. The cytotoxicity efficacy appeared remarkablely by MTT methods in HO-8910-Fcy::Fur when the final concentration of 5-FC >10 u mol/L. At 1000 u mol/L, 75 % of HO-8910-Fcy::Fur presented growth inhibition. Whereas the 5-FC had no evident cytotoxicity to normal HO-8910 at the same final concentration of 5-FC .4. A manifest apoptosis peak appeared prior to the normal Go/G] peak in HO-8910-Fcy::Fur .The percentage of apoptosis ofHO-8910-Fcy::Fur was 33.8% after the administration of 5-FC calculated by the flow cytometry, compared with 2.86% of the control.5.The expression of Fas and FasL showed positive in HO-8910-Fcy::Fur after the 96 hours administration of 5-FC compared with negative in normal HO-8910 by immunohistochemical assay.Conclusion:Fusion suicide gene Fcy::Fur combined with 5-fluorocytosine could significantly kill HO-8910, and the growth inhibition rate(GIR) increased remarkablely compared with the control. The percentage of apoptosis calculated by the flow cytometry was much higher than that of the control, so we could infer that the apoptosis might play a role in killing mechanism. Immunohistochemical assay demonstrated the upregulation of Fas and Fas-L after the administration of 5-FC, which suggested that the killing mechanism of Fcy::Fur/5-fluorocytosine therapies mightbe associated with apoptosis induced by Fas and Fas-L.更多还原