【作者】 李玉梅；

【导师】 林秋华；

【作者基本信息】 中南大学 ， 临床妇产科学， 2006， 博士

【摘要】 目的 构建可被放射线激活而转录的早期生长反应因子-1（Egr-1）启动子驱动的双自杀基因CDTK重组真核表达载体（pcDNA3.1-CMV-Egr-1-CDTK）；将其转染人宫颈癌细胞株Hela细胞，在⁶⁰C_O-γ射线照射后，研究其在宫颈癌细胞中的表达及在前体药物5-FU和GCV作用下体外对宫颈癌细胞的杀伤作用及旁观者效用；同时将转染了重组真核表达载体（pcDNA3.1-CMV-Egr-1-CDTK）的人宫颈癌细胞种植于Balb／c裸鼠皮下，建立裸鼠宫颈癌种植瘤模型，研究放射线诱导性自杀基因在射线作用下对体内宫颈癌的治疗作用。 方法 利用PCR扩增CD，TK基因，并将其构建成融合基因，连至pc DNA3.1质粒载体上，从家鼠基因组中扩增Egr-1启动子序列，并扩增CMV增强子插入Egr-1启动子构建成真核表达载体pcDNA3.1-CMV-Egr-1-CDTK。体外培养人宫颈癌细胞株Hela细胞，将真核表达载体pcDNA3.1-CMV-Egr-1-CDTK通过电转染方法转染人宫颈癌细胞hela细胞株，经放射线⁶⁰CO-γ照射并给予前体药物，通过RT-PCR，Western等方法检测转染细胞中融合基因CDTK的表达情况，MTT法检测细胞存活比率来评价我们构建的肿瘤自杀基因载体对Hela细胞的杀伤效应和旁观者效应以及对放射治疗的增敏性。将转染肿瘤自杀基因载体阳性的Hela细胞进行裸鼠体内致瘤，了解致瘤特性，在前体药物和放射线⁶⁰C_O-γ照射的不同组合治疗条件下，测量肿瘤大小，进行肿瘤组织病理切片检查，同时RT-PCR检测肿瘤组织内CDTK扩增片段。 结果 真核表达载体质粒经酶切和测序鉴定证明其方向正确。真核表达载体质粒转染Hela细胞，筛选后得到表达CDTK基因的Hela／CDTK细胞，RT-PCR显示长度为400bp的阳性条带，Western杂交技术检测结果可见一59KD大小的蛋白质，与CDTK基因序列分析的预期大小一致，证明该载体在Hela细胞能被转录和翻译成CDTK融合蛋白。MTT检测发现，转染pcDNA3.1-CMV-Egr-1-CDTK的细胞对放射线⁶⁰C_O-γ照射的敏感性明显增强。分组动物试验时，治疗前五组动物瘤体大小无差异，治疗的最后一天，阳性细胞瘤+前体药物+放射组肿瘤体积最小，与阳性细胞瘤+放射组肿瘤有显著性差异（P＜0.05），与阳性细胞瘤+PBS组肿瘤有极显著性差异（P＜0.01）。转染阳性细胞组病理检查细胞有大量坏死和溶解，对照组则可见细胞排列紧密未更多还原

【Abstract】 Objective Recombinant plasmid vector pcDNA3.1-CMV-Egr-1-CDTK was constructed by fusing of early growth respons-1（Egr-1） promoter to the double suicide gene CDTK gene; To transfer human cervical cancer cell lines （Hela） with recombinant plasmid vector pcDNA3.1-CMV-Egr-1-CDTK,after irradiated by ⁶⁰Co-γ rays ,the CDTK mRNA expression in the transfected cell line and the killing effects and the by-stander killing effects in vitro of double suicide genes on the cell lines were observed; The animal model of Balb/c nude mice was established by injected subcutaneously with exponentially growing transfected cell lines for further research of the killing effect on cervical cancer in vivo by radio-induced suicide gene after exposure to ⁶⁰Co-γ rays.Methods The CDTk gene were amplified with polymerase chain reaction （PCR） technique and were fused into CDTK gene, linked with plasmid vector pcDNA3.1, Egr-1 promotor and CMV enhancer were amplified and then inserted CMV enhance infront of Egr-1, aquired plasmid vector pcDNA3.1-CMV-Egr-1-CDTK. Which were transfected into cervical cancer cell lines with electroblot as a delivery system. After accepted with ⁶⁰Co-r rays irradiation, prodrugs（5-FC,GCV） was used in the cells ,RT-PCR, Western blot analysis were used to determine the CDTK mRNA expression in Hela cells which had been transfected by pcDNA3.1-CMV-Egr-1-CDTK plasmid vector. The viabilitys of cells were determined by the method of MTT .For the purpose ,we can research the killing effect and the by-stander killing effect of radio-induced double suicide gene on the cervical cancer cell lines in vitro and the sensitization to irradiation. The Balb/c nude mice were injected subcutaneously with pcDNA3.1-CMV-Egr-1-CDTK. The tumorigenesis characteristic were obtained. The animals were devided into five groups and after the therapy of irradiation and prodrugs respectively, the volume of tumor were observed. Furthermore, the pathological results of tumor was observed. At the same time, RT-PCR was used to detect the tumors CDTK amplification fragements.Results The successful construction of recombinant plasmid vector was certificated through enzyme cutting and sequencing. The length of Hela/CDTK gene was certificated by RT-PCR and Western blot, a 59KD protein was obtaind which was equal to the expection of CDTK gene sequencing. So proved the recombinant plasmid更多还原