辐射诱导重组质粒pegr-p在黑素瘤b细胞中的表达特性与凋亡作用

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1、汕头大学医学院硕士学位论文辐射诱导重组质粒pEgr-P16在黑素瘤B16细胞中的表达特性与凋亡作用2002级硕士研究生：周莉导师：黄天华教授ExpressionPropertiesandApoptosisEffectsofpEgr-P16RecombinantPlasmidsinMelanomaB16InducedbyIonizingIrradiation.Postgraduate:ZhouLiAdvisor:ProfessorHuangTianHua0Cells汕头大学医学院硕士学位论文目录符号说明„„„„„„„„„„

2、„„„„„„„„„„„„„„„„„„„(1)中文摘要„„„„„„„„„„„„„„„„„„„„„„„„„„„„„(2)英文摘要„„„„„„„„„„„„„„„„„„„„„„„„„„„„„(3)前言„„„„„„„„„„„„„„„„„„„„„„„„„„„„„(5)材料和方法„„„„„„„„„„„„„„„„„„„„„„„„„„„(14)结果„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„(23)讨论„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„(29)参考文献„„„„„„„„„„„„„„„„„„„„„„

3、„„„„„„„(33)致谢„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„„(38)1汕头大学医学院硕士学位论文符号说明AmpBSAcDNADEPCDMSOEgr-1FCMODPBSPCRAmpicillinBovineserumalbuminComplementaryDNADiethylpyrocarbonateDimethylsulfoxideEarlygrowthresponsegene-1FlowcytometryOpticdensityPhosphate-bufferedSalinePolymeras

4、echainreaction氨苄青霉素牛血清白蛋白互补DNA聚碳酸二乙酯二甲基亚砜早期生长反应-1基因启动子流式细胞术光密度磷酸盐缓冲液聚合酶链式反应EDTAEthyleneDiammineteTraaceticAcid乙二胺四乙酸rpmRT-PCRTBETrisCDKsrotationperminuteReversetranscriptionTris.ClborateEDTAtris(hydroxymethyl)aminomethancCyclin-dependentkinase每分钟转速反转录PCRTris-硼酸

5、盐缓冲液三羟甲基氨基甲烷细胞周期素依赖性激酶MTTMethlthiazolyltetrazolium噻唑蓝法reductionassayhGygmlminsTEhourgraygrammilliliterminutesecondTris.ClEDTA2小时戈瑞（吸收剂量单位）克毫升分钟秒Tris-EDTA缓冲液汕头大学医学院硕士学位论文辐射诱导重组质粒pEgr-P16在黑素瘤B16细胞中的表达特性与凋亡作用摘要背景和目的：根据电离辐射可诱导早期生长反应因子-1(Egr-1)基因启动子激活、启动下游基因表达的机制和

6、抑癌基因p16抑瘤作用，研究pEgr-p16重组质粒在转染的黑素瘤B16细胞中的辐射诱导表达特性，验证其联合放射治疗体外抑制B16细胞增殖的作用，为临床上对黑色素瘤进行基因-放射联合治疗奠定基础。材料和方法：在已构建了含有放射敏感性启动子的Egr-1和人源性p16基因重组表达质粒pEgr-p16基础上，将脂质体包裹的pEgr-p16重组质粒，转染B16细胞株中；采用定量PCR方法检测不同剂量X射线诱导后p16的表达和同一剂量X射线诱导下p16的表达时程；采用流式细胞术检测细胞凋亡率的变化；用MTT法检测pEgr-p16基因协

7、同放疗治疗后B16细胞的存活情况。结果：研究结果表明pEgr-p16重组质粒转染B16细胞，不同剂量X射线均可诱导p16表达增强，为假照组的3.78~6.67倍(P<0.01)；2GyX射线照射后，p16表达随时间延长而逐渐增强，在照射后72h达到最高值。p16基因联合放射可诱导B16细胞凋亡，凋亡率高于单纯照射组和单纯基因诱导组(P<0.05~0.01)；转染pEgr-p16质粒的细胞经2GyX射线照射，8d后细胞数明显低于同时间其它实验组(P<0.05~0.001)。结论：pEgr-p16基因-放射联合治疗有明显的抑制黑

8、素瘤B16细胞生长的作用，其作用优于单纯给予射线或基因转染。本研究结果为探索新的肿瘤治疗方案，提高肿瘤放射治疗效果具有重要的理论和实际意义。关键词：pEgr-p16基因；Egr-1启动子；基因－放射联合治疗；B16细胞；黑素瘤3汕头大学医学院硕士学位论文ExpressionProperti