【作者】 周忆新；

【导师】 杨吉成；

【作者基本信息】 苏州大学 ， 免疫学， 2007， 硕士

【摘要】 目的:构建裂解型腺病毒载体,研究其体外对黑色素瘤细胞及小鼠腹水型肝癌的抑制作用。方法:利用本室已构建的pUCm-T-E1A质粒为模板进行PCR,将目的片段用HindⅢ、EcoRV双酶切后连接到带有GFP标记的pAdTrack-CMV质粒上,PmeI线性化重组质粒pAdTrack-CMV-E1A与腺病毒质粒pAdeasy-1共转化BJ5183细菌,获得重组腺病毒表达载体pAdeasy-1-pAdTradck-CMV-E1A经pacI线性化后转染QBI-293A细胞;收获病毒重组子。将获得的重组病毒子感染A375黑色素瘤细胞。然后分别用RT-PCR、流式细胞仪及MTT法等方法鉴定E1A基因的表达并且研究其对小白鼠腹水型肝癌的抑制及其免疫功能的影响。结果:成功构建了裂解型腺病毒Ad-E1A,可显著裂解A375黑色素瘤细胞,抑制细胞生长。Ad-E1A可体内抑制小鼠腹水型肝癌的生长,且对小鼠的免疫器官没有抑制,同时可提高中性粒细胞和单核细胞的数目。结论:裂解型腺病毒Ad-E1A可在体外裂解黑色素瘤细胞,抑制人黑色素瘤细胞生长。体内抑制小鼠腹水型肝癌H22的生长,并可以提高小白鼠免疫功能。更多还原

【Abstract】 Objective:To construct The oncolytic adenovirus Ad-E1A and explore the effect of E1A on A375 cells.It inhibit H22 tumor growth in vivo and enhance the immune function of BALB/c mice.Methods:The pAdTrack-CMV-E1A was constructed with pUCm-T-E1A recombined plasmid by PCR as template,enzyme digestion and ligation.The pAdTrack-CMV-E1A lineared by PmeI was co-transducted into BJ5183 with pAdeasy-I.Analysis of PCR and DNA sequencing was carried out to demonstrate the sequence of the piasmid.The pAdeasy-1-pAdTrack-CMV-E1A recombined adenovirus vector was leared with PacI and then transfected into QBI-293A cells.The Ad-E1A recombined adenovirus was obtained and was used to infect A375 cells.Identification of Ad-E1A mRNA by RT-PCR was carried out to demonstrate the expression of Ad-E1A in A375 cells.The effect of Ad-E1A on A375 was studied by MTT,flow cytometry.And study the effect of Ad-E1A on H22 tumor growth in vivo and the immune function of BALB/c mice.Results:The recombined oncolytic adenovirus Ad-E1A was obtained. The oncolytic adenovirus inhibit H22 ascitic hepatoma obviously,have no side effect on immune organ and enhance the numbers of monocyte and neutrophil. Conclusion:The oncolytic adenovirus Ad-E1A is constructed succefully.The oncolytic adenovirus Ad-E1A inhibites A375 obviously.It inhibit H22 tumor growth in vivo and enhance the immune function of BALB/c mice.更多还原