【作者】 许子志；

【导师】 王川；

【作者基本信息】 福建医科大学 ， 肿瘤学， 2009， 硕士

【摘要】 [目的]:构建Id1 miRNA真核表达载体,并成功转染乳腺癌MDA-MB-231细胞株,筛选稳定表达的乳腺癌细胞株,观察对乳腺癌细胞的对Id 1、MMP 9表达的影响。[方法]:利用Invitrogen公司网上BLOCK-iT miR RNAi设计工具,设计针对Id 1基因（ID:3397）的两个转录版本mRNA（NM₀02165.2和NM₁81353.1）都有效的干扰序列。将干扰序列插入pcDNA6.2-GW/EmGFP-miR表达载体,构建Id1miRNA真核表达载体,通过脂质体转染法转染到乳腺癌MDA-MB-231细胞株,以杀稻瘟霉素筛选稳定表达的细胞株。经RT-PCR方法鉴定其对Id1、MMP9表达的影响。[结果]:成功构建Id1 miRNA真核表达载体,经电泳及测序鉴定正确,转染乳腺癌MDA-MB-231细胞系。经过2W杀稻瘟霉素筛选,获得稳定表达的细胞系。经RT-PCR方法鉴定,结果表明转染Id1 miRNA真核表达载体的乳腺癌MDA-MB-231细胞株其Id 1、MMP 9表达水平低于对照组,且Id 1、MMP 9表达水平正相关。未转染组与阴性对照转染组无明显差异。[结论]:本实验成功构建Id1 miRNA真核表达载体,并成功转染乳腺癌MDA-MB231细胞株,筛选出稳定表达Id1 miRNA的乳腺癌细胞株,证实干扰有效,并且Id1与mmp9表达相关,为将来进一步研究他们之间的生物学行为差异,或详细了解其分子作用机制奠定了基础。更多还原

【Abstract】 [Purpose]: To construct the miRNA eukaryotic expression vector of Id1 and transfect the MDA-MB-231 breast cancer cell line,and finally select the stabilized expressed cells lines. To explore the relationship between Id 1 and MMP 9 expression in breast cancer line MDA-MB-231 .[Method]: The miRNA array of Id1 gene was designed and insert into pcDNA6.2-GW/EmGFP-miR expression vector to construct the Id1 miRNA eukaryotic expression vector. The liposome infection protocol was used to transfect the MDA-MB-231 cell lines. Use Blasticidin to select stabilized expressed cell lines.the level of Id1 and MMP 9 was detected using RT-PCR methods.[Result]: The eukaryotic expression vector of Id1 miRNA was successfully constructed After Identifying by electrophoresis and measuring array, the MDA-MB-231 breast cancer cell line was tranfected. We acquire the stabilized expressed cell lines through 2W Blasticidin selection. As a result of RT-PCR Identification, the level of Id1 and MMP 9 in Id1 miRNA transfeced cell line is lower than that in the control groups. While there is no difference between the not transfected and negative control transfected cell line.[Conclusion]:The experiment successfully construct the eukaryotic expression vector of Id1 miRNA and successfully transfect the MDA-MB-231 breast cancer cell line and finally select the stabilized expressed cells lines.It prove that Id1and MMP9 are closely related.更多还原