肿瘤细胞外泌体中融合基因的检测

摘要

Objective:To explore whether exosomes originated from tumor cells included parent cells-derived fusion gene mRNA.Methods:Firstly,exosomes were extracted from the supematant ofH3122 cells by the ExoQuick kit and western blot analysis was adopted to verify that exosomes were successfully extracted.Secondly,Total RNA was extracted from both the cell lysate and exosomes of H3122 cells and reverse transcription was performed.Subsequently,qualitative PCR of cDNA was performed with the primer used to amplify EML4-ALK variant 1.Finally,the PCR products were sent to be sequenced and we blasted and analyzed the sequencing results.Results:We successfully exacted exosomes produced from the H3122 cells,and detected EML4-ALK mRNA in the exosomes from the H3122 cells.The sequencing results showed that both the H3122 cells and the exosomes derived from the H3122 cells harbored EML4-ALK fusion variant 1,which also demonstrated that we could detect the mRNA or gene fusion of the tumor cells through the exosomes from the tumor cells.Conclusion:Exosomes secreted by tumor cells not only contain parent cells-derived mRNA but also reflect some biological characteristics including gene fusion of the parent cells.%目的:探究肿瘤细胞分泌的外泌体中是否可以检测到来源于源细胞的融合基因mRNA.方法:培养人非小细胞肺癌NCI-H3122细胞,采用外泌体试剂盒提取细胞上清中的外泌体,并用Western Blot实验验证外泌体是否提取成功.分别提取外泌体以及H3122细胞中的总RNA,将RNA反转录为cDNA,通过PCR反应扩增v1型EML4-ALK融合基因片段,经琼脂糖凝胶电泳确定目的条带后,将两种PCR产物送公司进行基因测序,最后对测序结果进行比对分析.结果:从H3122细胞上清中成功提取了外泌体,并且在外泌体中检测到来源于H3122细胞的mRNA;从H3122细胞外泌体中检测到EML4-ALK融合基因,并发现外泌体中的EML4-ALK融合基因的融合形式为V1,与在H3122细胞中检测到的EML4-ALK融合基因的融合形式完全相同.结论:肿瘤细胞分泌的外泌体中可以检测到肿瘤细胞来源的mRNA,并且外泌体中的mRNA可以反映肿瘤细胞mRNA的基因融合情况等生物学特性.