利用抑制性消减杂交技术筛选太空诱变宫颈癌细胞的差异表达基因-病理学与病理生理学专业论文.docxVIP

硕士学位论文 硕士学位论文 中文摘要 位素标记探针和反向Northern Dot Blot技术可高通量、高灵敏的对 消减文库进行筛选。 3．太空特殊环境可影响肿瘤细胞的细胞骨架、 线粒体、转录调控、核酸代谢等相关基因表达的改变。4．对太空特 殊环境影响肿瘤细胞生物学行为的初步探讨丰富了太空生物学研究 内容，相信随着研究的进一步深入，将为肿瘤发生发展的机制研究提 供一个新的视角，为肿瘤的治疗开辟一个新的道路。 关键词：空间特殊环境，宫颈癌，抑制性消减杂交，差异表达基因 Ⅱ 硕士学位论文 硕士学位论文 英文摘要 ABSlRACT Objecfive：To isolate genes expressed differentially fTom strains subclonal 48A9 Caski cell line by Exposing to the Space Environment and to explain the molecular mechanisms ofthe changes at the gene level． Methods： Super SMART CDNA synthesis and suppression subtractive hybridization(SSH)were performed t0 isolate differentially expressed cDNA fragments from strains subclonal 48A9 cellline．cDNA from the 48A9 cell line were used as‘rester．the other from the normal Caski cell line as“driver．Subtractive products were directly inserted into T／A cloning vectofand then transformed into host bacteria,to set up a subtractive cDNA library of specially or highly expressed genes in f strains subclonal 48A9 cell line．1110∞clones were screened and identified by reverse Northern dot blot technique which the probes were labeled with’2E Positive clones were sequenced and compared with known sequenCOS in the public databases of GenBank／EMBL／DDBJ using NCBI BLAST for homology analysis．11le unknown fragments were then submitted to GenBanL Results：mRNA were directly extracted and purified in good quality． Double strand cDNA were reverse transcripted integmtedly,and then cut by Rsa I into evgn length short segments．Ligation was identified highly effective．After two hybridizations，a subtractive library of differentially expressed genes in strains subclonal 48A9 cell line was constructed successfully by SSH．Nested-PCR Was specificial，which amplified the differentially expressed genes exponentially．