CAJ | 학술논문

目的构建氡染毒小鼠肺及支气管组织差异表达基因的cDNA文库,并对其进行初步鉴定和同源性分析.方法采用SR-NIM02型氡室对小鼠进行吸入染毒后,常规饲养3个月,分别提取和纯化染毒组(30工作水平月,WLM)与对照组(0.02 WLM)小鼠的肺及支气管组织的总RNA,采用Super SMART技术和抑制性差减杂交技术(SSH),构建氡染毒后肺及支气管组织的正、反向差减杂交的cDNA文库;常规连接pGEM-T-easy载体,转化DH5α感受态细胞,巢式PCR鉴定;对阳性克隆测序,并与GeneBank数据库进行BLAST同源性比对,进行初步功能分类.结果共获得克隆460个,其中含有插入片段的克隆146个,经BLAST同源性比对后有48个正向差减cDNA片段与61个反向差减cDNA片段与GeneBank中的序列有不同程度的同源性.结论成功构建了氡染毒小鼠肺支气管差异表达cDNA文库,染毒后小鼠肺及支气管组织中某些基因会发生差异表达,其中部分基因可能参与细胞凋亡和周期调控、机体免疫调节及细胞间信号传导等过程.
목적 구건동염독소서폐급지기관조직차이표체기인적cDNA문고,병대기진행초보감정화동원성분석.방법 채용SR-NIM02형동실대소서진행흡입염독후,상규사양3개월,분별제취화순화염독조(30공작수평월,WLM)여대조조(0.02 WLM)소서적폐급지기관조직적총RNA,채용Super SMART기술화억제성차감잡교기술(SSH),구건동염독후폐급지기관조직적정、반향차감잡교적cDNA문고;상규련접pGEM-T-easy재체,전화DH5α감수태세포,소식PCR감정;대양성극륭측서,병여GeneBank수거고진행BLAST동원성비대,진행초보공능분류.결과 공획득극륭460개,기중함유삽입편단적극륭146개,경BLAST동원성비대후유48개정향차감cDNA편단여61개반향차감cDNA편단여GeneBank중적서렬유불동정도적동원성.결론 성공구건료동염독소서폐지기관차이표체cDNA문고,염독후소서폐급지기관조직중모사기인회발생차이표체,기중부분기인가능삼여세포조망화주기조공、궤체면역조절급세포간신호전도등과정.
Objective To construct and identify differentially expressed cDNA library in lung and bronchus of mice exposed to radon.Methods 2 week old,weishing(18-22)g,male BALB/c mice were placed in a SR-NIM02 radon chamber.One group of mice was exposed to radon,which was equivalent to the accumulative dose of 30 WLM.The control group was about 0.02 WLM.To construct a subtracted cDNA library enriched with differentially expressed genes,the Super SMART technique and the suppression subtractive hybridization(SSH)were performed.The obtained forward and revere cDNA fragments were directly inserted into pGEM-T-easy vector and transformed into E.coli DH5a.The inserts in plasmid were amplified by nested polymerasc chain reaction(PCR).and some of which were sequenced.In the end these sequences were BLASTed with GeneBank.Results 146 of 460 clones obtained randomly were positive clones contained(1000-1500)bp inserted cDNA fragments.The forward and reverse subtracted cDNA library in lung and bronchus of mice exposed to radon was constructed,and 48 up-regulation and 61 down-regulation eDNA sequences selected were homologous with GeneBank in different extent.Conclusions The subtracted cDNA library in lung and bronchus of mice exposed to radon is successfully constructed,and genes that differentially expressed are identified.some genes might have rclation with the immunity.cell cycle and apoptosis.