国际生物医学工程杂志
國際生物醫學工程雜誌
국제생물의학공정잡지
INTERNATIONAL JOURNAL OF BIOMEDICAL ENGINEERING
2014年
4期
210-213,232,后插4
,共6页
陈青青%杨冰%赵秀娟%马芹%刘超%梁绍平%闵琦%吴少华%孟歆怿
陳青青%楊冰%趙秀娟%馬芹%劉超%樑紹平%閔琦%吳少華%孟歆懌
진청청%양빙%조수연%마근%류초%량소평%민기%오소화%맹흠역
外周T细胞淋巴瘤%zeste基因增强子同源物2%慢病毒载体%RNA干扰
外週T細胞淋巴瘤%zeste基因增彊子同源物2%慢病毒載體%RNA榦擾
외주T세포림파류%zeste기인증강자동원물2%만병독재체%RNA간우
Peripheral T-cell lymphomas%Enhancer of zeste homolog 2%Lentiviral vector%RNA interference
目的 外周T细胞淋巴瘤(PTCL)源于成熟的胸腺后淋巴细胞,是一类少见的生物学行为及临床表现均高度异质性的疾病,且预后差.包装zeste基因增强子同源物2(EZH2)RNA干扰(RNAi)慢病毒载体,可为今后相关实验研究奠定基础.方法 靶向EZH2基因的短发夹RNA(EZH2-shRNA)质粒转化感受态细菌,筛选阳性克隆,经测序鉴定正确后通过脂质体将慢病毒三质粒系统共转染293T细胞,进行慢病毒包装.病毒载体感染Hut78细胞后,观察感染效率,并用反转录定量PCR(RT-qPCR)和Western Blot检测EZH2mRNA和蛋白表达水平.结果 成功包装了慢病毒表达载体,对Hut78细胞的感染效率在95％以上.RT-qPCR和Western Blot检测结果显示,EZH2基因在靶细胞中的表达水平降低.结论 成功包装并鉴定了EZH2基因RNAi慢病毒表达载体.
目的 外週T細胞淋巴瘤(PTCL)源于成熟的胸腺後淋巴細胞,是一類少見的生物學行為及臨床錶現均高度異質性的疾病,且預後差.包裝zeste基因增彊子同源物2(EZH2)RNA榦擾(RNAi)慢病毒載體,可為今後相關實驗研究奠定基礎.方法 靶嚮EZH2基因的短髮夾RNA(EZH2-shRNA)質粒轉化感受態細菌,篩選暘性剋隆,經測序鑒定正確後通過脂質體將慢病毒三質粒繫統共轉染293T細胞,進行慢病毒包裝.病毒載體感染Hut78細胞後,觀察感染效率,併用反轉錄定量PCR(RT-qPCR)和Western Blot檢測EZH2mRNA和蛋白錶達水平.結果 成功包裝瞭慢病毒錶達載體,對Hut78細胞的感染效率在95％以上.RT-qPCR和Western Blot檢測結果顯示,EZH2基因在靶細胞中的錶達水平降低.結論 成功包裝併鑒定瞭EZH2基因RNAi慢病毒錶達載體.
목적 외주T세포림파류(PTCL)원우성숙적흉선후림파세포,시일류소견적생물학행위급림상표현균고도이질성적질병,차예후차.포장zeste기인증강자동원물2(EZH2)RNA간우(RNAi)만병독재체,가위금후상관실험연구전정기출.방법 파향EZH2기인적단발협RNA(EZH2-shRNA)질립전화감수태세균,사선양성극륭,경측서감정정학후통과지질체장만병독삼질립계통공전염293T세포,진행만병독포장.병독재체감염Hut78세포후,관찰감염효솔,병용반전록정량PCR(RT-qPCR)화Western Blot검측EZH2mRNA화단백표체수평.결과 성공포장료만병독표체재체,대Hut78세포적감염효솔재95％이상.RT-qPCR화Western Blot검측결과현시,EZH2기인재파세포중적표체수평강저.결론 성공포장병감정료EZH2기인RNAi만병독표체재체.
Objective Peripheral T-cell lymphomas (PTCLs) originated from mature,post thymic T cells are a rare heterogeneous group of clinically aggressive non-Hodgkin lymphoma (NHL) with a dismal prognosis.This study was aimed to package and identify a lentiviral vector of RNA interference (RNAi) targeting enhancer of zeste homolog 2 (EZH2),would lay the foundation for the future study.Methods After transformation into competent E.coli bacteria,the candidate clones were identified by DNA sequencing.The EZH2-short hairpin RNA (shRNA) plasmid and the two packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamine 2000 to produce the lentiviral vector.The Hut78 cells were infected with the lentiviral vector obtained and the transfection efficiency was assessed under the fluorescent microscope.Then the expression of EZH2 in the transfected cells was determined by RT-qPCR and Western Blot.Results The lentiviral RNAi vector for the EZH2 gene was packaged successfully.Strong red fluorescence was observed in the Hut78 cells under the fluorescent microscope after co-transfection of the cells with the 3 plasmids of the lentiviral vector.The transfection efficiency of the collected virus exceeded 95％ in the Hut78 cells.The lentiviral vector significantly inhibited the expression of EZH2 at both the mRNA and protein levels.Conclusions The lentiviral RNAi vector of EZH2 has been successfully packaged and identified.
