浙江大学学报（医学版）
浙江大學學報（醫學版）
절강대학학보（의학판）
JOURNAL OF ZHEJIANG UNIVERSITY MEDICAL SCIENCES
2014年
2期
193-199
,共7页
杨幼萍%丁燕%王继荣%曾玲晖%林红霞%朱杨丽%吴宏卫%王若燕%张建民%应荣彪
楊幼萍%丁燕%王繼榮%曾玲暉%林紅霞%硃楊麗%吳宏衛%王若燕%張建民%應榮彪
양유평%정연%왕계영%증령휘%림홍하%주양려%오굉위%왕약연%장건민%응영표
整合素类%磷酸转移酶类%前列腺肿瘤%白细胞介素类%基因, 肿瘤抑制%慢病毒属/遗传学%遗传载体%基因表达
整閤素類%燐痠轉移酶類%前列腺腫瘤%白細胞介素類%基因, 腫瘤抑製%慢病毒屬/遺傳學%遺傳載體%基因錶達
정합소류%린산전이매류%전렬선종류%백세포개소류%기인, 종류억제%만병독속/유전학%유전재체%기인표체
Integrins%Phosphotransferases%Prostatic neoplasms%Interleukins%Genes,tumor suppressor%Lentivirus/genetics%Genetic vectors%Gene expression
目的：建立整合素相关激酶（ ILK）基因敲降和黑色素瘤分化相关基因（ mda7）过表达慢病毒包装表达系统。方法：针对人ILK基因序列，设计基因敲降靶点序列（A、B、C），通过限制性内切酶HpaⅠ和XhoⅠ双酶切、T4 DNA连接酶连接，将ILK插入慢病毒载体pSicoR-eGFP，构建siILK-pSicoR-eGFP重组质粒；根据人mda7基因序列，设计引物扩增mda7全长，并插入慢病毒载体pLVX-Puro，构建mda7-pLVX-Puro重组质粒。经双酶切及测序鉴定正确后通过脂质体将慢病毒四质粒系统共转染人胚肾细胞系293 T细胞，进行慢病毒包装并测定病毒滴度、观察感染效率。各组病毒载体转染PC-3细胞后，用定量PCR和蛋白质印迹法检测ILK基因和mda7 mRNA转录水平及蛋白表达水平。通过MTT法和Transwell实验考察ILK和mda7对PC-3细胞增殖和迁移的影响。结果：成功构建ILK基因敲降及mda7过表达的慢病毒载体，四质粒系统共转染293 T细胞后可见大量绿色荧光染色阳性细胞。浓缩病毒后293 T细胞的感染效率在90％以上，并能高效率感染PC-3前列腺癌细胞。 ILK-A-pSicoR-eGFP和ILK-B-pSicoR-eGFP组ILK干扰效果最佳（P＜0．05），mda7的表达水平远高于对照组（P＜0．05），且持续稳定表达至少1个月。 ILK和mda7对PC-3细胞的增殖在96 h内有明显抑制作用，并对其迁移亦有显著抑制（均P＜0．05）。结论：成功构建并鉴定人ILK 基因敲降和mda7过表达慢病毒载体，可为探讨ILK和mda7基因在肿瘤细胞中的生物学功能提供良好工具，也为探索安全、高效的肿瘤治疗途径奠定实验基础。
目的：建立整閤素相關激酶（ ILK）基因敲降和黑色素瘤分化相關基因（ mda7）過錶達慢病毒包裝錶達繫統。方法：針對人ILK基因序列，設計基因敲降靶點序列（A、B、C），通過限製性內切酶HpaⅠ和XhoⅠ雙酶切、T4 DNA連接酶連接，將ILK插入慢病毒載體pSicoR-eGFP，構建siILK-pSicoR-eGFP重組質粒；根據人mda7基因序列，設計引物擴增mda7全長，併插入慢病毒載體pLVX-Puro，構建mda7-pLVX-Puro重組質粒。經雙酶切及測序鑒定正確後通過脂質體將慢病毒四質粒繫統共轉染人胚腎細胞繫293 T細胞，進行慢病毒包裝併測定病毒滴度、觀察感染效率。各組病毒載體轉染PC-3細胞後，用定量PCR和蛋白質印跡法檢測ILK基因和mda7 mRNA轉錄水平及蛋白錶達水平。通過MTT法和Transwell實驗攷察ILK和mda7對PC-3細胞增殖和遷移的影響。結果：成功構建ILK基因敲降及mda7過錶達的慢病毒載體，四質粒繫統共轉染293 T細胞後可見大量綠色熒光染色暘性細胞。濃縮病毒後293 T細胞的感染效率在90％以上，併能高效率感染PC-3前列腺癌細胞。 ILK-A-pSicoR-eGFP和ILK-B-pSicoR-eGFP組ILK榦擾效果最佳（P＜0．05），mda7的錶達水平遠高于對照組（P＜0．05），且持續穩定錶達至少1箇月。 ILK和mda7對PC-3細胞的增殖在96 h內有明顯抑製作用，併對其遷移亦有顯著抑製（均P＜0．05）。結論：成功構建併鑒定人ILK 基因敲降和mda7過錶達慢病毒載體，可為探討ILK和mda7基因在腫瘤細胞中的生物學功能提供良好工具，也為探索安全、高效的腫瘤治療途徑奠定實驗基礎。
목적：건립정합소상관격매（ ILK）기인고강화흑색소류분화상관기인（ mda7）과표체만병독포장표체계통。방법：침대인ILK기인서렬，설계기인고강파점서렬（A、B、C），통과한제성내절매HpaⅠ화XhoⅠ쌍매절、T4 DNA련접매련접，장ILK삽입만병독재체pSicoR-eGFP，구건siILK-pSicoR-eGFP중조질립；근거인mda7기인서렬，설계인물확증mda7전장，병삽입만병독재체pLVX-Puro，구건mda7-pLVX-Puro중조질립。경쌍매절급측서감정정학후통과지질체장만병독사질립계통공전염인배신세포계293 T세포，진행만병독포장병측정병독적도、관찰감염효솔。각조병독재체전염PC-3세포후，용정량PCR화단백질인적법검측ILK기인화mda7 mRNA전록수평급단백표체수평。통과MTT법화Transwell실험고찰ILK화mda7대PC-3세포증식화천이적영향。결과：성공구건ILK기인고강급mda7과표체적만병독재체，사질립계통공전염293 T세포후가견대량록색형광염색양성세포。농축병독후293 T세포적감염효솔재90％이상，병능고효솔감염PC-3전렬선암세포。 ILK-A-pSicoR-eGFP화ILK-B-pSicoR-eGFP조ILK간우효과최가（P＜0．05），mda7적표체수평원고우대조조（P＜0．05），차지속은정표체지소1개월。 ILK화mda7대PC-3세포적증식재96 h내유명현억제작용，병대기천이역유현저억제（균P＜0．05）。결론：성공구건병감정인ILK 기인고강화mda7과표체만병독재체，가위탐토ILK화mda7기인재종류세포중적생물학공능제공량호공구，야위탐색안전、고효적종류치료도경전정실험기출。
Objective: To construct and identify lentiviral vector containing human ILK-shRNA and mda7 gene.Methods:Based on the human ILK gene sequences , RNAi target sequences were designed and cloned into the lentiviral vector pSicoR -eGFP by restriction endonuclease HpaⅠand XhoⅠdouble digestion and T 4 DNA ligase ligation . Based on the human mda7 gene sequences , PCR primers were designed to clone the full-length mda7, and were cloned into the lentiviral vector pLVX-Puro.After the candidate clones were identified by DNA sequencing , the recombinant plasmid and the three packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamine 2000 to produce the lentiviral particles .Human prostate cancer PC-3 cells were infected with the constructed lentiviral vector .The ILK and mda7 expression levels in PC-3 cells were quantified by qPCR and Western blot , respectively .The effect of ILK and mda7 on proliferation and migration of PC-3 cells were assessed by MTT method and Transwell assay, respectively.Results: ILK-pSicoR-eGFP and mda7-pLVX-Puro lentiviral vectors were successfully constructed .Strong green fluorescence was observed in the 293T cells under the fluorescent microscope after co-transfection of 293T cells with 4 plasmids of lentiviral vector .The transfection efficiency of the collected virus exceeded 90%in the 293T cells and the PC-3 cells were infected with the lentiviral particles with high efficiency .The A and B lentiviral vector inhibited the expression of ILK at both the mRNA and protein levels in PC-3 cells significantly .The mda7-pLVX-Puro lentiviral vector increased the expression of mda 7 in PC-3 cells, and the ability was maintained for one month.Within 96 h, ILK and mad7 significantly inhibited the proliferation and migration of PC-3 cells ( Ps <0.05 ) . Conclusion: The lentiviral vectors of ILK knockdown and mda 7 over-expression have been successfully constructed and identified . The recombinant lentivirus can efficiently infect human prostate cancer PC -3 cells, in which ILK expression is inhibited and mda 7 is over-expressed .
