国际医药卫生导报
國際醫藥衛生導報
국제의약위생도보
INTERNATIONAL MEDICINE & HEALTH GUIDANCE NEWS
2013年
16期
2474-2478
,共5页
黄柏强%李二毛%孔冠群%黄志平
黃柏彊%李二毛%孔冠群%黃誌平
황백강%리이모%공관군%황지평
腺病毒%NK4%PUMA%胰腺癌
腺病毒%NK4%PUMA%胰腺癌
선병독%NK4%PUMA%이선암
Adenovirus%PUMA gene%NK4 gene%Pancreatic cancer
目的 制备同步表达HGF/NK4基因和PUMA基因的重组腺病毒,感染胰腺癌细胞后检测基因的有效表达.方法 将PUMA基因和HGF/NK4基因以串联方式依次克隆至腺病毒穿梭载体,构建重组穿梭载体pAdTrack-PUMA-NK4,转化大肠杆菌BJ5183并在细菌内与腺病毒载体同源重组制备重组腺病毒载体pAd-PUMA-NK4.PacⅠ酶切后转染至HEK293细胞包装得到重组腺病毒.一定滴度的腺病毒感染人胰腺癌细胞株,荧光定量RT-PCR检测细胞中NK4和PUMA基因mRNA表达水平.结果 双酶切重组pAd-PUMA-NK4穿梭载体鉴定证实目的基因PUMA和HGF/NK4正确克隆；重组腺病毒载体经PacⅠ酶切得到特异的酶切产物转染HEK293细胞后成功包装出重组腺病毒；病毒感染胰腺癌细胞后3～9d内PUMA和NK4基因的表达水平较高,整体持续表达可维持2w.结论 成功构建了同时表达NK4和PUMA基因的重组腺病毒载体并包装出相应的重组腺病毒颗粒,该腺病毒能有效感染胰腺癌细胞并高表达NK4和PUMA基因,这为进一步确定NK4和PUMA基因的功能及指导双基因联合治疗胰腺癌提供了理论依据和必要的材料.
目的 製備同步錶達HGF/NK4基因和PUMA基因的重組腺病毒,感染胰腺癌細胞後檢測基因的有效錶達.方法 將PUMA基因和HGF/NK4基因以串聯方式依次剋隆至腺病毒穿梭載體,構建重組穿梭載體pAdTrack-PUMA-NK4,轉化大腸桿菌BJ5183併在細菌內與腺病毒載體同源重組製備重組腺病毒載體pAd-PUMA-NK4.PacⅠ酶切後轉染至HEK293細胞包裝得到重組腺病毒.一定滴度的腺病毒感染人胰腺癌細胞株,熒光定量RT-PCR檢測細胞中NK4和PUMA基因mRNA錶達水平.結果 雙酶切重組pAd-PUMA-NK4穿梭載體鑒定證實目的基因PUMA和HGF/NK4正確剋隆；重組腺病毒載體經PacⅠ酶切得到特異的酶切產物轉染HEK293細胞後成功包裝齣重組腺病毒；病毒感染胰腺癌細胞後3～9d內PUMA和NK4基因的錶達水平較高,整體持續錶達可維持2w.結論 成功構建瞭同時錶達NK4和PUMA基因的重組腺病毒載體併包裝齣相應的重組腺病毒顆粒,該腺病毒能有效感染胰腺癌細胞併高錶達NK4和PUMA基因,這為進一步確定NK4和PUMA基因的功能及指導雙基因聯閤治療胰腺癌提供瞭理論依據和必要的材料.
목적 제비동보표체HGF/NK4기인화PUMA기인적중조선병독,감염이선암세포후검측기인적유효표체.방법 장PUMA기인화HGF/NK4기인이천련방식의차극륭지선병독천사재체,구건중조천사재체pAdTrack-PUMA-NK4,전화대장간균BJ5183병재세균내여선병독재체동원중조제비중조선병독재체pAd-PUMA-NK4.PacⅠ매절후전염지HEK293세포포장득도중조선병독.일정적도적선병독감염인이선암세포주,형광정량RT-PCR검측세포중NK4화PUMA기인mRNA표체수평.결과 쌍매절중조pAd-PUMA-NK4천사재체감정증실목적기인PUMA화HGF/NK4정학극륭；중조선병독재체경PacⅠ매절득도특이적매절산물전염HEK293세포후성공포장출중조선병독；병독감염이선암세포후3～9d내PUMA화NK4기인적표체수평교고,정체지속표체가유지2w.결론 성공구건료동시표체NK4화PUMA기인적중조선병독재체병포장출상응적중조선병독과립,해선병독능유효감염이선암세포병고표체NK4화PUMA기인,저위진일보학정NK4화PUMA기인적공능급지도쌍기인연합치료이선암제공료이론의거화필요적재료.
Objective To construct replication deficient recombinant adenovirus co-expressing PUMA and NK4 genes using AdEasy-1 system,observe the expression of PUMA and NK4 genes in pancreatic cancer cell after andenovirus infection.Methods PUMA and HGF/NK4 gene were cloned to adenovirus shutter plasmid for the construction of recombinant plasmid pAdTrack-PUMA/NK4.The recombinant plasmid digested by PmeI was transformed into bacteria B J5183 containing backbone vector pAdEasy-1 for homologous recombination to obtain the recombinant adenoviral vector pAd-PUMA-NK4.The recombinant adenoviral vector was then transfected into HEK293 package cells to produce virus particles.The expression of NK4 and puma gene in cells were detected in human pancreatic cancer cells by qRT-PCR after adenovirus infection.Results The recombinant adenoviral vector was identified by PCR and restriction endonuclease digestion.High level expression of NK4 and PUMA mRNA was maintained for 6 days in SW1990 cells after adenovius infection.Conclusion The infection of Recombinant adenovirus Ad-PUMA-NK4 can mediate high level expression of NK4 and PUMA,it provides an important tool for further gene therapy of pancreatic cancer with them.
