生物化学与生物物理进展
生物化學與生物物理進展
생물화학여생물물리진전
PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS
2007年
5期
465-470
,共6页
薛志刚%李剑%尹彪%张雅坤%刘雄昊%潘乾%龙志高%戴和平%夏昆%邬玲仟%梁德生%夏家辉
薛誌剛%李劍%尹彪%張雅坤%劉雄昊%潘乾%龍誌高%戴和平%夏昆%鄔玲仟%樑德生%夏傢輝
설지강%리검%윤표%장아곤%류웅호%반건%룡지고%대화평%하곤%오령천%량덕생%하가휘
人源基因载体%人端粒酶逆转录酶(hTERT)%p53
人源基因載體%人耑粒酶逆轉錄酶(hTERT)%p53
인원기인재체%인단립매역전록매(hTERT)%p53
human-derived vector%human telomeras reverse transcriptase (hTERT)%p53
利用人源基因载体结合人巨细胞病毒(CMV)增强子,人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)启动子及肿瘤抑制基因p53构建肿瘤抑制载体,研究它们在肝癌细胞株Bel-7402中的功能.将CMV增强子(CMVe)与肿瘤特异性启动子hTERTp组合,分别结合GFP及精氨酸型和脯氨酸型肿瘤抑制基因p53,构建GFP表达载体pchEGFP以及p53表达载体pchp53Arg和pchp53Pro;脂质体2000转染肝癌细胞株Bel-7402;24 h后利用荧光显微镜以及流式细胞仪检测GFP的表达;RT-PCR、蛋白质印迹检测p53基因的表达;利用Hoechst 33258、Annexin V/PI染色,检测p53促使肿瘤细胞的促凋亡情况.结果表明:GFP荧光表达载体在Bel-7402细胞中能有效表达;RT-PCR及蛋白质印迹检测到精氨酸型和脯氨酸型p53基因在Bel-7402细胞内表达;Hoechst 33258染色及Annexin V/PI染色检测细胞凋亡实验没有得到明显的细胞凋亡结果.结果表明:实验所构建的肿瘤抑制载体在肝癌细胞Bel-7402能有表达,并能形成外源性p53蛋白,但没有检测到明显的p53促使肝癌细胞调亡的结果.这可能与p53对hTERT的负调控作用有关.
利用人源基因載體結閤人巨細胞病毒(CMV)增彊子,人耑粒酶逆轉錄酶(human telomerase reverse transcriptase,hTERT)啟動子及腫瘤抑製基因p53構建腫瘤抑製載體,研究它們在肝癌細胞株Bel-7402中的功能.將CMV增彊子(CMVe)與腫瘤特異性啟動子hTERTp組閤,分彆結閤GFP及精氨痠型和脯氨痠型腫瘤抑製基因p53,構建GFP錶達載體pchEGFP以及p53錶達載體pchp53Arg和pchp53Pro;脂質體2000轉染肝癌細胞株Bel-7402;24 h後利用熒光顯微鏡以及流式細胞儀檢測GFP的錶達;RT-PCR、蛋白質印跡檢測p53基因的錶達;利用Hoechst 33258、Annexin V/PI染色,檢測p53促使腫瘤細胞的促凋亡情況.結果錶明:GFP熒光錶達載體在Bel-7402細胞中能有效錶達;RT-PCR及蛋白質印跡檢測到精氨痠型和脯氨痠型p53基因在Bel-7402細胞內錶達;Hoechst 33258染色及Annexin V/PI染色檢測細胞凋亡實驗沒有得到明顯的細胞凋亡結果.結果錶明:實驗所構建的腫瘤抑製載體在肝癌細胞Bel-7402能有錶達,併能形成外源性p53蛋白,但沒有檢測到明顯的p53促使肝癌細胞調亡的結果.這可能與p53對hTERT的負調控作用有關.
이용인원기인재체결합인거세포병독(CMV)증강자,인단립매역전록매(human telomerase reverse transcriptase,hTERT)계동자급종류억제기인p53구건종류억제재체,연구타문재간암세포주Bel-7402중적공능.장CMV증강자(CMVe)여종류특이성계동자hTERTp조합,분별결합GFP급정안산형화포안산형종류억제기인p53,구건GFP표체재체pchEGFP이급p53표체재체pchp53Arg화pchp53Pro;지질체2000전염간암세포주Bel-7402;24 h후이용형광현미경이급류식세포의검측GFP적표체;RT-PCR、단백질인적검측p53기인적표체;이용Hoechst 33258、Annexin V/PI염색,검측p53촉사종류세포적촉조망정황.결과표명:GFP형광표체재체재Bel-7402세포중능유효표체;RT-PCR급단백질인적검측도정안산형화포안산형p53기인재Bel-7402세포내표체;Hoechst 33258염색급Annexin V/PI염색검측세포조망실험몰유득도명현적세포조망결과.결과표명:실험소구건적종류억제재체재간암세포Bel-7402능유표체,병능형성외원성p53단백,단몰유검측도명현적p53촉사간암세포조망적결과.저가능여p53대hTERT적부조공작용유관.
In order to study the tumor suppression effect of p53 with CMV enhancer and hTERT promoter mediated by human-derived vector pHrn in liver cancer cell Bel-7402, report plasmid pchEGFP, tumor suppressor plasmids pchp53Arg and pchp53Pro were constructed by inserting expression cassette CMVe+hTERTp+EGFP, CMVe+hTERTp+p53Arg and CMVe+hTERTp+p53Pro into pHrn respectively. 24 h after cell transfection by lipofectamine 2000, GFP expression pattern was analyzed through fluorescence microscope and flow cytometry; RT-PCR and Western blot were taken to study the p53 expression pattern. The cell apoptosis by Hoechst 33258 and Annexin V-FITC/PI staining was also studied. Results show that the expression of GFP and p53 protein in Bel-7402were detected, but apparent cell apoptosis could not be found. The recombinant p53 mediated by human-derived vector could express in Bel-7402, but no significant tumor suppression effect was detected, which might result from the down regulation effect of the wild type p53 on hTERT promoter.
