南京师大学报（自然科学版）
南京師大學報（自然科學版）
남경사대학보（자연과학판）
Journal of Nanjing Normal University (Natural Science Edition)
2015年
3期
64-70,79
,共8页
罗丽芸%白敏%张剑%曹令森%曹祥荣
囉麗蕓%白敏%張劍%曹令森%曹祥榮
라려예%백민%장검%조령삼%조상영
hAIF-1%腺病毒%增殖%迁移
hAIF-1%腺病毒%增殖%遷移
hAIF-1%선병독%증식%천이
hAIF-1%adenovirus%proliferation%migration
hAIF-1是一种炎症因子，本研究通过构建hAIF-1基因的重组腺病毒，探讨hAIF-1对A549细胞增殖和迁移能力的影响.用PCR的方法克隆hAIF-1基因编码序列，并亚克隆到腺病毒穿梭质粒载体pDC315-EGFP上，构建穿梭质粒pDC315-EGFP-hAIF-1，将穿梭质粒与腺病毒骨架质粒pBHGloxdeltaE13Cre共转染293细胞，利用AdMax腺病毒载体包装系统包装腺病毒，TCID50法测定病毒滴度，利用RT-PCR和Western Blot分析检测目的基因表达，用MTT法检测A549细胞的增殖，细胞划痕和Transwell实验检测A549细胞的迁移.结果表明，成功构建了表达hAIF-1基因的重组腺病毒，所得病毒滴度为2.5×108 pfu/mL；用hAIF-1重组腺病毒感染人A549细胞， RT-PCR和Western Blot鉴定显示细胞可过表达hAIF-1；MTT显示过表达hAIF-1可显著增强A549细胞增殖能力；细胞划痕和Tranwell实验证明hAIF-1能够促进A549细胞迁移.
hAIF-1是一種炎癥因子，本研究通過構建hAIF-1基因的重組腺病毒，探討hAIF-1對A549細胞增殖和遷移能力的影響.用PCR的方法剋隆hAIF-1基因編碼序列，併亞剋隆到腺病毒穿梭質粒載體pDC315-EGFP上，構建穿梭質粒pDC315-EGFP-hAIF-1，將穿梭質粒與腺病毒骨架質粒pBHGloxdeltaE13Cre共轉染293細胞，利用AdMax腺病毒載體包裝繫統包裝腺病毒，TCID50法測定病毒滴度，利用RT-PCR和Western Blot分析檢測目的基因錶達，用MTT法檢測A549細胞的增殖，細胞劃痕和Transwell實驗檢測A549細胞的遷移.結果錶明，成功構建瞭錶達hAIF-1基因的重組腺病毒，所得病毒滴度為2.5×108 pfu/mL；用hAIF-1重組腺病毒感染人A549細胞， RT-PCR和Western Blot鑒定顯示細胞可過錶達hAIF-1；MTT顯示過錶達hAIF-1可顯著增彊A549細胞增殖能力；細胞劃痕和Tranwell實驗證明hAIF-1能夠促進A549細胞遷移.
hAIF-1시일충염증인자，본연구통과구건hAIF-1기인적중조선병독，탐토hAIF-1대A549세포증식화천이능력적영향.용PCR적방법극륭hAIF-1기인편마서렬，병아극륭도선병독천사질립재체pDC315-EGFP상，구건천사질립pDC315-EGFP-hAIF-1，장천사질립여선병독골가질립pBHGloxdeltaE13Cre공전염293세포，이용AdMax선병독재체포장계통포장선병독，TCID50법측정병독적도，이용RT-PCR화Western Blot분석검측목적기인표체，용MTT법검측A549세포적증식，세포화흔화Transwell실험검측A549세포적천이.결과표명，성공구건료표체hAIF-1기인적중조선병독，소득병독적도위2.5×108 pfu/mL；용hAIF-1중조선병독감염인A549세포， RT-PCR화Western Blot감정현시세포가과표체hAIF-1；MTT현시과표체hAIF-1가현저증강A549세포증식능력；세포화흔화Tranwell실험증명hAIF-1능구촉진A549세포천이.
hAIF-1 is an inflammatory factor. The study was supposed to explore the effect of hAIF-1 on the proliferation and migration of A549 cells by the preparation of the recombinant adenovirus with hAIF-1 gene. The hAIF-1 gene was cloned by PCR and subcloned into the shuttle vector pDC315-EGFP. The shuttle plasmid and adenovirus genomic plas?mid pBHGloxdeltaE13Cre were co-transfected into 293 cells for packaging recombinant adenovirus with the help of the adenovirus packaging system AdMax. The titer of the virus was measured by TCID50. The expression of the target gene was identified by RT-PCR and Western Blot,and the effect of the target gene on the proliferation and migration of A549 cells was examined by MTT,wound healing and Transwell assay. The results showed that the AdDC315-EGFP-hAIF-1 was successfully constructed and the titer of the virus was 2.5×108 pfu/mL;Analysis of RT-PCR and Western Blot indi?cated that A549 cells could overexpress hAIF-1 after infected with the recombinant adenovirus;The MTT assay showed that hAIF-1 could increase the proliferative rate of A549 cells;The wound healing and Transwell assay proved that hAIF-1 could promote the migration of A549 cells.
