腺病毒介导的mIL-12基因转染H22细胞及对其生长的影响  

Transfection of mIL-12 gene mediated by adenoviral vector to H22 cells and its effect on the growth of H22 cells

作　　者：严林[1] 潘运龙[1] 覃莉[2] 王华曦[1] 

机构地区：[1]暨南大学附属第一医院普外科 [2]暨南大学医学院组织胚胎学教研室

出　　处：《暨南大学学报（自然科学与医学版）》2004年第6期675-680,共6页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：广东省自然科学基金资助项目(010367);广东省卫生厅科研项目(A1999294)

摘　　要：　目的:研究重组小鼠白介素-12腺病毒(AdvmIL-12)和携带增强型荧光蛋白腺病毒(Adv-EGFP)在小鼠肝癌细胞H22中的转染及对其生长的影响。方法:将mIL-12基因p35、p40的cDNA分别插入5型腺病毒的E1和E3区构建成AdvmIL-12,分离纯化后进行鉴定、病毒滴度检测;同法构建含增强型绿色荧光蛋白(EGFP)的重组腺病毒(Adv-EGFP)。在不同的感染复数(MOI)、感染时间(TOI)条件下用Adv-EGFP转染H22细胞,观察Adv-EGFP的转染率。用AdvmIL-12、Adv-EGFP分别转染H22细胞,ELISA法检测上清液中mIL-12的含量。改良MTT法检测AdvmIL-12、Adv-EGFP在体外对H22细胞生长的影响。结果:得到AdvmIL-12和Adv-EGFP,每毫升空斑形成单位(PFU/mL)分别为1×108、1×109。TOI为1、2、4、24h时,转染效率分别为87 67%、96 38%、96 43%和96 32%;MOI为0、50、100、200、500时,转染效率分别为0、89 29%、96 41%、96 72%和98 37%。106个AdvmIL-12/H22细胞上清液中mIL-12(48h)为(89 71±22 05)ng。AdvmIL-12和Adv-EGFP对H22细胞生长无抑制。结论:本实验成功构建了AdvmIL-12和Adv-EGFP,它们在体外能够转染H22细胞,并能表达mIL-12和EGFP,但不能抑制H22细胞的生长。感染复数为100、感染时间为2h是重组腺病毒转染H22细胞的最适条件。Aim: To study the transfection of AdmIL-12 and Ad-EGFP to H22 cells, which effect the growth change on H22 cells. Methods: The cDNA of p35 and p40 of mIL-12 gene were inserted into the region of E1 and E3 of Ad5 respectively. AdmIL-12 was valuated and viral tite was detected after AdmIL-12 was constructed. The recombinant adenovirus vector with enhanced green fluorescence protein gene (Ad-EGFP) was constructed by the same way. Ad-EGFP transfected H22 cells by different multiplicity of infection (MOI) and time of infection (TOI), then the transfection efficiency of Ad-EGFP was observed. H22 cells were transfected by AdmIL-12 and Ad-EGFP respectively, then the content of mIL-12 in the supernatant was detected by ELISA. The effects of AdmIL-12 and Ad-EGFP on H22 cells growth was detected by improved MTT. Results: AdmIL-12 and Ad-EGFP were obtained, and their plaque-forming units were 1×10~8/mL and 1×10~9/mL. The transfection efficiency of Ad-EGFP were 87.67%、96.38%、96.43% and 96.32% respectively when TOI was 1 h, 2 h, 4 h and 24 h. The transfection efficiency of Ad-EGFP were 0、89.29%、96.41%、96.72% and 98.37% respectively when MOI was 0, 50, 100, 200 and 500. The content of mIL-12 in the supernatant of AdmIL-12/H22 was (89.71±22.05) ng·48 h^(-1)·10~6 cells^(-1). AdmIL-12 and Ad-EGFP did not inhibit the growth of H22 cells. Conclusion:AdmIL-12 and Ad-EGFP were constructed successfully, and they can transfect H22 cells and express mIL-12 and EGFP respectively, but they do not inhibit the growth of H22 cells in vitro. That MOI is 100 and TOI is 2 hours, which are the best transfection condition of recombinant adenovirus on H22 cells.

关 键 词：白介素12 腺病毒 转染 H22细胞 

分 类 号：R735.7[医药卫生—肿瘤]