摘要
目的 构建重组vp3基因腺病毒pAD-vp3,观察其体内外对人乳腺癌细胞MCF-7的诱导凋亡作用.方法 克隆vp3基因,loxP法同源重组构建重组腺病毒载体pLP-AD-vp3（pAD-vp3）,转染293A细胞进行病毒包装,然后NIH3T3细胞测定病毒滴度;将腺病毒感染人乳腺癌细胞MCF-7,蛋白免疫印迹（Western blot）检测Apoptin蛋白表达,噻唑蓝（MTT）比色法检测细胞增殖抑制,48 h后流式细胞仪（FCM）法检测肿瘤细胞的凋亡,并应用表面增强激光解析电离-蛋白质飞行时间质谱仪（SELDI-TOF-MS）检测乳腺癌细胞MCF-7标志蛋白变化.建立乳腺癌细胞MCF-7裸鼠模型,分组给予vp3病毒治疗后测定抑瘤率,实时定量聚合酶链反应技术（real-time PCR）检测vp3基因表达,原位凋亡（TUNEL法）检测肿瘤细胞的凋亡情况,并检测肿瘤组织vp3基因表达.结果 重组腺病毒载体pAD-vp3经鉴定连接正确,转染293A后上清液中病毒滴度可达到3×108 pfu;MTT检测48 h细胞抑制率（62.3%）,明显高于正常对照组（P＜0.05）,转染48 h后Western blot可见Apoptin蛋白高表达.FCM检测出现凋亡峰,其凋亡百分率高达31.87%.SELDI检测可见2个蛋白质峰M_5480.8＋H和M_4967.4＋H在两组之间表达量差异有统计学意义（P＜0.05）.裸鼠实验可见pAD-vp3组抑瘤率分别为56.82%和69.53%,明显高于空白对照组（t=4.82,P＜0.01）,且vp3基因高表达,TUNEL检测可见pAD-vp3组和5-氟脲嘧啶（5-Fu）组细胞核呈棕黄色,低剂量和高剂量组凋亡指数与5-Fu组比较差异无统计学意义（t1=1.25,t2=0.86,均为P＞0.05）.结论 成功构建了pAD-vp3腺病毒,vp3基因在体内外均能有效地诱导人乳腺癌MCF-7细胞凋亡.
Objective To construct recombinant vp3 gene adenovirus pAD-vp3 and study its apoptosis inducing effect on human breast cancer MCF-7 cells. Methods vp3 gene was cloned and recom-bined into adenovirus vector pLP-AD-vp3 （pAD-vp3） at loxP site according to homologous recombination principle. pAD-vp3 was transformed into package cell line 293A and then into NIH3T3 cells for titer assay. The MCF-7 cells were transfected with pAD-vp3.Western blotting was used to detect the Apoptin protein expression. MTT assay was adopted to measure cellular proliferation and vp3 gene expression. Forty-eight h after transfection, flow cytometry （FCM） was used to examine apoptosis, and surface enhanced laser de-sorption ionization-time of flight-mass spectrometry （SELDI-TOF-MS） was used to assay protein profile. Nude mice model of MCF-7 cells was set up to observe the tumor inhibition rate of pAD-vp3, and real-time PCR and TUNEL assay were used to detect vp3 gene and apoptosis respectively. Results Recombinant adenovirus vector pAD-vp3 was successfully constructed. Virus titer was 3 x 108 pfu/ml in the 293A culture supernatant. Forty-eight h after transfection, cellular inhibition rate was 63.3% in MTT assay, higher than that in blank control （P 〈 0.05） , and Apoptin protein was highly expressed in test group by Western blotting. FCM assay showed apoptotic peaks with a percentage of 31.87%. SELDI-TOF-MS findings suggested that two protein peaks, M_5480.8 ＋ H and M_4967.4 ＋ H, had statistically significant difference between transfection group and control group （P 〈 0.05）. The tumor inhibition rate in pLVP3 group was 56.82% and 69.53% , significantly higher than that in control group （t - 4.82,P 〈 0.01） , with high mRNA expression level. TUNEL assay findings revealed that positive yellow stains were seen in pAD-vp3 group and 5-Fu positive control group without significant difference （t1 = 1.25, t2 = 0.86, P 〉 0.05）. Conclusion Recombinant adenovirus bearing vp3, pAD-vp3, was set up successfully. vp3 could induce apoptosis in MCF-7 cells in vivo and in vitro.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2010年第8期1082-1085,共4页
Chinese Journal of Experimental Surgery
