瘤内注射慢病毒载体介导的Bmi1-shRNA对A549皮下移植瘤的抑制作用

Intratumoral injection of recombinant lentivirus-mediated Bmi1-shRNA inhibits growth of A549 xenografts in mice

目的探讨瘤内注射慢病毒介导的原癌基因Bmi1的特异性短发夹RNA(Bmi1-shRNA)对人非小细胞肺癌免疫缺陷鼠皮下种植瘤生长的抑制作用。方法建立NOD/SCID(非肥胖糖尿病/重症联合免疫缺陷)小鼠人肺癌A549荷瘤模型,共27只,分为3组。其中治疗组移植瘤内注射含有Bmi1-shRNA的慢病毒载体,阴性对照组注射含有NC-shRNA的慢病毒载体,空白对照注射同剂量PBS。通过激光共聚焦观察移植瘤内绿色荧光蛋白(GFP)表达,RT-PCR、Western blot检测Bmi1mRNA、蛋白水平的表达,观察RNA干扰效果,测量肿瘤体积,计算肿瘤抑制率。结果将A549细胞移植到NOD/SCID鼠背部皮下,成瘤率为100%。激光共聚焦观察治疗组及阴性对照组皮下移植肿瘤组织中有GFP表达,而空白对照组未见GFP表达。治疗组NOD/SCID小鼠的肿瘤体积较阴性对照组和空白对照组显著缩小(P<0.05),肿瘤抑制率分别为52.9%和57.9%。Bmi1-shRNA治疗组中的Bmi1蛋白明显降低,相对空白对照组和阴性对照组,蛋白表达抑制率分别为51.1%、50.3%,差异有显著性(P<0.05)。结论利用慢病毒载体成功将Bmi1-shRNA导入移植瘤细胞内,检测到Bmi1蛋白表达的下调,且Bmi1的下调能显著抑制A549小鼠移植瘤的生长。

Objective To investigate the inhibitory effect of lentivirus-mediated Bmi1-shRNA on transplanted human non-small cell lung cancer（NSCLC）by intratumoral injection.MethodsRecombinant lentivirus containing Bmi1-shRNA was constructed.A model of subcutaneous implanted tumor was generated through injecting human lung cancer cell A549（107）into the NOD/SCID mice.The 27 established mice with a mass at a diameter of 4 to 5 mm were divided into 3 groups randomly and equally.Recombinant lentivirus containing Bmi1-shRNA（5×10^8 TU/ml,0.1 ml）,lentivirus containing NC-shRNA（5×10^8 TU/ml,0.1 ml,as negative controls）and PBS（0.1 ml,as blank control）was given the mice of the 3 groups respectively,once per 3 d,for 4 weeks.The size of the tumor mass were measured every 3 d.The mice were sacrificed in the end of 4 weeks＇ treatment.The expression of GFP in the xenografts was detected through laser scanning confocal microscope.RT-PCR and Western blot analysis were used to test the mRNA and protein expressions of Bmi1 in the xenografts.Results GFP was expressed in the tumor mass of the recombinant lentivirus groups,but not in the blank control.The average tumor volume in Bmi1-shRNA lentivirus group was significantly lower than that in the negative control and blank control groups（P〈0.05）with an inhibitory rate of 52.9 % and 57.9% respectively.RT-PCR and Western blotting showed that the mRNA and protein expression of Bmi1 was decreased in the Bmi1-shRNA lentivirus group（P〈0.05）.ConclusionIntratumoral injection of recombinant Bmi1-shRNA Lentivirus inhibits the growth and proliferation of A549 xenografts in the NOD/SCID mice,suggesting that Bmi1 might represent a therapeutic target in the genotherapies of non-small cell lung cancer.