Survivin T34A质粒/脂质体复合物抑制Lewis肺癌实验研究

Efficient inhibition of murine Lewis lung cancer by Survivin T34A plasmid/liposome complexes

目的本研究旨在观察及探讨Survivin基因突变体Survivin T34A对Lewis肺癌的影响和作用机制。方法构建Survivin T34A质粒/脂质体复合物(Lip-mS)及空载质粒/脂质体复合物(Lip-null);体外分别以Lip-mS及Lip-null转染Lewis肺癌细胞,以空白组(NS)作为对照组,利用流式细胞仪检测各组肿瘤细胞的凋亡率;体内试验使用C57BL/6小鼠建立Lewis皮下移植瘤动物模型,随机分为Lip-mS治疗组、Lip-null治疗组及NS组,观察各组小鼠肿瘤生长情况,通过对肿瘤组织CD31免疫组化、TUNEL荧光染色探讨SurvivinT34A基因的抗瘤机制。结果流式细胞仪检测体外转染不同质粒后各组肿瘤细胞的凋亡:NS组(8.7±0.5)%、Lip-null组(9.0±1.0)%、Lip-mS组(41.6±1.7)%,Lip-mS组相对于其他各组诱导凋亡作用显著(P<0.01);C57BL/6小鼠Lewis肺癌模型接受治疗后,计算各组小鼠抑瘤率(TIR),Lip-null组为负值,Lip-mS组为71.1%,分析小鼠肿瘤生长曲线,Lip-mS组较其他各组有显著抑瘤作用(P<0.05);肿瘤组织凋亡染色(TUNEL):Lip-mS组凋亡系数为(40.0±2.3)%,与NS组的(11.8±2.4)%、Lip-null组的(12.8±1.8)%比较有统计学差异(P<0.05);CD31免疫组化染色中肿瘤微血管密度:Lip-mS组为6.3±0.76,明显小于NS组的47.1±1.37和Lip-null组的45.8±1.18(P<0.01)。结论 SurvivinT34A质粒/脂质体复合物对Lewis肺癌治疗有效,能够诱导肿瘤细胞凋亡,抑制肿瘤微血管生成,从而控制肿瘤生长。

Objective This study was to explore the effects and mechanism of Survivin mutant Thr34→Ala(Survivin T34A)on Lewis lung cancer.Methods Plasmid encoding the phosphorylation defective dominant-negative mouse survivin threonine 34→alanine mutant(Survivin T34A)complexed to a DOTAP-chol liposome(Lip-mS)and a empty plasmid complexed to a DOTAP-chol liposome(Lip-null)were administered in Lewis Lung Carcinoma(LLC)cells and in mice bearing LLC tumors separately,compared with the control group(NS).The effects on apoptosis,tumor growth and angiogenesis were assessed.Results The proportion of sub-G1 cells of Lip-mS is(41.6±1.7)% in Lip-mS treatment group,it is significantly increased compared with(8.7±0.5)% of NS group and(9.0±1.0)% of Lip-null group(P<0.01).In mice bearing LLC tumors,the mice tumor inhibitory rate(TIR)of Lip-null group is negative,while Lip-mS group was 71.1%.From the tumor growth curve,the Lip-mS treatment group improved the antitumor activity and strengthen tumor regression when compared with the NS and Lip-null group(P<0.05).The apoptotic factor of Lip-mS group is(40.0±2.3)%,which is significantly higher than(12.8±1.8)% of Lip-null group and(11.8±2.4)% of NS group with TUNEL method(P<0.05).The microvessel density of Lip-mS is 6.3±0.76,which is significantly less than 47.1±1.37 of NS group and 45.8±1.18 of Lip-null group Conclusions These studies suggest that Survivin T34A plasmid/liposome complexes can cause increased apoptosis of tumor cells and inhibition of tumor angiogenesis,which may play important roles in anti-tumor effects in vivo.