慢病毒介导NK4过表达对人肺腺癌移植瘤生长的影响

The effect of lentivirus-mediated NK4 overexpression on the growth of lung adenocarcinoma xenograft tumor in nude mice

目的研究瘤内注射慢病毒载体介导的NK4过表达对肺腺癌移植瘤生长、远处转移以及微血管生成的影响。方法构建人肺腺癌A549裸鼠皮下移植瘤模型并按照随机数字表法分为磷酸盐缓冲液(PBS)组、LV-con组和LV-NK4组,每组5只。通过瘤内注射的方式分别向3组注射PBS(25μL)、阴性对照慢病毒载体LV-con(25μL,6×10^(8)TU/mL)和重组过表达慢病毒载体LV-NK4(25μL,6×10^(8)TU/mL),共注射2次,间隔3 d。观察裸鼠皮下移植瘤生长情况,测量瘤体积、瘤质量,计算抑瘤率。HE染色观察肿瘤组织和肺组织的形态变化。免疫组化染色检测c-Met、CD31表达,微血管密度(MVD)检测评估肿瘤血管生成情况。原位缺口末端标记(TUNEL)法检测瘤组织细胞凋亡情况。实时荧光定量聚合酶链反应(q PCR)和Western blot分别检测NK4 mRNA及蛋白表达水平。结果成功构建裸鼠肺腺癌移植瘤模型。小鼠荷瘤实验显示,LV-NK4组瘤体积、瘤质量均小于LV-con组和PBS组(P<0.05),而LV-con组和PBS组间差异无统计学意义。LV-NK4组抑瘤率为44.51%,LV-con组抑瘤率为10.35%。LV-NK4组肿瘤组织凋亡、坏死明显增多,c-Met蛋白表达量减少,微血管生成受到抑制。肺组织HE染色结果发现,LV-con组和PBS组可见肿瘤转移灶,LV-NK4组未见明显转移灶。LV-NK4组NK4 mRNA及蛋白表达水平均高于LV-con组和PBS组。结论慢病毒介导的NK4过表达可以通过抑制c-Met蛋白表达及微血管的形成,抑制裸鼠肺腺癌移植瘤的生长和肺部转移。

Objective To study the effect of NK4 overexpression mediated by intratumoral injection of lentivirus on the growth,invasion,metastasis and microangiogenesis of lung adenocarcinoma xenografts in nude mice.Methods A549 cells were implanted into the right flank of BALB/c mice.After modeling,the mice were randomly divided into three groups:the PBS group,the LV-con group and the LV-NK4 group,5 mice in each group.Mice in the three groups were received intratumoral injections of PBS(25μL),LV-con(25μL,6×10^(8) TU/mL)and LV-NK4(25μL,6×10^(8) TU/mL)respectively,twice at an interval of 3 days.The tumor volumes and weights of xenografts were measured,and the inhibitory rate of tumor was calculated at the end of the experiments.The tumor tissues and lung tissues in each group were observed by hematoxylin-eosin staining.The expressions of c-Met and CD31 were detected by immunohistochemistry,and microvessel density(MVD)was used to detect angiogenesis.The apoptosis of tumor cells in each group was detected by TUNEL.The expression levels of NK4 mRNA and protein in xenograft tumors were detected by real-time PCR and Western blot assay,respectively.Results The xenograft tumor model of human lung adenocarcinoma was established successfully.The volume of tumor was smaller in the LV-NK4 group than that of the LV-con group and the PBS group(P<0.05).There was no significant difference between the LV-con group and the PBS group.The tumor weight was significantly decreased in the LV-NK4 group compared with the LV-con group and the PBS group(P<0.05).The tumour suppression rate was 44.51%in the LV-NK4 group and 10.35%in the LV-con group.The necrosis and apoptosis in tumor tissue of nude mice were significantly more in the LV-NK4 group than those in the LV-con group and the PBS group.The expression of c-Met in tumor tissue was significantly decreased in the LV-NK4 group,and the microangiogenesis was inhibited.There were no obvious lung metastases in the LV-NK4 group while there were obvious metastases in the LV-con group and the PBS group.The expression levels of NK4 mRNA and protein were significantly up-regulated in the LV-NK4 group than those of the other two groups.Conclusion Lentivirus-mediated NK4 overexpression can inhibit the growth and lung metastasis of lung adenocarcinoma xenograft in nude mice by inhibiting the expression of c-Met protein and the formation of micro vessels.