小鼠结直肠癌半脾肝转移模型用于Hippo信号通路的研究

Study on Hippo signaling pathway in mouse liver metastasis semi-spleen model of colorectal cancer

目的:应用小鼠结直肠癌半脾切除肝转移模型研究Hippo信号通路的作用。方法:20只BALB/c小鼠随机分为假手术组和实验组。假手术组将小鼠开腹后关腹,实验组将小鼠结肠腺癌CT26细胞注入脾的下极后切除,建立肝转移模型。7 d后小鼠处死并解剖。ELISA法检测小鼠眼球血中肾上腺素(epinephrine,EPI)、胰高血糖素(glucagon,GC)、溶血磷脂酸(lysobisphosphatidic acids,LPA)和1-磷酸鞘氨醇(sphingosine-1phosphate,S1P)的含量;HE染色结果确定脾半切除肝移植瘤造模成功,免疫组织化学法对Yes相关蛋白1(yes-associated protein 1,YAP1)和磷酸化YAP1(phospho-yes-associated protein 1,p-YAP1)进行定位,蛋白质印迹法检测肝组织中YAP1和p-YAP1的表达水平。结果:小鼠结直肠癌半脾切除模型建立7 d后肝转移率为100%;假手术组血浆中EPI与GC的含量均明显高于实验组(P值均<0.01),实验组小鼠血浆中LPA与S1P的含量均明显高于假手术组(P值均<0.01);实验组肝组织HE染色结果显示典型的低分化腺癌特征,有核分裂现象。假手术组正常肝组织中YAP1蛋白表达在细胞质中[细胞核YAP1(-),细胞质YAP1(+)],p-YAP1蛋白在细胞质和细胞核中均有表达[细胞核p-YAP1(+),细胞质p-YAP1(+)];实验组肝肿瘤组织中YAP1蛋白在细胞质和细胞核中均有表达[细胞核p-YAP1(+),细胞质p-YAP1(+)],p-YAP1蛋白在细胞核与细胞质中均未见表达[细胞核p-YAP1(-),细胞质p-YAP1(-)]。YAP1在实验组肝肿瘤组织中的表达水平明显高于假手术组(P <0.01),p-YAP1在实验组肝肿瘤组织中的表达水平明显低于假手术组(P <0.01)。结论:成功建立结直肠癌肝转移模型,并发现EPI、GC与YAP1表达呈负相关,与p-YAP1表达呈正相关,LPA、S1P与YAP1呈正相关,与p-YAP1呈负相关。有望开发针对Hippo信号通路相关血清因子调节剂,进行靶向治疗研究。

Objective:To study the role of Hippo signaling pathway using mouse liver metastasis and semi-spleen model of colorectal cancer in vivo.Methods:Twenty BALB/c mice were randomly divided into sham operated group and experimental group.In the sham operation group,the abdomen of mouse was opened and closed.In the experimental group,the mouse colonic adenocarcinoma CT26 cells were injected into the lower pole of spleen,and excised to establish a liver metastasis model.After 7 days,the mice were sacrificed and dissected.The expression levels of epinephrine(EPI),glucagon(GC),lysobisphosphatidic acids(LPA)and sphingosine-1 phosphate(S1P)in plasma collected from the eyeballs of mice were detected by ELISA.The successful semi-spleen liver metastasis modeling of colorectal cancer was determined by HE staining.The yes-associated protein 1(YAP1)and phospho-YAP1(p-YAP1)were localizated by immunohistochemistry.The expression levels of YAP1 and p-YAP1 proteins in the liver tissues of mice were detected by Western blotting.Results:The liver metastasis rate was 100%after 7 days of spleen hemisection model establishment.The levels of plasma EPI and GC in the sham operated group were significantly higher than those in the experimental group(both P<0.01).The levels of plasma LPA and S1 P in the experimental group were significantly higher than those in the sham operated group(both P<0.01).The HE staining result of experimental group showed typical features of poorly differentiated adenocarcinoma with mitotic division.In the normal liver tissues of sham operated group,YAP1 protein expressed in the cytoplasm[nuclear YAP(-)and cytoplasmic YAP(+)],p-YAP1 protein expressed in the cytoplasm and nuclei[nuclear p-YAP1(+)and cytoplasmic p-YAP1(+)];In the tumor liver tissues of experimental group,YAP1 protein expressed in the cytoplasm and nuclei[nuclear YAP1(+)and cytoplasmic YAP1(+)],p-YAP1 protein was not expressed in the cytoplasm or nuclei(nuclear p-YAP1(-)and cytoplasmic p-YAP1(-))The expression level of YAP1 protein in the liver of experimental group was significantly higher than that in the sham operated group(P<0.01),while the expression level of p-YAP1 in the liver of experimental group was significantly lower than that in the sham operated group(P<0.01).Conclusion:The liver metastasis model of colorectal cancer is successfully established.The expressions of EPI and GC are negatively correlated with YAP1,and positively correlated with p-YAP1.The expressions of LPA and S1 P are positively correlated with YAP1,and negatively correlated with p-YAP1.It is expected to develop a targeted therapeutic study for Hippo signaling pathway-related blood factor modulators.