表面标志物Cripto-1表达对食管癌干细胞生长的作用机制研究

目的:探讨表面标志物Cripto-1(CR-1)的表达对食管癌干细胞生长的作用机制.方法:自食管癌Eca109细胞中分选Eca109干细胞,提取正常食管上皮细胞HEEC和Eca109细胞、Eca109干细胞总蛋白,采用Western-blot法检测CR-1表达水平;构建CR-1基因沉默载体CNE2/CR-1 -及过表达载体CNE1/CR-1 +,利用脂质体转染法将CNE2/CR-1 -和CNE1/CR-1 +分别转染进Eca109干细胞中,并设对照组,采用RT-PCR法检测CR-1表达;MTT法检测对照组、CNE2/CR-1 -组、CNE1/CR-1 +组Eca109干细胞增殖情况;流式细胞仪检测各组Eca109干细胞凋亡情况;Western-blot法检测各组细胞Akt、p-Akt蛋白表达情况.结果:Eca109细胞及Eca109干细胞中CR-1表达均明显高于HEEC(P<0.01).RT-PCR结果显示,CNE2/CR-1 -组CR-1表达水平明显低于对照组,CNE1/CR-1 +组明显高于对照组(P<0.01).MTT结果显示,CNE2/CR-1 -组细胞增殖明显低于对照组,CNE1/CR-1 +组明显高于对照组(P<0.01).流式细胞仪检测显示,CNE2/CR-1 -组细胞凋亡率明显高于对照组, CNE1/CR-1 +组细胞凋亡率明显低于对照组(P<0.01).Western-blot结果显示,CNE2/CR-1 -组p-Akt蛋白表达明显低于对照组,CNE1/CR-1 +组p-Akt蛋白表达明显高于对照组(P<0.01);而Akt蛋白表达未出现明显变化(P>0.05).结论:CR-1作为潜在的食管癌干细胞标记物,可能通过PI3K/Akt信号通路调控食管癌干细胞的增殖与凋亡.

Active mechanism of the surface marker CR-1 expression on the esophageal cancer stem cell growth

Objective:To investigate the active mechanism of the surface marker Cripto-1(CR-1) expression on esophageal cancer stem cell growth. Methods:Eca109 stem cells was isolated from Eca109 cells. The total proteins of HEEC,Eca109 cells and Eca109 stem cells were extracted,and the expression level of CR-1 was detected using Western-blot. The silencing CNE2/ CR1 - vector targeting CR-1 gene and overexpression vector CNE1/ CR-1 + were constructed,and transfected into the Eca109 stem cells using lipofectamine.The control group was set. The transfection efficiency was examined using RT-PCR. The proliferation of Eca109 stem cells in control group,CNE2/ CR-1 - and CNE1/ CR-1 + were detected using MTT. the prognosis of Eca109 stem cells in each group was examined using flow cytometer. The protein expression levels of Akt and p-Akt in each group were detected using Western-blot. Results:The expression levels of CR-1 in Eca109 cells and Eca109 stem cells were significantly higher than that in HEEC cells(P < 0. 01). RT-PCR results showed that the expression level of CR-1 in CNE2/ CR-1-group was significantly lower than that in control group,and the expression level of CR-1 in CNE1/ CR-1 + group was significantly higher than that in control group(P <0. 01). MTT assay results showed that the cell proliferation in CNE2/ CR-1 - group was significantly lower than that in control group,and which in CNE1/ CR-1 + group was significantly higher than that in control group(P <0. 01). Flow cytometry results showed that the apoptosis rate of cells in CNE2/ CR-1 - group was significantly higher than that in control group,which in CNE1/ CR-1 + group was significantly lower than that in control group(P <0. 01). Western-blot results showed that the expression level of p-Akt protein in CNE2/ CR-1 - was significantly lower than that in control group,and the expression level of p-Akt protein in CNE1/ CR-1 + group was significantly higher than that tin control group(P <0. 01). The expression change of Akt protein was not obvious ( P > 0. 05 ). Conclusions: The CR-1, anesophageal cancer stem cell marker,may regulate the proliferation and apoptosis of esophageal stem cell by PI3K/ Akt signaling pathway.