EpCAM特异性适配体对胰腺癌细胞的靶向识别及促凋亡研究

Specific Aptamer of EpCAM Recognize Pancreatic Cancer Cell Line PANC-1 and Induce Apoptosis in This Cell Line

目的:验证EpCAM分子特异性适配体对胰腺癌细胞的亲和性及对细胞凋亡的影响。方法:通过文献查询EpCAM分子特异性适配体,以流式细胞术验证适配体与胰腺癌PANC-1细胞的特异性亲和力;通过细胞免疫组化验证适配体与细胞结合的位点;通过流式细胞术检测适配体对细胞的凋亡影响。结果:选择文献报道的EpCAM分子特异性适配体EP166,在不破坏主要二级结构情况下做截断处理形成EP166s。通过流式细胞术验证发现EP166s与原适配体EP166均可以特异性识别胰腺癌PANC-1细胞,而与阴性对照的HEK293T细胞无特异性亲和力。细胞免疫荧光显示EP166s主要结合在细胞膜表面。凋亡检测结果发现EP166s在与PANC-1孵育12 h、24 h后均可促进细胞凋亡,且主要是细胞的早期凋亡,其所占百分比分别为12.2±0.20,27.13±0.36,而随机文库对照组的早期凋亡百分比分别为5.21±0.63,4.91±0.72;均值的配对T检验发现EP166s在孵育12 h和24 h后其早期凋亡所占百分数与随机文库组均有统计学差异,而且EP166s与细胞孵育12 h与24 h对凋亡的影响也有统计学差异,P≤0.01。结论:修饰后的适配体EP166s可以特异性识别胰腺癌PANC-1细胞,且结合位点位于细胞膜上;该适配体有促进PANC-1细胞凋亡的作用。

Objective： To verify the affinity of EpCAM specific aptamer on pancreatic cancer cell line PANC-1 and its effect on cell apoptosis. Methods： The specific aptamers of EpCAM were searched by literature from Pub Med. The specific affinity of aptamers binding to pancreatic cancer cell line PANC-1 was tested by flow cytometry. The binding sites of the aptamer to PANC-1 cells were verified by cellular immunohistochemistry. The apoptotic effect of aptamers in this cell line was examined by flow cytometry using Annexin V-FITC/PI Kit. Results： The EpCAM specific aptamer EP166 reported in the literature was selected to be cut off to form EP166 s without destroying the major secondary structure. Flow cytometry showed that both EP166 s and EP166 can specifically recognize pancreatic cancer cell line PANC-1,but not recognize human embryonic kidney cell line HEK293 T. Cellular immunofluorescence showed that EP166 s mainly binds to the cell membrane surface. Apoptosis test results showed that EP166 s promoted apoptosis after 12 h and 24 h of incubation with PANC-1,the percentages of the cell early apoptosis rate were 12.2±0.20 and 27.13±0.36,respectively. The percentages were 5.21±0.63,4.91±0.72 respectively in the random library control group. The paired T-test of the mean found that the percentages of early apoptosis of EP166 s with 12 h and 24 h incubation were statistically different from that of the random library group,and incubation with 12 h was statistically different from with 24 h,P ≤0.01. Conclusions： The modified EpCAM specific aptamer EP166 s can specifically recognize pancreatic cancer cell line PANC-1,and the binding site is located on the cell membrane. The aptamer EP166 s has the effect of promoting apoptosis of PANC-1.