上皮细胞黏附分子靶向的核酸适体药物偶连物用于胃癌靶向治疗的研究

A study on epithelial cell adhesion molecule targeted nucleic acid aptamer drug conjugate in gastric cancer targeted treatment

目的探讨上皮细胞黏附分子(EpCAM)靶向的核酸适体药物偶连物(SYL3C-MMAE)对人胃上皮细胞GES-1(以下简称GES-1细胞)以及人胃癌细胞AGS、MKN45(以下简称AGS、MKN45细胞)的亲和力以及毒性。方法采用实验研究方法。采用免疫组织化学染色检测胃癌组织中EpCAM的表达水平;采用实时荧光定量聚合酶链式反应法(PCR)检测胃癌组织中EpCAM的mRNA表达水平;应用蛋白免疫印迹法(Western blot)检测GES-1、AGS、MKN45细胞中EpCAM蛋白的表达水平;通过流式细胞仪检测SYL3C对3种细胞的亲和力;通过巯基的SYL3C序列马来酰亚胺反应合成SYL3C-MMAE;采用CCK8法检测药物对细胞的毒性;碘化丙啶法检测药物处理后细胞周期情况。观察指标:(1)EpCAM在胃癌中的表达情况。(2)靶向EpCAM的抗体与SYL3C对相关细胞系的亲和力。(3)药物合成情况。(4)药物毒性检测以及细胞周期抑制情况。正态分布的计量资料以x±s表示,多组间比较采用单因素方差分析,两两比较采用最小显著差异法检验;方差不齐者采用Welch't检验。偏态分布的计量资料以M(IQR)表示,组间比较采用配对秩和检验。计数资料以绝对数表示,组间比较采用配对χ^(2)检验。结果(1)EpCAM在胃癌中的表达情况。组织芯片免疫组织化学染色检测结果显示:35对胃癌及其癌旁组织(正常组织)中,胃癌组织EpCAM阳性率为82.9%(29/35),正常组织EpCAM阳性率为22.9%(8/35),两者比较,差异有统计学意义(P<0.05)。实时荧光定量PCR检测结果显示:12对胃癌组织及其癌旁组织中EpCAM的mRNA相对表达水平分别为1.23(4.13)和4.04(1.72),两组比较,差异有统计学意义(Z=-2.67,P<0.05)。Western blot检测结果显示:以AGS细胞中EpCAM蛋白表达量为标准,GES-1、AGS、MKN45细胞中,EpCAM蛋白的相对表达量分别为0、1.00、0.27。(2)靶向EpCAM的抗体与SYL3C对相关细胞系的亲和力。流式细胞仪检测结果显示:EpCAM抗体以及SYL3C均对AGS以及MKN45细胞有较好的亲和力,而对于GES-1细胞则没有亲和力。(3)药物合成情况。SYL3C与单甲基奥瑞他汀E(VcMMAE)连接合成质谱检测结果显示:合成完成后的药物原液于16355分子量位置出现强峰,符合SYL3C-MMAE复合物的预期分子量,提示SYL3C-MMAE合成成功。(4)药物毒性检测以及细胞周期抑制情况。CCK8法检测细胞活力结果显示:VcMMAE对GES-1、AGS、MKN45细胞半抑制浓度(IC50)分别为123.00、30.48、51.83 nmol/L。SYL3C-MMAE对于上述3种细胞的IC50分别为241.80、20.66、27.64 nmol/L。PI法检测细胞周期结果显示:VcMMAE与SYL3C-MMAE均能引起GES-1、AGS、MKN45细胞的细胞周期发生G2/M期阻滞。结论SYL3C-MMAE对于胃癌细胞有较好的亲和力,与VcMMAE比较,其在高效抑制胃癌细胞的同时对正常细胞的影响显著减轻。

Objective To investigate the affinity and toxicity of epithelial cell adhesion molecule(EpCAM)targeted nucleic acid aptamer drug conjugate SYL3C-MMAE on human gastric epithelial cells GES-1(hereinafter referred to as GES-1 cells)and human gastric cancer cells AGS and MKN45(hereinafter referred to as AGS cells and MKN45 cells).Methods The experimental study was conducted.The expression level of EpCAM in gastric cancer tissues was detected using immunohistochemistry.The mRNA expression level of EpCAM in gastric cancer tissues was detected using real-time fluorescence quantitative PCR(RT-PCR).The expression level of EpCAM protein in GES-1,AGS and MKN45 cells was detected using Western blot.The affinity of SYL3C on GES-1,AGS and MKN45 cells was detected using flow cytometry.SYL3C-MMAE was synthesized through a thiol-maleimide reaction.The toxicity of drugs on GES-1,AGS and MKN45 cells was detected using CCK-8 assay.The cell cycle condition of GES-1,AGS and MKN45 cells after drug treatment was detected using propidium iodide(PI)staining.Observation indicators:(1)expression of EpCAM in gastric cancer;(2)affinity of antibodies targeting EpCAM and SYL3C on GES-1,AGS and MKN45 cells;(3)situation of drug synthesis;(4)drug toxicity and inhibition of cell cycle.Measurement data with normal distribution were represented as Mean±SD.One-way ANOVA was used for comparison among multiple groups,and pairwise comparison was conducted using the least significant difference test.Comparison of unequal variances was conducted using the Welch't test.Measurement data with skewed distribution were represented as M(IQR),and comparison between groups was conducted using the paired rank sum test.Count data were described as absolute numbers,comparison between groups was conducted using the paired chi-square test.Results(1)Expression of EpCAM in gastric cancer.Results of immunohistochemistry on tissue microarrays showed that the positive rate of EpCAM was 82.9%(29/35)and 22.9%(8/35)in the 35 pairs of gastric cancer and its adjacent tissues(normal tissues),respectively,showing a significant difference between them(P<0.05).Results of RT-PCR showed that the mRNA relative expression levels of EpCAM was 1.23(4.13)and 4.04(1.72)in 12 pairs of gastric cancer and its adjacent tissues respectively,showing a significant difference between them(Z=-2.67,P<0.05).Results of Western blot showed that the relative expression levels of EpCAM protein in GES-1,AGS,and MKN45 was 0,1.00,and 0.27,respectively,with the expression level of EpCAM protein in AGS cells as the standard.(2)Affinity of antibodies targeting EpCAM and SYL3C on GES-1,AGS and MKN45 cells.Results of flow cytometry showed that antibodies targeting EpCAM and SYL3C had good affinity on AGS and MKN45 cells but no affinity on GES-1 cells.(3)Situation of drug synthesis.Results of mass spectrometry showed that the drug solution of compound formed by connecting SYL3C with monomethylorestatin E(VcMMAE)exhibited a strong peak at the molecular weight position of 16355,consistent with the expected molecular weight of the SYL3C-MMAE complex,indicating that SYL3C-MMAE was successfully synthesized.(4)Drug toxicity and inhibition of cell cycle.Results of CCK-8 assay showed that the half maximal inhibitory concentration(IC50)of VcMMAE on GES-1,AGS and MKN45 cells was 123.00,30.48 and 51.83 nmol/L,respectively.The IC50 of SYL3C-MMAE on GES-1,AGS and MKN45 cells was 241.80,20.66 and 27.64 nmol/L,respectively.Results of PI staining and flow cytometry showed that both VcMMAE and SYL3C-MMAE could induce G2/M phase blockage in the cell cycle of GES-1,AGS and MKN45 cells.Conclusion The SYL3C-MMAE has a good affinity on gastric cancer cells.Compared with VcMMAE,SYL3C-MMAE exhibits efficient inhibition on gastric cancer cells,but less influence on normal cells.