Zn3In2S6-CEA量子点荧光探针对结肠癌细胞株SW480靶向标记及生物毒性研究

Targeted labeling and biological toxicity of Zn3In2S6-CEA quantum dot fluorescent probe to colon cancer cell line SW480

目的量子点荧光探针是量子点-抗体复合物,可用于靶向探测肿瘤细胞并在特定条件下使肿瘤细胞发光成像,同时具有对结肠癌诊断及治疗双重功效。本研究通过Zn3In2S6-CEA量子点荧光探针对结肠癌细胞株SW480靶向标记,并进一步研究其细胞毒性。方法用免疫组化法鉴定结肠癌细胞株SW480表达癌胚抗原(carcino-embryonic antigen,CEA),合成水溶性量子点Zn3In2S6并与抗CEA抗体结合形成Zn3In2S6-CEA荧光探针。用Zn3In2S6-CEA荧光探针对结肠癌细胞株SW480进行荧光标记,并用免疫荧光法检测荧光信号。用MTT法和流式细胞术检测与Zn3In2S6-CEA荧光探针结合后结肠癌细胞的增殖抑制和凋亡情况。结果新型Zn3In2S6-CEA量子点荧光探针可有效与结肠癌细胞株SW480结合,分别在荧光显微镜和共聚焦显微镜下显示其对SW480细胞株成功标记。5%浓度组Zn3In2S6-CEA探针在2、24、48和72h细胞抑制率分别为0.002 262、0.101 111、0.213 652和0.301 923,CuInS2@ZnS-CEA探针分别为-0.014 933、0.111 301、0.235 352和0.303 073。细胞毒性实验析因设计的方差分析结果表明,在48h时,Zn3In2S6-CEA探针组细胞抑制率显著低于CuInS2@ZnS-CEA探针组,t=23.928,P<0.001;其他时间点两组细胞抑制率差异无统计学意义;且两探针组的细胞抑制率均随着时间的增大而增大,不同时间点之间的细胞抑制率差异有统计学意义,F=4 721.400 1,P<0.001。探针和时间对细胞抑制率的交互作用差异有统计学意义,F=17.366 0,P<0.001。模型拟合效果理想,R2=0.999。流式细胞术实验结果与MTT法实验结果基本一致。结论结肠癌细胞株SW480胞膜和胞质中大量表达CEA抗原。新合成Zn3In2S6-CEA探针对其胞质和胞膜有效标记后,证实在孵育到达一定时间点时,Zn3In2S6-CEA探针的细胞毒性相比CuInS2@ZnS-CEA探针较低。

OBJECTIVE Quantum dot fluorescent probes are synthesized by conjugating quantum dots with antibodies,which is named quantum dot-antibody complex.As it can be used to target and image tumor cells under specific conditions,it is named quantum dot fluorescent probe.It also has the dual efficacy of diagnosis and treatment of colon cancer.In this study,the Zn3 In2 S6-CEA quantum dot fluorescent probe was produced and used to target the colon cancer cell line SW480 and its cytotoxicity was further studied.METHODS Immunohistochemistry was performed to characterize the carcinoembryonic antigen（CEA）on the cytoplasm and envelope of colon cancer cells.The Zn3 In2 S6-CEA fluorescent probe was synthesized by combining water-soluble quantum dots Zn3 In2 S6 with anti-CEA antibody.Immunofluorescence was used to detect the fluorescence signaling of Zn3 In2 S6-CEA fluorescent probe labeled SW480 cells.The proliferation inhibition and apoptosis of Zn3 In2 S6-CEA fluorescent probe on colon cancer cells were measured with MTT assay and flow cytometry.RESULTS The new Zn3 In2 S6-CEA quantum dot fluorescent probe can effectively bind to the colon cancer cell line SW480 and intense fluorescence signaling can be observed on SW480 cell line under fluorescence microscope and confocal microscope,respectively.The cell inhibition rates of Zn3 In2 S6-CEA and CuInS2@ZnS-CEA probes at concentration of 5%were 0.002 262,0.101 111,0.213 652,0.301 923,and-0.014 933,0.111 301,0.235 352,and 0.303 073 at 2 h,24 h,48 h,and72 h,respectively.The Factorial analysis of variance of the cytotoxicity assay showed that the inhibition rate of the Zn3 In2 S6-CEA probe was significantly lower than that of the CuInS2@ZnS-CEA probe（t=23.928,P0.001）at 48 hrs,and there was no significant difference in cell inhibition rate between the two probes at the other time points,moreover the cell inhibition rate of the two probes was demonstrated in an increasing time-dependent manner with significant difference among different time points（F=4 721.400 1,P0.001）,The interaction between probes and time on cell inhibition was statistically significant（F=17.366 0,P0.001）.The model fitting effect was quite satisfactory,R2=0.999.Flow cytometry results were basically consistent with the MTT assay results.CONCLUSIONS CEA is highly expressed on the cell membrane and in the cytoplasm of colon cancer cell line SW480.The newly synthesized Zn3 In2 S6-CEA probe was used and successfully labeled to the SW480 cells.After incubation for a certain time point,the cytotoxicity of the Zn3 In2 S6-CEA probe was lower than that of the CuInS2@ZnS-CEA probe.