增强细胞缝隙连接功能对三阴性乳腺癌细胞放疗敏感性的研究

摘要

Objectvi e To investigate whether promoting gap junctions may contribute to the radiosensi-tivity in triple negative breast cancer( TNBC)cells.Methods HCC70(triple-negative),MCF-7(ER-posi-tive)or SK-BR3(HER2-positive )cells were transfected with pcDNA/5 -Cx43 expression plasmid using liposome 2000.The transfected cells were treated with various doses of radiation(0,5,10,15 Gy),and the level of Cx43 protein was determined by Western blot and the cell connectivity was determined by fluorescent tracer technique. Cell proliferation inhibition,clone formation ability and apoptosis were detected using MTT,clone formation assay, AnnexinV-FITC/PI double staining and flow cytometer,respectively.Results The level of Cx43 protein signifi-cantly increased in HCC 70 -Cx43 ,MCF-7 -Cx43 and SK-BR3 -Cx43 cells.After transfection the cells were treated with various doses of radiation,level of Cx43protein was gradually enhanced in dose dependent fashion .The re-sults form fluorescent tracer technique showed that fluorescence intensity was gradually elevated with increase of radiation doses.Cell viability and clone formation ability were decreased gradually in dose dependent manner in HCC70-Cx43 ,MCF-7 -Cx43 and SK-BR3-Cx 43 cells.Unexpectedly,the inhibitive effect of proliferation ability and clone formation ability in HCC70 -Cx43 cell was higher than in MCF-7 -Cx43 and SK-BR3 -Cx43 cells under same conditions.The results from AnnexinV-FITC/PI and flow cytometer showed that apoptosis rate was enhanced gradually accompanying with increase of radiation doses.Conclu sion Enhancing the function of cell gap junc-tions promoted radiosensitivity of breast cancer cells,particularly in TNBC cells.Radiation can strengthen cell gap junctions in breast cancer cell and cytotoxicity of TNBC cell can be enhanced by both synergistic effects.%目的：研究增强细胞缝隙连接功能对三阴性乳腺癌细胞放疗敏感性的影响。方法运用脂质体2000将pcDNA／5－Cx43表达载体转染HCC70（三阴性）、MCF－7（ ER阳性）和SK－BR3细胞（ HER－2阳性）；细胞经不同剂量电离辐射（0、5、10、15 Gy）照射后运用Western blot测定细胞中Cx43蛋白水平；荧光示踪法测定细胞连接功能，运用MTT法、克隆形成实验、AnnexinV－FITC／PI双染法和流式细胞仪分别测定细胞的增殖、克隆形成能力以及细胞凋亡情况。结果转染的HCC70－Cx43、MCF－7－Cx43和SK－BR3－Cx43细胞中Cx43蛋白水平显著增加，经不同剂量辐照后Cx43蛋白表达量逐渐增加，具有剂量依赖效应；荧光示踪结果显示随着电离辐射剂量的增加，荧光密度逐渐升高；HCC70－Cx43、MCF－7－Cx43和SK－BR3－Cx43细胞活性和克隆形成能力逐渐降低，具有剂量－时间依赖效应；相同情况下，HCC70－Cx43细胞的增殖能力和克隆形成能力的抑制作用高于MCF－7－Cx43和SK－BR3－Cx43细胞；AnnexinV－FITC／PI和流式细胞仪结果显示，随着辐照剂量的增加，细胞凋亡率逐渐升高。结论增强细胞缝隙连接功能可提高乳腺癌细胞，尤其是三阴性乳腺癌细胞的放疗敏感性；辐射能增强乳腺癌细胞的缝隙连接功能，二者协同作用增强三阴性乳腺癌的细胞毒性。