【摘要】 目的：研究叶绿素铜钠盐（chlorophyllin, CHL）对人胰腺癌细胞响应吉西他滨（gemcitabine,GEM）治疗敏感性的影响，以及对已经发生GEM耐药胰腺癌细胞的治疗作用。方法：用低剂量持续递增法诱导对GEM耐药的胰腺癌细胞MIA PaCa-2/GEM （MIA GR）。采用CCK-8法检测GEM和CHL对亲本MIA PaCa-2细胞（MIA WT）和MIA GR细胞的半数抑制浓度(half inhibitory concentration,IC₅₀)，并计算耐药指数；根据IC₅₀值，用不同倍数IC₅₀的CHL和（或）GEM分别干预MIA WT和MIA GR细胞，并通过CCK-8法检测CHL联合GEM干预下MIA WT和MIA GR细胞的生长抑制曲线；以IC₅₀值为CHL和（或）GEM的处理浓度分别干预MIA WT和MIA GR细胞，随后采用克隆形成实验检测对细胞增殖的影响，在荧光显微镜下观察对细胞毒性/活性的影响，并采用流式细胞术检测对细胞凋亡的影响，最后采用蛋白质印迹法检测CHL和（或）GEM干预对MIA GR细胞中耐药性相关分子P-糖蛋白（P-glycoprotein,P-gp）和核糖核苷酸还原酶调节亚基M2（ribonucleotide reductase regulatory subunit M2,RRM2），及敏感性相关分子脱氧胞苷激酶（deoxycytidine kinase,DCK）蛋白表达水平的影响。结果：MIA GR细胞经验证具有耐药性，在GEM干预48和96 h后相比同源野生型MIA WT细胞的耐药指数分别为549.1和667.9；与MIA WT细胞相比，CHL干预对MIA GR细胞的增殖抑制作用更加显著，并且CHL联合GEM相较于GEM单药可以发挥在MIA WT细胞（P<0.001）和MIA GR细胞（P<0.01）细胞中更加显著的胰腺癌细胞生长抑制效应。CHL能明显抑制MIA GR细胞的增殖，但联合GEM后在2株细胞中的抑制效果均更加显著（P<0.000 1）；另外，相较于GEM单药，联合CHL干预可以对MIA WT细胞和MIA GR细胞造成更明显的细胞毒性（P<0.000 1），且诱导更高比例的细胞凋亡。蛋白质印迹法检测结果表明，CHL干预会使MIA GR细胞中P-gp和RRM2蛋白的表达水平下调，同时使DCK的蛋白表达水平升高。结论：CHL可提高胰腺癌细胞对GEM的敏感性，同时可诱导已耐药的胰腺癌细胞对GEM的抵抗性降低。更多还原

【Abstract】 Objective: To investigate the effect of copper chlorophyllin sodium salt（CHL） on the sensitivity of human pancreatic cancer cells in response to gemcitabine（GEM） therapy and on the therapeutic effect on pancreatic cancer cells that have developed GEM resistance.Methods: MIA GR（a pancreatic cancer cell line resistant to GEM） was induced by a lowdose continuous incremental method, and the half inhibitory concentration(IC₅₀) of MIA WT and MIA GR to GEM treatment was detected by the CCK-8 method, and the resistance index was calculated; the difference in IC₅₀ of CHL on the two types of cells was detected by the CCK-8 method after treating MIA WT and MIA GR cells with different concentrations of CHL,CCK-8 method was used to detect the difference in IC₅₀ of CHL on the two types of cells; on the basis of IC₅₀, MIA WT and MIA GR cells were intervened with CHL and（or） GEM with different multiplicity of IC₅₀, respectively, and the growth inhibition curves of MIA WT and MIA GR cells were detected by the CCK-8 method under the intervention of CHL combined with GEM; After the intervention of MIA WT and MIA GR cells with CHL and（or） GEM at IC₅₀,respectively, the effects on the proliferation of the two different cells were detected using the clone formation assay; the effects on cytotoxicity/activity were observed under fluorescence microscopy; and the effects on apoptosis were detected using flow cytometry.Finally, western blotting was used to detect the effects of CHL and（or） GEM interventions on the drug resistance-associated molecules P-glycoprotein（P-gp） and ribonucleotide reductase regulatory subunit M2（RRM2） in MIA GR cells, the and sensitivity-related molecule deoxycytidine kinase（DCK） on protein expression levels.Results: MIA GR cells were verified to be well drug resistant, with resistance indices of 549.1 and 667.9 after 48 h and 96 h after GEM intervention compared to homologous wildtype MIA WT cells, respectively; CHL intervention inhibited the proliferation of MIA GR cells more significantly compared to that of MIA WT cells; and CHL in combination with GEM exerted a more significant growth inhibitory effect compared to GEM alone in both MIA WT cells（P<0.001） and MIA GR cells（P<0.01）. CHL significantly inhibited the tumor proliferation of MIA GR cells, and the inhibitory effect was more pronounced in both cells when combined with GEM（P < 0.000 1）; furthermore, compared to GEM alone, the intervention with CHL could cause more pronounced cytotoxicity（P < 0.000 1） in both MIA WT and MIA GR cells. caused more pronounced cytotoxicity（P < 0.000 1） and induced a higher percentage of apoptosis than GEM alone. The results of the western blotting assay showed that CHL intervention caused a decrease in the expression levels of P-gp and RRM2 proteins, as well as an increase in the protein expression level of DCK in MIA GR cells.Conclusion: CHL increases the sensitivity of pancreatic cancer cells to GEM and also induces a decrease in the resistance of drug-resistant pancreatic cancer cells to GEM.更多还原