摘要目的:探讨间充质干细胞条件培养基对多西紫杉醇(Doc)诱导的多倍体肺癌细胞衰老表型的影响。方法:人非小细胞肺癌1 299细胞株细胞分为3组，常规组、Doc(24 h)组和Doc(24 h)+3 d组。常规组为二甲基亚砜处理细胞24 h；Doc(24 h)组为100 nmol/L的Doc处理细胞24 h；Doc(24 h)+3 d组为100 nmol/L的Doc处理细胞24 h，更换新鲜的含10%胎牛血清的DMEM/F12培养基，继续培养3 d。Doc(24 h)+3 d组细胞分为2组，Doc组和Doc+CM组。Doc组为含10%胎牛血清的DMEM/F12培养基中培养4 d；Doc+CM组为含10%胎牛血清的DMEM/F12培养基和hUC-MSC-CM(1∶1)的混合培养基中培养4 d。Hoechst 33342染色观察细胞核形态，流式细胞术分析细胞DNA含量、线粒体膜电位及DNA损伤应答信号分子磷酸化组蛋白H2A.X(γ-H2A.X)的表达，衰老β-半乳糖苷酶染色试剂盒检测β-半乳糖苷酶活性，实时定量聚合酶链式反应(RTQ-PCR)检测衰老相关的炎性细胞因子白细胞介素6(IL-6)与白细胞介素8(IL-8)的信使核糖核酸(mRNA)表达水平。结果:Doc(24 h)组和Doc(24 h)+3 d组细胞核体积增大、DNA含量增加，常规组、Doc(24 h)组和Doc(24 h)+3 d组多倍体细胞百分比分别为(12.27±3.29)%、(27.13±6.02)%和(43.60±4.26)%，Doc(24 h)组和Doc(24 h)+3 d组细胞百分比明显高于常规组，差异有统计学意义( F＝33.91， P＝0.001)。常规组、Doc(24 h)组与Doc(24 h)+3 d组细胞密度分别为(8.18±0.54)×10 4/cm 2、(3.78±0.54)×10 4/cm 2及(0.75±0.08)×10 4/cm 2，Doc(24 h)组与Doc(24 h)+3 d组细胞密度显著低于常规组，差异有统计学意义( F＝212.55， P＝0.001)。常规组细胞β-半乳糖苷酶无活性，细胞染色阴性，Doc(24 h)组与Doc(24 h)+3 d组中观察到有呈蓝色的阳性染色细胞，β-半乳糖苷酶活性增强。流式细胞术分析线粒体膜电位，常规组、Doc组与Doc+CM组的JC-1单体百分比分别为(1.7±1.2)%、(48.2±12.9)%及(46.6±12.0)%，Doc组与Doc+CM组低电势细胞百分比明显高于常规组，差异有统计学意义( F＝20.15， P＝0.002)，Doc组细胞与Doc+CM组细胞线粒体膜电位比较，差异无统计学意义( F＝20.15， P＝0.854)。常规组、Doc组与Doc+CM组中γ-H2A.X表达阳性的细胞百分比分别为(54.9±3.2)%、(58.9±4.0)%及(60.6±5.4)%，差异无统计学意义( F＝1.40， P＝0.317)。Doc组与Doc+CM组γ-H2A.X荧光信号强度明显高于常规组，荧光信号右移，Doc组与Doc+CM组γ-H2A.X荧光信号强度没有差异，荧光信号没有偏移。Doc组IL-6与IL-8的mRNA表达水平是常规组的(7.22±0.34)倍及(157.64±7.49)倍，差异有统计学意义( t＝1.00， P＝0.001)。Doc+CM组IL-6与IL-8的mRNA表达水平是Doc组的(48±6)%及(43±3)%，差异有统计学意义( t＝1.00， P＝0.001)。 结论:Doc诱导人非小细胞肺癌1 299细胞株细胞产生多倍体肿瘤细胞，即多倍体肺癌细胞，多倍体肺癌细胞表现多种衰老表型特征，hUC-MSC-CM不改变Doc诱导的多倍体肺癌细胞衰老表型，但显著降低了炎性因子IL-6和IL-8的mRNA表达水平。

abstractsObjective:To investigate the effects of mesenchymal stem cells-conditioned medium on the senescence phenotype of polyploid lung cancer cells induced by docetaxel(Doc).Methods:Human non-small cell lung cancer cell line 1 299 cells were divided into three groups: routine group, Doc(24 h)group and Doc(24 h)+ 3 d group.In the routine group, the cells were treated with dimethyl sulfoxide for 24 hours.In Doc(24 h)group, the cells were treated with 100 nmol / L Doc for 24 h. In Doc(24 h)+ 3 d group, the cells were treated with 100 nmol / L Doc for 24 h, then continuing to culture for 3 days after replacing with fresh DMEM / F12 medium containing 10% fetal bovine serum.The cells of Doc(24 h)+ 3 d group were divided into two groups: Doc group and Doc+ CM group.In Doc group, the cells of Doc(24 h)+ 3 d group were cultured for 4 days in DMEM / F12 medium containing 10% fetal bovine serum.In Doc+ CM group, the cells of Doc(24 h)+ 3 d group were cultured for 4 days in mixed medium containing DMEM / F12 medium and hUC-MSC-CM(1∶1).The nuclear morphology was observed after staining with Hoechst 33342.DNA content, mitochondrial membrane potential and expression of DNA damage response signaling molecule(phosphorylated histone H2A.X, γ-H2A.X)were analyzed by flow cytometry.Activity of β-galactosidase was detected by Senescence β-Galactosidase Staining Kit.Themessenger Ribonucleic Acid(mRNA)expression levels of senescence-associated inflammatory cytokines interleukin-6(IL-6)and interleukin-8(IL-8)were detected by real-time quantitative polymerase chain reaction(RTQ-PCR).Results:Compared with routine group, the nuclear volume and DNA content in Doc(24 h)group and Doc(24 h)+ 3 d group were increased.The percentage of polyploid cells in routine group, Doc(24 h)group and Doc(24 h)+ 3 d group were(12.27±3.29)%, (27.13±6.02)% and(43.60±4.26)%, respectively.The percentage of polyploid cells in Doc(24 h)group and Doc(24 h)+ 3 d group was significantly higher than that in routine group( F=33.91, P=0.001).The cell density of routine group, Doc(24 h)group and Doc(24 h)+ 3 d group were(8.18±0.54)×10 4/cm 2, (3.78±0.54)×10 4/cm 2 and(0.75±0.08)×10 4/cm 2, respectively.The cell density of Doc(24 h)group and Doc(24 h)+ 3 d group was significantly lower than that of routine group( F=212.55, P=0.001).The activity of β-galactosidase was inactive in routine group because the cells were negative after staining, but the activity of β-galactosidase in Doc(24 h)group and Doc(24 h)+ 3 d group was increased because there were positive cells after staining.According to the analysis of mitochondrial membrane potential by flow cytometry, the percentage of JC-1 monomer in routine group, Doc group and Doc+ CM group was(1.7±1.2)%, (48.2±12.9)% and(46.6±12.0)%, respectively.The percentage of low potential cells in Doc group and Doc+ CM group was significantly higher than that in routine group( F=20.15, P=0.002).There was no significant difference on the mitochondrial membrane potential between Doc group and Doc+ CM group( F=20.15, P=0.854).The percentage of γ-H2A.X positive cells was(54.9±3.2)%, (58.9±4.0)% and(60.6±5.4)%, respectively.There was no significant difference among them( F=1.40, P=0.317).The intensity of γ-H2A.X fluorescence signal in Doc group and Doc+ CM group was significantly higher than that in routine group because the fluorescence signal shifted to right, but there was no difference between Doc group and Doc+ CM group because the fluorescence signal did not shift.The mRNA expression levels of IL-6 and IL-8 in Doc group were(7.22±0.34)times and(157.64±7.49)times higher than those in routine group, respectively( t=1.00, P=0.001).The mRNA expression levels of IL-6 and IL-8 in Doc+ CM group were(48±6)% and(43±3)% of those in Doc group, respectively( t=1.00, P=0.001). Conclusions:Doc induces human non-small cell lung cancer cell line 1 299 cells to form polyploid tumor cells, namely polyploid lung cancer cells, which show many characteristics of senescence phenotypes.hUC-MSC-CM does not affect the senescence phenotype of polyploid lung cancer cells induced by Doc, but significantly reduces the mRNA expression levels of inflammatory factors IL-6 and IL-8.