目的 探讨沉默甲壳质酶蛋白40(YKL-40)表达对子宫内膜癌顺铂(DDP)耐药细胞株Ishikawa/DDP的影响.方法 采用DDP长期浓度梯度递增法体外建立Ishikawa/DDP耐药细胞株,并检测多药耐药相关基因(MDR1)和Bcl-2、Bax和caspase-3的表达,采用MTT法检测Ishikawa细胞和Ishikawa/DDP细胞的增殖活性,计算半数抑制浓度(IC50).Ishikawa/DDP分别转染NC阴性序列(NC组)和siRNA YKL-40(si-YKL-40组),另设未转染细胞作为对照组,采用MTT法、划痕实验和Annexin V/PE双染法检测DDP对si-YKL-40组Ishikawa/DDP细胞增殖、迁移能力和凋亡的影响.结果 体外成功建立Ishikawa/DDP耐药细胞株,3. 125、6. 25、12. 5、25、50、100μmol/L DDP对Ishikawa/DDP细胞的增殖抑制率分别为(6. 93±2. 45)％、(8. 14±4. 50)％、(11. 37±4. 62)％、(15. 18±3. 97)％、(26. 29±5. 08)％、(41. 32±7. 64)％,明显低于Ishikawa细胞(P＜0. 05).DDP对Ishikawa和Ishikawa/DDP的IC50分别为14. 58μmol/L和116. 70μmol/L,Ishikawa/DDP的耐药指数为8. 004.QPCR检测结果显示,Ishikawa细胞YKL-40、MDR1、Bcl-2、Bax、caspase-3 mRNA表达量分别为0. 82±0. 15、0. 43±0. 11、1. 05±0. 23、1. 17±0. 20、0. 96±0. 18,而Ishikawa/DDP细胞分别为1. 87±0. 40、2. 34±0. 46、1. 52±0. 28、0. 72±0. 21、0. 49±0. 17,差异有统计学意义(P ＜0. 05).3. 125、6. 25、12. 5、25、50、100μmol/L DDP对si-YKL-40组细胞的增殖抑制率分别为(10. 95±2. 74)％、(18. 73±5. 30)％、(32. 79±5. 47)％、(52. 28±6. 58)％、(61. 73±5. 26)％、(65. 45±7. 33)％,明显高于对照组和NC组,差异有统计学意义(P＜0. 05).DDP对si-YKL-40组细胞的IC50为22. 19μmol/L.与对照组和NC组比较,si-YKL-40组细胞凋亡率升高、愈合率下降; YKL-40、MDR1、Bcl-2蛋白表达量下调,Bax和caspase-3蛋白表达量上调,差异有统计学意义(P＜0. 05).结论 沉默YKL-40可显著提高Ishikawa/DDP细胞株对DDP的敏感性,并促进细胞凋亡,其具体机制可能与下调MDR1、Bcl-2表达,及上调Bax和caspase-3表达有关.%Objective To investigate the effect of silenced chitinase 40 (YKL-40) expression on cisplatin-resistant endometrial cancer cell line Ishikawa/DDP. Methods Ishikawa/DDP drug-resistant cell lines were established by DDP long-term concentration gradient method in vitro, and the expression of MDR1, Bcl-2, Bax and caspase-3 were detected. The proliferation activity of Ishikawa cells and Ishikawa/DDP cells was detected by MTT method, and the half inhibitory concentration (IC50) was calculated. Ishikawa/DDP was transfected with NC negative sequence (NC group) and siRNA YKL-40 (si-YKL-40 group), respectively. The untransfected cells were set as blank control group. The effects of DDP on proliferation, migration and apoptosis of Ishikawa/DDP cells in siYKL-40 group were detected by MTT, scratch test and Annexin V/PE double staining. Results Ishikawa/DDP resistant cell lines were successfully established in vitro. The inhibitory rates of 3. 125, 6. 25, 12. 5, 25, 50 and 100 μmol/L DDP on proliferation of Ishikawa/DDP cells were (6. 93 ± 2. 45) ％, (8. 14 ± 4. 50) ％, (11. 37 ± 4. 62) ％, (15. 18 ± 3. 97) ％, (26. 29 ± 5. 08) ％, (41. 32 ±7. 64) ％, which were significantly lower than those of Ishikawa cells (P＜0. 05). The IC50 were 14. 58 μmol/L and 116. 70 μmol/L, respectively. The resistance index of Ishikawa/DDP was 8. 004. The expression of YKL-40, MDR1 and Bcl-2 in Ishikawa/DDP cells were 1. 87±0. 40, 2. 34±0. 46 and 1. 52±0. 28, which were higher than those in Ishikawa cells (P＜0. 05). The expression of Bax and caspase-3 in Ishikawa/DDP cells were 0. 72±0. 21 and 0. 49±0. 17, which were lower than those in Ishikawa cells (P＜0. 05). The inhibitory rates of 3. 125, 6. 25, 12. 5, 25, 50 and 100 μmol/L DDP on the proliferation of si-YKL-40 cells were (10. 95± 2. 74) ％, (18. 73± 5. 30) ％, (32. 79 ± 5. 47) ％, (52. 28 ± 6. 58) ％, (61. 73 ± 5. 26) ％, (65. 45 ± 7. 33) ％, respectively, which were significantly higher than those of blank control group and NC group (P＜0. 05). The IC50 of DDP on si-YKL-40 cells was 22. 19 μmol/L. Compared with the blank control group and NC group, the apoptotic rate increased and the healing rate decreased in the si-YKL-40 group, while the expression of YKL-40, MDR1 and Bcl-2 decreased, while the expression of Bax and caspase-3 increased (P＜0. 05).Conclusion Silencing YKL-40 can significantly increase the sensitivity of Ishikawa/DDP cell lines to cisplatin and promote cell apoptosis, which may be related to down-regulation of MDR1, Bcl-2 expression and up-regulation of Bax and caspase-3 expression.
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