[1]曲志梅,董 伟,刘艳萍.维奈克拉增强MDS 细胞系对地西他滨化疗敏感性机制的实验研究[J].现代检验医学杂志,2023,38(03):53-57+78.[doi:10.3969/j.issn.1671-7414.2023.03.010]
　QU Zhi-mei,DONG Wei,LIU Yan-ping.Experimental Study on the Mechanism of Venetoclax Enhancing the Sensitivity of MDS Cell Lines to Decitabine Chemotherapy[J].Journal of Modern Laboratory Medicine,2023,38(03):53-57+78.[doi:10.3969/j.issn.1671-7414.2023.03.010]
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维奈克拉增强MDS 细胞系对地西他滨化疗敏感性机制的实验研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年03期
页码:
53-57+78
栏目:
论著
出版日期:
2023-05-15

文章信息/Info

Title:
Experimental Study on the Mechanism of Venetoclax Enhancing the Sensitivity of MDS Cell Lines to Decitabine Chemotherapy
文章编号:
1671-7414（2023）03-053-06
作者:
曲志梅; 董 伟; 刘艳萍
（山东第一医科大学附属人民医院，济南 271199）
Author(s):
QU Zhi-mei;  DONG Wei;  LIU Yan-ping
（the People’s Hospital Affiliated to the First Medical University of Shandong, Jinan 271199, China）
关键词:
维奈克拉; 地西他滨; 骨髓增生异常综合征; 细胞凋亡; 自噬
分类号:
R551.3；R392.11
DOI:
10.3969/j.issn.1671-7414.2023.03.010
文献标志码:
A
摘要:
目的 研究维奈克拉（venetoclax，VCX）对骨髓增生异常综合征(myelodysplastic syndromes, MDS) 细胞系地西他滨（decitabine，DAC）化疗敏感性的可能作用机制。方法 CCK-8 法检测不同浓度VCX 对MDS 细胞（SKM-1 和MUTZ-1 细胞系）增殖活力的影响；将MUTZ-1 细胞根据不同处理分为4 组：对照组、VCX 组、DAC 组和VCX+DAC组；Annexin V-FITC/PI 法检测各组细胞凋亡率；Western blotting 检测细胞中凋亡相关蛋白[ 细胞色素C（cytochromeC），裂解型半胱天冬酶3（cleaved Caspase-3）表达水平]，B 细胞白血病/ 淋巴瘤2(B cell leukemia/lymphoma-2，Bcl-2) 与Bcl-2 相关蛋白X（Bax）比值；JC-1 线粒体膜电位检测试剂盒检测各组SKM-1 和MUTZ-1 细胞的线粒体膜电位；H2DCF-DA 荧光探针法检测细胞中活性氧（reactive oxygen species，ROS）含量；Western blotting 检测细胞中自噬相关蛋白Beclin1，P62 和LC3- Ⅱ /LC3- Ⅰ比值。结果 随VCX 浓度升高，MUTZ-1 细胞增殖活性明显降低，且呈浓度依赖性（F=0.003，P=0.001）。与对照组（4.28%±1.66%）相比，VCX 组（13.75%±3.02%），DAC 组（12.39%±4.16%）和VCX+DAC组（18.10%±3.50%）细胞凋亡率明显增高，差异有统计学意义（F=45.782，P<0.05）。与对照组（1.01±0.02，1.04±0.02，1.01±0.04） 相比，VCX 组（1.67±0.05，2.23±0.10，0.43±0.05），DAC 组（1.62±0.08，1.85±0.06，0.49±0.07） 和VCX+DAC 组（3.24±0.10，3.81±0.19，0.13±0.01） 细胞中凋亡相关蛋白cytochrome C，cleavedCaspase-3 蛋白表达及Bcl-2/Bax 比值明显升高，差异均有统计学意义（F=116.384，282.069，248.035，均P<0.05）。与对照组（2.05±0.34，8.78±1.37）相比，VCX 组（8.72±1.26，14.02±1.45），DAC 组（8.44±2.13，13.20±2.41）和VCX+DAC 组（15.66±2.90，26.45±1.53）细胞线粒体膜电位及ROS 含量明显升高，差异具有统计学意义（F=66.782，69.071，均P<0.05）。与对照组（1.05±0.04，1.02±0.08，1.01±0.07）相比，VCX组（1.62±0.15，2.60±0.19，0.56±0.15），DAC 组（1.67±0.17，2.45±0.20，0.54±0.14）和VCX+DAC 组（3.72±0.21，3.58±0.27，0.13±0.09）自噬相关蛋白Beclin1，LC3- Ⅱ /LC3- Ⅰ表达明显升高，而P62 蛋白表达则明显降低，差异均有统计学意义（F=118.257，209.422，236.92，均P<0.05）。与DAC 组比较，VCX+DAC 组细胞凋亡率、cytochrome C，cleaved Caspase-3，Beclin1 蛋白表达水平，LC3- Ⅱ /LC3- Ⅰ比值和ROS 含量均明显降低（t=2.473，28.564，17.291，16.115，7.021，9.319）；线粒体膜电位、Bcl-2/Bax 比值和P62 蛋白表达均明显升高（t=4.621，9.244，4.278），差异具有统计学意义（均P ＜ 0.05）。DAC 组和VCX 组细胞中上述检测指标差异均无统计学意义（均P ＞ 0.05）。结论 VCX 可能通过调节细胞凋亡、自噬和氧化应激来促进MDS 细胞对DAC 的化疗敏感性。
Abstract:
Objective To investigate the possible mechanism of action of venetoclax (VCX) susceptibility to chemotherapy in the myelodysplastic syndrome (MDS) cell line decitabine (DAC). Methods The CCK-8 method detected the effect of different concentrations of VCX on the proliferation and viability of MDS cells (SKM-1 and MUTZ-1 cell lines). MUTZ-1 cells were divided into 4 groups according to different treatments: control group, VCX group, DAC group and VCX+DAC group. The apoptosis rates of MUTZ-1 cells in each group were detected by Annexin V-FITC/PI method .Western blotting measured the ratio of apoptosis-associated protein (cytochrome C, cleaved Caspase-3) in cells, B-cell leukemia/lymphoma-2, Bcl-2, to Bcl-2-related protein X (Bax). Detection of mitochondrial membrane potential of SKM-1 and MUTZ-1 cells by JC-1 method.H2DCF-DA fluorescent probe method to detect the content of reactive oxygen species (ROS) in cells. Western blotting measured the ratio of autophagy-related proteins Beclin1, P62 and LC3-II./LC3-I. in cells. Results With the increase of VCX concentration, the proliferative activity of MUTZ-1 cells decreased significantly, and it was concentration-dependent. Compared with the control group (4.28%±1.66%), the apoptosis rate of VCX group (13.75%±3.02%), DAC group (12.39%±4.16%) and VCX+DAC group (18.10%±3.50%) was significantly higher, and the difference was statistically significant (F=45.782, P<0.05). Compared with the control group (1.01±0.02, 1.04±0.02, 1.01±0.04), the VCX group (1.67±0.05, 2.23±0.10, 0.43±0.05), the DAC group (1.62±0.08, 1.85±0.06, 0.49±0.07). In the VCX+DAC group (3.24±0.10, 3.81±0.19, 0.13±0.01), apoptosisrelated protein cytochrome C, cleaved Caspase-3 expression and Bcl-2/Bax ratio were increased significantly, the differences were statistically significant (F=116.384，282.069，248.035，all P<0.05). Compared with control group (2.05±0.34, 8.78±1.37), VCX group (8.72±1.26, 14.02±1.45), DAC group (8.44±2.13, 13.20±2.41), VCX+DAC group (15.66±2.90, 26.45±1.53) mitochondrial membrane potential and ROS contents were significantly increased，and the differences were statistically significant (F=66.782，69.071，all P<0.05). Compared with control group (1.05±0.04, 1.02±0.08, 1.01±0.07), VCX group (1.62±0.15, 2.60±0.19, 0.56±0.15), DAC group (1.67±0.17, 2.45±0.20, 0.54±0.14), in VCX+DAC group (3.72±0.21, 3.58±0.27, 0.13±0.09), the expression of autophagy related protein Beclin1, LC3- Ⅱ /LC3- Ⅰ was significantly increased, while the expression of P62 protein was significantly decreased, with statistical significance (F=118.257，209.422，236.92，all P<0.05). Compared with DAC group, apoptosis rate, cytochrome C, cleaved Caspase-3, Beclin1 protein expression level, LC3- Ⅱ /LC3- Ⅰ ratio and ROS content in VCX+DAC group were significantly decreased (t=2.473, 28.564, 17.291, 16.115, 7.021, 9.319). Mitochondrial membrane potential, Bcl-2/Bax ratio and P62 protein expression were significantly increased (t=4.621, 9.244, 4.278), the differences were statistically significant, respectively (all P<0.05). There was no statistical significance in the above indexes between DAC group and VCX group (all P>0.05). Conclusion VCX may promote chemotherapy sensitivity of MDS cells to DAC by regulating apoptosis, autophagy, and oxidative stress.

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备注/Memo

备注/Memo:
基金项目：山东省医药卫生科技发展计划项目（编写：2017WS137）：戊乙奎醚对脓毒症诱发的肺损伤的保护作用及其机制研究。
作者简介：曲志梅（1971-），女，学士学位，主管药师，研究方向；药理学基础实验，E-mail：CC9671a@163.com。
更新日期/Last Update: 2023-05-15