左布比卡因对子宫内膜癌细胞RL-952生长和线粒体膜电位的影响

Effects of levobupivacaine on the growth and mitochondrial membrane potential of endometrial cancer cells RL-952

目的研究左布比卡因对子宫内膜癌细胞RL-952增殖、凋亡和线粒体膜电位(MMP)的影响。方法将正常培养的RL-952细胞记为对照组,低、中、高剂量实验组用含100、200、400μmol·L^(-1)左布比卡因的培养液孵育RL-952细胞。通过细胞计数试剂盒、集落形成实验、JC-1法、流式细胞术检测布比卡因对RL-952细胞活力、克隆形成、线粒体膜电位(以红/绿荧光强度比值表示)以及凋亡的影响。用蛋白质印迹法检测Ki-67、增殖细胞核抗原(PCNA)、B细胞淋巴瘤-2(Bcl-2)、剪切的胱天蛋白酶-3(Cl-caspase-3)和Bcl-2相关X蛋白(Bax)蛋白水平。结果对照组和低、中、高剂量实验组RL-952细胞克隆形成数分别为(270.75±21.49)、(219.93±21.35)、(143.87±15.72)和(111.15±13.39)个;红/绿荧光强度比值分别为(100.00±4.86)%、(71.10±4.18)%、(58.39±7.28)%和(39.14±5.62)%;凋亡率分别为(4.12±0.67)%、(10.34±0.77)%、(15.95±1.32)%和(24.09±2.74)%。与对照组比较,低、中、高剂量实验组RL-952细胞活力(48 h、72 h)、克隆形成数、红/绿荧光强度比值以及Ki67、PCNA和Bcl-2蛋白水平显著降低(均P<0.05),凋亡率以及Cl-caspase-3、Bax蛋白水平显著升高(均P<0.05)。结论左布比卡因可降低子宫内膜癌细胞RL-952的线粒体膜电位水平,从而抑制细胞增殖并诱导凋亡。

Objective To explore the influence of levobupivacaine on the proliferation,apoptosis and mitochondrial membrane potential(MMP)of endometrial cancer cells RL-952.Methods The normal RL-952 cells were recorded as the control group,the low,medium and high dose experimental groups were incubated with medium containing 100,200,400μmol·L^(-1)levobupivacaine.The effect of bupivacaine on RL-952 cells viability,colony formation,mitochondrial membrane potential(expressed as the ratio of red/green fluorescence intensity),and apoptosis were detected by cell counting kit,colony formation assay,JC-1 method and flow cytometry,respectively.Western blotting were performed to calculate the levels of Ki-67,proliferating cell nuclear antigen(PCNA),B cell lymphoma-2(Bcl-2),cleaved-cysteine aspartic acid specific protease-3(Cl-caspase-3)and Bcl-associated X protein(Bax)protein.Results The clones formed number of RL-952 cell in control group,low,medium and high dose experimental groups were 270.75±21.49,219.93±21.35,143.87±15.72 and 111.15±13.39;the ratios of red/green fluorescence intensitywere(100.00±4.86)%,(71.10±4.18)%,(58.39±7.28)%and(39.14±5.62)%;the apoptosis rates were(4.12±0.67)%,(10.34±0.77)%,(15.95±1.32)%and(24.09±2.74)%.Compared with the control group,the RL-952 cell viability(48 h,72 h),the colony formation number,the ratio of red/green fluorescence intensity and the levels of Ki-67,PCNA and Bcl-2 protein in the low,medium and high dose experimental groups were significantly decreased(all P<0.05);the apoptosis rate and the levels of Cl-caspase-3 and Bax proteins were significantly increased(all P<0.05).Conclusion Levobupivacaine could reduce the mitochondrial membrane potential level of endometrial cancer cells RL-952,thereby inhibiting cell proliferation and inducing apoptosis.