洛铂对人肝癌细胞HepG2增殖及凋亡作用的实验研究

目的 探讨洛铂（LBP）对人肝癌HepG2细胞增殖、凋亡的影响及其可能机制。方法取对数生长期HepG2细胞，分别采用不同浓度LBP（0、2.5、5、10、20 μmol／L）处理48 h。普通光镜观察细胞形态学改变；MTS法检测细胞增殖，并计算半数抑制浓度（IC50）；Annexin Ⅴ-FITC／PI双染法及Hoechst 33258染色检测细胞凋亡；Western blotting检测Bax、Bak、Bcl-2、Bid、Bcl-XL、Survivin及PARP-1蛋白表达。结果 随着LBP浓度的升高HepG2细胞密度减少，离巢死亡细胞数目增多；光镜下可见典型核固缩、碎裂的凋亡细胞。LBP能显著抑制HepG2细胞增殖，且呈浓度和时间依赖性（P＜0.05）；2.5、5、10、20 μmol／L的LBP作用HepG2细胞48 h后的增殖率分别为（92.11±1.79）％、（65.87±1.78）％、（51.57±0.81）％及（33.11±1.47）％； LBP作用HepG2细胞48 h的IC50为13.28 μmol／L。2.5、5、10、20 μmol／L LBP作用48 h HepG2细胞的凋亡率分别为（11.64±0.85）％、（20.99±2.21）％、（33.02±2.30）％和（40.77±1.58）％，与LBP 0 μmol／L 的（3.29±0.43）％比较，差异有统计学意义（P＜0.05）。2.5、5、10、20 μmol／L LBP能够下调HepG2细胞中的Bcl-2、Mcl-1蛋白表达，上调Bax、Bid、PARP-1蛋白表达， 对Bcl-XL、Bak及Survivin蛋白表达无影响。结论 LBP能够诱导HepG2细胞凋亡、抑制细胞增殖，其可能机制与上调Bax、Bid和PARP-1蛋白以及下调Bcl-2、Mcl-1蛋白表达有关。

Experimental study of lobaplatin on the effect of the proliferation and apoptosis of human hepatocellular cercinoma cell line HepG2

Objective To investigate the effect of lobaplatin （LBP） on proliferation and apoptosis of human hepatic cancer cell line HepG2 and possible mechanisms. Methods Logarithmic growth phase HepG2 cells were used for study. Different concentrations of LBP （0， 2.5， 5， 10， 20 μmol／L） treated HepG2 cells for 48 hours. General morphological changes were observed under an optical microscope. MTS assay was used to detect the relative viability of HepG2 cells， and IC50 （50％ inbibiting concentration） was calculated. Hoechst 33258 staining and flow cytometry based on Annexin V-PI double staining were used to measure the apoptosis of HepG2 cells. Western blotting assay was used to detect the expression changes of Bax， Bak， Bcl-2， Bcl-XL，Mcl-1， Survivin and PARP-1 protein. Results Cell morphological observation showed LBP treated HepG2 cells for 48 hours with the increase of drug concentration the HepG2 cell density decreased， the number of swelling cytoplasm and floating rounded cells increased significantly. Hoechst33258 staining to test the typical apoptotic cells with nuclear fragmentation or nuclear condensation showed that LBP induced apoptosis， in a dose-dependent manner. MTS assay indicated LBP greatly inhibited HepG2 cell growth in a dose- and time-dependent manner. The proliferation rates of HepG2 cells treated with 2.5, 5, 10, 20 μmol/L LBP for 48 h were （92.11±1.79）％， （65.87±1.78）％，（51.57±0.81）％ and （33.11±1.47）％. After LBP treatment HepG2 cell for 48 hours the value of IC50 was 13.28 μmol／L. The apoptotic rates of HepG2 cells treated with 2.5, 5, 10, 20 μmol/L LBP for 48 h were （11.64±0.85）%, （20.99±2.21）%, （33.02±2.30）% and （40.77±1.58）%. The difference was significant between LBP treat group and control group （P＜0.05）. Western blotting analysis showed that 2.5, 5, 10, 20 μmol/L LBP down-regulated Bcl-2 and Mcl-1 protein expression， whereas upregulated Bax， Bid and PARP-1 expression. Bcl-XL， Bak and Survivin protein expression was not changed. Conclusion LBP significantly exerts anti-cancer effects through inducing cell growth inhibition and apoptosis， the possible mechanism is related to the regulation of apoptosis related proteins including Bax， Bid， Bcl-2 and Mcl-1.