紫杉醇与顺铂联合化疗对宫颈鳞癌细胞株HCE1作用的实验研究

目的 观察紫杉醇加顺铂联合化疗在宫颈鳞癌细胞株HCE1中的协同效应,指导进一步临床应用.方法 (1)培养宫颈鳞癌细胞株HCE1,观察活细胞生长情况并摄片.(2)实验分组及检测:①终浓度为20 μmol/L、40 μmol/L、80 μmol/L的紫杉醇培养HCE1细胞;②终浓度为20 μg/ml、40 μg/ml、80 μg/ml顺铂培养HCE1细胞;③紫杉醇联合顺铂培养HCE1细胞,通过MTT比色法检测培养24 h、48 h、72 h后细胞增殖活力,并计算细胞生长抑制率;流式细胞仪分别检测24 h、48 h、72 h三个不同时间点的细胞凋亡发生率,并分析细胞周期改变.结果 (1)紫杉醇处理后凋亡细胞明显增多,细胞缩小变圆,细胞膜出泡等.(2)经终浓度分别为20 μmol/L、40 μmol/L、80 μmol/L紫杉醇处理HCE1细胞24 h时,细胞抑制率分别为(9.5±0.66)%、(17.1±1.51)%、(33.3±1.77)%,48 h时,细胞抑制率分别为(28.0±2.27)%、(45.2±3.15)%、(66.0±2.95)%,72 h时,细胞抑制率分别为(39.4±2.81)%、(66.2±7.02)%、(81.5±1.78)%;经终浓度分别为20 μg/ml、40 μg/ml、80 μg/ml顺铂处理HCE1细胞24 h时,细胞抑制率分别为(3.6±0.56)%、(6.5±0.98)%、(14.1±2.52)%,48 h时,细胞抑制率分别为(6.5±0.46)%、(9.9±1.35)%、(20.0±3.05)%,72 h时,细胞抑制率分别为(14.1±3.05)%、(42.1±3.90)%、(59.4±4.26)%;联合处理组以48 h 20 μmol/L紫杉醇联合20 μg/ml顺铂对HCE1细胞的增殖抑制作用为例,细胞增殖抑制率为(30.2±3.34)%,综上可见紫杉醇和顺铂以时间和剂量依赖性特点抑制宫颈癌细胞的增殖,紫杉醇联合顺铂对人宫颈癌HCE1细胞的增殖抑制具有协同作用.(3)流式细胞仪细胞周期分析表明,紫杉醇处理组G1期细胞比例有所增加,S期细胞比例明显降低,顺铂处理组G1期细胞比例明显降低,反之S期细胞比例明显升高,紫杉醇联合顺铂处理组G1期细胞比例和S期细胞比例介于紫杉醇处理组和顺铂处理组之间.以20 μmol/L紫杉醇作用HCE1细胞48 h为例,G1期细胞比例和S期细胞比例分别为(86.0±3.01)%、(9.1±0.66)%,40 μg/ml顺铂作用HCE1细胞48 h G1期细胞比例和S期细胞比例分别为(2.1±0.26)%、(92.2±6.24)%,20 μmol/L紫杉醇联合40 μg/ml顺铂作用HCE1细胞48 h G1期细胞比例和S期细胞比例分别为(13.2±0.78)%、(80.5±2.46)%.(4)细胞凋亡分析表明,紫杉醇处理组细胞凋亡率较对照组升高,呈时间和剂量依赖性特点;顺铂处理组细胞凋亡率较对照组也升高,呈时间和剂量依赖性特点;紫杉醇联合顺铂处理组细胞凋亡率较单药处理组高.结论 紫杉醇与顺铂联合对宫颈癌细胞的抑制作用明显增强,提示紫杉醇可与顺铂对宫颈癌细胞增殖的抑制产生协同作用.

Experimental study of effect of paclitaxei combined with cisplatin on cervical carcinoma cell line HCE1 cell

Objective To study cervical carcinoma cell line HCE1 cell in the therapy of chemotherapy regimen of paclitaxel combined with cisplatin treated on. Methods （ 1 ） The human cervical carcinoma cell line HCE1 cells were cultured. （2）Essay grouping and measure： HCE1 cells were cultured and treated by paelitaxel with 20 μmol/L,40μmoL/L and 80 μmoL/L concentrations for 24 h respectively. Cell viability were measured by MIT assay, At the same time,HCE1 cells were cultured and treated by paelitaxel with 20μmol/L,40 μmol/L and 80 p, mol/L concentration for 24,48 and 72 h,by cisplatin with 20 μg/ml,40 μg/ml and 80 μg/ml concentration for 24,48 and 72 h, by paelitaxel combined with cisplatin for 24,48 and 72 h, the effect of paelitaxel on HCE1 cells were analyzed by MTF assay,respectively. HCE1 celis were cultured and treated by paelitaxel with 20 μmol/L,40 p, mol/L and 80 μmol/L concentrations for 24,48,72 h,the apoptotic rate of HCE1 cell were analyzed by flow cytometry after PI staining,respectively. Treated with 20 μmol/L,40 μmol/L and 80 μmol/L paelitaxel, 20 μg/ml, 40 μg/ml and 80 μg/ml cisplatin, paelitaxel combined with cisplatin for 24,48 and 72 h, the apoptotic rate and cell cycle distribution were analyzed by flow cytometry after PI staining, respectively. Results （ 1 ） HCE1 cells became round, small, cell shrinkage ＇,membrane blebbing and so on,after exposed to paelitaxel. （2）After treated with paelitaxel with concentration about 20 μmol/L, 40μmol/L, 80 μmol/L for 24 h, HCE1 cell inhibition rate discern was （ 9.5 ± O. 66） %, （ 17. 1 ± 1.51 ） % , （33.3 ± 1.77 ） % , after 48 h, HCE1 cell inhibition rate discern was （ 28.0 ± 2. 27 ） % , （45.2 ±3. 15 ） %, （ 66.0 ± 2. 95 ） %, after 72 h, HCE1 cell inhibition rate discern was （ 39.4 ± 2. 81 ） % , （ 66.2 ± 7.02 ） % , （81. 5 ± l. 78 ） %. After treated with eisplatin with concentration about 20 μg/m1,40 μg,/ml and 80 μg/ml for 24 h, HCE1 cell inhibition rate discern was （ 3.6 ± 0. 56 ） %, （ 6. 5 ± 0. 98 ） % , （ 14. 1 ± 2. 52 ） % , after 48 h, HCEI cell inhibition rate discern was（6. 5±0.46）%, （9. 9± 1.35）%, （20. 0±3.05）% ,after 72 h, HCE1 cell inhibition rate discern was（ 14. 1± 3.05）% , （42. 1 ± 3.90）% , （59.4 ± 4. 26）%. After treated with 20 μmol/L paelitaxel combined with 20 μg/ml cisplatin, HCE1 cell inhibition rate discern was （ 30.2 ±3.34 ） % , MTT assays showed that paelitaxel inhibited the growth of the cervical carcinoma cells by concentration and time dependent, and synergistic effect appeared when combined with cisplatin. （3）Flow cytometry results demonstrated that the cell cycle was redistributed： the G1-Phase cell fraction was conspicuous increased while the S-Phase cell fraction was significantly decreased after the cells were treated with paelitaxel（ P 〈 0.05 ）. However, the result was opposite after treated with eisplatin. When treated with both of them, GI -Phase and S-Phase cell fraction were between the values of single agent. For example, after treated with 20 μmol/L paelitaxel for 48 h, the G1-Phase cell fraction and the S- Phase cell fraction were （86.0 ± 3.01 ） % and （9. 1 ± 0. 66） % , after treated with 40 μg/ml cisplatin for 48 h, the G1- Phase cell fraction and the S-Phase cell fraction were （ 2. 1 ± O. 26 ） % and （92. 2 ± 6. 24 ） % , after treated with 20 μmol/L paelitaxel combine with 40 μg/ml cisplatin for 48 h,the GI-Phase cell fraction and the S-Phase cell fraction were（ 13.2 ±0. 78 ） % and （80. 5 ± 2. 46） %. （4） Both of paelitaxel and cisplatin could induce apoptosis in cervical carcinoma cells by a time and dose dependent way. The apoptotic index were significantly increased after treated with paelitaxel combined with cisplatin. Conclusions The apoptotic index is significantly increased after treated with paelitaxel combined with cisplatin, suggesting that the combination of them could exert synergistic antiproliferative effect on cervical carcinoma cells.