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目的探讨四个半LIM结构域蛋白1(FHL1)对肝癌细胞紫杉醇耐药的作用机制。方法按照FHL1的表达状态,设置敲低FHL1表达的肝癌细胞株为实验组,表达FHL1的肝癌细胞株为对照组,分别给予紫杉醇药物处理,并同时以奥沙利铂作为实验参照。细胞活性测定不同表达的细胞活性的改变,细胞克隆实验检测不同表达细胞的克隆形成。实验组和对照组的细胞克隆接种于裸鼠,并给予紫杉醇治疗,观察两组的成瘤情况以及对紫杉醇的反应。进一步的凋亡蛋白Caspase-3活性测定实验分别对两组Caspase-3活性进行测定。结果紫杉醇药物处理后,实验组和对照组的细胞活性分别为0.41±0.02和0.70±0.02(HepG2细胞);0.44±0.02和0.63±0.03(SMMC 7721细胞),差异均有统计学意义(均P<0.05),且加入奥沙利铂后,实验组和对照组的细胞活性分别为0.70±0.02和0.71±0.02(HepG2细胞);0.64±0.02和0.63±0.03(SMMC 7721细胞),差异无统计学意义(P>0.05)。实验组与对照组的克隆形成计数分别为93.52±14.58和302.48±21.56(HepG2细胞);55.14±10.82和192.28±19.47(SMMC 7721细胞),差异均有统计学意义(均P<0.05);在裸鼠实验中,实验组与对照组肿瘤体积分别为0.02±0.02和1.89±0.51,差异统计学意义(P<0.05)。此外,Caspase-3活性实验显示,实验组与对照组Caspase-3表达分别为10.27±0.47和26.72±0.54,差异有统计学意义(P<0.05);加入抑制药Z-VAD-FMK后,实验组与对照组Caspase-3表达分别为8.87±0.36和29.96±0.68,差异有统计学意义(P<0.05)。结论 FHL~(-1)是紫杉醇耐药基因,FHL~(-1)敲低后,紫杉醇的抗肿瘤作用增强,促进Caspase-3激活,紫杉醇诱导的细胞凋亡作用增强。
Objective To investigate the mechanism of four and a half LIM domain protein 1 (FHL1) on paclitaxel resistance in hepatocellular carcinoma cells. Methods According to the expression of FHL1, FHL1 knockdown HCC cell lines were selected as experimental group and FHL1-expressing hepatocellular carcinoma cell lines as control group. Paclitaxel was administered to patients with oxaliplatin as control group. Cell viability was measured for changes in cell activity of differently expressed cells. Cell cloning experiments were performed to detect clonogenicity of different expressed cells. Cell clones of the experimental and control groups were inoculated into nude mice and treated with paclitaxel. The tumorigenicity and response to paclitaxel in both groups were observed. Caspase-3 activity assay was further used to determine the activity of Caspase-3 in two groups. Results After paclitaxel treatment, the cell viability of the experimental and control groups were 0.41 ± 0.02 and 0.70 ± 0.02 (HepG2 cells), 0.44 ± 0.02 and 0.63 ± 0.03 (SMMC 7721 cells), respectively, and the differences were statistically significant (P <0.05), and the cell viability of experimental group and control group were 0.70 ± 0.02 and 0.71 ± 0.02 (HepG2 cells); 0.64 ± 0.02 and 0.63 ± 0.03 (SMMC 7721 cells) after addition of oxaliplatin, the difference was not statistically significant Significance (P> 0.05). The colony formation counts of experimental group and control group were 93.52 ± 14.58 and 302.48 ± 21.56 (HepG2 cells), 55.14 ± 10.82 and 192.28 ± 19.47 (SMMC 7721 cells), respectively, with statistical significance (all P <0.05) The nude mice experiment, the experimental group and the control group tumor volume were 0.02 ± 0.02 and 1.89 ± 0.51, the difference was statistically significant (P <0.05). In addition, the Caspase-3 activity assay showed that the expression of Caspase-3 in experimental and control groups was 10.27 ± 0.47 and 26.72 ± 0.54, respectively, with statistical significance (P <0.05). After addition of Z-VAD-FMK, The expression of Caspase-3 in group and control group were 8.87 ± 0.36 and 29.96 ± 0.68 respectively, with significant difference (P <0.05). Conclusions FHL ~ (-1) is a paclitaxel resistance gene. After FHL ~ (-1) knockdown, the antitumor effect of paclitaxel is enhanced, the activation of Caspase-3 is promoted and the paclitaxel-induced apoptosis is enhanced.