亚毒性剂量THP联合TRAIL对前列腺癌细胞Du145体外生长影响的研究

发布时间：2018-11-05 19:09

【摘要】：目的：通过研究亚细胞毒性剂量的化疗药物吡柔比星（THP）和肿瘤坏死因子相关的凋亡诱导配体（TRAIL）对前列腺癌体外生长的影响，探讨两药单独用药和联合用药对人前列腺癌细胞Du145体外生长的影响，为TRAIL抗肿瘤作用的临床应用提供一个新的实验依据，可能为激素难治性前列腺癌的治疗寻求一种新的方案。方法：常规方法培养前列腺癌细胞Du145，给予不同浓度的THP和TRAIL，分为单用THP组、单用TRAIL组、THP+TRAIL联合组、对照组和空白组。各组影响因素作用24小时后，采用MTT比色法测定不同药物浓度作用后，各组细胞生长抑制率，同时选定THP的亚毒性剂量。倒置相差显微镜下观察各组细胞形态学变化以及生长情况，再采用Annexin V/PI双染色法进行流式细胞术检测前列腺癌细胞Du145的凋亡率。各组药物刺激后，提取Du145细胞胞浆蛋白，，采用Western-Blot法进行蛋白印迹显影，检测各组细胞胞浆蛋白内caspase-3的表达。结果： 1、THP和TRAIL对人前列腺癌细胞株Du145均有抑制生长作用，THP的抑制作用较强，Du145细胞对其相对敏感。TRAIL虽有抑制作用，但Du145细胞对TRAIL并不敏感。 2、亚毒性剂量的THP联合不同药物浓度的TRAIL对Du145细胞有较强的抑制作用，并且呈现出较高的协同作用，其协同因子均大于1,最大协同因子CF=1.38807。 3、倒置相差显微镜下观察不同药物浓度刺激下，细胞出现不同程度的死亡。见附图。 4、流式细胞术检测发现药物刺激后出现不同程度的凋亡，THP+TRAIL联合组凋亡率最高。同时证明了协同杀伤作用是通过诱导细胞凋亡实现的。 5、采用Western-Blot法检测药物刺激后Du145细胞胞浆蛋白内caspase-3蛋白的表达，显影后可见THP+TRAIL联合组刺激后的细胞胞浆蛋白内caspase-3蛋白明显高于单用药物组和对照组。见附图。结论：THP和TRAIL对人前列腺癌细胞株Du145的生长均有抑制作用，但Du145细胞对TRAIL并不敏感。亚细胞毒性的THP联合TRAIL可提高对Du145细胞的抑制作用。亚细胞毒性的THP联合TRAIL作用于Du145细胞，表现出明显的高诱导细胞凋亡作用，增加了Du145细胞凋亡率。亚细胞毒性的THP联合TRAIL作用于Du145细胞所表现的高诱导细胞凋亡作用可能与细胞胞浆蛋白中caspase3蛋白的高表达有关。
[Abstract]:Aim: to investigate the effects of subcytotoxic dose of pirarubicin (THP) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the growth of prostate cancer in vitro. To explore the effect of two drugs on the growth of human prostate cancer cell line Du145 in vitro, and to provide a new experimental basis for the clinical application of TRAIL anti-tumor effect, and to seek a new scheme for the treatment of steroid refractory prostate cancer. Methods: prostate cancer cells Du145, were treated with different concentrations of THP and TRAIL,. They were divided into three groups: single THP group, TRAIL group combined with, THP TRAIL group, control group and blank group. The cell growth inhibition rate and the subtoxic dose of THP were determined by MTT colorimetry. The morphologic changes and growth of prostate cancer cells were observed under inverted phase contrast microscope. The apoptosis rate of prostate cancer cell line Du145 was detected by flow cytometry with Annexin V/PI double staining. The cytoplasmic proteins of Du145 cells were extracted after drug stimulation, and the expression of caspase-3 in cytoplasmic proteins was detected by Western-Blot assay. Results: 1 both THP and TRAIL could inhibit the growth of human prostate cancer cell line Du145, THP had a strong inhibitory effect, and Du145 cells were relatively sensitive to it. Although TRAIL had inhibitory effect, Du145 cell was not sensitive to TRAIL. 2. The subtoxic dose of THP combined with different concentrations of TRAIL has a strong inhibitory effect on Du145 cells, and shows a high synergistic effect, its synergistic factors are all greater than 1, and the maximum synergistic factor CF=1.38807. is more than 1. 3. The cell death was observed under inverted phase contrast microscope under different drug concentration. See attached figure. 4. Flow cytometry showed that the apoptotic rate of, THP TRAIL combined with drug stimulation was the highest. At the same time, it is proved that the synergistic killing effect is achieved by inducing cell apoptosis. 5. Western-Blot assay was used to detect the expression of caspase-3 protein in cytoplasmic protein of Du145 cells after drug stimulation. The expression of caspase-3 protein in cytoplasmic protein of the combined THP TRAIL group was significantly higher than that of the single drug group and the control group. See attached figure. Conclusion: both THP and TRAIL can inhibit the growth of human prostate cancer cell line Du145, but Du145 cells are not sensitive to TRAIL. Subcytotoxic THP combined with TRAIL increased the inhibitory effect on Du145 cells. The effect of subcytotoxic THP combined with TRAIL on Du145 cells showed highly induced apoptosis and increased the apoptosis rate of Du145 cells. The highly induced apoptosis of Du145 cells induced by subcytotoxic THP combined with TRAIL may be related to the high expression of caspase3 protein in cytoplasmic proteins.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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