【摘要】：目的：通過研究亞細胞毒性劑量的化療藥物吡柔比星（THP）和腫瘤壞死因子相關的凋亡誘導配體（TRAIL）對前列腺癌體外生長的影響，探討兩藥單獨用藥和聯(lián)合用藥對人前列腺癌細胞Du145體外生長的影響，為TRAIL抗腫瘤作用的臨床應用提供一個新的實驗依據(jù)，可能為激素難治性前列腺癌的治療尋求一種新的方案。 方法：常規(guī)方法培養(yǎng)前列腺癌細胞Du145，給予不同濃度的THP和TRAIL，分為單用THP組、單用TRAIL組、THP+TRAIL聯(lián)合組、對照組和空白組。各組影響因素作用24小時后，采用MTT比色法測定不同藥物濃度作用后，各組細胞生長抑制率，同時選定THP的亞毒性劑量。倒置相差顯微鏡下觀察各組細胞形態(tài)學變化以及生長情況，再采用Annexin V/PI雙染色法進行流式細胞術(shù)檢測前列腺癌細胞Du145的凋亡率。各組藥物刺激后，提取Du145細胞胞漿蛋白，，采用Western-Blot法進行蛋白印跡顯影，檢測各組細胞胞漿蛋白內(nèi)caspase-3的表達。 結(jié)果： 1、THP和TRAIL對人前列腺癌細胞株Du145均有抑制生長作用，THP的抑制作用較強，Du145細胞對其相對敏感。TRAIL雖有抑制作用，但Du145細胞對TRAIL并不敏感。 2、亞毒性劑量的THP聯(lián)合不同藥物濃度的TRAIL對Du145細胞有較強的抑制作用，并且呈現(xiàn)出較高的協(xié)同作用，其協(xié)同因子均大于1,最大協(xié)同因子CF=1.38807。 3、倒置相差顯微鏡下觀察不同藥物濃度刺激下，細胞出現(xiàn)不同程度的死亡。見附圖。 4、流式細胞術(shù)檢測發(fā)現(xiàn)藥物刺激后出現(xiàn)不同程度的凋亡，THP+TRAIL聯(lián)合組凋亡率最高。同時證明了協(xié)同殺傷作用是通過誘導細胞凋亡實現(xiàn)的。 5、采用Western-Blot法檢測藥物刺激后Du145細胞胞漿蛋白內(nèi)caspase-3蛋白的表達，顯影后可見THP+TRAIL聯(lián)合組刺激后的細胞胞漿蛋白內(nèi)caspase-3蛋白明顯高于單用藥物組和對照組。見附圖。 結(jié)論：THP和TRAIL對人前列腺癌細胞株Du145的生長均有抑制作用，但Du145細胞對TRAIL并不敏感。亞細胞毒性的THP聯(lián)合TRAIL可提高對Du145細胞的抑制作用。亞細胞毒性的THP聯(lián)合TRAIL作用于Du145細胞，表現(xiàn)出明顯的高誘導細胞凋亡作用，增加了Du145細胞凋亡率。亞細胞毒性的THP聯(lián)合TRAIL作用于Du145細胞所表現(xiàn)的高誘導細胞凋亡作用可能與細胞胞漿蛋白中caspase3蛋白的高表達有關。
[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.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.25

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