【作者】 李炎生；

【导师】 肖亚军；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 第一部分良性永生化前列腺上皮细胞LH/LE对TRAIL不同敏感性及其机制研究目的肿瘤坏死因子相关凋亡诱导配体(Tumor necrosis factor-related apoptosis-inducing ligand, TRAIL)可选择性诱导某些肿瘤细胞凋亡,而对绝大多数正常细胞无毒性作用,这一特性使TRAIL在抗肿瘤治疗中具有很好的应用前景。本研究是为了明确良性永生化前列腺上皮细胞LH／LE对TRAIL的不同敏感性及其机制。方法正常前列腺上皮细胞通过导入猿猴空泡病毒40(SV40)大T抗原和端粒酶末端转移酶催化亚单位(hTERT),使其永生化,得到LH细胞；在这基础上又导入SV40小T抗原片段,得到LE细胞。TRAIL作用于这两种细胞后,MTT法检测细胞活力,显微镜下观察细胞形态学改变和流式细胞术检测细胞凋亡状态。Western blot检测PP2A和c-Fos的蛋白表达。TRAIL处理LH和LE细胞后检测PP2A酶活性。SPSS 13.0软件进行统计学分析。结果LH细胞对TRAIL耐受,LE细胞对TRAIL敏感。TRAIL作用后大约45%的LE细胞发生凋亡,而TRAIL作用后的LH细胞发生凋亡的比例很低。Western blot结果显示,PP2A蛋白表达量在LH细胞与LE细胞间未见明显差异,而PP2A的酶活性在LH细胞中明显高于LE; c-Fos蛋白表达在LE细胞中明显高于LH细胞。结论SV40 ST赋予了良性永生化前列腺上皮细胞LE细胞TRAIL敏感性表型。第二部分奥克代酸诱导TRAIL。耐受的良性永生化前列腺上皮细胞LH细胞获得TRAIL敏感表型的研究目的研究奥克代酸(Okadaic acid, OA)诱导TRAIL耐受的LH细胞获得TRAIL敏感性及其机制。方法蛋白磷酸酶2A(PP2A)抑制剂OA处理TRAIL耐受的良性永生化前列腺上皮细胞LH细胞后,TRAIL作用于处理组和对照组,MTT法检测细胞活力,细胞形态学和流式细胞术染色检测细胞凋亡状态。Western blot检测4PP2A和·c-Fos的蛋白表达。酶活性试剂盒检测PP2A酶活性。用SPSS 13.0进行统计学分析。结果显示经OA诱导后的LH细胞对TRAIL敏感,细胞形态学可见大量的细胞发生凋亡,流式细胞检测显示经OA诱导TRAIL作用后的LH细胞有大约32%的细胞发生凋亡,而未经OA诱导TRAIL作用后的LH细胞中,凋亡细胞的比例很低。Western blot结果显示,经OA诱导后LH细胞中PP2A蛋白表达量明显减少,PP2A酶活性明显下降,而c-Fos蛋白表达量经OA诱导后明显增多。结论OA诱导TRAIL耐受的LH细胞获得TRAIL敏感表型。第三部分A-Fos逆转奥克代酸诱导TRAIL耐受的LH细胞向TRAIL敏感转化的研究目的明确c-Fos在良性永生化前列腺上皮细胞对TRAIL敏感性中的作用及其机制。方法A-Fos质粒转染LH细胞,奥克代酸(Okadaic acid, OA)处理后,TRAIL作用于各实验组,MTT法检测细胞活力,细胞形态学和流式细胞术检测细胞凋亡状态。Western blot检测PP2A和c-Fos的蛋白表达。用SPSS 13.0进行统计学分析。结果MTT法检测细胞活力显示,经OA诱导后的LH细胞对TRAIL敏感,转染A-Fos质粒的LH细胞虽然经OA诱导但对TRAIL敏感性下降；流式细胞术显示：未经OA诱导TRAIL作用后的LH细胞中凋亡细胞的比例很低,经OA诱导TRAIL作用后的LH细胞后有大约51%的细胞发生凋亡；而经OA诱导TRAIL作用后,转染A-Fos质粒的LH细胞的细胞凋亡率低于未转染A-Fos质粒的LH细胞；Western blot结果显示,经OA诱导后未转染组和A-Fos转染组中PP2A蛋白表达量明显减少,而c-Fos蛋白表达量增多；且A-Fos转染组中c-Fos蛋白表达量低于未转染组。结论OA诱导TRAIL耐受的LH细胞获得TRAIL敏感表型,其机制可能通过减少PP2A酶表达,抑制PP2A酶活性,增加c-Fos表达等途径发挥作用。而A-Fos可能通过抑制c-Fos表达,从而逆转OA诱导TRAIL耐受的LH细胞向TRAIL敏感转化。c-Fos作为促凋亡因素,是TRAIL诱导凋亡所必须的。更多还原

【Abstract】 PART 1:Sensitivity and mechanism of the benign prostatic immortalized cells to TRAIL-induced apoptosisObjective Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it induces apoptosis in cancer cells but spares most normal cells. To determine whether benign prostatic immortalized cells have variable sensitivity to TRAIL-induced apoptosis and explore the possible mechanism.Methods Normal prostatic epithelial cells immortalized by combination of the early region of simian virus 40 large T antigen and a catalytic subunit of telomerase (hTERT) and then followed by the early region of simian virus 40 small T antigen (LE) were treated with different doses of TRAIL. MTT assay was used to detect the cell viability. Flow cytometry and cell morphology were performed to examine apoptotic percentages of treated cells. Western blot were used to detect the expression of PP2A and c-Fos. The PP2A activity of LH and LE cells were determined by phosphatase assays after treatment with TRAIL.Statistic analyses were performed using SPSS software version 13.0.Results After treated with increasing doses of TRAIL, LH cells were resistant to TRAIL, whereas LE cells were sensitive to TRAIL. Nearly 45% of LE cells had undergone apoptosis after administrated with TRAIL, whereas the LH cells showed much lower percentages of cell death. Western blot results revealed that PP2 A protein expressions showed no significant differences between LH cells and LE cells with/without TRAIL treatment; the protein expression of c-Fos/AP-1 significantly increased in TRAIL-sensitive LE cells.There is decreased PP2A activity in LE cells compared to LH cells. And the PP2A activity in both LH and LE cells did not change when treated with TRAIL.Conclusion SV40 ST endues the immortalized benign prostatic epithelial cells LE cells with TRAIL-sensitive phenotype. PART 2:Okadaic acid Endues the TRAIL-resistant Benign Immortalized Prostatic Epithelial Cells LH with TRAIL-sensitive PhenotypeObjective To determine whether okadaic acid changes LH cells from TRAIL-resistant to TRAIL-sensitive and its possible mechanism.Methods The TRAIL-resistant Benign Immortalized Prostatic Epithelial Cells (LH) were treated with different doses of TRAIL after treated by Okadaic acid, an inhibitor of PP2A. MTT assay was used to detect the cell viability. Flow cytometry and cell morphology were performed to examine apoptotic percentages of treated cells. Western blot were used to detect the expression of PP2A and c-Fos. The PP2A activity of LH and LE cells were determined by phosphatase assays after treatment with TRAIL. Statistic analyses were performed using SPSS software version 13.0.Results OA combined with TRAIL significantly increased apoptosis in the TRAIL-resistant LH cells as shown by Annexin V and cell viability assays. Western blot results revealed that OA alone or OA combined with TRAIL not only decreased the protein expression of PP2A, but also increased the protein expression of c-Fos. In accordance with our immunoblot findings, OA alone or OA combined with TRAIL decreased PP2A activity.Conclusion Okadaic acid endued the TRAIL-resistant Benign Immortalized Prostatic Epithelial Cells LH with TRAIL-sensitive Phenotype. PART 3:A-Fos converts the LH cells from a relatively TRAIL-sensitive to a TRAIL-resistant phenotype after treatment by OA combined with TRAILObjective To explore the effect of c-Fos on the benign prostatic immortalized cells sensitive to TRAIL and its possible mechanism.Methods The TRAIL-resistant Benign Immortalized Prostatic Epithelial Cells (LH) were treated with OA, TRAIL or in combination with OA and TRAIL after transfection with/without the A-Fos vector. MTT assay was used to detect the cell viability. Flow cytometry and cell morphology were performed to examine apoptotic percentages of treated cells. Western blot were used to detect the expression of PP2A and c-Fos. Statistic analyses were performed using SPSS software version 13.0.Results OA did not enhance the ability of TRAIL to promote apoptosis like before.Western blot results revealed that the activity of c-Fos/AP-1 was inhibited by the dominant negative form A-Fos.Conclusion ectopic expression of the dominant-negative form of c-Fos, A-Fos, converted the LH cells from a relatively TRAIL-sensitive to a TRAIL-resistant phenotype after treatment by OA combined with TRAIL.更多还原