【作者】 胡晓勇；

【导师】 陈晓春；

【作者基本信息】 华中科技大学 ， 外科学， 2006， 博士

【摘要】 一、PED/PEA-15在前列腺癌中的表达及意义目的检测PED/PEA-15在人前列腺癌组织和细胞(PC-3)中的表达,探讨PED/PEA-15在前列腺癌中表达的意义。方法应用免疫组化SP法检测前列腺癌和正常前列腺组织中PED/PEA-15的表达;用RT-PCR法检测PC-3细胞中PED/PEA-15的表达。结果免疫组化和RT-PCR均显示PED/PEA-15在前列腺癌中高表达;正常前列腺组织没有或只有少量很弱的表达。结论抗凋亡因子PED/PEA-15的表达对前列腺癌的发展有重要作用。二、PED/PEA-15真核表达载体的构建及对前列腺癌细胞凋亡的影响目的构建PED/PEA-15真核表达载体,探讨PED/PEA-15对PC-3细胞凋亡的影响。方法用RT-PCR法从PC-3中扩增出PED/PEA-15全长基因,以pEGFP-N1为载体构建其真核表达质粒。以脂质体法转染PED/PEA-15真核表达载体至前列腺癌细胞(PC-3)中,用流式细胞和MTT法检测PED/PEA-15对PC-3细胞凋亡和生长的影响。结果酶切和DNA测序证实PED/PEA-15的真核表达载体构建成功。转染PED/PEA-15真核载体的PC-3细胞凋亡下调,且对肿瘤杀伤因子KillerTRAIL的敏感性亦下降。结论抗凋亡因子PED/PEA-15可阻抑前列腺癌细胞(PC-3)的凋亡,PED/PEA-15的表达对前列腺癌的发展有重要作用。三、siRNA沉默PED/PEA-15对前列腺癌细胞凋亡的影响目的通过构建PED/PEA-15特异的siRNA载体,探讨PED/PEA-15对前列腺癌细胞(PC-3)凋亡的影响。方法利用Invivogen公司的软件辅助设计PED/PEA-15特异性siRNA序列并体外合成后,克隆入真核表达载体psiRNA-hH1neo。以脂质体法转染psiRNA-PED/PEA-15至PC-3细胞中,以RT-PCR和Western blot法检测psiRNA-PED/PEA-15对PED/PEA-15表达的影响;用MTT和流式细胞法检测其对PC-3细胞凋亡的影响。结果酶切和DNA测序证实合成的siRNA基因序列正确,并已被准确克隆入psiRNA-hH1neo载体。psiRNA-PED/PEA-15可特异性抑制PC-3细胞中PED/PEA-15的表达。转染psiRNA-PED/PEA-15的PC-3细胞凋亡显著增加(P<0.05)结论PED/PEA-15可阻抑前列腺癌细胞凋亡,PED/PEA-15的表达对前列腺癌的发展有重要作用。四、抗凋亡因子XIAP和PED/PEA-15在前列腺癌(PC-3)中的表达及对细胞凋亡的影响目的检测抗凋亡因子XIAP与PED/PEA-15在前列腺癌细胞(PC-3)中的表达,探讨二者对前列腺癌细胞凋亡的影响。方法应用半定量RT-PCR法检测前列腺癌细胞(PC-3)中PED/PEA-15和XIAP的表达。设计并构建PED/PEA-15和XIAP特异的siRNA载体,以脂质体法转染二者的siRNA载体至前列腺癌细胞(PC-3)中,半定量RT-PCR法检测特异siRNA载体对PED/PEA-15和XIAP转录的影响;光镜观察细胞形态改变;流式细胞法检测细胞凋亡的变化。结果半定量RT-PCR显示PED/PEA-15和XIAP均在前列腺癌细胞(PC-3)中高表达。酶切和DNA测序证实XIAP和PED/PEA-15的siRNA载体构建成功。共转染XIAP和PED/PEA-15的siRNA载体入PC-3细胞,可导致XIAP和PED/PEA-15的转录抑制,并增加PC-3细胞对阿霉素的敏感性凋亡亦明显增加,处理组凋亡率为79%对照组为46%(p<0.05)。结论PED/PEA-15和XIAP在前列腺癌细胞的凋亡中有重要作用。六、Omi/HtrA2促前列腺癌细胞(PC-3M)凋亡的实验研究目的检测人前列腺癌细胞(PC-3M)中Omi/HtrA2的表达情况,并构建其小干扰RNA (siRNA)表达载体,探讨Omi/HtrA2对PC-3M凋亡的影响。方法以细胞免疫组化和RT-PCR法检测Omi/HtrA2在前列腺癌细胞系(PC-3M)和正常前列腺细胞中的表达。利用Invivogen公司的软件辅助设计Omi/HtrA2特异性siRNA序列,体外合成后将其克隆入真核表达载体psiRNA- hH1neo。以脂质体法转染psiRNA-Omi/HtrA2载体至PC-3M中,以RT-PCR和Western blot法检测psiRNA-Omi/HtrA2对Omi/HtrA2转录和表达的沉默效应。用MTT和流式细胞法检测siRNA导致Omi/HtrA2基因沉默后,PC-3M细胞凋亡的变化。结果细胞免疫组化和RT-PCR均显示Omi/HtrA2在PC-3M细胞中高表达。酶切和DNA测序证实合成的siRNA基因序列正确,并已被准确克隆入psiRNA-hH1neo载体中。psiRNA-Omi/HtrA2载体可特异性抑制PC-3M细胞中Omi/HtrA2的表达。转染psiRNA-Omi/HtrA2载体的PC-3M细胞凋亡下调。结论促凋亡因子Omi/HtrA2可导致前列腺癌细胞凋亡,Omi/HtrA2的表达对前列腺癌的发展有重要作用。七、Omi/HtrA2抑制PED/PEA-15促前列腺癌细胞(PC-3)凋亡的研究背景与目的促、抑凋亡因子间的相互作用与肿瘤的发生、发展密切相关,Omi/HtrA2是新近发现的一种凋亡调节因子,PED/PEA-15是一种广泛表达的抗凋亡蛋白,本研究旨在探讨Omi/HtrA2对PED/PEA-15表达和前列腺癌细胞(PC-3)凋亡的影响。方法构建Omi/HtrA2的表达载体和siRNA载体,并用脂质体法将二载体分别转染至PC-3细胞中,Western blot和ELISA法检测Omi/HtrA2对PED/PEA-15表达和细胞凋亡的影响;caspase-8检测试剂盒检测PED/PEA-15对caspase-8活性的影响;Western blot、RT-PCR检测Omi/HtrA2特异siRNA序列对其转录、翻译的影响,流式细胞法检测siRNA导致Omi/HtrA2基因沉默后,PC-3细胞凋亡的变化。结果酶切和DNA测序证实Omi/HtrA2的表达载体和siRNA载体构建成功。通过转染Omi/HtrA2表达载体高表达Omi/HtrA2可抑制PED/PEA-15表达,并增加肿瘤细胞的凋亡率;抑制PED/PEA-15的表达可提高caspase-8活性。siRNA导致Omi/HtrA2基因沉默后PC-3细胞对顺铂的敏感性降低。结论Omi/HtrA2可通过抑制抗凋亡蛋白PED/PEA-15表达而在PC细胞凋亡中发挥重要作用。八、高表达Omi/HtrA2对PED/PEA-15表达缺陷的前列腺癌细胞凋亡的影响目的探讨Omi/HtrA2促前列腺癌细胞凋亡机制,找寻前列腺癌治疗新方法。方法分别构建促凋亡因子Omi/HtrA2的真核表达载体、抗凋亡蛋白PED/PEA-15的特异siRNA载体。用脂质体法将PED/PEA-15的siRNA载体转染入前列腺癌细胞(PC-3)中,经G418筛选获得抗性亚克隆细胞株,RT-PCR、Western blot法对亚克隆细胞株进行PED/PEA-15基因鉴定。将Omi/HtrA2表达载体转染入亚克隆细胞株,流式细胞仪检测高表达Omi/HtrA2对亚克隆细胞株凋亡的影响。结果酶切和DNA测序证实Omi/HtrA2表达载体和PED/PEA-15特异siRNA载体构建成功。G418筛选出的亚克隆细胞株中PED/PEA-15的表达极弱。在亚克隆细胞株中高表达Omi/HtrA2细胞凋亡率较高、XIAP的抑制亦更明显。结论抗凋亡蛋白PED/PEA-15的表达可阻抑Omi/HtrA2促前列腺癌细胞凋亡之作用。更多还原

【Abstract】 1. Expression and significance of PED/PEA-15 in prostate cancer Objective To study the expression of PED/PEA-15 in prostate cancer, and interpret the role of it in the occurrence and development of prostate cancer. Methods The expression of PED/PEA-15 in prostate cancer cell (PC-3) and normal prostate tissue were respectively assayed by means of RT-PCR and immuneohistochemistry techniques. Results PED/PEA-15 high express in PC-3 cells, 73.17% prostate cancer tissue express PED/PEA-15, by contrast, the normal prostate tissue showed no or weak expression of it. Conclusions Anti-apoptotic factor PED/PEA-15 can inhibit apoptosis, and its expression might be involved in prostate cancer development.2. The effect of PED/PEA-15 on prostate cancer cells (PC-3) apoptosis Objective To study the effect of PED/PEA-15 on human prostate carcinoma cells (PC- 3) apoptosis through constructing its eukaryotic expressing vectors. Methods PED/PEA-15 gene was amplified by means of RT-PCR technique and was cloned into pEG FP-N1 vect- or. The constructed vector was transiently transfected into PC-3 cells under the induction of liposome. Using flow cytometry technique to determine cancer cells apoptosis. Results Enzyme digestion analysis and DNA sequencing confirmed that the PED/PEA-15 eukaryo- tic expression vector was constructed successfully. High expression of PED/PEA-15 can inhibit PC-3 cells apoptosis and degrade KillerTRAIL’s proapoptotic effect. Conclusions PED/PEA-15 can inhibit PC-3 cells’apoptosis, and its expression might be involved in pr- ostate cancer’s development.3. Effect of small interference RNA targeting PED/PEA-15 on the apoptosis of prostate cancer cell (PC-3) Objective To approach the effect of PED/PEA-15 on prostate cancer cell (PC-3) apopto- sis by means of constructing PED/PEA-15 specific siRNA vector. Methods Using the s- oftware of Invivogen company, computer aided designing PED/PEA-15 specific siRNA s- equence. The synthesized siRNA sequence was cloned into psiRNA-hH1neo vector. The constructed psiRNA-PED/PEA-15 was transiently transfected into PC-3 cells and the inh- ibited effect of psiRNA-PED/PEA-15 on expression of PED/PEA-15 was detected using RT-PCR and Western blot, the effect of psiRNA-PED/PEA-15 on cancer cells’apoptosis was determined by flowcytomtry and MTT techniques. Results Enzyme digestion analy- sis and DNA sequencing confirmed that the PED/PEA-15 specific siRNA expression vect- or was successfully constructed. The apoptosis of PC-3 cells and the expression of PED/P- EA-15 were both down-regulated after the transfection of psiRNA-PED/PEA-15. Conclu- sions PED/PEA-15 can inhibit the apoptosis of PC-3 cells, and that its expression might be involved in prostate cancer development.4. Effect of PED/PEA-15 and XIAP on prostate cancer cell (PC-3) apoptosis Objective To study the effect of antiapoptosis factors PED/PEA-15 and XIAP on prosta- te cancer cells (PC-3) apoptosis. Methods Expression of XIAP and PED/PEA-15 in pro- state cancer cells (PC-3) were respectively assayed using RT-PCR technique. PED/PEA-15 and XIAP specific siRNA vectors were designed and constructed and then were transientl- y cotransfected into PC-3 cells under induction of liposome. Effects of siRNA sequences on PED/PEA-15 and XIAP transcription were assayed by RT-PCR technique, effects of XIAP and PED/PEA-15 on cancer cell apoptosis was determined by flow cytometry and microscope observation. Results PED/PEA-15 and XIAP were both highly expressed in PC-3 cells. Enzyme digestion analysis and DNA sequencing confirmed that PED/PEA-15 and XIAP specific siRNA expression vectors were constructed successfully. The designed siRNA sequences of PED/PEA-15 and XIAP could specificly inhibit theirs transcription. The cotransfected with PED/PEA-15 and XIAP specific siRNA vectors PC-3 cells were more sensitive to doxorubicin and its apoptosis rate also increased significantly. Conclus- ions PED/PEA-15 and XIAP might be involved in the development of prostate cancer. 5. Immunohistochemical analysis of Omi/HtrA2 expression in prostate cancer and benign prostatic hyperplasiaTo study the expression and significance of the serine protease Omi/HtrA2 in p- rostate cancer and benign prostatic hyperplasia. The expression of Omi/HtrA2 was assayed by means of immunohistochemical technique in prostate cancer, benign prostatic hyperpla- sia and normal prostate tissue specimens. Omi/HtrA2 mRNA levels of in vivo prostate can- cer and benign prostatic hyperplasia were assayed by semi-quantitative RT-PCR technique. Omi/HtrA2 expression was positive in 30 of 42 prostate cancer specimens, and the positive rate was lower in well differentiated group than in poorly and moderately differentiated gr- oups (P < 0. 05). By contrast, the cells in benign prostatic hyperplasia and normal prostate groups showed no or weak expression of Omi/HtrA2. The Omi/HtrA2 mRNA level of pro- state cancer is much higher than benign prostatic hyperplasia. Prostate cancer cells in vivo may need Omi/HtrA2 expression for apoptosis, and Omi/HtrA2 might be involved in pros- tate cancer development.6. Experimental study on Omi/HtrA2 promoting prostate cancer cell (PC-3M) apopt- osisObjective To study the expression of Omi/HtrA2 in human prostate cancer cell line (PC- 3M) and to approach the effect of Omi/HtrA2 on PC-3M apoptosis by means of construct- ing the Omi/HtrA2 specific siRNA expressing vector. Methods The expression of Omi/- HtrA2 in human prostate cancer cell line (PC-3M) was detected by means of RT-PCR and immunohistochemical techniques. According to the software of Invivogen company, com- puter aided design Omi/HtrA2 specific siRNA sequence and then synthesized and cloned into the expression vector psiRNA-hH1neo. The constructed psiRNA-Omi/HtrA2 was tra- nsiently transfected into human prostate cancer cell line (PC-3M) and the inhibited effect of psiRNA-Omi/HtrA2 on expression of Omi/HtrA2 in PC-3M cells was detected using RT-PCR and Western Blot; the apoptosis of cancer cells was determined by flow cytome- try and MTT techniques. Results The expression level of Omi/HtrA2 was up-regulated in PC-3M cells. Enzyme digestion analysis and DNA sequencing confirmed that the Omi/- HtrA2 specific siRNA expression vector was constructed successfully. The apoptosis of PC-3M and the expression of Omi/HtrA2 were down-regulated in prostate cancer cells af- ter the transfection of psiRNA-Omi/HtrA2. Conclusions prostate cancer cells may need Omi/HtrA2 expression for apoptosis, and Omi/HtrA2 might be involved in prostate cancer development.7. Omi/HtrA2 promotes prostate cancer cell death by degrading the anti-apoptotic protein PED/PEA-15BACKGROUND & OBJECTIVE: Abnormal apoptosis is one of the key factors for the development of neoplasms. Omi/HtrA2 is a novel gene involved in regulation of apoptosis. PED/PEA-15 is a ubiquitously expressed cytosolic protein exerting a broad anti-apoptotic action. This study was designed to explore the effect of Omi/HtrA2 on PED/PEA-15 expr- ession and prostate cancer cells (PC-3) apoptosis. METHODS: Omi/HtrA2 expression and specific siRNA vectors were respectively constructed and were transiently transfected into PC-3 cells under induction of liposome. Effect of Omi/HtrA2 on PED/PEA-15 expression and PC-3 cells apoptosis was assayed by Western blot and ELISA techniques. Caspase-8 activity was assayed by caspase-8 colorimetric assay kit which can be inhibited by PED/P- EA-15. Using RT-PCR and Western blot techniques assayed the effect of Omi/HtrA2 spec- ific siRNA sequence on its transcription and translation. Effect of Omi/HtrA2 inhibited by siRNA on PC-3 cells apoptosis was determined by flowcytometry technique. RESULTS: Enzyme digestion analysis and DNA sequencing confirmed that the Omi/HtrA2 expression and specific siRNA vectors were successfully constructed. High express PED/PEA-15 can inhibit caspse-8 activity and hinder cancer cells apoptosis. The PC-3 cells transfected with Omi/HtrA2 specific siRNA vectors were less sensitive to Cisplatin. CONCLUSIONS: Omi/HtrA2 can inhibit PED/PEA-15 expression and might play an important role in the development of prostate cancer.8. Effect of high expressed Omi/HtrA2 on PED/PEA-15 knockout prostate cancer cell apoptosis.Objective To study the mechanism of Omi/HtrA2 promoting prostate cancer cells apopt- osis and find out the new method of prostate cancer gene treatment. Methods Omi/Htr- A2 eukaryotic expression and PED/PEA-15 specific siRNA vectors were respectively con- structed. Under the induction of liposome, PED/PEA-15 specific siRNA vector was transf- ected into prostate cancer cell line (PC-3). After being selected by G418, the subclone cell line was obtained. Cellular gene silence of PED/PEA-15 was identified by RT-PCR and Western blot techniques. Omi/HtrA2 eukaryotic expression vector was transfected into su- bclone cells to high express it under induction of liposome. Effect of Omi/HtrA2 on subcl- one cells apoptosis was assayed by flow cytometry and Western blot technique. Results Enzyme digestion analysis and DNA sequencing confirmed that Omi/HtrA2 eukaryotic ex- pression and PED/PEA-15 specific siRNA vectors were successfully constructed. RT-PCR and Western blot revealed that there was no PED/PEA-15 expression in subclone cells, by the contrast, high level mRNA and protein of PED/PEA-15 was detected in PC-3 cells (P < 0. 01). High express Omi/HtrA2 in subclone cells can increase its apoptosis. Conclusions PED/PEA-15 can affect Omi/HtrA2 proapoptotic function. Omi/HtrA2 might be involved in prostate cancer development.更多还原