【作者】 徐晓峰；

【导师】 周四维；

【作者基本信息】 华中科技大学 ， 外科学， 2006， 博士

【摘要】 目的研究PAR基因（Prostate Androgen Regulated gene，PAR）在前列腺癌雄激素依赖性和雄激素非依赖性细胞株间表达的差异。方法选取前列腺癌雄激素依赖性细胞株LNCaP和雄激素非依赖性细胞株PC3，抽提细胞基因组，用逆转录聚合酶链反应分别检测两株细胞PAR基因mRNA表达的差异。结果雄激素非依赖性前列腺癌PC3细胞株PAR基因mRNA的表达水平是雄激素依赖性前列腺癌LNCaP细胞株的4倍。结论PAR基因在雄激素非依赖性前列腺癌细胞特异性高表达，为以后的雄激素非依赖性前列腺癌的发病机制和治疗的实验研究提供了线索。第二部分雄激素对前列腺癌雄激素受体阳性和雄激素受体阴性细胞株PAR基因表达影响的研究目的研究雄激素对前列腺癌雄激素受体（Androgen Receptor，AR）阳性LNCaP细胞株和雄激素受体阴性PC3细胞株PAR基因表达的影响。方法以细胞计数分别检测不同浓度的双氢睾酮（Dihydrotestosterone，DHT）对LNCaP、PC3细胞增殖的刺激效应，以RT-PCR分别检测不同浓度的双氢睾酮对LNCaP、PC3细胞PAR基因mRNA表达水平的影响。。结果低浓度（0.001nM-1nM）双氢睾酮刺激可促进LNCaP细胞增殖，使PAR基因mRNA表达水平上调，此效应在0.1nM浓度时达最大（细胞数量、PAR基因mRNA表达水平分别为对照组的（170.74±0.78）％、（272.42±8.24）％，P＜0.05）；高浓度（10nM、100nM）双氢睾酮对LNCaP细胞生长和PAR基因mRNA表达起抑制作用。双氢睾酮对PC3细胞生长及其PAR基因mRNA表达无明显影响（P＞0.05）。结论双氢睾酮可调节AR阳性前列腺癌细胞PAR基因的表达。第三部分雄激素影响PAR基因表达机制的研究目的：研究PAR基因对雄激素反应的机理，探讨通过抑制PAR基因治疗雄激素非依赖性前列腺癌的可能性。方法：以RT-PCR分别检测不同浓度的双氢睾酮和其拮抗剂联合作用对LNCaP细胞PAR基因mRNA表达水平的影响。构建野生型AR表达载体pcDNA3-AR并转染PC3细胞株（PC3-AR），双氢睾酮作用后以RT-PCR检测细胞株PAR基因mRNA表达的变化。结果：10μM氟他胺可抑制双氢睾酮对PAR基因mRNA表达的调节作用。AR阴性的细胞株PC3转染AR表达载体pcDNA3-AR，双氢睾酮可调节其PAR基因mRNA的表达。结论：双氢睾酮可能通过雄激素受体调节PAR基因的表达，PAR可能是雄激素—雄激素受体通路下游的前列腺癌特异性癌基因。第四部分下调PAR基因表达对前列腺癌细胞株恶性表型影响的研究目的：用RNA干扰技术下调PAR基因的表达，并探讨其对前列腺癌细胞株PC3恶性表型的影响及其机理。方法：构建针对PAR基因的shRNA表达载体psiRNA-PAR1、psiRNA-PAR2和psiRNA-PAR3，转染PC3细胞，以RT-PCR验证其对PAR基因表达抑制的有效性，用细胞计数、软琼脂克隆形成实验、流式细胞术研究PAR基因表达下调对PC3细胞生长的抑制作用及其机理。结果：3组shRNA表达载体均可抑制PC3细胞PAR基因表达，使细胞生长速率明显减慢，增殖受抑制，软琼脂克隆形成能力降低，其中psiRNA-PAR1的抑制效应最明显，并在转染48h后对PAR基因表达的抑制率达高峰，为81.18±1.68％，此时的流式细胞术检测表明细胞周期阻滞于G₂-M期，凋亡增加。结论：PAR基因表达下调，明显地抑制PC3细胞的生长，主要是通过诱导细胞G₂-M期阻滞和凋亡实现的；PAR基因可能是一个新的与雄激素非依赖性前列腺癌细胞恶性表型相关的癌基因，有望作为基因及药物治疗雄激素非依赖性前列腺癌的新靶点。更多还原

【Abstract】 Part OneDetection of differential expression of PAR between androgen-independent prostate cancer cell and androgen-dependentprostate cancer cell Aim: To study the difference of PAR expression betweenandrogen-independent prostate cancer cell and androgen-dependent prostate cancer cell.Methods: The difference of PAR expression between LNCaP and PC3 cells wasdetected by RT-PCR.Results: PAR expression was higher in PC3 cells than in LNCaP cells by 3-fold.Conclusion: PAR gene specifically overexpressed in androgen-independent prostatecancer cell.Part Two Study of the effects of androgen on PAR gene expression in AR-positiveprostate cancer cell and AR-negative prostate cancer cellAim: To investigate the effects of dihydrotestosterone(DHT) on PAR gene expression in AR-positive prostate cancer cell and AR-negative prostate cancer cell.Methods: Effects of DHT at different concentrations on proliferation of LNCaP andPC3 cells were studied by means of cell counts. PAR mRNA of LNCaP cells after DHTstimulation was detected by RT-PCR.Results: DHT at low concerntrations(0.001nM-lnM) stimulated proliferation andupregulated PAR mRNA expression of LNCaP cells. Maximum effect occurred at0.1 nM DHT(P<0.05).Whereas DHT at concemtrations greater than 1nM inhibited theLNCaP cells proliferation and its PAR mRNA expression. DHT had no significant effecton proliferation and PAR mRNA expression of PC3 cells(P>0.05).Conclusion: DHT modulates proliferation and PAR mRNA expression of LNCaPcells in a dose-related manner.Part ThreeStudy of the mechanism of dihydrotestosterone regulating PAR geneexpressionAims: To investigate the mechanism of dihydrotestosterone(DHT) on PARgene expression.Methods: PAR mRNA of LNCaP cells after DHT stimulation with its antagonistflutamide were detected by RT-PCR. DHT effects on PAR expression were evaluated byRT-PCR in PC3 cells stably transfected with vector containing wild-type AR.Results: The androgen receptor (AR) antagonist flutamide inhibited the stimulatoryeffect induced by DHT. Reintroduction of AR into PC3 cells by stable transfectionrestored the androgen effect on PAR up-regulation.Conclusion: DHT regulates PAR mRNA expression in an AR dependent pathway. PARgene is a potential target of gene and drugs therpy for androgen resistant prostate cancer. Part FourStudy of the effects of PAR gene down-regulation on malignant phenotype of prostate cancer cell lineAims: To investigate the effects and mechanism of downregulated PARexpression on proliferation of PC3 cells by using RNA interference (RNAi).Methods: Suppression of PAR expression was achieved by transfection of PC3 cellswith short hairpin RNA (shRNA) expression vectors targeted against PAR, which werenamed as psiRNA-PAR1, psiRNA-PAR2 and psiRNA-PAR3. The inhibitory effectswere confirmed by RT-PCR. The growth characteristics of PC3 transfectants wereanalysed by cell counts, colony formation in soft agar and flow cytometry.Results: The expression of PAR was suppressed by three shRNA expression vectors.PsiRNA-PAR1 was shown to inhibit the PAR expression most efficiently, with theinhibitory rate of 81.18 ± 1.68% peaking at 48 hours after transfection. PC3transfectants exhibited a decreased cell proliferation in cell culture and a low efficiencyof colony formation in soft agar. Flow cytometry revealed a cell cycle G2-M arrest andinduction of apoptosis.Conclusion: Downregulated PAR expression inhibits the growth of PC3 cells, throughinducing G2-M arrest and activating apoptotic pathway. As a potential proto-oncogenewhich trigger and/or maintain malignant proliferation, PAR may be a very importanttarget in the gene therapy in the future.更多还原