【作者】 吴学杰；

【导师】 杨罗艳；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 研究背景:前列腺癌是男性泌尿生殖系统常见的恶性肿瘤,近年来我国前列腺癌发病率逐渐上升,前列腺癌目前多采用雄激素全阻断为主的内分泌治疗,但多数病人在经过内分泌治疗后,常常转为非雄激素依赖性,成为激素难治性前列腺癌,出现复发或多发转移。传统的手术、放疗、化疗和激素疗法对激素难治性前列腺癌治疗效果不佳,目前国内外许多学者都在探求基因水平的调控方法和寻找治疗的新靶点,以期在前列腺癌的治疗上取得突破。近年研究证明细胞膜鞘磷脂的衍生物包括神经酰胺(Cer)、鞘氨醇(Sp)、1-磷酸鞘氨醇(S1P)等多种代谢产物,在调节细胞增殖和凋亡的动态平衡中起着关键的作用。而鞘氨醇激酶(SPK)是维持细胞内上述物质平衡的重要限速酶,也是细胞增殖及存活的重要调控因子。鞘氨醇激酶信号通路与细胞的凋亡、生长、增殖密切相关,SPK1是其主要功能酶。Cer和Sp是细胞增殖的负调控因子,能够抑制细胞生长、促进细胞凋亡,S1P则刺激细胞生长、抑制细胞凋亡。SPK1磷酸化Sp生成S1P,S1P通过细胞内外作用机制调节细胞生长和凋亡。对SPK1进行调控,将影响细胞内Cer、Sp、S1P的水平,细胞内Cer、Sp、S1P的水平的高低,将影响细胞的生长、增殖和凋亡。RNA干涉(RNAi)是近期发展起来的一种新的基因治疗和诊断技术,RNA干涉是指在多种生物细胞内,由双链RNA介导同源序列mRNA的特异性降解,从而导致基因沉默的现象。我们设想通过RNA干涉的方法,导致SPK1基因沉默,进而抑制SPK1的表达,调节神经酰胺、鞘氨醇、1-磷酸鞘氨醇在细胞内的水平,诱导非激素依赖性前列腺癌细胞的凋亡,以探讨对非激素依赖性前列腺癌细胞的基因治疗。目的:通过检测SPK1信号分子在非激素依赖性前列腺癌细胞Du145中的表达情况,以及评价重组腺病毒Ad-H1-SPK1介导,RNA干涉Du145细胞SPK1基因,对SPK1和抗凋亡蛋白Mcl-1表达的影响;探讨重组腺病毒Ad-H1-SPK1介导,干涉SPK1对细胞凋亡的作用及可能的机制,为非激素依赖性前列腺癌细胞的基因治疗寻找新的靶点。方法:我们选择非激素依赖性前列腺癌细胞株Du145,作为我们的实验研究对象;构建并制备了携带SPK1特异干涉序列的重组腺病毒Ad-H1-SPK1,作为介导对SPK1基因进行RNA干涉的研究工具;我们利用RT-PCR的方法,检测Du145细胞株中SPK1信号分子的表达;用腺病毒Ad-GFP作为参照,用不同滴度的腺病毒对Du145细胞进行感染,检测腺病毒对Du145的感染效率;我们用Ad-H1-SPK1感染Du-145细胞,并以不含SPK1特异干涉序列腺病毒Ad-H1作为对照,Western blot检测SPK1的表达,并用Western blot检测重要抗凋亡蛋白Mcl-1的表达,评价干涉SPK1对SPK1、Mcl-1蛋白表达的影响;用MTT的方法检测干涉SPK1,对阿霉素抑制前列腺癌细胞Du-145增殖的影响;用流式细胞仪检测细胞的凋亡情况,评价干涉SPK1对喜树碱诱导Du-145细胞凋亡的影响。结果:我们用RT-PCR的方法,在非激素依赖性前列腺癌细胞株Du-145中检测到了SPK1信号分子的表达;我们测定腺病毒对Du-145细胞的感染效率为99.92%(MOI=100),完全可以满足后续实验的要求;用Ad-H1-SPK1感染Du-145细胞,介导对SPK1进行干涉,SPK1干涉后,SPK1、Mcl-1蛋白表达明显受到抑制,Ad-H1-SPK1+阿霉素组细胞生存率低于对照Ad-H1+阿霉素组,48小时为对照组的71.2%,72小时为对照组的61.3%:用流式细胞仪检测Du145细胞的凋亡,Ad-H1-SPK1+喜树碱组的平均细胞凋亡率58.3%,高于对照组Ad-H1+喜树碱组29.1%,经统计学处理,差异有显著性(P＜0.01)。结论:我们的研究结果表明,SPK信号通路可以调节非激素依赖性前列腺癌细胞Du145的凋亡,靶向SPK1的RNA干扰可以抑制Du145细胞的SPK1以及重要抗凋亡蛋白Mcl-1的表达,增加阿霉素对Du145细胞增殖的抑制,增加喜树碱诱导的Du145细胞的凋亡。SPK信号通路可能为前列腺癌基因治疗提供新的靶点。更多还原

【Abstract】 BackgroundProstate cancer is one of the most common malignant tumor of the male urogenital system. In China, it’ s mobidity has been rising yearly in the recent decade . Endocrine therapy of androgen blockade is the mainly treatment for prostate cancer at present. But most patients after endocrine therapy usually change into androgen-independent and become hormone refractory prostate cancer ,recurrence or metastasis .Traditional surgery, radiotherapy, chemotherapy and endocrine therapy are ineffective for hormone refractory prostate cancer. Many scholars explore at the level of gene regulation and control to the treatment of prostate cancer,and look for a new target for treatment to make a breakthrough . In recent years, studies have proved that membrane sphingomyelin derivatives include ceramide (Cer), sphingosine (Sp), sphingosine-1- phosphate (S1P) and other metabolites .They plays a key role in the dynamic equilibrium regulation of cell proliferation and apoptosis . The sphingosine kinase (SPK) is important rate-limiting enzyme to maintain the intracellular balance of these substances. It was important regulatory factors of cell proliferation and survival. The cell levels of these sphingolipid metabolites are regulated by sphingosine kinase (SPK) which phosphorylates Sp to form S1P and regulates its cellular levels. Sphingosine kinase signaling pathway are closely related with cell apoptosis, growth and proliferation.RNA interference (RNAi) is recently developed a new gene therapy and diagnostic technology. RNA interference happen in a variety of biological cells .It is special phenomenon that double-stranded RNA mediated homologous sequence specific mRNA degradation and led to gene silencing. We envisage that RNA interference cause SPK1 gene silencing, thereby inhibiting the expression of SPK1, regulating ceramide, sphingosine-l-phosphate,sphingosine intracellular level, Induced apoptosis of hormone-independent prostate cancer cell. It was explored for hormone-independent prostate cancer gene therapy.OBJICTIVER: SPK1 signal molecule expression were examined in the hormone-independent prostate cancer cells line Du145. SPK1 and Myeloid cell leukemia-1 (Mcl-1) expression were examined after recombinant adenovirus Ad-H1-SPK1 mediated SPK1 interference on Dul45 cell .It was discussed that SPK1 interference play role on Du145 cell apoptosis and possible mechanism. We want to look for a new target for hormone-independent prostate cancer gene therapy.Method: The hormone-independent prostate cancer Du145 cell was selected as experimental subject. Recombinant adenovirus Ad-H1-SPK1 which carry SPK1 specific interference sequence was built and prepared, as an research tool to mediat RNA interference for SPK1 gene. RT-PCR method was used to measure SPK1 signal molecule expression on Du145. Infected efficiency of Du145 was tasted with adenovirus Ad-GFP as a reference. Adenovirus Ad-H1 which not to carry SPK1 specific interference sequence was as control. SPK1 and Mcl-1 expression was measured by Western blotting. Influence of SPK1 and Mcl-1 protein expression were evaluated after SPK1 interference. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide( MTT) dye reduction assay. Cell viability was assessed by MTT after the indicated times in Dul45 cells treated with doxorubicin 1.0μg/ml and infected by adenovirus Ad-H1 or Ad-H1-SPK1. Doxorubicin-induced proliferation inhibition on Du145 was evaluate after SPK1 interference by MTT. The percentages of apoptotic cell were determined by flow cytometry analysis. Camptothecin-induced apoptosis of Du145 was evaluated after SPK1 interference.Result: The results showed that Du145 cell expressed SPK1 signal molecule. Infected efficiency of Dul45 cells by the adenovirus is 99.92% percent(MOI=100) and meet the need of the follow-up experiment. SPK1 and Mcl-1 protein expression were significantly inhibited. Cell viability of (Ad-H1-SPK1 and doxorubicin 1μg/ml )group was lower control group (Ad-H1 + doxorubicin 1μg/ml), 48 hours for 71.2% of control and 72 hours 61.3% of control. Average cell apoptosis rate of (Ad-H1-SPK1 and camptothecin 1μM) group (58.3%) was higher than (Ad-H1 and camptothecin 1μM )group(29.1%).The difference between grops was significant (P<0.01).Conclusion: Taken together ,these results show that SPK signaling pathway can regulate prostate cancer cell apoptosis, SPK1 interference can enhance the hormone-independent prostate cancer cells Du145 docrobicin-induced proliferation and camptothecin-induced apoptosis. These results suggest that SPK1 may be a potential new therapeutic target in hormone-independent prostate cancer cell.更多还原