【作者】 施朝龄；

【导师】 王伟华；

【作者基本信息】 吉林大学 ， 外科学， 2011， 博士

【摘要】 作用的体外实验研究前列腺癌的发病率一直在欧美国家居高不下,目前仍高居男性恶性肿瘤发病率第二位。前列腺癌的发病原因复杂,且潜伏时间较长,患者往往无明显临床症状。近些年我国前列腺癌的发病率也呈逐年上升趋势,且多以中晚期患者居多,约有三分之一的患者出现骨转移症状。在过去的10年间,前列腺癌的治疗出现了新的思路,由以往只注重肿瘤治疗本身而改变为既治疗肿瘤同时又注重调节患者体内微环境。基于对肿瘤细胞与体内微环境间相互作用的新认识,进展性前列腺癌的治疗方法发生了改变。前列腺癌细胞和患者机体微环境内不同的细胞相互作用后,肿瘤细胞的生长得以增强,这一过程以破骨细胞的加入更为显著。这种肿瘤生长的过程是与患者自身产生的细胞因子类和炎症趋化因子类物质有关系的。越来越多的研究表明单核细胞趋化蛋白-1(MCP-1)在前列腺癌发展和转移过程中发挥重要作用。日前有国外学者指出MCP-1不但具有募集单核细胞的能力,还可以促进前列腺癌细胞增殖和转移。有学者指出MCP-1在前列腺癌转移中起到正性调节作用。单核细胞趋化蛋白-1也称趋化因子配体2(Chemokine ligand 2, CCL2)被认为是和骨转移发生有着最密切的关系。骨髓成骨细胞、内皮细胞、间质细胞以及前列腺癌细胞均可产生MCP-1,同时MCP-1与调控肿瘤相关性巨噬细胞和促进破骨细胞成熟也有密切关联。另外,MCP-1与其受体(CCR2)结合后可通过细胞的自分泌和旁分泌行为诱导前列腺癌细胞发生增殖、迁移和侵袭。MCP-1通过PI3K/AKT相关机制激活survivin基因,从而降低前列腺癌细胞的凋亡率。双链RNA对基因表达的阻断作用被称为RNA干扰,也被称为RNAi。双链RNA经酶切后会形成很多siRNA;信使RNA,即nRNA中的同源序列可以同这些siRNA的序列发生互补结合,从而致使mRNA失去应有的功能,使nRNA不能翻译产生蛋白质,达到基因沉默的效果。RNAi的治疗前景潜力巨大。许多疾病在基因病因学已经具有理论依据,并且有些疾病已经在体外试验和体内试验的模型中通过RNAi技术行靶向治疗。RNAi作为阻断肿瘤发生有关基因的有力手段已经越来越被重视。本研究是基于以上理论依据应用RNAi技术将MCP-1基因阻断,探索MCP-1在前列腺癌细胞生长、增殖、转移等方面的作用,并试图阐述以上机制。目的：本实验选用具有高度转移特性的人前列腺癌PC-3M细胞,并依托RNAi技术构建了pFIV-H1/U6-MCP-1 siRNA质粒(pFIV-si-MCP-1质粒)。探讨MCP-1在前列腺癌进展和转移过程中作用,同时应用pFIV-si-MCP-1质粒行RNAi抑制PC-3M细胞中MCP-1基因的实验,观察前列腺癌细胞的形态学变化及相关基因表达变化,以及前列腺癌细胞凋亡率变化,尝试为前列腺癌的治疗提供新线索。方法：设立control组,si-scramble组,MCP-1组和si-MCP-1组,si-scramble组和si-MCP-1组分别予以细胞转染pFIV-si-scramble质粒和pFIV-si-MCP-1质粒,MCP-1组给予50ng/ml的MCP-1细胞培养液。采用MTT细胞比色法观察各组肿瘤细胞的抑制率变化；应用细胞划痕实验观察肿瘤细胞迁移能力变化；应用Transwell细胞侵袭实验观察肿瘤细胞侵袭能力变化；ELISA检测肿瘤细胞内MCP-1蛋白含量变化；应用TUNEL和Annexin V-FITC流式细胞术检测细胞凋亡率变化；RT-PCR和Western blot检测VEGF.Caspase-3、MMP-9及MCP-1基因的转录与表达。结果：本研究结果表明,与control组和si-scramble组比较,MTT细胞比色法试验中观察到MCP-1具有肿瘤细胞增殖的作用,MCP-1组细胞增殖率可达30.44±0.12%,而pFIV-si-MCP-1质粒可明显抑制肿瘤细胞增殖,si-MCP-1组细胞抑制率为56.9±0.10%；细胞划痕实验观察到MCP-1可促进肿瘤细胞迁移,而pFIV-si-MCP-1质粒可明显抑制肿瘤细胞迁移；Transwell细胞侵袭实验观察到MCP-1可增强肿瘤细胞侵袭能力,MCP-1组穿过Matrigel胶的肿瘤细胞数量明显增多(P<0.01),而pFIV-si-MCP-1质粒可明显抑制肿瘤细胞侵袭能力,肿瘤细胞转染pFIV-si-MCP-1质粒后侵袭细胞数量显著减少(P<0.01)；应用TUNEL及Annexin V-FITC处理各组细胞后激光共聚焦显微镜下观察和流式细胞仪检测均显示MCP-1可减少肿瘤细胞凋亡,而pFIV-si-MCP-1质粒可促进肿瘤细胞凋亡,各组细胞凋亡率分别为5.4±1.12％(control组),7.0±3.24％(si-scramble组),1.0±0.59%(MCP-1组)和40.8±3.02％(si-MCP-1组)；ELISA测定各组细胞蛋白中MCP-1含量分别为,63.5±11.60 pg/μg(control组),64.9±11.07 pg/μg(si-scramble组),88.1±8.45pg/μg(MCP-1组)和44.5±11.29 pg/μg(si-MCP-1组),结果显示MCP-1具有自身促进产生作用,pFIV-si-MCP-1质粒可减少MCP-1产生；RT-PCR与Western blot结果提示MCP-1可使Caspase-3基因转录与翻译下调,同时促进MMP-9、VEGF及MCP-1本身基因转录与翻译上调,pFIV-si-MCP-1质粒则可使Caspase-3基因转录与翻译上调,同时MMP-9、VEGF及MCP-1本身基因转录与翻译下调。结论：1.实验证明MCP-1具有促进PC-3M细胞增殖作用和减少凋亡,并且增加PC-3M细胞侵袭能力的作用。2.上述作用与MCP-1上调VEGF、MMP-9表达和下调Caspase-3表达有关。3.实验证明pFIV-si-MCP-1质粒对PC-3M细胞具有降低肿瘤细胞侵袭能力和促进凋亡作用。4.上述作用与MCP-1表达受抑制后VEGF、MMP-9表达下调和Caspase-3表达上调有关。更多还原

【Abstract】 The incidence of prostate cancer has been high in Europe and America,it is still rankedsecond in the incidence of male cancer. The etiology of prostate cancer is complex and the latency is long, patients are often no obvious clinical symptoms. In recent years the incidence of prostate cancer is also increasing, about one-third of the patients had symptoms of bone metastases.In the past 10 years, there is a new way in the treatment of prostate cancer. Focus only on cancer treatment in the past change to the treatment of tumors at the same time as focus on regulation in microenvironment in patients. Based on the new understanding between in vivo microenvironment of tumor cells and the interaction, the treatment of prostate cancer has changed. Recently, monocyte chemoattractant protein-1 (MCP-1) has been shown to play an important role in the progression and metastasis of prostate cancer. Foreign scholars have recently found MCP-1 not only has raised the ability of monocytes, but also promote the proliferation and metastasis of prostate cancer. Some scholars have pointed out that the MCP-1 in prostate cancer metastasis positive regulatory role played. Monocyte chemoattractant protein-1 is cons-idered to have the most closely related to the occurrence of bone metastasis. Osteoblasts,endothelial cells, stromal cells and prostate cancer cells can produce MCP-1.MCP-1 can also regulate tumor-associatiated macrophages and promote osteoclastmaturation.In addition,MCP-1 and its receptor(CCR2) complex can induce prostate cancer cellsproliferation, migration and invasion. MCP-1 can activate the survivin gene through PI3K/AKT mechanisms to reduce prostate cancer cell apoptosis. Double-stranded RNA blocks gene expression known as RNA interference effect,also knowm as RNAi.Double-stranded RNA will be digested to many siRNA.Messenger RNA of homologous sequence can occur with these siRNA sequences,and then the mRNA lose their funcion,so translation of mRNA can not produce proteins.RNAi as a powerful tumor blocking tool for the gene has been more and more attention. Objective:In this experiment, a highly metastatic human prostate cancer PC-3M cells, and based on RNAi technology to build pFIV-H1/U6-MCP-1 siRNA vector (pFIV-si-MCP-1 vector). Our aim was to investigate the mechanisms underlying the functional role of MCP-1 in prostate cancer progression and metastasis., while application pFIV-si-MCP-1 vector for RNAi inhibition, attempt to provide a new treatment for prostate cancer clues. Methods:The control groups, si-scramble group, MCP-1 group and the si-MCP-1 group give to the appropriate disposal respectively.Using MTT assay to observe the inhibitory rate of tumor cells in changes.Using scratch experiments to observed cell migration changes. Using Transwell cell invasion assay to observe tumor cell invasion changes. Using ELISA to detect tumor cells MCP-1 protein content. Using TUNEL and Annexin V-FITC flow cytometry to cell observe apoptosis rate changes. Using RT-PCR and Western blot to detect VEGF, Caspase-3, MMP-9 and MCP-1 gene transcription and expression.Results:With the control group and the si-scramble group, the results show that MCP-1 was observed with tumor cell proliferation in the MTT test, cell proliferation rate of MCP-1 group was 30.4±0.12%.The pFIV-si-MCP-1 vector can inhibit tumor cell proliferation, the cell inhibition rate of si-MCP-1 group was 56.9±0.10%. Observed in cell scratch experiment MCP-1 can promote tumor cell migration, and pFIV-si-MCP-1 vector could inhibit tumor cell migration. In the transwell cell invasion assay MCP-1 can enhance the invasive ability of tumor cells, MCP-1 group significantly increased the number of plastic through the Matrigel (P<0.01), while the pFIV-si-MCP-1 vector can inhibit tumor cell invasion, Tumor cells transfected pFIV-si-MCP-1 vector significantly reduced the number of invasive cells (P<0.01). In TUNEL and Annexin V-FITC test in each group were observed under confocal laser microscopy and flow cytometry have shown that MCP-1 can reduce the tumor cell apoptosis, and pFIV-si-MCP-1 can promote tumor cell to apoptosis, the apoptosis rate in each group were 5.4±1.12%(control group),7.0±3.24% (si-scramble group),1.0±0.59%(MCP-1 group) and 40.8±3.02%(si-MCP-1 group). In the ELISA assay, the concentrations of MCP-1 were,63.5±11.60 pg/μg (control group),64.9±11.07 pg/μg (si-scramble group),88.1±8.45 pg/μg (MCP-1 group) And 44.5±11.29 pg/μg (si-MCP-1 group), MCP-1 showed an effect with its own promotion, pFIV-si-MCP-1 MCP-1 vector can reduced MCP-1. RT-PCR and Western blot results suggest that MCP-1 can downregulates Caspase-3 gene transcription and translation, while promoting MMP-9, VEGF and MCP-1 gene transcription and translation, pFIV-si-MCP-1 vector can be The Caspase-3 gene transcription and translation upregulates, and downregulates MMP-9, VEGF and MCP-1 gene transcription and translation. Conclusion:1. Experiments show that MCP-1 can promote the proliferation of PC-3M cells and reduced apoptosis, and increase the invasive ability of PC-3M cells.2. The reason of these effects was MCP-1 increased with VEGF, MMP-9 expression and decreased the expression of Caspase-3.3. pFIV-si-MCP-1 vector can reduce PC-3M cell invasion and promote apoptosis. 4. The reason of these effects was VEGF, MMP-9 expression decreased and the expression of Caspase-3 upregulated after MCP-1 expression was inhibited.更多还原