【作者】 杜岳峰；

【导师】 曾甫清；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 ㈠CXCR4/SDF-1通路在前列腺癌靶向性转移转移中作用的初步研究目的初步探讨趋化因子CXC受体4/基质细胞衍生因子1(CXCR4/SDF-1)通路在前列腺癌(PCa)靶向性转移中的作用。方法逆转录-链式聚合酶反应(RT-PCR)法和蛋白质斑迹法(Western Blot)法分别检测PCa细胞系LNCaP和PC-3m中CXCR4 mRNA和蛋白表达,免疫组化检测正常前列腺组织和前列腺癌组织CXCR4蛋白的表达。RT-PCR法检测并比较成人骨、淋巴结、肺、脑、肝组织中SDF-1 mRNA表达,酶联免疫吸附法(ELISA)检测人成骨细胞(HOB)条件培养基上清中SDF-1蛋白水平。观察外源性SDF-1对PCa细胞体外黏附、侵袭和迁移的影响及CXCR4抗体对其效应。结果两种PCa细胞系均有CXCR4 mRNA和蛋白表达,前列腺癌组织高表达CXCR4蛋白。成人骨、淋巴结、肺、脑、肝组织均有SDF-1 mRNA表达,半定量分析显示淋巴结和骨组织表达量最高;HOB细胞培养上清中可测及较高浓度的SDF-1蛋白。外源性SDF-1可促进两种PCa细胞黏附、迁移和侵袭,呈剂量依赖关系,原代HOB培养72小时收集的条件培养基亦可促进PCa细胞体外迁移能力;CXCR4抗体则可抑制上述效应。结论PCa细胞高表达具有活化功能状态的CXCR4,PCa常见的转移靶器官则高表达SDF-1;SDF-1可促进PCa细胞体外转移过程,且可被CXCR4抗体阻断,CXCR4/SDF-1通路可能在PCa靶向性转移中发挥重要作用。㈡携带报告基因EGFP的CXCR4 RNA干扰质粒的构建及其对前列腺癌细胞CXCR4基因表达的抑制作用目的研究RNA干扰(RNA interference, RNAi)表达载体对前列腺癌细胞趋化因子CXC受体4(CXCR4)基因的抑制作用。方法CXCR4特异性小干扰RNA(small interfering RNA, siRNA)转入带有增强型绿色荧光蛋白(EGFP)和启动子U6的Pgensil-1质粒构建针对CXCR4基因的RNAi质粒表达载体,脂质体法转染前列腺癌PC-3m、LNCaP细胞株,应用RT-PCR、Western Blot检测其对CXCR4 mRNA及蛋白表达的影响。结果构建的表达质粒在前列腺癌PC-3m、LNCaP细胞株中均抑制了CXCR4 mRNA及蛋白表达。与空白载体组细胞作对照,前者小干扰RNA (siRNA)在对CXCR4 mRNA的抑制率24小时、48小时分别为87.81±10.20%、56.10±9.32%,后者为56.93±8.78%、49.24±11.23% ,蛋白抑制率24小时前者为64.71±6.68%,后者为58.66±11.56%。结论携带有siRNA-CXCR4序列的RNAi表达载体可以有效地抑制前列腺癌细胞CXCR4基因的表达,为后续实验奠定基础。㈢hTERT启动子调控的CXCR4-siRNA逆转录病毒载体的构建及其体外靶向抑制hTERT+前列腺癌细胞生物学效应研究目的:构建人端粒酶逆转录酶(hTERT)启动子调控的趋化因子受体4(CXCR4)的RNA干扰逆转录病毒载体,并研究其在hTERT+前列腺癌细胞、乳腺癌细胞中的表达。方法:用PCR扩增CXCR4特异性小干扰RNA(small interfering RNA, siRNA),转入带有增强型绿色荧光蛋白(EGFP)和启动子U6的Pgensil-1质粒,以hTERT启动子代替U6启动子,再将重组基因片段导入到逆转录病毒真核表达载体PLXSN中,ERFP基因片段代替EGFP基因片段,行限制性酶切鉴定及测序。转染PA317病毒包装细胞,收获病毒上清分别转染前列腺癌细胞PC-3m、LNCaP及人胚肺成纤维细胞MRC5和人乳腺癌细胞MCF7,RT-PCR、Western Blot检测CXCR4 mRNA及蛋白表达。观察外源性SDF-1对CXCR4基因表达抑制后PCa细胞体外黏附、侵袭和迁移的影响。结果:限制性酶切、测序鉴定该重组质粒,成功构建了PLXSN/ERFP-hTERT-siCXCR4。与空载体组比较,MCF7、PC-3m、LNCaP细胞中CXCR4-siRNA对CXCR4 mRNA的48小时抑制率分别为(88.31±9.20)%、(87.20±10.34)%、(76.40±9.77)%,CXCR4蛋白的48小时抑制率分别为(82.60±7.26)%、(80.23±4.54)%、(84.09±3.37)%。MRC5细胞中PLXSN/ERFP-hTERT-siCXCR4对CXCR4 mRNA及蛋白不抑制。CXCR4基因敲低后,外源性SDF-1促进两种PCa细胞黏附、迁移和侵袭的能力下降。结论:该逆转录病毒系统中hTERT启动子调控的下游RNA干扰序列可选择性地在hTERT+前列腺癌细胞、乳腺癌细胞中表达,在hTERT- MRC5细胞不表达,具有显著的靶向性。敲低CXCR4基因表达可抑制前列腺癌细胞体外黏附、侵袭、转移。㈣hTERT启动子调控的CXCR4-siRNA逆转录病毒载体抑制前列腺癌细胞骨转移的体内实验研究目的:构建可荧光示踪的前列腺癌骨转移动物模型,并探讨抑制CXCR4基因表达后对前列腺癌体内骨转移的影响。方法:选用6-8周龄体重为20-22克NOD/SCID雄性小鼠,利用外科植入术将成人骨组织(HAB)植入小鼠皮下。2-3周后,将携有ERF(PDsRED2)基因及干扰序列CXCR4-siRNA的重组逆转录病毒转染前列腺癌细胞PC-3m与LNCaP,收获稳定表达红色荧光基因(ERFP)的前列腺癌细胞并配置成细胞悬液,通过小鼠尾静脉注入小鼠体内。2周后开始通过整体荧光显像系统观察小鼠体内移植人骨组织处前列腺癌转移瘤细胞ERFP的表达。对比CXCR4基因敲低组和对照组前列腺癌骨转移瘤的骨转移率、体积、湿重、存活时间。HE染色镜检转移瘤组织切片。结果:两种前列腺癌细胞CXCR4基因敲低组骨转移瘤的骨转移率、体积、湿重均低于对照组,而存活时间高于对照组(基因未敲低组)。组织切片显示干扰CXCR4基因表达后前列腺癌骨转移瘤较基因未敲低组肿瘤组织分化程度高。结论:成功构建了可体外无创性动态观察的稳定的前列腺癌骨转移动物模型,证实敲低CXCR4基因表达可抑制体内前列腺癌骨转移并抑制前列腺癌细胞恶性分化。更多还原

【Abstract】 ⒈The effect of CXCR4/SDF-1 pathway on prostate cancer targeted metastasisObjective To investigate preliminarily the role of CXCR4/SDF-1 in prostate cancer (PCa) targeted metastasis. Methods CXCR4 mRNA and protein expression in two PCa cancer cell lines, LNCaP and PC-3m, were examined by reverse transcript-PCR、western bloting and prostate cancer tissue was detected by immunohistochemistry. SDF-1 mRNA expressions in human bone, lymph node, lung, brain and liver tissues were determined by RT-PCR and SDF-1 secretary level in the culture medium of primary human osteoblasts (HOB) was detected by in ELISA. The effect of exogenous recombinant SDF-1 on PCa cells adhesion, transendothelial migration and invasion, and the effect of anti-CXCR antibody in this procedure were observed using an in vitro Transwell and Matrigel models.Results Both CXCR4 mRNA and protein expressed in LNCaP and PC-3m. CXCR4 protein expressed highly in prostate cancer tissues. Meanwhile, SDF-1 mRNA expressed in divers human tissues and semi-quantitative analysis illustrated that most highly in lymph node and bone. It is also demonstrated that SDF-1 were detected in the culture medium of HOB cells. In addition, pretreatment of exogenous SDF-1 enhanced significantly the in vitro adhesion, invasion and migration of both cell lines with a dose-dependent manner, and 50% primary osteoblast conditioned medium collected at 72h increased as well in vitro PCa cell invasion. Such performances could be blocked by administration of anti-CXCR4 antibody.Conclusion PCa Cells highly express functional CXCR4 and the most frequent metastasis organs intensively express SDF-1. SDF-1 can obviously increase the in vitro adhesion, migration and invasion of PCa cells while anti-CXCR4 antibody can block such effect. CXCR4/SDF-1 pathway may therefore play a of very importance role in PCa targeted metastasis. ⒉The Inhibitory Effects of siRNA Expression Vector on CXCR4 Expression of Prostate carcinoma cell linesObjective To investigate the inhibitory effects of RNAi (RNA interference, RNAi) expression vector on CXCR4 expression of Prostate carcinoma cell lines. Methods CXCR4 targeting siRNA gene was inserted into the Pgensil-1 plasmid containing U6 promoter and EGFP and siRNA expression vectors were constructed to be aimed directly at CXCR4 gene. The recombinants were transfected into Prostate carcinoma cell line, PC-3m and LNCaP, with liposomes.The expression of CXCR4 was detected by RT-PCR and Western Blot. Results Expression vectors could reduced the expressions of CXCR4 mRNA and protein in the PC-3m and LNCaP cells. Compared with NC, the former, the ratio of inhibition of the expression of CXCR4 mRNA was 87.81±10.20%、56.10±9.32% in 24th hour and 48th hour, the latter,was 56.93±8.78%、49.24±11.23% , respectively. The ratio of inhibitory of the expression of CXCR4 protein was 64.71±6.68%、58.66±11.56% in 24th hour respectively . Conclusion The RNAi expression vectors containing CXCR4-siRNA sequence can effectively inhibit the expression of CXCR4 gene, which lays a foundation for post study.⒊Establishment of RNA interfering retrovirus vector targeting CXCR4 gene driven by hTERT promoter and its biological effects on hTERT+ prostate cancer cells in vitroObjective: To construct RNA interfering retrovirus vector targeting CXCR4 gene driven by human telomerase reserve transcriptase gene promoter and investigate CXCR4 expression in hTERT+ carcinoma cells. Methods: To clone the CXCR4 targeting siRNA gene by PCR. The PCR products were inserted into the Pgensil-1 plasmid containing U6 promoter and EGFP.U6 promoter was replaced by hTERT promoter. Then, the recombinant EGFP-hTERT-siCXCR4 fragment was sub-cloned into PLXSN and EGFP was replaced by ERFP.Then the recombinant was evaluated by restriction enzyme and sequencing. The virus obtained from transfected PA317 cells was transfected respectively into PC-3m、LNCaP、MCF7、MRC5 cell. The expression of CXCR4 mRNA and protein was detected by RT-PCR and Western Blot. The effect of exogenous recombinant SDF-1 on adhesion, transendothelial migration and invasion of PCa cells interferenced by CXCR4-siRNA were observed using an in vitro Transwell and Matrigel models. Results: The expression of CXCR4 mRNA and protein in MCF7、PC-3m and LNCaP were reduced by the recombinant PLXSN-DsRED2-hTERT-siCXCR4 constructed successfully. The inhibitory rates of the expression of CXCR4 mRNA and protein were(88.31±9.20)%、(87.20±10.34)%、(76.40±9.77)% and (82.60±7.26)%、(80.23±4.54)%、(84.09±3.37)% at 48th hour respectively. CXCR4 mRNA and protein can not be inhibited by PLXSN-hTERT-siCXCR4 in MRC5 cell. The ability of exogenous SDF-1 enhancing adhesion, invasion and migration of prostate cancer cells in vitro descended. Conclusion: The downstream CXCR4-siRNA controlled by hTERT promoter in retrovirus system can be expressed selectively in hTERT+ carcinoma cells, but not in hTERT- cells, which showed the obvious targeting character. Knocking down CXCR4 gene can inhibit significantly PCa adhesion, invasion and migration of prostate cancer cells in vitro.4. The study on inhibition of prostate carcinoma metastasis to bone by using retrovirus-mediated CXCR4-siRNA driven by hTERT promoter in vivoObjective: To establish a stable prostatic carcinoma osseous metastasis NOD/SCID mouse model expressing enhanced red fluorescent protein and investigate effect of prostate carcinoma metastasis to bone by inhibiting CXCR4 expression. Methods: 6-8 monthsold, male mice were selected and implanted with human adult bone (HAB) by surgical implantation subcutaneously. Then about 2-3 weeks later, human prostatic carcinoma cell lines PC-3m and LNCaP, transfected by retroviral containing ERFP gene and interference sequence, were harvested and injected into the NOD/SCID mice through the lateral tail vein.2 weeks after injection, all NOD/SCID mice were started to be observed under the whole-body fluorescent imaging system to look for the sites with the expression of ERFP. Then, to 2 prostate carcinoma cell line, ratio of osseous metastasis、humid weight、volume and time of surviving were contrasted between interferenced group and uninterferenced group. Tissue slices of metastasic tumor used HE dyeing were detected. Results: To 2 prostate cancer cell lines PC-3m、LNCaP interferenced by CXCR4-siRNA,ratio of osseous metastasis、humid weight、volume were all lower than those of uninterferebnced prostate cancer lines, but time of surviving was longer and histodifferentiation degree of osseous metastasic tumor was superer. Conclusion: A stable prostate cancer osseous metastasis model expressing enhanced red fluorescent protein (ERFP) observed dynamically in vitro in a noninvasive way was established. Knocking down expression of CXCR4 gene can inhibit prostate cancer metastasis to bone and inhibit cancer cell maligdifferentiation in vivo.更多还原