【作者】 刁鑫伟；

【导师】 陈正堂；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2009， 博士

【摘要】 研究背景前列腺癌是好发于老年男性的恶性肿瘤,欧美多发。在我国,随着人口老龄化趋势加快及PSA血清学检测的普及,其发病呈上升趋势。前列腺癌易发生远处转移,尤其是骨转移,各种治疗效果不理想,患者受癌性疼痛困扰,死亡率较高。因此,阐明前列腺癌骨转移的分子机制,对前列腺癌转移的预防和治疗有重要意义。趋化因子SDF-1是趋化因子CXC亚家族成员之一,主要在骨髓基质及内皮细胞表达,CXCR4是SDF-1的特异性受体,在神经、血管及造血等组织中组成性表达。SDF-1通过与CXCR4结合,对造血干/祖细胞、肝星状细胞有趋化作用,对诱导造血细胞的归巢及体内再分布有重要意义。近期研究证实SDF-1/CXCR4生物轴与多种肿瘤的播散和转移密切关联。并提出肿瘤细胞基于趋化因子以实现器官特异性转移的理论模型,认为高表达趋化因子受体的肿瘤细胞能向高表达相应趋化因子配体的器官进行选择性转移,但其确切机制仍不清楚。ICAM-1、VCAM-1作为炎性粘附分子主要介导炎细胞-血管黏附作用。ICAM-1配体为淋巴细胞功能相关抗原-1(LFA-1),是β2整合素家族中的成员,而VLA-4(CD49d)是整合素β1亚家族重要成员之一,是VCAM-1配体。SDF-1可以通过促使LFA-1在淋巴细胞膜表面重分布,提高LFA-1的黏附能力,促进炎细胞与血管内皮的黏附。而ICAM-1与LFA-1结合,介导免疫细胞向组织迁移、细胞与细胞外基质的粘附等作用。近年来发现ICAM-1、VCAM-1在许多肿瘤的表达明显高于相应正常组织,炎症分子介导的细胞-血管黏附作用与肿瘤的侵袭及转移有明显相似性,提示炎症分子可能以促进炎性进展的方式在在肿瘤侵袭及其转移方面起重要作用。基于国外研究进展,在本研究中我们用逆转录病毒作为载体,将CXCR4基因转染前列腺癌细胞,增强CXCR4表达水平,在体外通过微孔隔离室将前列腺癌细胞株PC-3与高表达SDF-1α的骨髓内皮细胞BMEC-1共培养,分析前列腺癌细胞对骨髓内皮细胞的迁移效应。将转染CXCR4基因前列腺癌细胞株种植于SCID小鼠前列腺包膜下,观察前列腺癌的骨转移效应,分析SDF-1/ CXCR4信号转导通路在前列腺癌转移及粘附过程中的作用及分子机理,为前列腺癌骨转移机制提供新的实验依据。方法1.收集148例手术切除及穿刺确诊前列腺癌存档蜡块。应用免疫组化方法检测前列腺癌组织、前列腺增生组织中CXCR4蛋白的表达,分析其不同表达与年龄、雄激素受体(AR)、病理分级和转移等临床特征的关系。进一步用免疫组化方法CD34标记计数肿瘤间质微血管密度(MVD),观察前列腺癌转移相关分子VEGF及MMP-9与微血管密度的关系。2.分离健康人外周血单个核细胞,提取总RNA,以其为模板, RT-PCR法扩增CXCR4,获取CXCR4基因全长序列,装入带有绿色荧光蛋白(GFP)的逆转录病毒质粒pLEGFP-N1,用脂质体法转染PC-3细胞,采用实时定量PCR和Western blotting观察CXCR4表达情况,并通过体外细胞-基质粘附试验和Transwells小室检测肿瘤细胞体外侵袭能力的变化。3.趋化因子SDF-1诱导培养CXCR4稳定转染PC-3细胞24h后,实时定量RT-PCR和Western blotting方法检测粘附分子LFA-1α/ICAM-1、VLA-4/VCAM-1及mRNA及蛋白表达情况,实时定量PCR和EMSA方法检测HIF-1α及P65NF-κB的mRNA水平及DNA结合活性。探讨前列腺癌细胞SDF-1/CXCR4生物轴功能增强对上述分子表达的影响。4.接种人前列腺癌PC-3、转染CXCR4的PC-3以及LNcap细胞于雄性SCID小鼠皮下,建立SCID小鼠人前列腺癌移植模型及原位转移模型,观察小鼠活动、移植成瘤率、肿瘤生长及形态学特征,以免疫组化检测CXCR4、前列腺癌特异性抗原PSA以及转移相关因子VEGF和MMP-9的表达情况。并观察CXCR4转染对荷瘤SCID小鼠转移情况及对骨转移灶微环境的影响。结果1. 148例前列腺癌组织中CXCR4的表达率显著高于癌旁正常前列腺组织和前列腺增生组织(P<0.001)。前列腺癌组织中CXCR4蛋白的高表达与病理分级和远处转移有关;前列腺癌间质MVD值随肿瘤分化程度减低而增高;与患者年龄无关,转移组较无转移组MVD值增高,AR阴性组较阳性组MVD值增高,随CXCR4、VEGF及MMP-9蛋白表达逐渐增强MVD值增高。2.成功构建pLEGFP-CXCR4载体,转染人前列腺癌细胞PC-3 72h后,CXCR4 mRNA及蛋白质水平明显上调, PC-3细胞的体外侵袭及迁移能力明显增强(CXCR4转染组与对照组比较,P<0.01,89.33±11.67 vs 59.33±7.27)。3.给予趋化因子SDF-1予CXCR4稳定转染PC-3细胞24h后, P38 mRNA和蛋白的表达与对照组相比显著增强(P<0.01,n=3),明显促进磷酸化P38蛋白的表达(P<0.01,n=3),且磷酸化P38与P38比值明显增高,提示pLEGFP-CXCR4转染PC-3细胞P38蛋白活性增强;与对照组相比PC-3细胞ICAM-1蛋白的表达显著增高(p<0.01,n=3),且mRNA水平增高(P<0.05,n=3);转染CXCR4的PC-3细胞VCAM-1mRNA的表达显著增高(P<0.01),VCAM-1蛋白水平增高(P<0.05,n=3); LFA-1αmRNA及蛋白水平显著增高(P<0.01,,n=3);VLA-4mRNA水平变化不显著(P>0.05,n=3),VLA-4蛋白水平显著增高(P<0.01,n=3);实时定量PCR检测P65NF-κB的mRNA水平显著增高(P<0.01,n=3),HIF-1α的mRNA水平显著升高(P<0.01,n=3),EMSA检测HIF-1α及P65NF-κB蛋白DNA结合活性显著增强(P<0.01,n=3)。4. PC-3/PEGFP-CXCR4细胞组与PC-3、LNcap细胞组相比较,移植瘤细胞形态、生长特性和PSA等未见明显改变。转移相关基因VEGF和MMP-9的表达在未转染CXCR4转移瘤及转染CXCR4组中明显增高(P<0.05 )。瘤结节包埋于尿道部皮下,形成一种模仿人类前列腺癌骨转移的动物模型,观察发现各组均未出现侵犯前列腺及生殖腺组织。SCID鼠移植瘤实验观察发现与对照组比较,转染CXCR4荷瘤SCID小鼠瘤细胞成瘤时间短,瘤体积大,转移灶多。骨转移主要位于腰柢椎,而且转染CXCR4组软骨内成骨及钙化明显,骨小梁增多,新生血管增多,髓腔内单核细胞增多。结论1.前列腺癌中CXCR4表达增高,可协同肿瘤间质趋化因子SDF-1促进前列腺癌的侵袭转移。CXCR4高表达提示肿瘤具有更高的侵袭转移能力。CXCR4的蛋白表达与间质微血管密度及VEGF及MMP-9蛋白的表达呈正相关。2. CXCR4转染能够明显提高CXCR4 mRNA及蛋白水平,提高人前列腺癌细胞体外侵袭能力。3. CXCR4转染PC-3细胞可以不通程度上调LFA-1α/ICAM-1、VLA-4/VCAM-1及P38MAPK的mRNA mRNA及蛋白的表达水平,促进P38MAPK的磷酸化,上调HIF-1Α及P65NF-κB的mRNA水平,显著增强HIF-1α及P65NF-κB蛋白的DNA结合活性。4.前列腺癌细胞SCID小鼠腹腔移植模型,模拟了人前列腺癌转移的生物学行为,较好地保持了人前列腺癌的生物学特性,是研究前列腺癌转移机制及生物治疗较理想的动物模型。PLEGFP-CXCR4转染促进癌细胞生长及转移,参与骨的重塑更显著。更多还原

【Abstract】 Backgroud and objectiveProstate cancer(PCa)is the most common malignant tumors in eld men, it occurred more in Europe and the United States, with the trend of aging and popular of detection serological PSA in China. Incidences of PCa appear upward trend, As a result of PCa tend to distant metastases, especially bone metastases, all kinds of treatment result is not satisfactory, Unbearable pain troubled Patients with PCa,and result in higher mortality. Therefore it is important to clarify the molecular mechanism of bone metastasis of PCa for preventing and treatment of PCa metastases.Chemokine SDF-1, as a member of the CXC chemokine subfamily, mainly expressed in bone marrow stromal and endothelial cells, CXCR4, the specific receptor of SDF-1, mainly expressed in the nerves, blood vessels and blood components. SDF-1 binding with CXCR4 is important to chemotactic effect on inducting hematopoietic cells homing and redistribution for hematopoietic stem / progenitor cells, hepatic stellate cells in vivo. Recent research has shown that SDF-1/CXCR4 biological axis related closely with invasion and metastasis in a variety of tumor. They assumed, Base on theory model of tumor cells depending on chemokines to achieve organ-specific metastasis, that high expressing of chemokine receptors in the tumor cells metastasis to organs of high expression of the corresponding chemokine ligand. But the mechanism is still unclear.ICAM-1, VCAM-1 as inflammatory adhesion molecules, mainly mediated adhesion of inflammatory cells to vascular. Ligand of lymphocyte function associated antigen -1 (LFA-1) for ICAM-1, a members theβ2 integrin subfamily。VLA-4(CD49d), as an important member of the integrinβ1subfamily, is receptor of VCAM-1. SDF-1 can improve adhesion ability of LFA-1 to promote inflammation and vascular endothelial cell adhesion by promoting LFA-1 redistribution at membrane surface of lymphocyte. And ICAM-1 binding to LFA-1 mediated migration of immune cell to the organization, cells adhesion to extracellular matrix, and so on. In recent years, ICAM-1, VCAM-1 are found higher expression significantly in many tumors than the corresponding normal tissue, the adhesions of cells to vascular mediated by inflammation molecules are obviously similar to invasion and metastasis of tumor, it suggest that inflammatory molecules may be plays an important role to promot tumor invasion and metastasis as the way of inflammation.Based on research overseas, in this study, We transfect CXCR4 gene to PCa cells by retroviral vectors, enhance the expression level of CXCR4, analysis effect of PCa cells migration to bone marrow endothelial cell by co-culture PCa cells line PC-3 and bone marrow endothelial cells BMEC-1, which SDF-1αexpressed in high level, in Transwell chamber in vitro. Cultivate PCa cells line, which Transfected with CXCR4 gene, into capsule of the prostate in SCID mice, observate effects of bone metastasis of prostate cancer, analysis the role and molecular mechanism of SDF-1 / CXCR4 signaling pathway in prostate cancer adhesion and metastasis, to provide new experimental evidence for the mechanism of bone metastasis of prostate cancer.Method1. Collectiing 148 cases archived wax blocks of surgical resection and puncture, which diagnosised prostate cancer. Detect expression of CXCR4 protein in prostate cancer tissue and benign prostatic hyperplasia tissues by immunohistochemistry, analysis relations of CXCR4 expression , clinical characteristics, the age difference, Androgen receptor (AR), pathological grade and metastasis. Further count microvessel density (MVD) of CD34 marked, observe relations prostate cancer metastasis-associated Molecules VEGF, MMP-9 with microvessel density in prostate cancer tissues.2. extracte of total RNA in mononuclear cells Separated from healthy human peripheral blood, RT-PCR amplify CXCR4 as RNA extracted a template, to obtain full-length CXCR4 gene sequence, restructur it into retrovirus vectors pLEGFP-N1,which with a green fluorescent protein (GFP), then transfected to PC-3 cells with lipofectamine, observe the expression of CXCR4 by real-time quantitative PCR and Western blotting, and detecte the invasive ability of tumor cells through cell - matrix adhesion by Transwells chamber testing in vitro. 3. Prostate cancer cells line PC-3 transfected CXCR4 gene stably treated with Chemokine SDF-1 for 24 hr, Detect mRNA and protein expression adhesion molecules LFA-1α/ ICAM-1, VLA-4/ VCAM-1 and Kinase P38MAPK by real-time quantitative PCR and Western blot; detect mRNA level and DNA binding activity of HIF-1αand P65NF-κB by real-time quantitative PCR and EMSA. Investigate affect enhanced functions of SDF-1/CXCR4 biological axis in prostate cancer cells on the expression of Moleculars appeal.4. Inoculated human prostate cancer cells line PC-3, PC-3 cells transfected CXCR4 gene and LNcap cells in subcutaneously of male SCID mice, establish transplanted human prostate cancer model of SCID mice And in situ model of tumor metastasis, observe the activities, the rate of tumor incidence, tumor growth and morphological characteristics. Detect expression of CXCR4, prostate cancer-specific antigen (PSA), as well as metastasis-associated factors VEGF and MMP-9 by Immunohistochemistry. Observe impact of cancer cells metastasis and the bone micro-environment in tumor-bearing SCID mice after CXCR4 gene transfection.Result1. Expressing of CXCR4 in Prostate cancer tissues was significantly higher than that in normal prostate tissues adjacent to cancer and benign prostatic hyperplasia (P<0.01) in 148 cases. Expression of CXCR4 protein relate with histological grade and distant metastasis in human Prostate cancer tissues. Values of MVD in Prostate cancer interstitial increased with reduce the degree of tumor differentiation. unrelated with the patient’s age, the metastasis group have higher MVD values than in non-metastasis group, the values of MVD in AR-negative group are increased than in the value of AR-positive group, values of MVD increased as CXCR4, VEGF and MMP -9 protein expression gradually upregulated.2. pLEGFP-CXCR4 vector constructed successfully, which transfected into human prostate cancer cells line PC-3 after 72h, levels of CXCR4 mRNA and protein were significantly up-regulated, invasiveness and Migration of cells increased markedly(Compared with the control group, P<0.01, 89.33±11.67 vs 59.33±7.27)in vitro.3. Prostate cancer cells line PC-3 transfected CXCR4 gene stably treated with Chemokine SDF-1 for 24 hr significantly increase the expression P38MAPK mRNA and protein significantly (P<0.01, n=3), expression of phospho-P38 protein significantly increased (P<0.01, n=3). And Ratio of phospho-P38 and P38 increased, suggeste that CXCR4 gene transfection promote activity of P38 protein; Compared with the control group, expression of ICAM-1 protein in PC-3 cells increased Significantly(p<0.01, n=3), levels of ICAM-1 mRNA increased significantly (P<0.05, n=3); levels of VCAM-1 mRNA increased significantly (P<0.01, n=3), expression of VCAM-1 protein increased also (p<0.01, n=3) in PC-3 cells transfected CXCR4 gene ; levels of LFA-1αmRNA and protein increased significantly (P <0.01,n=3); levels of VLA-4 mRNA have no significant changes (P>0.05, n=3), levels of VLA-4 protein increased significantly (P<0.01, n=3).Compared with the control group, evels of P65NF-κB and HIF-1αmRNA in PC-3 cells transfected CXCR4 gene increased significantly (P<0.01, n=3), DNA binding activity of HIF-1αand P65NF-κB protein increased significantly detected by EMSA (P <0.01, n=3).4. Compared with PC-3/PEGFP-CXCR4 cells and PC-3, LNcap cells groups, significant Differences has not been found in prostate cancer lines in morphology of transplant tumor tissues, growth characteristics and the levels of PSA. Expression of VEGF and MMP-9 in groups of metastatic tumors tissues without CXCR4 transfected and PC-3/PEGFP-CXCR4 groups was significantly higher then two other groups (P<0.05). To simulate human In situ prostate cancer bone metastasis animal model by embedding tumor nodules in the subcutaneous urethra of SCID mice, that each group had not found invasion of the prostate tissues and gonad organizations. Observe SCID mice transplanted tumor transfected CXCR4 gene experiments, Compared with the control group, group of tumor transfected CXCR4 gene SCID mouse generate into a tumor need shorter time, tumor sizes biger, number of metastases more. Sites of cancer bone metastases are mainly in the lumbar vertebral. Dochondral osteogenesis Cartilage and calcification in CXCR4 transfection groups increased, and trabecular bone apparent increased, angiogenesis increased, medullary cavity mononucleosis increased.Conclusion1. Increased expression of CXCR4 in PCa can coordinated chemokine SDF-1 which from Stromal of tumor to promotes invasion and metastasis. high expression of CXCR4 in PCa cells make tumor has a higher capacity of invasion and metastasis. CXCR4 protein expression, MVD in stromal, VEGF and MMP-9 protein expression was positively correlated. 2. CXCR4 transfection can significantly increase the levels of CXCR4 mRNA and protein in prostate cancer cells line, enhance capacity of human prostate cancer cell invasion induced by SDF-1 in vitro.3. CXCR4 transfection PCa cells line PC-3 can increase levels of LFA-1α/ICAM-1, VLA-4/ VCAM-1 and P38MAPK the mRNA and protein in varying degrees, and promote the P38MAPK phosphorylation, increase levels of HIF-1αand the P65NF-κB mRNA, increased DNA binding activity of HIF-1αand P65NF-κB protein significantly.4. Human Prostate cancer SCID mouse transplantation model simulate biological behavior of metastasis for human prostate cancer, maintain a better biological characteristics of human prostate cancer, it is a excellent animal model to study metastasis mechanism and the biological treatment of prostate cancer. Transfection CXCR4 gene into prostate cancer cells promote growth and metastasis of cancer cells in SCID mice, and bone remodeled more significantly, when bone metastasis occur.更多还原