【作者】 周宇；

【导师】 王禾； 秦卫军；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 博士

【摘要】 前列腺癌是欧美男性病死率第二位恶性肿瘤,在我国其发病率也逐年升高。超过80%的晚期前列腺癌患者会发生骨转移。骨转移会严重影响前列腺癌患者的生活质量并且是前列腺癌患者死亡的主要原因。骨性转移可引起疼痛、病理性骨折、高钙血症、脊髓及其它神经受压等并发症,而这些并发症都是由于骨质破坏造成的。抑制骨质破坏的特异性治疗可以明显提高患者的生活质量并延长患者的生存时间。肝素酶1(HPSE1)是一种能切开乙酰肝素链的内切糖苷酶,而乙酰肝素链可以和许多生物活性分子结合,发挥生物调节功能。以往研究发现肝素酶1在多种肿瘤组织中高表达,而且与肿瘤的预后密切相关。另外肝素酶1在肿瘤细胞中过表达可以促进肿瘤的增殖、新生血管生成和向远处转移并且能抑制肿瘤细胞凋亡。最近的研究发现肝素酶1还与骨形成和骨折愈合相关,并且能促进骨髓瘤细胞向骨组织转移。但有关肝素酶1与前列腺癌及与前列腺癌骨转移所导致的骨破坏间的关系尚不清楚。研究目的:本实验拟建立肝素酶1过表达的前列腺癌细胞,并观察肝素酶1对前列腺癌细胞在体内外增殖、侵袭及对骨组织破坏作用的影响。研究方法:1.将插入人肝素酶1基因cDNA序列的真核表达载体pIRES2 -EGFP-HPSE1采用脂质体转染前列腺癌细胞系PC-3,并用G418稳定筛选得到过表达肝素酶1的细胞克隆,命名为PC-3-HPSE1。同时将pIRES2-EGFP空载体转染PC-3细胞并稳定筛选作为对照,命名为PC-3-EGFP。采用实时定量PCR、Western-blot和间接免疫荧光检测肝素酶1 mRNA和蛋白在两组细胞内的表达,并用ELISA法检测两组细胞内肝素酶1的酶活性。2.采用MTT法检测两组细胞在体外增殖情况,并用Transwell小室实验检测两组细胞在体外的侵袭力。建立裸鼠皮下移植瘤模型,定期观测肿瘤生长情况。6周后HE染色观察肿瘤组织形态及了解其它脏器的转移情况。采用免疫组化法检测肿瘤组织内肝素酶1的表达情况。3.采用胫骨内注射法建立裸鼠前列腺癌骨转移模型,定期观察裸鼠肿瘤生长、活动能力和肿瘤大小。4周后X线平片观察胫骨骨质破坏情况。取胫骨标本,肿瘤组织HE染色观察肿瘤形态,骨组织经脱钙后,HE染色观察肿瘤细胞在骨组织内生长情况及骨组织破坏程度。采用图形分析软件测量胫骨骨皮质面积,比较骨组织破坏程度。耐酒石酸酸性磷酸酶(TRAP)染色检测破骨细胞,比较两组破骨细胞的活性。研究结果:1.成功建立了稳定表达肝素酶1的PC-3细胞,实时定量PCR结果显示实验组PC-3-HPSE1细胞内肝素酶1的mRNA表达水平比对照组PC-3-EGFP细胞高8倍。Western-blot检测结果显示PC-3-HPSE1细胞内可检测到明显的条带,大小约为50kDa,与活性肝素酶1蛋白大小一致,而PC-3-EGFP细胞则检测不到。间接免疫荧光结果显示PC-3-HPSE1细胞内可见较强的红色荧光,而PC-3-EGFP细胞仅能观测到很弱的荧光。ELISA法检测细胞内肝素酶1活性结果显示PC-3-HPSE1细胞内肝素酶酶活性明显高于PC-3-EGFP细胞。2. MTT法检测细胞体外增殖能力,结果显示两组细胞体外增殖能力无显著差异。Transwell小室实验结果显示PC-3-HPSE1细胞穿膜细胞数明显多于PC-3-EGFP细胞。成功建立了两组细胞的裸鼠皮下移植瘤模型,结果显示PC-3-HPSE1细胞从接种细胞到可于皮下触及肿瘤包块所需时间明显短于PC-3-EGFP细胞,在多个时间点PC-3-HPSE1细胞形成的肿瘤体积明显大于PC-3-EGFP细胞组。PC-3-HPSE1细胞组发现肺转移6例,而PC-3-EGFP细胞组仅发现2例肺转移。免疫组化染色显示PC-3-HPSE1细胞组肿瘤组织肝素酶1表达水平较高,而PC-3-EGFP细胞组肿瘤组织肝素酶1表达水平很低。3.成功采用胫骨内注射法建立了裸鼠前列腺癌骨转移模型,每只裸鼠都在注射肿瘤的左后肢形成肉眼可见的肿瘤。X线平片显示所有的动物模型均发生了溶骨性骨破坏,且PC-3-HPSE1组骨破坏程度高于PC-3-EGFP组。PC-3-HPSE1组有3例发生病理性骨折,而PC-3-EGFP组没有发生病理性骨折。HE染色显示骨皮质发生了不同程度的破坏,骨组织内可见少量肿瘤细胞,在骨破坏区可见大量破骨细胞。用图像分析软件分析裸鼠胫骨骨皮质面积,结果显示PC-3-EGFP组骨皮质面积明显小于PC-3-EGFP组。抗酒石酸酸性磷酸酶(TRAP)染色结果显示PC-3-HPSE1组破骨细胞活性明显强于PC-3-EGFP组。结论:1.通过Real-Time PCR、Western-blot和间接免疫荧光检测,证实成功建立了过表达肝素酶1的前列腺癌细胞PC-3-HPSE1,PC-3-HPSE1细胞外源性表达的肝素酶1蛋白具有肝素酶酶活性。并且PC-3-HPSE1细胞在裸鼠体内生长时也能稳定表达肝素酶1。2.虽然肝素酶1过表达不能促进PC-3细胞在体外的增殖能力,但能明显促进PC-3细胞在体内的增殖能力。并能明显提高PC-3细胞在体内外的侵袭及发生转移的能力。3.肝素酶1过表达可以促进PC-3细胞在骨组织内的生长,同时也可以提高PC-3细胞诱导破骨细胞形成的能力,增加破骨细胞的活性,从而促进PC-3细胞对骨组织的破坏作用。因此肝素酶1可能是前列腺癌骨转移治疗的一个潜在靶点。更多还原

【Abstract】 Prostate cancer is the second leading cause of death in America and is becoming more common in China. Over 80% of advanced prostate cancer patients suffer from bone metastasis. Bone metastasis significantly decreases the patients’quality of life, and is the most common cause of death for prostate cancer patients. Complications of metastatic bone disease include pathological fractures, pain, cranial nerve palsies, spinal cord compression, and bone marrow suppression, most of which are caused by bone destruction. Treatment of bone metastasis includes chemotherapy, radiotherapy and bone specific therapy aimed to prevent bone destruction, which can prolong the patients’life span and improve quality of life. Based on this hypothesis, anti-osteoclast agents such as bisphosphonates are used to prevent bone lesions in many different cancers including prostate cancer.Heparanase is an endoglycosidase that cleaves heparin sulfate, which binds many different bioactive molecules. This enzyme has an important role in both normal and pathologic processes, especially in angiogenesis and metastasis of tumors. Previous studies revealed elevated heparanase expression in human breast, bladder, pancreas, prostate tumors, which correlated with advanced tumor progression and metastasis. Overexpression of heparanase in breast cancer cells accelerates cell proliferation and survival in vitro and promotes angiogenesis and invasion in vivo. In contrast, reducing heparanase levels in lymphoma cells with anti-heparanase ribozyme or siRNA-mediated gene-silencing vectors alters tumorigenic properties of the cells.Recent studies indicate that myeloma cells overexpressing heparanase can promote spontaneous metastasis to bone. Another study proved that heparanase was involved in the process of bone formation. However, it is unknown what role heparanase plays in bone destruction in prostate cancer patients. Objective:To established heparanase over-expressing PC-3 cells and evaluate the effects of elevated heparanase expression on bone destruction and invasiveness of prostate cancer cells.Methods:1. The pIRES2-EGFP/HPSE1 plasmid containing heparanase cDNA (HPSE1) and the empty pIRES2-EGFP vector as a control, were transfected into PC-3 human prostate cancer cells using the Lipofectin reagent. Cells stably expressing the plasmid were selected with G418. These two groups of cells are referred to as PC-3-HPSE1 and PC-3-EGFP. Transfected cells were maintained routinely in the selection medium to avoid overgrowth of non-transfected cells. Expression of heparanase1 was detected by REAL-TIME PCR, Western-blot and indirect Immunofluorescence Assay. Heparanase activity was measured by Heparan Deagrading Enzyme Assay.2. In vitro cell growth was investigated by MTT assay and in vitro cell invasiveness was investigated by Transwell assay. Tumorigenicity assay was performed in nude mice model to assess the malignant behavior of cells. Tumor diameter was recorded using caliper regularly. H&E staining was used for histological verification of tumor growth and metastasis. To determine if heparanase expression was maintained in vivo, immunohistochemistry of the tumors were performed using rabbit anti-human heparanase polyclonal antibody.3. Animal model of bone metastasis was established using direct tibia injection. Twenty-eight days after injection, the animals were anesthetized and a flat plate radiograph was taken. After sacrifice, the tumor-bearing leg was harvested, and the hindlimb tumor diameter was recorded using caliper. H&E staining was used for histological verification of tumor growth and bone destruction. The area of cortical bone present in each specimen was calculated using image analysis software. To detect osteoclasts, tartrate-resistant acid phosphatase (TRAP) staining was performed on sections of bone.Results:1. To investigate the role of heparanase in bone metastasis of prostate cancer, we successfully established human prostate cancer PC-3 cells over-expressing heparanase, referred to hereafter as PC-3-HPSE1 cells. PC-3 cells transfected with empty vector, PC-3-EGFP cells, were used as the negative control. Real time RT-PCR revealed an 8 fold increase in relative expression of heparanase mRNA in PC-3-HPSE1 cells compared to the PC-3-EGFP cells. Western blot analysis revealed no detectable heparanase protein in PC-3-EGFP cells, whereas there was a distinct heparanase protein band in the PC-3-HPSE1 cells. Indirect immunofluorescence labeling showed a very weak signal of heparanase in PC-3-EGFP cells, whereas there was much stronger staining in PC-3-HPSE1 cells. Heparan Deagrading Enzyme Assay showed higher heparanase activity in PC-3-EGFP cells than in PC-3-EGFP cells.2. Overexpressing heparanase did not alter the growth of the cells in vitro even after 72 hours. The invasion assay showed that more PC-3-HPSE1 cells traveled through the filter than in either of the control groups. Six weeks after the injection, each mouse formed a visible tumor. The mean tumor diameter of the PC-3-EGFP group was significantly smaller than that of the PC-3-HPSE1 group, which indicated that cells with higher heparanase level proliferated more quickly in vivo. Six cases of lung metastasis were found in the PC-3-HPSE1 group, while only two cases were found in the PC-3-EGFP group. Immunohistochemistry of tumors demonstrated that tumors of PC-3-EGFP cells were weakly positive for heparanase expression, while tumors of PC-3-HPSE1 cells are strongly positive for heparanase.3. Four weeks after the injection, radiographs of the mice showed that all animals in both groups formed osteolytic lesions characterized by obliteration of the partial proximal tibia. each mouse formed a visible tumor in the hindlimb which was injected with tumor cells. No pathologic fracture was found in the PC-3-EGFP group, while there were three cases of pathologic fracture in the PC-3-HPSE1 group. H&E staining of histological sections from the tibias in both groups revealed there was eradication of the proximal tibia in both groups, but in the PC-3-EGFP group, the cortical structure of the tibias was better preserved. Histomorphometric analysis revealed significantly less cortical bone destruction in the PC-3-EGFP group than that in the PC-3-HPSE1 group. TRAP staining showed fewer TRAP-positive cells in the PC-3-EGFP group than in the PC-3-HPSE1 group.Conclusions:1. PC-3-HPSE1 cells could express high level of heparanase both in vitro and in vivo.2. Although heparnase could not promote proliferation of tumor cells in vitro, it can promote cell proliferation and invasiveness in vivo.3. Heparanase could promote tumor cell growth in bone tissue and bone destruction caused by prostate cancer cells. Targeting heparanase in tumors may be an effective means of controlling bone metastasis in prostate cancer patients.更多还原