【作者】 常青；

【导师】 秦仁义；

【作者基本信息】 华中科技大学 ， 外科学， 2006， 博士

【摘要】 胰腺癌是人体最常见的恶性肿瘤之一，其患病率在世界范围呈上升趋势。胰腺癌由于恶性程度高、手术切除率低、早期诊断困难，治疗的效果不尽人意。近年来各种手术方式的改进以及化疗、放疗方案等综合措施的引进，手术并发症和死亡率有所降低，但5年生存率仍提高不多。随着免疫和分子生物学在肿瘤的发生、发展和转移机制研究的不断深入，为胰腺癌的治疗提供了新的希望。近年来发现，缺氧是影响化疗效果而且干扰了肿瘤对放疗的反应的一个重要因素。胰腺癌血供很差，在严重缺氧时还能继续增殖的原因可能在于持续缺氧的状态诱导了胰腺癌中缺氧诱导因子（HIF）-1α的表达。研究表明，血管内皮生长因子（VEGF）和糖酵解过程中多种关键酶基因都是受HIF-1α调控的，HIF-1α通过促进肿瘤组织的血管生成和能量代谢加速肿瘤组织的生长，同时也促进了肿瘤的侵袭和转移。HIF-1α在胰腺癌中广泛表达，对胰腺癌的血管生成、发生发展起到了很大的作用。而已有实验表明，人类凋亡抑制蛋白（human inhibitor of apoptosis protein，hIAP）家族成员之一survivin在胰腺癌中高表达，且与化疗敏感性有关；整合素家族中β₁-integrin在胰腺癌侵袭、转移过程中也起着重要的作用。本研究旨在观察反义HIF-1α对胰腺癌生长、转移、化疗敏感性的影响及其与survivin、β₁-integrin的关系。本实验共分为以下四部分。第一部分缺氧诱导因子-1α在胰腺癌中的表达和意义目的本研究旨在检测胰腺癌中缺氧诱导因子-1α（HIF-1α）的表达，探讨HIF-1α的表达与临床特征的关系，从而深入探讨其在胰腺癌发生发展中的作用。材料和方法运用免疫组织化学技术（streptavidin-peroxidase，SP）检测研究48例胰腺癌及癌旁组织中HIF-1α的表达。结果1．48例胰腺癌标本中27例表达HIF-1α蛋白，而在癌旁组织未检测到HIF-1α的表达（P＜0.01）。2．HIF-1α蛋白的表达与胰腺癌患者的性别、年龄和肿瘤大小无关，与胰腺癌的分化程度及是否转移有关（P＜0.05）。结论1．HIF-1α在胰腺癌中高度表达，在癌旁胰腺组织未见表达，可能与胰腺癌的发生发展或与治疗效果有关，但其具体作用及作用机制均有待进一步深入的研究。2．HIF-1α与胰腺癌患者预后的密切相关。第二部分重组表达质粒pcDNA3.1（+）／反义HIF-1α的构建及其在人胰腺癌细胞株BxPc-3中的稳定表达目的第一部分显示，胰腺癌高表达HIF-1α蛋白，本部分拟构建HIF-1α基因的反义表达核苷酸，经脂质体转染人胰腺癌细胞株BxPc-3，建立稳定表达反义HIF-1α基因的细胞模型，为反义封闭治疗奠定基础。方法1．采用PCR方法扩增pCR3.1-FLAG-HIF-1α质粒中反义HIF-1α基因功能片段。2．定向克隆至真核表达载体pcDNA3.1（+）上。3．转染至BxPc-3细胞内，经G418筛选，建立稳定表达HIF-1α反义基因的细胞株。4．RT-PCR和Western blot分析HIF-1α的表达，建立稳定表达HIF-1α反义基因的细胞株。结果1．酶切鉴定，凝胶电泳分析，两条带大小分别为721bp和5.4kb，与预期结果符合。2．脂质体包裹转染BxPc-3后，RT-PCR和Western blot分析证实实验组HIF-1αmRNA表达为0.2367±0.0094，蛋白表达为0.1798±0.0063，与对照组相比明显降低（P＜0.05）。结论1．本研究成功构建了HIF-1α的反义核苷酸，与预期结果符合。2．经脂质体途径转染BxPc-3细胞，该反义核苷酸能够抑制HIF-1α的表达。3．该反义核苷酸的成功构建为下一步实验研究奠定了基础。第三部分反义HIF-1α增加化疗药物敏感性的实验研究目的本研究拟利用构建的反义HIF-1α，转染BxPc-3细胞后，观察BxPc-3细胞对化疗药物敏感性的变化及其与survivin的关系。方法1．将HIF-1α反义核苷酸转染至BxPc-3细胞，经G418筛选后，建立稳定表达反义HIF-1α的BxPc-3细胞株，加入不同浓度的氟尿嘧啶，阿霉素，吉西他宾。2．MTT检测各组的增殖活性。3．运用流式细胞仪检测各组细胞凋亡指数。4．RT-PCR和Western blot分析各组survivin的表达。结果1．与对照组相比，实验组的生长抑制率、凋亡率与剂量成正比，高剂量引起高抑制（P＜0.05）。2．实验组survivin的mRNA表达为0.4601±0.0122，蛋白表达为0.3659±0.0117，与对照组相比明显降低（P＜0.05）。结论转染反义HIF-1α基因可明显增强氟尿嘧啶，阿霉素，吉西他宾对胰腺癌细胞的杀伤力，并伴有survivin表达量的明显下调，这表明反义HIF-1α可能通过阻断survivin的表达而增强胰腺癌对化疗的敏感性，因此，阻断HIF-1α的表达可望成为胰腺癌基因治疗的新途径。第四部分反义HIF-1α对胰腺癌生长、转移影响的实验研究目的本研究拟利用构建的反义HIF-1α，转染BxPc-3细胞后，观察其对胰腺癌生长、转移的影响及其与β₁-integrin的关系。方法1．将HIF-1α反义核苷酸转染至BxPc-3细胞，经G418筛选后，建立稳定表达反义HIF-1α的BxPc-3细胞株，Transwell侵袭室方法检测BxPc-3细胞侵袭能力。2．RT-PCR和Western blot分析各组β₁-integrin的表达。3．裸鼠皮下接种各组处理细胞8周后，观察其肿瘤生长情况。结果1．实验组迁移的细胞数为110.64±16.4，远少于对照组（P＜0.05）。2．实验组β₁-integrin的mRNA表达为0.2568±0.0115，蛋白表达为0.2962±0.0167，与对照组相比明显降低（P＜0.05）。3．实验组肿瘤的体积、重量、生长速度远低于对照组（P＜0.05）。结论转染反义HIF-1α基因可明显抑制胰腺癌细胞的侵袭力，并伴有β₁-integrin表达量的明显下调且抑制了皮下移植瘤的生长，这表明反义HIF-1α可能通过阻断β₁-integrin的表达而抑制胰腺癌的生长和转移。因此，阻断HIF-1α的表达可望成为胰腺癌基因治疗的新途径。本课题提出以下新见解：1．HIF-1α在胰腺癌中高度表达，在癌旁胰腺组织未见表达，提示HIF-1α在胰腺癌的发生发展或或治疗效果中发生重要的作用。2．本文探讨了HIF-1α的表达与胰腺癌患者临床特征的关系，结果显示HIF-1α蛋白的表达与胰腺癌患者的性别、年龄和肿瘤大小无关，而与胰腺癌的分化程度及是否转移有关，提示HIF-1α与胰腺癌患者预后的密切相关。3．本研究成功构建了HIF-1α的反义核苷酸，与预期结果符合。经脂质体途径转染BxPc-3细胞，可在BxPc-3中稳定表达，该反义核苷酸能够抑制HIF-1α的mRNA和蛋白的表达。4．缺氧状态下，反义HIF-1α基因可明显增强氟尿嘧啶，阿霉素，吉西他宾对胰腺癌细胞的杀伤力，并伴有survivin表达量的明显下调，这表明反义HIF-1α可能通过阻断survivin的表达而增强胰腺癌对化疗的敏感性。5．缺氧状态下，反义HIF-1α基因可明显抑制胰腺癌细胞的侵袭力，并伴有β₁-integrin表达量的明显下调且抑制了皮下移植瘤的生长，这表明反义HIF-1α可能通过阻断β₁-integrin的表达而抑制胰腺癌的生长和转移。因此，阻断HIF-1α的表达可望成为胰腺癌基因治疗的新途径。更多还原

【Abstract】 Hypoxia-inducible factor 1 （HIF-1） is a heterodimeric complex, composed of an α and a β subunit, both belonging to the basic helix-loop-helix （bHLH）/Per-aryl hydrocarbon receptor nuclear translocator-Sim （PAS） family of transcription factors. The bHLH domain mediates dimerization and DNA binding in many transcription factors. PAS is an additional dimerization motif. Whereas HIF-1β is a common subunit of multiple bHLH proteins, HIF-1α is the unique, O₂-regulated subunit that determines HIF-1 activity. Although HIF-1β is quite stable in normoxic conditions, HIF-1α is extremely unstable and quickly degraded by the ubiquitin-proteasome system. HIF-1α plays an important role in solid tumor formation in vivo by promoting angiogenesis and anaerobic metabolism. Several studies have demonstrated that HIF-1α can be detected in most types of human tumors examined, including bladder, breast, colon, glial, hepatocellular, ovarian, pancreatic, prostate, renal and oesophageal tumors. The expression of HIF-1α is inducible by oxygen concentrations below 6%, and the activation of hypoxia-responsive elements by HIF-1 has been reported to correlate with decreasing oxygen concentrations in vitro.Survivin, a member of the inhibitors of apoptosis protein （IAP） family, is characterized by a unique structure that discriminates it from other members of the IAP family. It contains only a single BIR repeat and lacks a carboxy terminal RING finger domain. Survivin is expressed in the G₂/M phase of cell cycle in a cycle-regulated manner. It directly binds to and inhibits both Caspase-3 and Caspase-7 activity, leading to arrest of apoptosis. Survivin expression is not detectable in differentiated normal adult cells of any organ, but it is abundantly expressed in embryonic tissues and in a wide range of cancer tissues including neuroblastoma, cholangiocarcinoma, colorectal, stomach, breast and uterine cervical carcinomas. It has been demonstrated recently that survivin is also frequently expressed in malignant pancreatic ductal tumors and pancreatic adenocarcinoma. Furthermore, the prognostic value of survivin expression has been reported in several human cancers. Studies demonstrating resistance of survivin-transfected cells to anticancer drug-induced apoptosis and sensitization to chemotherapy by survivin antisense treatment have shown that survivin is implicated in sensitization to chemotherapy.The integrin family of cell-surface heterodimeric glycoproteins composed of a and β subunits function primarily as receptors for extracellular matrix ligands, which regulate many aspects of cell physiology, including morphology, adhesion, migration, proliferation, and differentiation. Integrins may function by forming focal adhesions at the site of the cytoplasmic membrane processes. Many cancers show abnormalities of integrin function as a result of transformation by oncogenes. More importantly, the growth of several tumors depends on β₁-integrin function. Previous studies have demonstrated that β₁-integrin is a poor prognostic factor in patients with cancers. Evidences show an essential role of β₁-integrins in invasion of pancreatic carcinoma cells and also suggest subtle regulatory mechanisms of cell invasion.On the other hand, the prognosis for patients with pancreatic cancer remains poor, prompting the search for new treatment strategies. Although treatments such as pre-operative chemotherapy and chemoradiation therapy are currently used for patients with advanced pancreatic cancer, their results are not satisfactory. Even in early-stage disease, we have experienced many patients who developed local recurrence of tumor or distant metastasis within a short period after operation.In pancreatic cancer, the relationship of HIF-1α, survivin and β₁-integrin is not very clear at present. This study aimed to observe the effect of antisense HIF-1α on progression, metastasis and chemosensitivity of pancreatic cancer. Part 1 Expression and significance of HIF-1α in pancreatic cancer ObjectiveTo investigate the expression of HIF-1α, the relationship with clinical features, and determine the role in the development of pancreatic cancer.MethodsThe expression of HIF-1α in tumor tissues and non-cancerous adjacent pancreas tissues was detected by SP immunohistochemical technique.ResultsHIF-1α was expressed in 27 of 48 cases （56.3%）. In contrast, no expression of HIF-1α in adjacent histologically noncancerous pancreas was detectable （P<0.01） . Expression of HIF-1α had no relationship with ages, sexes and tumor sizes of pancreatic cancer patients. But the status of HIF-1α was significantly correlated with metastatic status and differentiation level （P<0.05）.ConclusionHigh expression of HIF-1α in pancreatic cancer suggests it may play an important role in the development of pancreatic cancer. Therefore, assessment of HIF-1α expression might be useful for predicting the prognosis of pancreatic cancer patients. Part 2 Reconstruction of eukaryotic gene expression plasmid pcDNA3.1（+） /antisense HIF-1α and its stable expression in human pancreatic cancer cell line BxPc-3ObjectiveTo further explore the role of HIF-1α, antisense plasmid of HIF-1α was constructed, and transfected into human pancreatic cancer cell line BxPc-3.MethodsAntisense HIF-1α DNA structure was amplified by PCR and inserted into eukaryotic plasmid pcDNA3.1（+）. The expression plasmid pcDNA3.1（+）/antisense HIF-1α was transfected into human pancreatic cancer cell line BxPc-3 through Dosper liposomal transfection reagent. Cells stably expressing antisense HIF-1α were screened with G418. BxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O₂ for 4 hours. The expression of HIF-1α was detected by RT-PCR and Western blot.ResultsThe constructed expression plasmid was analyzed with restriction enzymes and gel electrophoresis. Two fragments at 721bp and 5.4kb, were respectively found, which was consistent with what expected. Eukaryotic plasmid was transfected into BxPc-3 cells through Dosper liposomal transfection reagent. HIF-1α mRNA （0.2367±0.0094） and protein （0.1798±0.0063） expression in the experimental group were significantly depressed by this plasmid, compared with the control （P<0.05）.ConclusionBxPc-3 with stable antisense HIF-1α expression was successfully constructed, which could be applied to explore further. Part 3 An antisense plasmid targeting HIF-1α expression sensitizes pancreatic cancer cells to chemotherapyObjectiveThe apoptosis induced by chemotherapeutic agent was inhibited through HIF-1α expression. Antisense plasmid of HIF-1α was constructed and transfected into BxPc-3. The change of sensitizion to chemotherapy and the relationship with survivin was detected.MethodsBxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O₂ for 4 hours. Different dosages of chemotherapy agents （5-fluorouracil, doxorubicin, and gemcitabine） were added into BxPc-3 cells after antisense HIF-1α transfected. The growth inhibition rates were measured by MTT, apoptosis rates were measured by flow cytometry and the expression of survivin was detected by RT-PCR and Western blot.ResultsHigher dosages (100, 200, and 400 mg/L of 5-fluorouracil; 0.05, 0.075, and 0.1 mg/L of doxorubicin; and 10^-9, 10^-8, and 10^-7 mol/L of gemcitabine) caused a greater increase of inhibition in the experimental group than in control （P<0.05）. survivin mRNA （0.4601±0.0122） and protein （0.3659±0.0117） expression in the experimental group were markly down-regulated compared with the control （P<0.05）.ConclusionOur data demonstrate that antisense HIF-1α inhibits expressions of survivin, enhancing apoptosis in human pancreatic cancer cells and thus the sensitivity to chemotherapy was increased. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy. Part 4 An antisense plasmid targeting HIF-1α expression restrains the progression and metastasis of pancreatic cancerObjectiveTo observe the effect of antisense HIF-1α on the progression and metastasis of pancreatic cancer and the relationship with β₁ integrin.MethodsBxPc-3 cells transfected with antisense HIF-1α plasmid were exposed to 0.5% O₂ for 4 hours. The expression of β₁ integrin was detected by RT-PCR and Western blot. The migration of BxPc-3 cells was assayed using transwell cell culture chambers. Subcutaneous transplantation of BxPc-3 cells in nude mice for 8 weeks was to assess progression and metastasis of pancreatic cancer.Resultsβ₁ integrin mRNA （0.2568±0.0115） and protein （0.2962±0.0167） expression in the experimental group were markly down-regulated compared with the control （P<0.05）.The number of migrated BxPc-3 cells in the experimental group was 110.6±16.4, far less than in control （P<0.05）. In vivo, the tumor size and weight in the experimental group were significantly lower than those in control （P<0.05）.ConclusionOur data demonstrate that antisense HIF-1α inhibits expressions of β₁ integrin, restraining the progression and metastasis of pancreatic cancer. Therefore, HIF-1α may play a very important role in progression and metastasis of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy. In summary, the following points of view outcome:1. High expression of HIF-1α in pancreatic cancer, but not in non-cancerous adjacent pancreas tissues, suggests it may play an important role in the development of pancreatic cancer.2. The relationship between HIF-1α expression and the clinical features of pancreatic cancer was discussed. Expression of HIF-1α had no relationship with ages, sexes and tumor sizes of pancreatic cancer patients. But the status of HIF-1α was significantly correlated with metastatic status and differentiation level. Therefore, assessment of HIF-1α expression might be useful for predicting the prognosis of pancreatic cancer patients.3. BxPc-3 with stable antisense HIF-1α expression was successfully constructed. The mRNA and protein expression of HIF-1α was successfully inhibited by the antisense HIF-1α plasmid under hypoxia.4. Under hypoxia, antisense HIF-1α inhibits expressions of survivin, enhancing apoptosis in human pancreatic cancer cells and thus the sensitivity to chemotherapy was increased. Therefore, HIF-1α may play a very important role in chemosensitivity of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy.5. Under hypoxia, antisense HIF-1α inhibits expressions of β₁ integrin, restraining the progression and metastasis of pancreatic cancer. Therefore, HIF-1α may play a very important role in progression and metastasis of human pancreatic cancer. Blocking HIF-1α in pancreatic cancer cells may offer an avenue for gene therapy.更多还原