【作者】 牛卫博；

【导师】 牛军； 徐克森；

【作者基本信息】 山东大学 ， 外科学， 2010， 博士

【摘要】 研究背景和意义整合素是细胞表面黏附分子家族成员,介导细胞间及细胞与细胞外基质(ECM)的黏附反应,影响细胞基因的表达及细胞内信号传递,调节细胞的各种生物学行为。其中αvβ6是一种特殊类型的整合素亚型,在正常上皮组织、上皮良性肿瘤组织中不表达,在多种恶性上皮源性肿瘤组织中诱导表达,在肿瘤增殖、侵袭和转移中发挥了重要调控作用。目前在口腔、皮肤鳞癌、乳腺癌、结直肠癌、胃癌、胰腺癌、卵巢及富颈癌中均发现αvβ6高表达,在结肠癌领域对αvβ6的研究更为深入细致。前期研究表明：通过转基因诱导表达αvβ6整合素,可使结肠癌细胞分泌MMP-9增多,加速破坏细胞外基质,促进结肠癌肝脏转移。β6链胞内功能段与细胞外信号调节激酶(ERK)之间具有生理性直接连接。αvβ6在结肠癌标本侵袭边缘高表达,在肿瘤内部低表达,作为影响结肠癌预后的重要危险因子,与结肠癌进展密切相关。但是整合素αvβ6对胰腺癌恶性生物学行为、化疗耐药及预后的影响,目前国内外尚未见报道。胰腺癌是预后最差的消化系统恶性肿瘤之一,由于它的高侵袭性和高转移率,使80%以上的病人就诊时已失去了根治性切除的机会,而且胰腺癌对现有的化疗、放疗及免疫治疗均不敏感。在过去20年中胰腺癌5年生存率无显著变化,仍在5%以下。探讨整合素αvβ6对胰腺癌增殖、侵袭、转移、凋亡、化疗耐药及预后的影响,有助于深入理解胰腺癌恶性进展的分子生物学机制,以αvβ6为靶点进行高效无毒的靶向治疗,对于提高胰腺癌的疗效及改善预后将起到重要的促进作用。第一部分整合素αvβ6在胰腺癌组织中的表达及对预后的影响目的研究整合素αvβ6及其相关因子TGF-β1在胰腺导管腺癌组织中的表达情况,与临床病理特征间的关系及对患者预后的影响。方法选择92例胰腺导管腺癌病人的手术病理标本,10例尸解正常胰腺组织作为对照,制作组织芯片；免疫组化染色检测αvβ6、TGF-β1在胰腺癌组织中的表达情况；回顾性分析病人的临床病理资料并随访生存情况,探讨整合素αvβ6、TGF-β1在胰腺癌中的表达情况及其相关性,与临床病理特征之间的关系；通过生存分析,判断其表达高低对患者预后的影响。结果整合素αvβ6在正常胰腺组织中不表达,在胰腺导管腺癌组织中的阳性表达率为56.5%,其表达高低与肿瘤大小(P=0.041),TNM分期(P=0.027)及淋巴结转移(P=0.030)成正相关。TGF-β1在胰腺癌组织中的阳性表达率为66.3%,其表达高低与肿瘤分化不良及TNM分期密切相关。Spearman相关检验证实αvβ6与TGF-β1在胰腺癌中的表达呈正相关关系。Kaplan-Meier生存曲线显示αvβ6高表达的病人中位生存时间较αvβ6低表达的病人明显缩短(P=0.001),单变量及多变量生存分析显示整合素αvβ6是影响胰腺癌预后的独立不良指标。结论整合素αvβ6在胰腺导管腺癌组织中呈现高表达并与TGF-β1的表达情况成正相关,是影响病人预后的一种重要危险因素。意义揭示了整合素αvβ6和TGF-β1在胰腺癌组织中的表达情况及其相关性,与各临床病理特征之间的关系；首次证实整合素αvβ6是影响胰腺癌预后的一个独立不良指标,为更好地判断患者预后并以αvβ6作为靶点对胰腺癌进行靶向治疗提供了实验及理论依据。第二部分整合素αvβ6对胰腺癌细胞增殖、侵袭及转移的影响目的研究整合素αvβ6在胰腺癌细胞中的表达情况及对细胞增殖、侵袭及转移能力的影响。方法选取三种胰腺腺癌细胞系,RT-PCR、流式细胞术检测整合素αvβ6在这三种细胞中的表达高低；选取αvβ6高表达的胰腺癌细胞,应用针对αvβ6的功能性阻断单抗10D5封闭细胞中αvβ6的功能后,MTT法检测细胞增殖能力的变化；Transwell侵袭小室法检测细胞侵袭能力的变化；明胶酶谱及蛋白印迹法检测细胞MMP-9分泌水平的变化；通过比较实验组和对照组之间各组数据的差异,分析整合素αvβ6所起的重要作用。结果RT-PCR及流式细胞检测从转录及蛋白水平证实整合素αvβ6在胰腺癌细胞系PANC-1中高表达,在Capan-2中较高表达,在CFPAC-1中低表达；选取PANC-1及Capan-2细胞应用10D5封闭细胞中αvβ6的功能后,细胞增殖率仅为对照组细胞的(61.94±3.62)%和(67.35±5.02)%；侵袭细胞数目由71±9／HP,36±6／HP分别减少到19±5／HP,13±4／HP；如先用抗MMP-9的单抗Ab-1预处理细胞,则各组的侵袭细胞数减少到相似的水平(20±4／HP,12±5／HP),提示胰腺癌细胞通过重建基底膜的侵袭能力是由整合素αvβ6介导的,并依赖于MMP-9的活性；明胶酶谱及蛋白印迹结果显示阻断细胞中αvβ6的功能后,PANC-1及Capan-2细胞的MMP-9分泌量分别减少到对照组细胞的(9.28±3.11)%和(6.35±2.46)%。结论整合素αvβ6对胰腺癌细胞的体外增殖、侵袭及MMP-9分泌水平起着重要的促进作用。阻断了细胞中αvβ6的功能,就可以显著抑制细胞的增殖、侵袭能力及转移潜能。意义首次检测了整合素αvβ6在不同胰腺癌细胞系的表达情况,揭示了αvβ6在胰腺癌细胞增殖、侵袭及转移过程中的重要作用,为针对αvβ6通过单克隆抗体对胰腺癌进行靶向治疗提供了确切的实验依据。第三部分整合素αvβ6对胰腺癌细胞凋亡及化疗敏感性的影响目的研究整合素αvβ6在胰腺癌细胞凋亡及化疗敏感性中所起的重要作用方法构建针对αvβ6的小片段干扰RNA (small interfering RNA, siRNA),选取干扰效率最强的αvβ6 siRNA,采用脂质体2000将其转染入胰腺癌细胞PANC-1,无效干扰片段RNA转染组作为对照；RT-PCR、Western Blotting检测细胞中αvβ6表达的沉默效果;MMT法检测细胞增殖状态；流式细胞术检测细胞周期分布及细胞凋亡Annexin V-FITC试剂盒测定细胞凋亡率的变化；以吉西他滨为化疗药物,检测抑制αvβ6表达后胰腺癌细胞化疗敏感性及凋亡改变；应用Caspase-3检测试剂盒测定细胞内Caspase-3活性的变化；通过建立BALB／c裸鼠胰腺癌皮下种植瘤模型,比较各组细胞的成瘤能力及αvβ6对肿瘤生长的影响。结果αvβ6 siRNA转染入PANC-1细胞后48小时,细胞内αvβ6的表达在mRNA和蛋白水平均被显著抑制,并持续到转染后96小时。沉默αvβ6表达后PANC-1细胞呈现时间依赖性的增殖抑制；转染后96小时,有41%的PANC-1细胞阻滞在G2／M期,细胞凋亡率升高到13.5±2.3%；吉西他滨可以诱导PANC-1细胞凋亡,应用siRNA沉默αvβ6表达可以提高细胞对吉西他滨的化疗敏感性,增强吉西他滨诱导的Caspase-3介导的细胞凋亡。成瘤实验见αvβ6 siRNA转染组细胞形成肉眼可见的肿瘤时间平均为(15±4.6)d,较对照组(9±2.7)d明显延长。种植后6周,αVβ6 siRNA转染组形成肿瘤的平均体积较阴性转染组明显缩小(170±32mm3vs 760±48mm3,P<0.01)。结论整合素αvβ6在抑制胰腺癌细胞凋亡及增强吉西他滨化疗耐药性中起着重要作用,通过RNA干扰沉默αvβ6表达可以显著抑制胰腺癌细胞体内外增殖,引起细胞周期G2／M阻滞,诱导细胞凋亡及增强吉西他滨化疗敏感性。整合素αVβ6作为胰腺癌靶向治疗新的潜在靶点,有着广阔的临床应用前景。意义首次构建了靶向αvβ6基因的小干扰RNA并将其成功转染入PANC-1胰腺癌细胞,结果显著抑制了胰腺癌细胞的体内外生长,引起细胞周期G2／M阻滞,诱导细胞凋亡并增强了吉西他滨化疗敏感性,为以αvβ6为靶点对胰腺癌进行基因治疗提供了一条新的途径。更多还原

【Abstract】 Background and significanceIntegrins are a family of cell surface adhesion molecules which mediate cell-cell and cell-extracellular matrix (ECM) interactions, influence the expression of cellular gene, and trigger intracellular signaling pathways to modulate cellular biological behavior. Among the various members of integrin family,αvβ6 appears to be directly implicated in the cancer progression. As an epithelial-specific integrin,αvβ6 usually is not constitutively expressed in normal epithelium and benign epithelial tumor tissues. Induced expression of avP6 has been observed in a variety of epithelial malignancies including breast, colorectum, stomach, pancreas, ovary, cervical cancers as well as oral and skin squamous cell carcinomas. There is increasing evidence to suggest that integrinαvβ6 plays important roles in tumor proliferation, invasion, and metastasis, especially in the field of colon cancer. Our previous study discovered that transfection ofαvβ6 gene could remarkably increase the MMP-9 secretion of colon cancer cells, promote the degradation of ECM, and mediate the potentials of colon cancer cells to metastasize to the liver. There is a direct binding between avP6 and ERK, and this novel binding defines a direct signal pathway for avP6 to regulate the progression of the malignant tumors. High avP6 expression was detected at the invading edges of colon cancer specimens, and low avP6 expression within the interior of tumor masses. Previous study has confirmed that high avP6 expression in human colon carcinoma is a prognostic indicator of poor survival. However, the effects of avP6 expression on biological behavior of pancreatic carcinoma cells, chemoresistance, and prognosis have not been studied up to now.Pancreatic cancer is one of the malignancies with the worst prognosis because of aggressive invasion, early metastasis, and almost complete resistance to existing chemotherapeutic agents, radiation therapy, and immunotherapy. Most patients can’t receive curative surgical resection at diagnosis and the five-year survival rate of patients remains less than 5%in the past 20 years. The aim of this study is to investigate the roles of integrinαvβ6 in cell proliferation, invasion, metastasis, apoptosis, chemoresistance and prognostic significance in pancreatic carcinoma, which would help us to further understand the molecular biologic mechanisms of the malignant progression of pancreatic carcinoma. Targeting therapy directed againstαvβ6 has the potential to enhance therapeutic efficacy for aggressive pancreatic carcinoma and might ultimately improve the patient’s clinical outcome.Part IThe expression and prognostic significance of integrinαvβ6 in pancreatic ductal adenocarcinoma tissuesObjective To investigate clinicopathologic significance and prognostic value ofαvβ6 and TGF-β1 expression in pancreatic ductal adenocarcinoma tissues.Methods We generated the microarray of 92 surgically resected pancreatic ductal adenocarcinoma specimens,10 normal autopsy pancreatic tissues were used as controls. The expression status ofαvβ6 and TGF-β1 was examined by immunohistochemical staining. Clinical and pathological data were retrospectively analyzed. We compared the staining results with clinicopathologic characteristics and patients survival. Univariate and multivariate survival analyses were performed to assess their prognostic value. The correlation between avP6 and TGF-β1 expression were also analyzed.Results Normal pancreatic epithelium did not exhibit any staining forαvβ6. In pancreatic carcinomas, positive staining of avP6 was detected in 52 patients (56.5%) and highαvβ6 expression was associated with larger tumor size (P=0.041), advanced TNM stage (P =0.027) and lymph node metastasis (P=0.030). TGF-β1 stained the cytoplasms of tumor cells in 61 cases (66.3%) and high TGF-β1 expression was correlated with poor tumor differentiation and advanced TNM stage. Furthermore, the results of Spearman correlation test showed that avP6 expression correlated positively with TGF-β1 expression. The patients with lowαvβ6 expression had much longer median survival time than those with highαvβ6 expression (P=0.001). The results of univariate and multivariate survival analyses indicated that highαvβ6 expression was an independent unfavorable prognostic factor in pancreatic ductal adenocarcinoma tissues.Conclusions The concomitant expression ofαvβ6 and TGF-β1 is likely to play an important role in the progression of pancreatic carcinoma. High expression of avP6 is an independent unfavorable prognostic indicator in pancreatic carcinoma tissues.Significance We revealed the expression status of integrinαvβ6 and TGF-β1 and their correlation in pancreatic carcinoma tissues, compared the results with various clinicopathologic parameters and patients’survival. High expression ofαvβ6 was defined as an independent unfavorable prognostic indicator in pancreatic carcinoma for the first time. These findings might have potential value for the judgment of patient’s prognosis and targeted therapy directed against avP6.PartⅡThe effects of integrinαvβ6 on the proliferation, invasion and metastasis of pancreatic carcinoma cellsObjective To study the important roles of integrinαvβ6 on the proliferation, invasion and metastasis of pancreatic carcinoma cells.Methods we examined the expression status of avP6 in three pancreatic adenocarcinoma cell lines by RT-PCR and Flow cytometry. PANC-1 and Capan-2 cells were selected to determine the effects of function-blocking mAb againstαvβ6 (10D5) on the cellular malignant behavior. MTT assays were performed to test the changes of cell proliferation. Transwell invasion assays were performed to test the changes of invasive capability. To determine whether blockingαvβ6 function would affect MMP-9 secretion, we performed gelatin zymography and Western blotting analysis. The function of integrinαvβ6 was analyzed via comparing the differences between the data of treated cells and control cells.Results High expression of avP6 at both mRNA and protein levels was observed in PANC-1 cells. Capan-2 cells showed relatively high expression ofαvβ6, while CFPAC-1 cells showed lowαvβ6 expression. Therefore, PANC-1 and Capan-2 cells were selected for subsequent applications. After incubation with 10D5 for 24h, the proliferation rate of PANC-1 and Capan-2 cells decreased to (61.94±3.62)%and (67.35±5.02)%respectively compared to the control cells. The number of invasive PANC-1 and Capan-2 cells dramatically decreased after blockingαvβ6 function with 10D5 (71±9/HP versus 19±5/HP, 36±6/HP versus 13±4/HP, respectively). Furthermore, pretreatment with anti-MMP-9 antibody Ab-1 greatly reduced the number of invasive cells in both cell lines (20±4/HP, 12±5/HP, respectively) at similar levels in the absence and presence of 10D5 or control IgG2a, indicating that the invasion ability of pancreatic carcinoma cells through reconstituted basement membrane was mediated through integrinαvβ6, and the MMP-9 activity is essential for cell invasion. The results of gelatin zymography and Western blotting showed that after suppressingαvβ6 function with 10D5, the MMP-9 secretion levels in PANC-1 and Capan-2 cells decreased to (9.28±3.11)%and (6.35±2.46)%respectively when compared to those of control cells.Conclusions Integrinαvβ6 plays important roles in promoting the proliferation, invasion, and MMP-9 secretion of pancreatic carcinoma cells. Blocking the function ofαvβ6 could inhibit cellular proliferation, invasive, and metastatic capabilities. Significance We tested the expression status ofαvβ6 in three pancreatic carcinoma cell lines, revealed the important roles of avP6 on the proliferation, invasion and metastasis of pancreatic carcinoma cells, and provided the exact experimental evidence for the targeted therapy againstαvβ6 for pancreatic carcinoma.PartⅢThe effects of integrin avP6 on cell apoptosis and chemosensitivity of pancreatic carcinoma cellsObjective To study the important roles of integrin avP6 on cell apoptosis and chemosensitivity to gemcitabine in pancreatic carcinoma cells.Methods We designed and constructed three small interfering RNA (siRNA) against the different positions ofαvβ6 open reading frame. After testing for the most effective targeting sequence, theαvβ6 siRNA was transfected into PANC-1 pancreatic carcinoma cells using the Lipofectamine TM 2000 reagent. A scrambled version ofαvβ6 siRNA was used as a control siRNA. RT-PCR and Western blotting analyses were performed to determine the silencing effect ofαvβ6 expression. MTT assays were performed to detect the changes of cell proliferation. The effects ofαvβ6 siRNA transfection on cell cycle distribution and cell apoptosis were determined by flow cytometry. To quantify apoptosis, cells were stained with an Annexin V-FITC apoptosis assay kit. Gemcitabine was used as chemotherapeutics, and we detected the changes of chemosensitivity and gemcitabine-induced apoptosis after silencingαvβ6 expression by siRNA. Caspase 3 activity was measured using a fluorometric protease assay kit. The effects of avP6 on tumorigenicity and tumor growth in vivo were assessed by subcutaneous implantation of avP6 siRNA and control siRNA transfected PANC-1 cells in athymic nude mice.Results 48 hours following siRNA transfection, the expression ofαvβ6 in PANC-1 cells was markedly suppressed at both mRNA and protein levels byαvβ6 siRNA but not control siRNA, and inhibitive effect persisted 96h after transfection. Silencing of avP6 expression inhibited the cell proliferation in a time-dependent manner. At 96h post-transfection,41%ofαvβ6 siRNA-transfected cells were arrested in G2/M phase, and the apoptotic rate of avP6 siRNA-treated cells was increased to 13.5±2.3%when compared with control siRNA-treated cells. Gemcitabine treatment could induce the apoptosis of PANC-1 cells. Silencing avP6 expression with siRNA significantly enhanced gemcitabine sensitivity and increased gemcitabine-induced caspase-mediated apoptosis. Tumorigenicity assay showed that the average time for the macroscopic tumor formed from theαvβ6 siRNA-transfected cells was about (15±4.6) days, while it was about (9±2.7) days from the control siRNA-transfected cells. At 6 weeks after implantation, the average volume of tumor formed from the avP6 siRNA-transfected cells was significantly decreased compared with that formed from control siRNA transfected cells (170±32mm3 vs 760±48mm3, P<0.01).Conclusions Integrin avP6 plays important roles in inhibiting cell apoptosis and enhancing chemoresistance to gemcitabine in pancreatic carcinoma cells. Silencing ofαvβ6 expression by siRNA significantly inhibited cell growth in vitro and in vivo, caused cell cycle arrest at G2/M phase, induced cell apoptosis, and enhanced gemcitabine sensitivity. Integrinαvβ6 is a potential therapeutic target for highly-resistant pancreatic carcinoma and has promising perspective for clinical application.Significance The siRNA against integrin avP6 was successfully constructed and transfected into PANC-1 pancreatic carcinoma cells for the first time. Silencing ofαvβ6 expression by siRNA significantly inhibited cell growth in vitro and in vivo, resulted in cell cycle G2/M arrest, induced cell apoptosis, and enhanced chemosensitivity to gemcitabine. These findings might provide a new approach for the gene therapy directed against integrinαvβ6 for pancreatic carcinoma.更多还原