annexin Ⅰ对胰腺癌细胞生物学行为影响的体内研究

目的 探讨annexin Ⅰ在胰腺癌发生过程中的作用.方法 应用RNA干扰技术,敲低annexin Ⅰ基因在mRNA水平的表达,并经Western blot检测证实.分别接种人胰腺癌Suit-Ⅱ细胞及转染annexin Ⅰ-siRNA2、annexin Ⅰ-siRNA3、annexin Ⅰ-siRNAN的Suit-Ⅱ细胞,建立裸鼠胰腺癌移植瘤模型,观察胰腺癌细胞的成瘤能力、肿瘤生长速度的变化,测定肿瘤体积和瘤重.结果 (1)Westernblot检测结果显示,转染pSilencer-annexin Ⅰ-siRNA1的Suit-Ⅱ细胞annexin Ⅰ表达显著降低,转染pSilencer-annexin Ⅰ-siRNA2和pSilencer-annexin Ⅰ-siRNA3的Suit-Ⅱ细胞annexin Ⅰ的表达几乎被完全抑制.(2)接种转染annexin Ⅰ-siRNA2、annexin Ⅰ-siRNA3细胞组的肿瘤生长速度较接种亲本Suit-Ⅱ细胞组明显减慢,肿瘤生长被抑制,抑制率分别达76.6%和68.4%.接种肿瘤细胞后44 d,接种转染annexin Ⅰ-siRNA2细胞组、接种转染annexin Ⅰ-siRNA3细胞组的瘤重分别为0.8987和0.8992 g,显著低于接种亲本Suit-Ⅱ细胞组和接种转染annexin Ⅰ-siRNAN细胞组(分别为2.5866和2.4070 g,P<0.001).结论 annexin Ⅰ基因在胰腺癌发生过程中起到了促进胰腺癌细胞生长增殖、增强胰腺癌细胞成瘤能力的重要作用,可作为基因治疗的潜在靶点.     

靶向Survivin的siRNA对胰腺癌细胞PC-2的放射增敏作用

目的采用RNA干扰技术阻断Survivin基因的表达,观察其诱导胰腺癌细胞凋亡及对放射的增敏作用。方法构建靶向Survivin的siRNA真核表达载体,采用lipofectamineTM2000转染胰腺癌细胞PC-2,采用半定量RT-PCR、免疫组化技术检测转染前后PC-2细胞Survivin基因表达的变化;采用流式细胞术检测其诱导PC-2细胞凋亡及对放射的增敏作用。结果靶向Survivin的序列特异性的siRNA可以有效地抑制PC-2细胞Survivin基因的表达,在mRNA水平其表达抑制率为81.25%;在蛋白质水平其表达抑制率为74.24%;转染靶向Survivin的siRNA真核表达载体48h后可以诱导7.03%的细胞凋亡;阻断Survivin基因的表达,可以显著提高PC-2细胞对放射的敏感性,靶向Survivin的siRNA联合放射可以诱导14.58%的细胞凋亡。结论本研究所构建的靶向Survivin的siRNA真核表达载体可以有效、特异地阻断Survivin基因的表达,阻断Survivin基因的表达可以诱导一定程度的PC-2细胞的自发凋亡,并显著增强其对放射的敏感性。靶向Survivin的RNA干扰技术在胰腺癌的基因治疗中具有重要的潜在价值。     

敲低PRPF19对胰腺癌细胞增殖、迁移和侵袭的影响

目的探讨敲低PRPF19之后对胰腺癌细胞增殖、迁移和侵袭能力的影响。方法利用GEPIA等数据库分析PRPF19在胰腺癌及其正常组织中的表达差异;通过Western blot和qRT-PCR检测胰腺癌细胞中PRPF19蛋白和mRNA的表达水平;小干扰RNA(siRNA)技术沉默胰腺癌细胞中PRPF19的表达,并通过Western blot和qRT-PCR验证其敲低效率;CCK-8、克隆形成以及Transwell实验检测敲低PRPF19对胰腺癌细胞增殖、克隆形成以及迁移和侵袭能力的影响。结果GEPIA分析显示,与正常胰腺组织相比,PRPF19在胰腺癌组织中高表达;与正常胰腺细胞相比,PRPF19在MIA PaCa-2、PANC-1等多种胰腺癌细胞系中高表达(P<0.05);与对照组相比,敲低PRPF19后,胰腺癌细胞的增殖速率明显下降,克隆形成数量、细胞迁移和侵袭数量均减少(P<0.05)。结论敲低PRPF19可抑制胰腺癌细胞的增殖、迁移和侵袭能力,PRPF19可能作为胰腺癌的一个癌基因发挥重要作用。     

RNA干扰沉默DcR3对人胰腺癌细胞化疗药物敏感性影响的实验研究

背景与目的：大部分胰腺癌具有高表达诱骗受体-3(decoy receptor 3,DcR3)的特征,而后者与FasL凋亡途径相关,可能导致胰腺癌对化疗耐药。该研究旨在探讨RNA干扰沉默DcR3基因对人胰腺癌细胞化疗药物敏感性的影响及其可能机制。方法：构建带有DcR3-siRNA序列的稳定表达质粒,通过Lipofectamine^TM2000转染至人胰腺癌细胞AsPC-1细胞株,筛选出转染后稳定低表达DcR3的胰腺癌细胞,同时设未转染对照组(control组)和转染阴性质粒对照组(mock组)。应用ELISA和蛋白［质］印迹法(Western blot)检测各组AsPC-1细胞中DcR3的蛋白表达;MTT实验检测各组AsPC-1细胞对吉西他滨的敏感性;流式细胞术检测各组AsPC-1细胞凋亡情况;Western blot和实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测各组AsPC-1细胞中FasL、Caspase-8、Caspase-3蛋白和mRNA的表达。结果：转染DcR3-siRNA后AsPC-1细胞中DcR3蛋白较其他对照组明显降低;转染DcR3-siRNA后AsPC-1细胞对吉西他滨的敏感性显著增加;沉默DcR3基因可以上调FasL、Caspase-8和Caspase-3的表达并促进吉西他滨诱导的细胞凋亡。结论：RNA干扰沉默DcR3基因可激活FasL/Caspase凋亡途径,促进肿瘤细胞凋亡,增加人胰腺癌AsPC-1细胞对化疗药物的敏感性。     

胰腺癌TFPI-2蛋白表达与细胞凋亡

目的:探讨胰腺癌组织中细胞凋亡与TFPI-2基因表达的相互关系,研究TFPI-2对胰腺癌细胞凋亡的影响.方法:应用免疫组化技术检测TFPI-2基因在50例胰腺癌及9例正常胰腺组织中的表达,并应用TUNEL法检测细胞凋亡,计算凋亡指数;体外将TFPI-2基因真核表达载体转染人胰腺癌细胞株Panc-1细胞,DNA片段化实验检测细胞凋亡.结果:TFPI-2在胰腺癌中的表达低于正常胰腺组织,TFPI-2指数分别为73.28±9.63和81.42±12.25,P＜0.05,差异有统计学意义.TFPI-2的表达及细胞凋亡与临床分期及转移有关,Ⅰ、Ⅱ期及无转移的胰腺癌组织中TFPI-2指数及AI值均高于Ⅲ、Ⅳ期及有转移的胰腺癌组织,TFPI-2阳性组织中的AI值高于TFPI-2阴性组织的AI值,P＜0.05,差异有统计学意义;DNA片段化实验证实TFPI-2能诱导Panc-1细胞凋亡.结论:胰腺癌组织中细胞凋亡与TFPI-2的表达水平有关,TFPI-2可能在诱导胰腺癌细胞凋亡中起重要作用.     

重组人可溶性凋亡素-2在人喉癌株中表达及其体外诱导凋亡作用的实验研究

目的:研究凋亡因子TRAIL结合端粒酶启动子对肿瘤的特异性杀伤作用。方法:刺激人外周血淋巴细胞增殖,提取总RNA。采用RTPCR扩增IL2信号肽基因和TRAIL基因的融合基因,并克隆入真核表达载体pGL3181hTERT肿瘤特异性端粒酶启动子的下游,构建TRAIL基因的重组真核表达载体pGL3181hTERT/TRAIL。用Westernblot鉴定喉癌细胞株Hepa2中表达产物。将该重组载体经阳离子脂质体转染入人喉癌细胞株Hepa2中,用电镜观察细胞的形态变化,用流式细胞仪技术(FCM)分析转染后细胞凋亡率及细胞周期的变化。结果:PCR扩增得到了613bp的cDNA片断。与GenBank中报道的IL2信号肽和TRAIL凋亡诱导功能区cDNA序列完全一致,成功构建了重组真核表达载体pGL3181hTERT/TRAIL。Westernblot结果显示,获得的瞬时转染TRAIL在喉癌细胞Hepa2中特异表达,且能诱导其凋亡。结论:重组真核表达载体pGL3181hTERT/TRAIL的成功构建,为肿瘤基因治疗的靶向性提供了可行的理想工具。     

细胞外基质蛋白Del-1在胰腺癌中高表达及其对胰腺癌细胞失巢凋亡的抑制作用北大核心CSCD

目的 :观察细胞外基质蛋白Del-1(developmental endothelial locus-1)在胰腺癌组织及细胞中的表达情况,并探讨Del-1蛋白对胰腺癌细胞增殖和凋亡的影响及其分子机制。方法:采用免疫组织化学法检测81例胰腺导管腺癌组织及39例正常胰腺组织中Del-1蛋白的表达水平,并分析Del-1表达与胰腺癌患者临床病理参数和生存期的关系。采用实时荧光定量PCR和蛋白质印迹法检测Del-1在正常胰腺细胞hTERT-HPNE及6种胰腺癌细胞(AsPC1、BxPC3、CFPAC-1、HPAC、PANC1和SW1990)中的表达水平。应用10和100nmol/L重组Del-1蛋白分别处理胰腺癌PANC1和AsPC1细胞,然后采用CCK-8、FCM和蛋白质印迹法分别检测细胞增殖、饥饿诱导凋亡、失巢凋亡以及Bcl-2和Bax蛋白表达水平的变化。结果:胰腺导管腺癌组织中Del-1的高表达率为80.2%(65/81),明显高于正常胰腺组织的10.3%(4/39),2者差异有统计学意义(P<0.001)。胰腺导管腺癌组织中Del-1表达与肿瘤大小及神经侵袭与否存在相关性(P值均<0.05),而与肿瘤位置和TNM分期等无关(P值均>0.05)。KaplanMeier分析结果显示,Del-1高表达组患者的中位生存期明显低于Del-1低表达组(P<0.01)。相对于正常胰腺细胞hTERT-HPNE,所有6种胰腺癌细胞(AsPC1、BxPC3、CFPAC-1、HPAC、PANC1和SW1990)的Del-1 mRNA和蛋白均呈高表达(P值均<0.05)。重组Del-1蛋白处理后,胰腺癌PANC1和AsPC1细胞的增殖及凋亡没有明显变化(P值均>0.05),但其失巢凋亡能力被明显抑制(P值均<0.05)。重组Del-1蛋白处理组细胞中抗凋亡蛋白Bcl-2的表达水平明显升高,而促凋亡蛋白Bax的表达水平明显降低(P值均<0.05)。结论:细胞外基质蛋白Del-1在胰腺癌中异常高表达,可保护肿瘤细胞避免失巢凋亡,其机制可能与改变凋亡相关蛋白的表达有关。     

TFPI-2基因克隆及其在胰腺癌细胞中的表达

目的克隆人TFPI-2基因全长cDNA,构建其真核表达载体,并将其转染到人胰腺癌细胞Panc-1中,检测其表达。方法用RT-PCR法从人胎盘组织中扩增人TFPI-2基因,并将其与真核表达载体pEGFP-C1连接,构建真核表达载体pEGFP-C1-TFPI-2。将构建的重组载体转染到人胰腺癌细胞系Panc-1细胞,Westernblot检测TFPI-2在Pane-1细胞中的表达。结果RT-PCR成功的扩增出一条约708bp的片断,扩增片断与载体连接后,经限制性内切酶酶切电泳分析和DNA序列测定证实该基因已经成功构建到载体上,转染Panc-1细胞后,荧光显微镜观察到稳定转染细胞发出较强绿色荧光,Westernblot技术证明TFPI-2基因能在Panc-1细胞中稳定表达。结论成功构建了人TFPI-2基因的真核表达载体pEGFP-C1-TFPI-2,获得了稳定表达TFPI-2的Panc-1细胞,证实TFPI-2基因能够在人胰腺癌细胞系Panc-1中高效、稳定的表达,为进一步研究其胰腺癌迁移、浸润及转移中的作用打下基础。     

Livin靶向siRNA对大肠癌细胞凋亡的诱导作用

目的:构建Livin靶向小干扰RNA(siRNA)重组表达载体,观察其诱导大肠癌细胞的凋亡效应.方法:构建Livin靶向siRNA重组表达栽体并转染结肠癌细胞,RT-PCR和蛋白质印迹法检测Livin的表达,MTT法检测siRNA对细胞增殖的抑制作用,流式细胞仪检测细胞的凋亡效应.结果:经酶切鉴定和测序结果证实,Livin靶向sirNA重组表达载体构建成功,它对大肠癌细胞Livin mRNA和蛋白的表达抑制率分别为30.18%和28.88%,P<0.05,肿瘤细胞的生长受到明显抑制,细胞凋亡率为(13.36±1.45)%,P<0.05.结论:Livin靶向siR-NA重组表达载体构建成功,能有效抑制Livin基因表达并能显著诱导肿瘤细胞凋亡.     

人胰腺癌细胞外基质蛋白基因表达的研究

胰腺癌组织具有较强的结缔组织反应特性，细胞外基质蛋白（Extracellularmatrixproteins，ECM）诸成分，在RNA和蛋白水平异常表达，具有一定的分子生物学意义。用Northernblotting方法，显示了胰腺组织及其癌变标本中ECM的基因表达（Geneexpression），以证实胰腺癌细胞本身能合成共产生这些细胞间质成分。结果显示：胰腺癌组织与胰腺癌细胞株（PaTu8988、PaTu8902）中均有ECM的基因表达，在RNA水平，Ⅳ型胶原和层粘素LN的表达较显著。正常组织中ECM的基因表达，在RNA水平，纤粘素FN和层粘素LN低水平表达，Ⅰ、Ⅲ、Ⅳ型胶原更低。提示ECM可能在肿瘤的浸润、转移过程中起一定的作用。     

Annexin II expression is reduced or lost in prostate cancer cells and its re-expression inhibits prostate cancer cell migration

While studying Bim, a BH3-only proapoptotic protein, we identified an approximately 36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven prostate cancer cell lines. The approximately 36 kDa protein was subsequently identified as annexin II by proteomic approach and confirmed by Western blotting using an annexin II-specific antibody. Conventional and 2D SDS-PAGE, together with Western blotting, also revealed reduced or lost expression of annexin I in prostate cancer cells. Subcellular localization studies revealed that in NHP cells, annexin II was distributed both in the cytosol and underneath the plasma membrane, but not on the cell surface. Prostate cancer cells showed reduced levels as well as altered expression patterns of annexin II. Since annexins play important roles in maintaining Ca(2+) homeostasis and regulating the cytoskeleton and cell motility, we hypothesized that the reduced or lost expression of annexin I/II might promote certain aggressive phenotypes of prostate cancer cells. In subsequent experiments, we indeed observed that restoration of annexin II expression inhibited the migration of the transfected prostate cancer cells without affecting cell proliferation or apoptosis. Hence, our results suggest that annexin II, and, likely, annexin I, may be endogenous suppressors of prostate cancer cell migration and their reduced or lost expression may contribute to prostate cancer development and progression.     

The proteasome inhibitor MG132 induces apoptosis in human pancreatic cancer cells

The ubiquitin-proteasome system plays a critical role in the regulation of programmed cell death. Proteasome inhibitors induce apoptosis in various cancer cells and have antitumor effects in murine tumor models. In the present study, we investigated whether the cell-permeable proteasome inhibitor MG132 (carbobenzoxyl-L-leucyl-L-leucyl-L-leucinal) reduced the growth of a human pancreatic cancer cell line through induction of apoptosis in vitro. The effects of MG132 (0.125-1.000 microM) on the growth of the human pancreatic cancer cell line BxPC-3 were analyzed by cell count and MTT assay. Apoptosis was determined by FACS analysis after annexin V and propidium iodide staining and the enrichment of intracellular nucleosomes. The proteasome inhibitor MG132 decreased cell growth of the human pancreatic cancer cell line BxPC-3 in a dose- and time-dependent manner. This effect was at least in part mediated by the induction of apoptosis. A combination therapy with standard cytotoxic agents and proteasome inhibitors could potentially be a novel therapeutic strategy in treatment of pancreatic cancer.     

Lactate dehydrogenase A is overexpressed in pancreatic cancer and promotes the growth of pancreatic cancer cells

The prognosis for pancreatic cancer is very poor, and developing new therapeutic strategies for this cancer is needed. Recently, the Warburg effect (aerobic glycolysis) has attracted much attention for its function in the tumorigenesis. Lactate dehydrogenase A (LDHA) executes the final step of aerobic glycolysis and has been reported to be involved in the tumor progression. However, the function of LDHA in pancreatic cancer has not been studied. Here, we found that the expression of LDHA was elevated in the clinical pancreatic cancer samples. Forced expression of LDHA promoted the growth of pancreatic cancer cells, while knocking down the expression of LDHA inhibited cell growth dramatically. Moreover, silencing the expression of LDHA inhibited the tumorigenicity of pancreatic cancer cells in vivo. Mechanistically, knocking down the expression of LDHA activated apoptosis pathway. Taken together, our study revealed the oncogenic role of LDHA in pancreatic cancer and suggested that LDHA might be a potential therapeutic target.     

GSK1838705A对胰腺癌细胞增殖和凋亡的影响

目的:研究胰岛素样生长因子1受体抑制剂GSK1838705A对人胰腺癌细胞的抗肿瘤作用.方法:Cell Counting Kit-8(CCK-8)方法检测不同浓度GSK1838705A对胰腺癌CAPAN-2细胞增殖能力的影响;流式细胞术检测GSK1838705A对CAPAN-2细胞凋亡的影响;Western blot方法检测GSK1838705A作用于胰腺癌CAPAN-2细胞后Bax和Cytochrome C蛋白表达水平的变化;划痕试验观察GSK1838705A处理CAPAN-2细胞后对细胞生长及迁移的影响.结果:CCK-8检测结果表明不同浓度GSK1838705A能够不同程度抑制胰腺癌CAPAN-2细胞的增殖,当药物浓度达10 μmol/L时,其抑制率可达98.29％;流式结果显示,GSK1838705A可诱导CAPAN-2细胞发生凋亡,随着GSK1838705A浓度的增加,诱导凋亡作用增强;Western blot结果显示,Bax和Cytochrome C蛋白表达水平升高;划痕试验结果表明,不同浓度GSK1838705A能够不同程度抑制CAPAN-2细胞的增殖和迁移.结论:胰岛素样生长因子1受体抑制剂GSK1838705A在体外可显著抑制胰腺癌CAPAN-2细胞的增殖,诱导细胞凋亡并抑制细胞迁移,在胰腺癌细胞中发挥着显著的抗肿瘤作用.     

Enhanced expression of annexin II in human pancreatic carcinoma cells and primary pancreatic cancers

Annexin II is a calcium and phospholipid binding protein anda substrate for protein-tyrosine kinases. Recent investigationshave revealed involvement of annexin II in DNA synthesis andcell proliferation. Increased levels of annexin II are observedin cancer cells and tissues. To investigate the expression ofannexin II in pancreatic adenocarcinoma cells and primary tumors,we measured the levels of annexin II mRNA and protein in normalhuman pancreas, five established human pancreatic adenocarcinomacell lines, three primary pancreatic cancers and one metastatictumor. All five cell lines examined had 5- to 15-fold higherlevels of annexin II as compared to normal pancreas. Significantelevations (2-to 8-fold) of annexin II expression were observedin the three primary pancreatic tumors and one metastatic tumorexamined. Immunocytochemical analysis indicates that the increasedexpression of annexin II is limited to proliferating ductularadenocarcinoma, and annexin II expression co-localizes withcells that express PCNA. In normal pancreas, annexin II expressionis seen in ductal and ductular cells and no expression is seenin acinar or islet cells. We conclude from these findings thatannexin II has a role in cell proliferation and its regulationis altered in pancreatic cancer.     

BCL2 expression correlates with metastatic potential in pancreatic cancer cell lines

BACKGROUND: Programmed cell death (termed apoptosis) regulates normal tissue homeostasis. Loss of local paracrine signals and intercellular adhesion molecules are potent inducers of apoptosis and thereby eliminate normal cells that may have escaped beyond the confines of the local organ environment. Dysregulation in the expression of the BCL2 gene family, the prototypic regulators of apoptosis, is a common occurrence in cancer and imparts resistance to standard triggers of apoptosis. Therefore, the authors sought to examine whether abnormal BCL2 gene family expression correlated with resistance to apoptosis and increased metastatic potential in pancreatic carcinoma. METHODS: The authors examined BCL2 expression and apoptotic sensitivity in three panels of human pancreatic cancer cell lines that possess varying metastatic potential. Stable transfectants were generated that overexpress BCL2. These transfectants were then analyzed for differences in metastasis formation in athymic mice. RESULTS: Among the isogenic panels of pancreatic cancer cell lines, BCL2 expression levels correlated with metastatic potential. Highly metastatic variants of each family of cell lines were more resistant to induction of apoptosis. Finally, using the BCL2 transfectant in a xenograft model, elevated BCL2 expression led to a higher incidence of metastases. CONCLUSIONS: The authors conclude that increased BCL2 expression correlates with apoptotic resistance and metastatic potential; dysregulation of BCL2 expression may be involved in the metastatic progression of pancreatic carcinoma.     

ADH-1 suppresses N-cadherin-dependent pancreatic cancer progression

Pancreatic cancer is one of the most aggressive malignant diseases. We recently reported that N-cadherin plays a key role in tumor progression and metastasis in pancreatic cancer. For this study, we sought to determine if an N-cadherin-blocking peptide (ADH-1) could prevent N-cadherin-mediated tumor progression in a mouse model for pancreatic cancer. The effect of ADH-1 on N-cadherin-mediated cell scattering and migration on collagen I was examined using pancreatic cancer cells. We also examined the influence of ADH-1 on cell apoptosis. Furthermore, in vivo animal studies were performed using orthotopic injection of N-cadherin overexpressing BxPC-3 cells with or without ADH-1 treatment. BxPC-3 and Capan-1 cells exhibited increased expression of N-cadherin in response to collagen I. This increase in N-cadherin promoted cell scattering and migration in response to collagen I. ADH-1 prevented these changes, but did not inhibit upregulation of N-cadherin. TUNEL assays and immunoblots for caspase-3 showed that ADH-1 induced apoptosis in a concentration dependent and N-cadherin dependent manner in pancreatic cancer cells. ADH-1 treatment resulted in significant reductions in tumor growth and lung metastasis in a mouse model for pancreatic cancer. The N-cadherin antagonist, ADH-1 has significant antitumor activity against N-cadherin-expressing cells using in vitro assays and in an orthotopic mouse model for pancreatic cancer, raising the possibility that N-cadherin antagonists have therapeutic potential for the treatment of pancreatic cancer in humans.