靶向Ang2的RNAi慢病毒载体的构建及其对恶性黑色素瘤细胞中Ang2基因表达的影响

目的 构建携带促血管生成素2-小干扰RNA（Ang2-siRNA）慢病毒载体,观察其对恶性黑色素瘤细胞中Ang2基因表达的干扰作用.方法 将经XbaⅠ酶切电泳鉴定的带有加强绿色荧光蛋白的转移质粒（pNL-EGFP）载体与pSilencer 1.0-U6启动子-促血管生成素2-小干扰RNA（pSilencer 1.0-U6-Ang2-siRNA）重组质粒连接,产生加强绿色荧光蛋白的转移质粒-U6启动子-促血管生成素2-Ⅰ（pNL-EGFP-U6-Ang2-Ⅰ）、加强绿色荧光蛋白的转移质粒-U6启动子-促血管生成素2-Ⅱ（pNL-EGFP-U6-Ang2-Ⅱ）慢病毒转移质粒,电泳筛选阳性克隆,测序鉴定.用连接成功的慢病毒转移质粒、水疱性口炎病毒G蛋白（pVSVG）包膜质粒和pHelper包装质粒共转染293T细胞,产生pNL-EGFP-U6-Ang2-Ⅰ、pNL-EGFP-U6-Ang2-Ⅱ慢病毒.收集病毒上清,测定病毒滴度.将收集的病毒上清感染恶性黑色素瘤细胞,通过实时荧光定量RT-PCR测定抑制Ang2基因表达的效率.结果 酶切电泳与测序鉴定证实成功构建了Ang2-SiRNA慢病毒载体,293T细胞测定病毒原液滴度为8.0×103/ml.实时荧光定量RT-PCR结果显示：Ang2-siRNA慢病毒载体感染恶性黑色素瘤细胞,抑制了恶性黑色素瘤细胞中Ang2基因的表达（P＜0.05）.结论 成功构建了Ang2-SiRNA慢病毒载体,体外研究显示Ang2-SiRNA慢病毒载体能抑制恶性黑色素瘤细胞中Ang2 mRNA的表达,为下一步进行裸鼠恶性黑色素瘤移植瘤生长的干预实验奠定基础,为肿瘤的基因治疗提供实验依据.     

Ang2-siRNA慢病毒干预裸鼠移植性恶性黑色素瘤的研究

目的研究血管生成素2小干扰RNA（Ang2-siRNA）对裸鼠移植性恶性黑色素瘤血管形成及其生长的影响。方法建立人恶性黑色素肿瘤裸鼠异种移植瘤模型。在体内使用pNL-EGFP—Ang2-siRNA慢病毒干扰裸鼠移植性恶性黑色素瘤Ang2基因的表达,观察统计各组肿瘤（空白组、空载组、实验组）生长情况。应用实时荧光定量RT—PCR检测肿瘤组织中Ang2基因的mRNA表达水平;CD34单克隆抗体作为血管内皮标志物,免疫组织化学法检测其表达,以评价肿瘤微血管密度。结果成功建立了恶性黑色素瘤裸鼠异种移植瘤模型,实验组肿瘤Ang2基因mRNA水平、微血管密度、肿瘤体积显著小于空白组和空载组（P〈0．01）,空白组和空载组之间无统计学意义（P〉0．05）。结论pNL—EGFP—Ang2-siRNA慢病毒能沉默Ang2的表达,显著抑制恶性黑色素瘤血管的形成和肿瘤的生长,可能为临床恶性黑色素肿瘤的基因治疗开辟新的途径。     

靶向下调FOXM1 基因表达对乳腺癌MDA-MB-231 细胞增殖的影响

目的：探讨靶向抑制FOXM1 对乳腺癌细胞增殖能力的影响,为乳腺癌的个性化靶向治疗提供理论依据。方法：利用重组真 核转录载体pSilencer1.0-U6-FOXM1-shRNA,脂质体法转染乳腺癌细胞株MDA-MB-231,下调其FOXM1基因表达。采用四甲基 偶氮唑盐(MTT) 比色法、平板克隆形成实验观察细胞增值曲线以及克隆形成能力;采用实时定量- 聚合酶链反应(Real-time qPCR)、蛋白免疫印迹法（Western blot）分别检测FOXM1 基因在mRNA、蛋白水平的表达变化。结果：重组载体pSilencer1. 0-U6-FOXM1-shRNA转染MDA-MB-231细胞后,与对照组相比,增殖速率明显下降（P<0.05）,平板克隆形成显著减少（P<0.05）, 重组载体转染后显著抑制MDA-MB-231 细胞中FOXM1 基因在mRNA、蛋白水平的表达。结论：沉默FOXM1 基因对乳腺癌细胞 株MDA-MB-231 生长具有抑制作用,为阐明乳腺癌发病机制提供了新的切入点,也为临床抑制肿瘤生长提供了新的作用靶点。     

RNA干扰抑制SiHaB2细胞中Bcl-2基因的表达

Bcl-2基因作为细胞凋亡的一个潜在抑制剂调节细胞的死亡,抑制恶性肿瘤细胞中Bcl-2基因的表达可促进肿瘤细胞的凋亡。采用RNA干扰技术,合成了含有21个核苷酸的小双链干扰RNA（siRNA．Bcl-2）,并构建了含有19个核苷酸基因的质粒载体（pSilencer2．1-U6-Bcl-2）,把合成的siRNA．Bcl-2和pSilencer2．1-U．Bcl-2分别转导入Bcl-2高表达的细胞株SiHaB2中,通过Western印迹检测,免疫荧光法检测及DNA梯（ladder）检测,可观察到在导入siRNA．Bcl-2和pSilencer2．1-U．Bcl-2的SiHaB2细胞被培养72h后,可以明显抑制Bcl-2蛋白的表达。     

KLF2与血管静止

血管生成素-1（Ang1）根据细胞中其受体Tie2的状况,既可调节血管静止状态,也可调节血管的生成。与细胞互作的Tie2可以启动静止状态,而与细胞外介质（ECM）互作的Tie 2则可刺激血管的生成,与细胞互作的Ang1-Tie2复合体可以诱导转录因子KLF2（Krvppel-like factor 2）的表达。     

RNA干扰敲减FucT VII表达抑制人结肠癌细胞HT-29和HUVECs的粘附能力

 探讨利用RNA干扰（RNAi）技术抑制岩藻糖基转移酶Ⅶ（FucT Ⅶ）表达对人结肠癌细胞HT-29与人脐静脉内皮细胞（HUVEC）粘附能力的影响及其机制.本课题构建3对针对FucT Ⅶ基因的RNAi表达载体,并将其转染入人结肠癌细胞HT-29,Western 印迹检测FucT Ⅶ及其下游产物sLeX蛋白的变化;实时PCR 检测FucT Ⅶ mRNA表达的变化;玫瑰红染色法检测RNAi 对HT-29与HUVECs细胞粘附能力的影响.结果显示,3对FucT Ⅶ siRNA表达载体均可有效抑制HT-29细胞FucT Ⅶ mRNA和蛋白表达,以pSilencer 2.0 FucT Ⅶ 2最为有效;与空白细胞组比较,转染pSilencer 2.0-FucT Ⅶ的HT-29细胞表面sLeX表达水平明显下降,以pSilencer 2.0-FucT Ⅶ 2最为显著;RNA干扰FucT Ⅶ表达后HT 29细胞和HUVEC之间的粘附能力明显受到抑制.研究表明,RNAi靶向沉默HT-29细胞中FucT Ⅶ基因表达可显著降低其下游产物sLeX的合成,进而抑制HT-29细胞与HUVECs的粘附能力.     

EGFL7 基因RNAi慢病毒表达载体的构建及鉴定

目的：构建人类表皮生长因子域7（EGFL7）基因RNA干扰（RNAi）重组慢病毒表达载体。方法：参照小分子干扰RNA 的设 计原则,应用OligoDesigner 3.0 软件设计三条靶向人EGFL7 基因的RNA干扰序列（hEGFL7-RNAi）,并将其分别插入含有绿色 荧光蛋白（GFP）的慢病毒载体pLV3 中,获得重组质粒,与包装质粒pRsv-REV、pMDlg-pRRE 和pMD2G共同转染293T细胞,包 装产生重组慢病毒,培养72 h后,应用qRT-PCR 检测慢病毒感染人脐静脉内皮细胞（HUVEC）后靶基因mRNA 的水平,以评价 其基因沉默效果。结果：筛选出3 条人EGFL7 基因的RNAi 序列,分别包装出重组慢病毒,其滴度分别为1× 108、2× 108和5× 108TU/mL,将其转染入HUVEC 后,EGFL7mRNA表达均受到明显抑制(P<0.05)。结论：成功筛选出三条针对人EGFL7基因的 RNAi有效靶序列,并成功构建重组慢病毒表达载体,证明该序列可沉默HUVECs 中EGFL7 基因的表达。     

RNA干扰技术对肝癌细胞内源survivin基因表达的影响

应用RNA干扰技术（RNAi）研究针对凋亡抑制因子survivin的siRNA抑制肝癌细胞株内源survivin基因的表达.转染重组质粒pshRNA-survivin至肝癌细胞株SMMC-7721,通过免疫荧光、蛋白质印迹和半定量RT-PCR检测survivin蛋白表达及mRNA转录水平的变化.结果表明：构建的三种重组质粒pshRNA-survivin1/2/3均能明显抑制survivin基因的表达;应用免疫荧光检测survivin基因的表达,转染重组质粒pshRNA-survivin的实验组survivin荧光强度明显低于转染载体pTZU6＋1和pshRNA-GFP对照组;蛋白质印迹结果表明,重组质粒pshRNA-survivin明显抑制survivin蛋白的表达,抑制率为62%～78％,通过半定量RT-PCR检测到survivin基因mRNA转录明显减少,抑制率为57%～64％.由上述结果可以得出结论：重组质粒pshRNA-survivin可明显抑制SMMC-7721细胞内源survivin的表达和mRNA的转录,为survivin介导的肿瘤基因沉寂疗法提供实验基础.     

MEKK2基因siRNA重组腺病毒表达载体的构建及其对MEKK2的表达抑制

设计并合成针对人MEKK2基因3个不同部位siRNA靶点的模板DNA序列,将合成的互补片段退火后克隆入pRNAT-H1.1/Adeno穿梭载体中,并使其在大肠杆菌BJ5183中与腺病毒骨架质粒pAdEasy-1进行同源重组。将经鉴定正确的重组腺病毒质粒转染293A细胞,包装得到具有感染能力的pAd-MEKK2-siRNA重组腺病毒。病毒体外转导人胃腺癌AGS细胞,Western blot印迹法检测其对MEKK2表达的抑制。经酶切和测序鉴定均证实pAd-MEKK2-siRNA重组腺病毒载体构建成功,其插入序列正确无误。Western blot印迹检测结果显示,重组腺病毒表达载体可抑制MEKK2基因的表达,以pAd-MEKK2-siRNA2（针对MEKK2 cDNA 992-1 010的片段）抑制效果为最佳,pAd-MEKK2-siRNA1（针对MEKK2 cDNA 1 456-1 474的片段）和pAd-MEKK2-siRNA3（针对MEKK2 cDNA 1 351-1 369的片段）则未见明显的抑制效果。DNA Ladder和细胞存活测定结果表明,敲减MEKK2的表达后,AGS细胞接受H2O2刺激后的凋亡受到较强抑制、细胞存活数增加,明显高于野生型细胞和转导siRNA阴性对照腺病毒细胞接受H2O2刺激后的,差异具有统计学意义（P〈0.05）,而后两者之间差异无统计学意义（P〉0.05）。该研究成功地构建了针对MEKK2基因的siRNA重组腺病毒载体,为进一步深入研究MEKK2基因在人胃腺癌细胞中的作用和功能奠定了基础。     

shRNA靶向干扰COX-2抑制人肝癌裸鼠皮下移植瘤及其血管生成

目的:探讨重组干扰质粒pshRNA-COX-2对人肝癌细胞Hep3B裸鼠皮下移植瘤生长和肿瘤血管生成的抑制作用。方法:重组干扰质粒pshRNA-COX-2转染Hep3B细胞并筛选后,RT-PCR和Western blot检测COX-2mRNA和蛋白表达,RT-PCR检测VEGFmRNA表达。将被成功转染的Hep3B细胞种植于裸鼠皮下,测量肿瘤大小,4周后处死裸鼠,免疫组织化学法检测肿瘤组织中COX-2蛋白表达和肿瘤微血管密度(MVD)。结果:与未转染细胞相比,干扰组COX-2mRNA和蛋白表达抑制率分别为65.3%和52.8%(P<0.05),干扰组VEGFmRNA表达抑制率为56.5%(P<0.05)。干扰组瘤体大小明显小于阴性组和空白组(P<0.01)。干扰组COX-2得分和MVD均明显低于阴性组和空白组(P<0.01)。结论:pshRNA-COX-2通过抑制COX-2表达明显抑制人肝癌细胞Hep3B裸鼠皮下移植瘤生长和肿瘤血管生成。     

Tie2受体研究进展及其在抗肿瘤治疗中的应用

Tie2是胚胎血管发育和肿瘤血管形成都需要的内皮细胞酪氨酸激酶受体,血管生成素(Ang)是其配体。正常成人组织中,Ang／Tie2受体水平较低,用于维持成熟的血管结构;一般癌组织中Ang／Tie2的表达较为活跃。本综述了Ang／Tie2的结构和功能研究的最新进展,Ang／Tie2在血管形成中的重要调节作用,以及可溶性Tie2在治疗肿瘤方面的前景。     

Characterization and expression of a novel alternatively spliced human angiopoietin-2

Angiopoietin-2 (Ang2) is a naturally occurring antagonist of angiopoietin-1 (Ang1) that competes for binding to the Tie2 receptor and blocks Ang1-induced Tie2 autophosphorylation during vasculogenesis. Using the polymerase chain reaction, we isolated a cDNA encoding a novel shorter form of Ang2 from human umbilical vein endothelial cell cDNA and have designated it angiopoietin-2(443) (Ang2(443)), because it contains 443 amino acids. Part of the coiled-coil domain (amino acids 96-148) is absent in Ang2(443) because of alternative splicing of the gene. Like Ang2, recombinant Ang2(443) expressed in COS-7 cells is secreted as a glycosylated homodimeric protein. Recombinant Ang2(443) binds to the Tie2 receptor but does not induce Tie2 phosphorylation. Pre-occupation of Ang2(443) on Tie2 inhibits Ang1 or Ang2 binding and inhibits Ang1-induced phosphorylation. Expression of Ang2(443) mRNA is detectable in primary endothelial cells, several nonendothelial tumor cell lines, and primary tumor tissues. Interestingly, two cervical carcinoma cell lines express relatively moderate levels of Ang2(443) mRNA and protein. Macrophages express mainly Ang2 mRNA, but the expression of Ang2(443) mRNA is temporarily up-regulated during macrophage differentiation. These results suggest that Ang2(443) is a functional antagonist of Ang1 and could be an important regulator of angiogenesis during some tumorigenic and inflammatory processes.     

Increasing Ang1/Tie2 expression by simvastatin treatment induces vascular stabilization and neuroblast migration after stroke

In this study, we tested the hypothesis that the Angiopoietin 1 (Ang1)/Tie2 pathway mediates simvastatin-induced vascular integrity and migration of neuroblasts after stroke. Rats were subjected to 2 hrs of middle cerebral artery occlusion (MCAo) and treated, starting 1 day after stroke with or without simvastatin (1 mg/kg, daily) for 7 days. Simvastatin treatment significantly decreased blood–brain barrier (BBB) leakage and concomitantly, increased Ang1, Tie2 and Occludin expression in the ischaemic border (IBZ) compared to the MCAo control group. Simvastatin also significantly increased doublecortin (DCX, a marker of migrating neuroblasts) expression in the IBZ compared to control MCAo rats. DCX was highly expressed around vessels. To further investigate the signalling pathway of simvastatin-induced vascular stabilization and angiogenesis, rat brain microvascular endothelial cell (RBMEC) culture was employed. The data show that simvastatin treatment of RBMEC increased Ang1 and Tie2 gene and protein expression and promoted phosphorylated-Tie2 activity. Simvastatin significantly increased endothelial capillary tube formation, an index of angiogenesis, compared to non-treated control. Inhibition of Ang1 or knockdown of Tie2 gene expression in endothelial cells significantly attenuated simvastatin-induced capillary tube formation. In addition, simvastatin significantly increased subventricular zone (SVZ) explant cell migration compared to non-treatment control. Inhibition of Ang1 significantly attenuated simvastatin-induced SVZ cell migration. Simvastatin treatment of stroke increases Ang1/Tie2 expression and thereby reduces BBB leakage and promotes vascular stabilization. Ang1/Tie2 expression induced by simvastatin treatment promotes neuroblast micro-vascular coupling after stroke.     

LRP5 Regulates Development of Lung Microvessels and Alveoli through the Angiopoietin-Tie2 Pathway

Angiogenesis is crucial for lung development. Although there has been considerable exploration, the mechanism by which lung vascular and alveolar formation is controlled is still not completely understood. Here we show that low-density lipoprotein receptor-related protein 5 (LRP5), a component of the Wnt ligand-receptor complex, regulates angiogenesis and alveolar formation in the lung by modulating expression of the angiopoietin (Ang) receptor, Tie2, in vascular endothelial cells (ECs). Vascular development in whole mouse lungs and in cultured ECs is controlled by LRP5 signaling, which is, in turn, governed by a balance between the activities of the antagonistic Tie2 ligands, Ang1 and Ang2. Under physiological conditions when Ang1 is dominant, LRP5 knockdown decreases Tie2 expression and thereby, inhibits vascular and alveolar development in the lung. Conversely, when Ang2 dominates under hyperoxia treatment in neonatal mice, high LRP5 and Tie2 expression suppress angiogenesis and lung development. These findings suggest that the LRP5-Tie2-Ang signaling axis plays a central role in control of both angiogenesis and alveolarization during postnatal lung development, and that deregulation of this signaling mechanism might lead to developmental abnormalities of the lung, such as are observed in bronchopulmonary dysplasia (BPD).     

Differential function of Tie2 at cell-cell contacts and cell-substratum contacts regulated by angiopoietin-1

Tie2 belongs to the receptor tyrosine kinase family and functions as a receptor for Angiopoietin-1 (Ang1). Gene-targeting analyses of either Ang1 or Tie2 in mice reveal a critical role of Ang1-Tie2 signalling in developmental vascular formation. It remains elusive how the Tie2 signalling pathway plays distinct roles in both vascular quiescence and angiogenesis. We demonstrate here that Ang1 bridges Tie2 at cell-cell contacts, resulting in trans-association of Tie2 in the presence of cell-cell contacts. In clear contrast, in isolated cells, extracellular matrix-bound Ang1 locates Tie2 at cell-substratum contacts. Furthermore, Tie2 activated at cell-cell or cell-substratum contacts leads to preferential activation of Akt and Erk, respectively. Microarray analyses and real-time PCR validation clearly show the differential gene expression profile in vascular endothelial cells upon Ang1 stimulation in the presence or absence of cell-cell contacts, implying downstream signalling is dependent upon the spatial localization of Tie2.     

Enhanced expression of angiopoietin-2 and the Tie2 receptor but not angiopoietin-1 or the Tie1 receptor in a rat model of myocardial infarction

Modulation of Tie2 receptor activity by angiopoietin ligands is crucial for angiogenesis, blood vessel maturation, and vascular endothelium integrity. The role of the angiopoietin (Ang) and Tie system in myocardial infarction is not well understood. To investigate the participation of the Ang/Tie in myocardial infarction, adult Sprague-Dawley rats with ligation of the left anterior descending coronary artery to induce myocardial infarction were studied. Ang1, Ang2, Tie1, and Tie2 were measured immediately after ligation of the coronary artery, and at 6 h, 1 and 3 days, and 1, 2, 3 and 4 weeks after ligation by Northern blotting, Western blotting, and immunohistochemical staining. Ang2 mRNA significantly increased from 2 weeks (2.1-fold) to 4 weeks (2.9-fold) after the infarction in the left ventricular free wall. Tie2 mRNA increased significantly from 1 week (2.1-fold) to 4 weeks (3.8-fold) after the infarction. Ang2 protein also significantly increased from 3 days (1.9-fold) to 4 weeks (3-fold) after the infarction in the left ventricular free wall. Tie2 protein increased 2.4-fold at 3 weeks and 2.8-fold at 4 weeks after the infarction. Neither Ang1 nor Tie1 mRNA or protein showed any significant change at any time point after the infarction. The ratio of Ang2/Ang1 mRNA and protein in the study group was higher than that in the control group. Ang2 and Tie2 expression in nonischemic myocardium showed no significant change. Immunohistochemical study also showed increased immunoreactivity of Ang2 and Tie2 at the infarct border. In conclusion, Ang2 and Tie2 expressions significantly increased both spatial and temporal patterns after myocardial infarction in the rat ventricular myocardium, while Ang1 and Tie1 receptor expression did not.     

Effects of angiopoietins-1 and -2 on the receptor tyrosine kinase Tie2 are differentially regulated at the endothelial cell surface

Angiopoietin-1 (Ang1) and Ang2 are ligands for the receptor tyrosine kinase Tie2. Structural data suggest that the two ligands bind Tie2 similarly. However, in endothelial cells Ang1 activates Tie2 whereas Ang2 can act as an apparent antagonist. In addition, each ligand exhibits distinct kinetics of release following binding. These observations suggest that additional factors influence function and binding of angiopoietins with receptors in the cellular context. Previous work has shown that Ang1 binding and activation of Tie2 are inhibited by Tie1, a related receptor that complexes with Tie2 in cells. In this study we have investigated binding of Ang1 and Ang2 to Tie2 in endothelial cells. In contrast to Ang1, binding of Ang2 to Tie2 was found to be not affected by Tie1. Neither PMA-induced Tie1 ectodomain cleavage nor suppression of Tie1 expression by siRNA affected the ability of Ang2 to bind Tie2. Analysis of the level of Tie1 co-immunoprecipitating with angiopoietin-bound Tie2 demonstrated that Ang2 can bind Tie2 in Tie2:Tie1 complexes whereas Ang1 preferentially binds non-complexed Tie2. Stimulation of Tie1 ectodomain cleavage did not increase the agonist activity of Ang2 for Tie2. Similarly, the Tie2-agonist activity of Ang2 was not affected by siRNA suppression of Tie1 expression. Consistent with previous reports, loss of Tie1 ectodomain enhanced the agonist activity of Ang1 for Tie2. Importantly, Ang2 was still able to antagonize the elevated Ang1-activation of Tie2 that occurs on Tie1 ectodomain loss. Together these data demonstrate that Ang1 and Ang2 bind differently to Tie2 at the cell surface and this is controlled by Tie1. This differential regulation of angiopoietin binding allows control of Tie2 activation response to Ang1 without affecting Ang2 agonist activity and maintains the ability of Ang2 to antagonize even the enhanced Ang1 activation of Tie2 that occurs on loss of Tie1 ectodomain. This provides a mechanism by which signalling through Tie2 can be modified by stimuli in the cellular microenvironment.