转化生长因子β1和核心蛋白聚糖在胰腺星状细胞中的表达

背景：胰腺星状细胞(PSCs)在胰腺纤维化的进程中具有重要作用，因此有必要对影响该细胞活化的细胞因子进行研究。目的：建立一种从大鼠中分离、培养PSCs的成熟方法，研究原代培养2～3代的传代PSCs中转化生长因子(TGF)-β。及其拮抗剂核心蛋白聚糖的mRNA(转录水平)和蛋白(翻译水平)的表达和分布。方法：取大鼠胰腺组织进行酶消化和不连续密度梯度离心以获得原代PSCs。采用免疫细胞化学染色检测PSCs中α-平滑肌肌动蛋白(SMA)、结蛋白(desmin)和纤维连接蛋白(FN)的表达；采用免疫细胞化学染色和原位杂交方法检测传代PSCs中TGF-β1和核心蛋白聚糖的蛋白和mRNA的表达。结果：体外成功分离并培养PSCs后，免疫细胞化学染色结果显示该细胞可活化，细胞质中出现α-SMA、desmin和FN阳性表达。在传代PSCs中，TGF-β1 mRNA在细胞核和细胞质内均有表达，其蛋白则主要在细胞质内表达；核心蛋白聚糖则无论mRNA或蛋白均主要在细胞核内表达。结论：大鼠PSCs可成功地在体外分离并培养。在培养过程中，该细胞可自发活化并向成纤维样细胞分化。PSCs可自分泌TGF-β1和核心蛋白聚糖，并可能在基因转录水平进行自身调控，从而对胰腺纤维化的形成产生一定影响。     

Circulating Vascular Endothelial Growth Factor in Patients With Colorectal Cancer

Objective : The expression of vascular endothelial growth factor (VEGF), a glycoprotein that selectively promotes proliferation of endothelial cells, has been associated with cancer development. The aim of the present study was to determine whether serum levels of VEGF correlate with disease progression in patients with colorectal cancer. Methods : VEGF levels were measured by a highly sensitive enzyme-linked immunosorbent assay in sera from 67 patients with colorectal cancer, 14 patients with colorectal adenomas, and 72 healthy volunteers, and in tissue homogenates from 10 patients with colorectal cancer. Results : Serum VEGF levels were significantly higher in patients with colorectal cancer than in patients with colorectal adenomas or in normal controls (   p < 0.01  ). In patients with colorectal cancer, serum VEGF levels were significantly associated with Dukes stage (   p < 0.01  ) and with carcinoembryonic antigen levels (   r = 0.725  ,   p < 0.001  ). Patients with hepatic and/or lymph node metastasis had higher serum VEGF levels than those without. Surgical resection of the colorectal tumor led to a decrease in serum VEGF levels whether or not metastasis was present (   p < 0.05  ). The tumor-bearing tissue contained significantly more VEGF than normal-appearing mucosa (   p < 0.05  ). Conclusions : VEGF is involved in the development of colorectal cancer. Measurement of VEGF in the serum may be a useful noninvasive clinical marker for evaluating the disease status.     

Pancreatic Stellate Cells Increase the Invasion of Human Pancreatic Cancer Cells through the Stromal Cell-Derived Factor-1/CXCR4 Axis

Aim: Both pancreatic stellate cells (PSCs) and the stromal cell-derived factor-1(SDF-1)/CXCR4 receptor ligand system have important roles in pancreatic cancer progression. This study set out to detect if PSCs express SDF-1 and promote the invasion of pancreatic cancer through the SDF-1/CXCR4 receptor ligand axis. Methods: RT-PCR was performed to detect the expression of SDF-1 and CXCR4 in PSCs, pancreatic cancer lines and cancer tissue samples. ELISA was used to investigate the concentration of SDF-1 in PSC supernatants. An MTT assay was applied to detect the proliferation of pancreatic cancer cells. A transwell chamber migration assay was employed to detect the migration of AsPC-1 cells. An in vitro invasion assay was used to detect the invasion of AsPC-1 cells. Results: CXCR4 expression was detected in PSCs; AsPC-1, SW1990 and BxPC-3 cancer cells; and cancer tissues. SDF-1 was detected in PSCs and cancer tissues, but not in AsPC-1, SW1990 and BxPC-3 cells. SDF-1α protein was found in PSC supernatants. PSC-conditioned media can promote the proliferation, migration and invasion of pancreatic cancer cells. SDF-1 neutralizing antibody or AMD3100 can significantly inhibit these promotive effects. Conclusion: PSCs can secrete SDF-1 and increase the invasion of pancreatic cancer cells through the SDF-1/CXCR4 axis.     

Synergistic Growth Inhibitory Effects of the Dual Endothelin-1 Receptor Antagonist Bosentan on Pancreatic Stellate and Cancer Cells

Pancreatic stellate cells (PSC) play a key role in pancreatic fibrosis. Activation of PSC occurs in response to pro-fibrogenic stimuli and is maintained by autocrine loops of mediators, such as endothelin (ET)-1. Here, we have evaluated effects of the dual ET receptor antagonist bosentan in models of pancreatic fibrogenesis and cancer. Cell culture studies revealed that PSC and DSL6A pancreatic cancer cells expressed both ET-1 and ET receptors. Bosentan efficiently inhibited proliferation of both cell types and collagen synthesis in PSC. Expression of the myofibroblastic marker α-smooth muscle actin, connective tissue growth factor, and ET-1 itself in PSC was reduced, while expression of matrix metalloproteinase-9 was enhanced. Like PSC, DSL6A cells secrete less ET-1 when cultured with bosentan. In a rat model of pancreatic fibrosis, chronic pancreatitis induced by dibutyltin dichloride, a tendency towards a diminished disease progression was observed in a subgroup of rats with less severe disease. Together, our results indicate that bosentan exerts antifibrotic and antitumor effects in vitro. Its efficiency in vivo warrants further investigation.     

Pancreatic Cancer: The Role of Pancreatic Stellate Cells in Tumor Progression

Pancreatic ductal adenocarcinoma is an aggressive and highly lethal disease frequently characterized by a dense stromal or desmoplastic response. Accumulating evidence exists that tumor desmoplasia plays a central role in disease progression and that e.g. activated pancreatic stellate cells (PSCs) are responsible for the excess matrix production. The mechanisms underlying the tumor versus stroma interplay are complex. Pancreatic cancer cells release mitogenic and fibrogenic stimulants, such as transforming growth factor β₁ platelet-derived growth factor (PDGF), sonic hedgehog, galectin 3, endothelin 1 and serine protease inhibitor nexin 2, all of which may promote the activated PSC phenotype. Stellate cells in turn secrete various factors, including PDGF, stromal-derived factor 1, epidermal growth factor, insulin-like growth factor 1, fibroblast growth factor, secreted protein acidic and rich in cysteine, matrix metalloproteinases, small leucine-rich proteoglycans, periostin and collagen type I that mediate effects on tumor growth, invasion, metastasis and resistance to chemotherapy. This review intends to shed light on the mechanisms by which PSCs in the stroma influence pancreatic cancer development. The increased understanding of this interaction will be of potential value in designing new modalities of targeted therapy.     

Phosphatidylethanol Mediates its Effects on the Vascular Endothelial Growth Factor via HDL Receptor in Endothelial Cells

Background:  Previous epidemiological studies have shown that light to moderate alcohol consumption has protective effects against coronary heart disease but the mechanisms of the beneficial effect of alcohol are not known. Ethanol may increase high density lipoprotein (HDL) cholesterol concentration, augment the reverse cholesterol transport, or regulate growth factors or adhesion molecules. To study whether qualitative changes in HDL phospholipids mediate part of the beneficial effects of alcohol on atherosclerosis by HDL receptor, we investigated whether phosphatidylethanol (PEth) in HDL particles affects the secretion of vascular endothelial growth factor (VEGF) by a human scavenger receptor CD36 and LIMPII analog-I (CLA-1)-mediated pathway.
Methods:  Human EA.hy 926 endothelial cells were incubated in the presence of native HDL or PEth-HDL. VEGF concentration and CLA-1 protein expression were measured. Human CLA-1 receptor-mediated mechanisms in endothelial cells were studied using CLA-1 blocking antibody and protein kinase inhibitors.
Results:  Phosphatidylethanol-containing HDL particles caused a 6-fold increase in the expression of CLA-1 in endothelial cells compared with the effect of native HDL. That emergent effect was mediated mainly through protein kinase C and p44/42 mitogen-activated protein kinase pathways. PEth increased the secretion of VEGF and that increase could be abolished by a CLA-1 blocking antibody.
Conclusions:  High density lipoprotein particles containing PEth bind to CLA-1 receptor and thereby increase the secretion of VEGF from endothelial cells. Ethanol-induced protective effects against coronary heart disease may be explained, at least partly, by the effects of PEth-modified HDL particles on VEGF via CLA-1-mediated mechanisms in endothelial cells.     

葡萄糖对血管内皮细胞小凹蛋白1和血管内皮生长因子表达的影响

为了探讨高浓度葡萄糖损伤血管内皮细胞及其对小凹蛋白-1和血管内皮生长因子表达的影响。将人脐静脉内皮细胞株ECV304．分别培养在对照组和含5.5mmol／L、11.1mmol／L、22.0mmol／L、33.0mmol／L葡萄糖的培养基中。经葡萄糖培养24h后，噻唑蓝法测定细胞增殖活性，硝酸还原酶法测定培养上清液中一氧化氮浓度，免疫组织化学和免疫印迹方法检测细胞中小凹蛋白-1和血管内皮生长因子的表达。结果发现，随着葡萄糖浓度的增加，内皮细胞增殖活性呈浓度依赖性抑制(r=-0.776，P=0.000)；一氧化氮浓度呈浓度依赖性增加(r=0.698，P=0.000)；小凹蛋白-1和血管内皮生长因子为棕黄色颗粒，主要分布于胞浆中；血管内皮生长因子的表达呈浓度依赖性增加(r=0.645，P=0.009)；小凹蛋白-1的表达也呈浓度依赖性增加(r=0.808，P=0.000)。提示高糖可诱导血管内皮细胞的血管内皮生长因子和小凹蛋白-1的表达，此变化可能与糖尿病患者高糖致血管病变有关。     

血管内皮生长因子与心肌缺血

血管内皮生长因子（VEGF）是内皮细胞特异性的促有丝分裂原,属分泌性的糖蛋白。有促进内皮细胞增生,增强血管通透性;加速新血管形成的作用。VEGF通过与其特异性受体结合发挥生物学作用。VEGF在生理和病理状态下新生血管形成中起重要作用。VEGF及其受体在缺血和／或缺 氧的 心肌细胞中表达升高。VEGF蛋白应用于心肌缺血可以通过促进缺血区新生血管形成,提高侧枝血流量而改善心功能。VEGF基因治疗心肌缺血具有广阔应用前景。     

碱性成纤维细胞生长因子对大鼠心脏微血管内皮细胞血管形成及血管内皮生长因子受体1的影响

目的探讨碱性成纤维细胞生长因子对诱导大鼠心脏微血管内皮细胞形成管腔样结构的影响,以及对血管内皮生长因子受体Flt-1的作用。方法分离、培养大鼠心脏微血管内皮细胞。应用Matrigel检测不同浓度碱性成纤维细胞生长因子作用后心脏微血管内皮细胞形成管腔的情况。逆转录聚合酶链反应检测各组血管内皮细胞Flt-1mRNA的表达量。结果应用Matrigel可诱导心脏微血管内皮细胞管腔样结构形成,随着碱性成纤维细胞生长因子浓度升高(0、51、0和20μg/L),管腔样结构形成的数量逐渐增多,当碱性成纤维细胞生长因子浓度达到40μg/L时管腔样结构的数量有所减少(P<0.05)。Flt-1 mRNA的表达量随碱性成纤维细胞生长因子作用浓度的升高(0、5、10和20μg/L)而逐渐增高,当碱性成纤维细胞生长因子浓度达到40μg/L时其mRNA水平有所降低(P<0.05)。结论应用Matrigel可在体外诱导培养的大鼠心脏微血管内皮细胞形成管腔样结构。碱性成纤维细胞生长因子对大鼠心脏微血管内皮细胞形成管腔样结构有促进作用,并在一定范围内呈剂量依赖性关系。碱性成纤维细胞生长因子影响大鼠心脏微血管内皮细胞膜受体Flt-1的表达,在一定范围内亦呈剂量依赖性,且这种剂量依赖性同微血管内皮细胞管腔样结构形成的数量一定范围内呈现一致性。     

醛固酮对人脐静脉内皮细胞血管内皮生长因子表达的影响

目的醛固酮对人脐静脉内皮细胞增殖的影响及其机制。方法应用MTT法测试细胞的生长曲线,检测不同浓度的醛固酮对人脐静脉内皮细胞的增殖,流式细胞仪检测醛固酮对人脐静脉内皮细胞的凋亡,半定量RT-PCR及Western印迹分别检测人脐静脉内皮细胞的血管内皮生长因子mRNA水平及蛋白水平的表达。结果①MTT比色法显示,醛固酮对人脐静脉内皮细胞增殖具有抑制作用且呈浓度依赖性;②当醛固酮浓度增加到800μg/L后,其增殖抑制率不再升高,而此浓度时人脐静脉内皮细胞的凋亡率达到峰值的26.5%±3.3%;③经过醛固酮处理,人脐静脉内皮细胞的血管内皮生长因子mRNA及蛋白水平的表达量均显著下降。结论醛固酮通过降低人脐静脉内皮细胞的血管内皮生长因子表达抑制人脐静脉内皮细胞增殖并促进人脐静脉内皮细胞凋亡。     

Vascular Endothelial Growth Factor Levels in Bile Distinguishes Pancreatic Cancer from Other Etiologies of Biliary Stricture: A Pilot Study

Background

Determining the benign or malignant nature of biliary strictures can be challenging. Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis.

Objective

The purpose of this study was to investigate whether VEGF levels in bile aspirated during endoscopic retrograde cholangiography (ERCP) can distinguish pancreatic cancer from other causes of biliary stricture.

Methods

Bile was directly aspirated in 53 consecutive patients from March 2012 to October 2012 during ERCP from the common bile duct including 15 with pancreatic cancer, 18 with primary sclerosing cholangitis (PSC), nine with cholangiocarcinoma (CCA), and 11 with benign biliary conditions (sphincter of Oddi and choledocholihiasis). Levels of VEGF in bile were measured. The diagnostic performance was then validated in a second, independent validation cohort of 18 patients (pancreatic cancer n = 10, benign n = 8).

Results

A total of 53 consecutive patients were recruited. The median bile VEGF levels were significantly elevated in patients with pancreatic cancer (1.9 ng/ml (interquartile range [IQR] 0.7, 2.2) compared to those with benign biliary conditions (0.3 ng/ml [IQR 0.2, 0.6]; p < 0.001), PSC (0.7 ng/ml [IQR 0.5, 0.9]; p = 0.02) or CCA (0.4 ng/ml [IQR 0.1, 0.5]; p < 0.001). A VEGF cut-off value of 0.5 ng/ml distinguished pancreatic cancer from CCA with a sensitivity and specificity of 93.3 and 88.9 %, respectively, and area under curve (AUC) of 0.93, and from benign conditions with a sensitivity and specificity of 93.3 and 72.7 %, respectively, with AUC of 0.89. The diagnostic accuracy of biliary VEGF was confirmed in the second independent validation cohort.

Conclusions

This study suggests that measurement of biliary VEGF-1 levels distinguishes patients with pancreatic cancer from other etiologies of biliary stricture. This may be particularly relevant in approaching patients with indeterminate biliary stricture.     

干细胞联合血管内皮生长因子治疗缺血性脑损伤

最近的动物实验研究表明,神经发生和血管生成是卒中恢复的重要机制.干细胞移植能改善卒中后神经功能缺损,而血管内皮生长因子也可通过促进血管生成改善卒中后神经功能.文章综述了二者联合应用治疗缺血性脑损伤的研究进展.     

Insulin-Like Growth Factor 1 Receptor Promotes the Growth and Chemoresistance of Pancreatic Cancer

Background

Insulin-like growth factor 1 receptor (IGF1R) plays important roles in the progression of pancreatic cancer. However, the underlying mechanism remains unclear.

Aims

The purpose of this study was to investigate the effects of IGF1R knockdown on the proliferation, apoptosis and chemosensitivity of pancreatic cancer cells, and explore the possible mechanisms.

Methods

Pancreatic cancer cells expressing IGF1R shRNA were established, and the cell proliferation, colony formation, and chemosensitivity to gemcitabine were examined in vitro. The activation of AKT and NF-κB was detected by Western blot analysis and luciferase assay, respectively. Xenograft mice models were established to evaluate the in vivo anti-tumor effects of IGF1R knockdown.

Results

IGF1R knockdown notably inhibited pancreatic cancer cell proliferation and colony formation, induced apoptosis, and inhibited xenograft tumor growth. Moreover, IGF1R knockdown significantly enhanced chemosensitivity to gemcitabine in pancreatic cancer cells, and this was correlated with the inhibition of PI3K/AKT and NF-κB pathways.

Conclusions

IGF1R knockdown suppresses tumor growth and enhances chemosensitivity in pancreatic cancer via the inhibition of PI3K/AKT and NF-κB pathways, and is a promising approach to overcome the chemoresistance of pancreatic cancer.     

睾酮通过上调血管内皮生长因子表达促进外周血内皮祖细胞增殖

目的 探讨雄激素对外周血内皮祖细胞(PB-EPC,)增殖能力的影响及其可能机制.方法 将健康志愿者外周血经密度梯度离心法分离的单个核细胞接种至人纤维连接蛋白包被的培养板中,EGM-2MV培养7天后,多波长激光共聚焦显微镜鉴定FITC标记的荆豆凝集素和Dil标记的乙酰化低密度脂蛋白双染色阳性为PB-EPC.将贴壁细胞分为5组,前4组分别加入0、1、10、100nmol/L睾酮,第5组加入10 nmol/L雄激素受体阻断剂氟他胺干预3h后,再加10 nmol/L睾酮干预.培养48 h后,MTT比色法检测各组PB-EPC的增殖能力.实时定量PCR检测血管内皮生长因子(VEGF) mRNA的表达变化,ELISA检测VEGF分泌量的变化.结果 睾酮呈浓度依赖性促进EPC增殖,雄激素受体阻断剂氟他胺完全阻断睾酮对EPC的促进作用.与空白对照组相比,睾酮在mRNA和蛋白水平上调PB-EPC的VEGF表达,氟他胺可阻断此作用.结论 睾酮通过雄激素受体途径上调VEGF的表达,促进PB-EPC增殖.