细胞外基质金属蛋白酶诱导分子（CD147）在胰腺癌细胞及胰腺星状细胞中的表达

目的：探讨细胞外基质金属蛋白酶诱导分子（CD147）在胰腺癌细胞（Panc-1）及胰腺星状细胞（PSCs）的表达。方法：应用QRT—PCR,免疫细胞化学和免疫印迹分析方法检测Panc-1和PSCs细胞中EMMRPIN的表达,应用脱糖基化试剂N—glycosidase F及Endoglycosidase H鉴定CD147糖基化形式。结果：CD147在Panc-1和PSCs细胞质膜及细胞质中高表达,通过脱糖基化法首次鉴定出胰腺癌细胞及胰腺星状细胞中CD147不同的糖基化修饰。结论：CD147的糖基化修饰具有细胞特异性,可能与细胞恶性程度相关。     

胰腺星状细胞的调控及转归

活化的胰腺星状细胞(pancreatic stellate cells,PSCs)是胰腺炎致胰腺纤维化的主要效应细胞。近年来,学者普遍认为,胰腺纤维化早期阶段是动态可逆的,因此,若在胰腺损伤的早期阶段,抑制PSCs的增殖、迁移,减少损伤部位PSCs的数目,降低细胞外基质(extracellular matrix,ECM)的生成,将可能逆转胰腺纤维化。该文以PSCs为靶点阐述了抗胰腺纤维化的新策略。     

油红O染色鉴定胰腺星状细胞

目的胰腺星状细胞(pancreatic stellate cells,PSCs)是一类胞浆内含有Vitamin A脂滴的特殊类型的细胞。油红O能够使脂肪组织及细胞内的脂滴着色。本研究的目的是采用油红O染色的方法对分离培养的PSCs进行鉴定。方法采用Histodenz密度梯度离心分离提取PSCs。异丙醇配制油红O染液,培养的PSCs应用油红O染液染色。结果油红O染色后可清晰显示PSCs内的脂滴。结论油红O染色可以特异性地显示脂滴,是鉴别PSCs的有效方法。     

高糖高胰岛素促进小鼠胰腺星状细胞活化和半乳凝素-3表达

该研究观察了高糖高胰岛素对小鼠胰腺星状细胞(pancreatic stellate cells,PSCs)活化、增殖、细胞外基质(extracellular matrix,ECM)合成和半乳凝素-3(galectin-3,Gal-3)表达的影响。分离PSCs并培养至3~5代后进行实验。PSCs干预分为低糖对照组(5 mmol/L葡萄糖)、高糖组(25 mmol/L葡萄糖)、高胰岛素组(5 mmol/L葡萄糖+100 nmol/L胰岛素)、高糖高胰岛素组(25 mmol/L葡萄糖+100 nmol/L胰岛素)。细胞免疫荧光检测胰岛素受体(insulin receptor,IR)和胰岛素样生长因子-1型受体(insulin like growth factor-1 receptor,IGF-1R)在PSCs的表达;MTT法检测PSCs增殖;RT-PCR和Western blot测定平α-滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、I型胶原(type I collagen,Col I)、纤连蛋白(fi bronectin,Fn)和Gal-3的m RNA和蛋白质水平。结果发现,PSCs细胞表达IR和IGF-1R;与低糖对照组相比,高糖组、高胰岛素组、高糖高胰岛素组均诱导PSCs活化、增殖并促进Col I、Fn生成和Gal-3表达,其中以高糖高胰岛素组最为显著。以上结果说明,2型糖尿病高糖、高胰岛素微环境可能促进PSCs活化、增殖、ECM生成和Gal-3表达,在一定程度上可导致胰腺纤维化。     

N-糖基化修饰在髓性白血病耐药中的作用

目的研究N-糖基化修饰、糖基因表达调控在髓性白血病耐药中的作用,明确N-糖基化修饰、糖基因与白血病耐药的相关性,从而为预测和诊断髓性白血病耐药性,寻求逆转药物提供新策略和靶点。方法通过修饰白血病耐药细胞株的N-糖基化（衣霉素Tunicamycin和PNGase F处理）,Western Blot检测Pgp、CD147糖蛋白的表达水平;MTT法检测N-糖基化修饰前后髓性白血病耐药细胞株的生长情况及对化疗药物的敏感性,观察上述细胞膜型N-糖基化修饰后对化疗药物耐药性的影响;进一步通过RNA干扰技术干预差异表达的糖基因,MTT法检测干扰前后白血病耐药细胞株的生长情况及对化疗药物的敏感性,观测糖基因的表达调控对髓性白血病耐药的影响。结果 NB4/ADR细胞经N-糖基化修饰后,P-gp、CD147糖蛋白的表达水平发生改变,同时该细胞的药物敏感性也增强（P〈0.05）;当通过RNA干扰技术特异性使NB4/ADR细胞中B3GNT8和ST8SIA4表达下调时,该细胞的药物敏感性增强（P〈0.05）。结论髓性白血病细胞株中N-糖基化修饰、糖基因的改变均与白血病多药耐药具有相关性,为预测和诊断髓性白血病耐药性,寻求逆转药物提供新策略和靶点。     

结缔组织生长因子在胰腺组织纤维化中的作用及机制探讨

目的 观察胰腺纤维化后结缔组织生长因子(Connective tissue growth factor,CTGF)在胰腺组织内的表达;进一步研究参与CTGF作用于胰腺星状细胞(pancreatic stellate cells,PSCs)的分子信号调控通路.方法 建立大鼠胰腺纤维化动物模型,HE染色、天狼猩红染色和免疫组织化学染色等方法观察胰腺纤维化后PSCs的活化情况及CTGF在胰腺组织的表达.Real-time RT PCR检测CTGF的基因表达.Western Blot检测PSCa内α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)及胶原蛋白Ⅰ(CollagenⅠ)水平.结果 胰腺组织纤维化后,PSCs大量活化,并显著表达CTGF.CTGF作用后,PSCs内CTGF mRNA、α-SMA和Collagen Ⅰ的合成均有显著增加,在给予不同的细胞信号通路阻断剂后,PSCs内α-SMA的合成有显著下降,而Collagen Ⅰ的降低没有表现出统计学差异.结论 CTGF参与了胰腺纤维化的调控,MAPK和PI3-K信号通路均参与了CTGF的调控作用.     

Notch3对促进胰腺星形细胞活化的基因表达及信号通路的影响

目的:分离培养小鼠胰腺星形细胞(PSCs),检测Notch3对促进PSCs活化的基因表达及信号通路的影响.方法:对小鼠PSCs进行分离培养及传代.采用免疫荧光染色检测活化的小鼠PSCs中α-SMA,fibronectin及collagen Ⅰ的表达;细胞分组为空白对照组(MOCK组),阴性对照组(转染Notch3 si...     

人成骨肉瘤细胞CD147基因编码区的克隆和序列测定

克隆人成骨肉瘤细胞CD147基因编码区并进行序列测定。以人成骨肉瘤细胞系MG63的mRNA为模板,采用RT-PCR方法得到人CD147的编码区cDNA,克隆至载体pcDNA3．1( )中,酶切鉴定后进行序列分析。结果获得人CD147编码区基因和重组质粒pcDNA3．1( )／asCD147,并经DNA序列分析证实其序列和献报道一致。以上结果证明了CD147在成骨肉瘤细胞中也有表达,为进一步进行其结构和功能研究打下了基础。     

人CD147的克隆表达及其对成纤维细胞基质金属蛋白酶表达的影响

CD147可以促进基质金属蛋白酶(MMPs)的表达,与肿瘤的生长和浸润有关。为了研究CD147在大肠癌中的作用,利用RT-PCR从一健康人克隆了cd147基因,测序发现该基因存在两个碱基突变,其中C634T造成了CD147跨膜区212位氨基酸由L突变为F。分别构建CD147的原核(pGEX-5x-147)和真核(pEGFP-147)表达系统,在宿主菌BL21和CCL229细胞中均获得了稳定表达。Western印迹显示原核表达产物比真核CD147分子量小,说明原核CD147缺乏糖基化。荧光显微镜显示真核CD147表达定位于CCL229细胞膜,表明突变L212F不影响CD147的膜定位。用明胶电泳检测表达的CD147对MMPs表达的影响,结果显示原核产物不能诱导MMPs表达上调,而真核产物能够明显诱导MMPs表达上调,说明糖基化对于CD147活性是必需的,真核系统能够表达具有生物功能的CD147,并且突变L212F不会影响蛋白质的活性。     

白介素-17A在小鼠慢性胰腺炎中的作用研究

目的:研究白细胞介素-17A在小鼠慢性胰腺炎模型中的表达及其对小鼠星状细胞的影响。方法:建立雨蛙肽诱导的小鼠实验性慢性胰腺炎动物模型,利用实时荧光定量PCR、ELISA、免疫组化和Western-blot等手段检测白介素-17A在雨蛙肽诱导的小鼠慢性胰腺炎模型中的表达变化及免疫活性。用重组白介素-17A作用于小鼠胰腺星状细胞,检测其对星状细胞活化的作用,并进一步探究其对促胰腺纤维化炎症因子白介素-6、白介素-1β、TGF-β在mRNA水平的表达变化。结果:慢性胰腺炎胰腺组织中白介素-17A受体IL-17RA及IL-17RC mRNA水平的表达较正常胰腺明显升高,慢性胰腺炎小鼠胰腺组织中IL-17A蛋白水平较正常小鼠明显升高,CP小鼠血清中IL-17A蛋白水平(56.40±10.50 pg/L)较NC组(27.88±5.74pg/L)亦明显升高,IL-17A在正常胰腺组织中鲜有表达(8.9±2.72%),而在CP组织中呈强阳性表达(55.84±5.71%),其免疫活性主要定位于间质炎性细胞及导管样复合体中;重组白介素-17A可促进小鼠星状细胞活化,并直接诱导星状细胞表达白介素-6、白介素-1β以及TGF-β等促纤维化细胞因子。结论:白介素-17A在雨蛙肽诱导的小鼠慢性胰腺炎模型中表达上调,并可能通过诱导小鼠星状细胞表达促炎细胞因子白介素-6、白介素-1β和TGF-β,促进胰腺星状细胞活化以及胰腺纤维化。     

Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells

The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated β-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not altered by treatment with anti-TGF-β-neutralizing antibody, excluding a central role of TGF-β in this process. In conclusion, PSCs promoted EMT in pancreatic cancer cells suggesting a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells.     

虎杖提取物对人胰腺癌细胞系Panc-1增殖与凋亡的影响

目的:通过虎杖提取物干预人胰腺癌细胞系Panc-1,探讨虎杖提取物对人胰腺癌细胞增殖凋亡表型的影响。方法:制备不同浓度(0、10、50、100、150、200μg/m L)的虎杖提取物,将各个浓度的虎杖提取物分别加入待处理的人胰腺癌Panc-1细胞系中持续培养24 h后,利用CCK-8(cell counting kit-8)法检测细胞株Panc-1的增殖活性;将100μg/m L虎杖提取物处理人胰腺癌细胞系Panc-124 h后,利用流式细胞术(FCM)检测其细胞周期及凋亡分布;100μg/m L虎杖提取物处理人胰腺癌细胞株Panc-124 h后,提取细胞总RNA及总蛋白,后续利用实时荧光定量PCR及Western blot分别检测人胰腺癌细胞株Panc-1增殖标志基因PCNA、CDK2及凋亡标志基因BAD、BAX的转录和翻译水平。结果:CCK-8结果表明虎杖提取物对人胰腺癌细胞系Panc-1细胞增殖的抑制率随浓度增加;流式细胞术结果显示虎杖提取物抑制人胰腺癌细胞增殖促进其凋亡;荧光定量PCR和Western blot结果显示虎杖提取物能使人胰腺癌细胞增殖标志基因PCNA,CDK2表达量下降,凋亡标志基因BAD,BAX表达量上升。结论:虎杖提取物能够抑制人胰腺癌细胞系Panc-1细胞增殖并促进其凋亡。     

CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells

Pancreatic stellate cells (PSCs) play a crucial role in the aggressive behavior of pancreatic cancer. Although heterogeneity of PSCs has been identified, the functional differences remain unclear. We characterized CD271⁺ PSCs in human pancreatic cancer. Immunohistochemistry for CD271 was performed for 31 normal pancreatic tissues and 105 pancreatic ductal adenocarcinomas (PDACs). We performed flow cytometry and quantitative RT-PCR, and assessed CD271 expression in PSCs isolated from pancreatic tissues and the changes in CD271 expression in PSCs cocultured with cancer cells. We also investigated the pattern of CD271 expression in a SCID mouse xenograft model. In the immunohistochemical analyses, the CD271-high staining rates in pancreatic stroma in normal pancreatic tissues and PDACs were 2/31 (6.5%) and 29/105 (27.6%), respectively (p = 0.0069). In PDACs, CD271⁺ stromal cells were frequently observed on the edge rather than the center of the tumors. Stromal CD271 high expression was associated with a good prognosis (p = 0.0040). Flow cytometric analyses demonstrated CD271-positive rates in PSCs were 0–2.1%. Quantitative RT-PCR analyses revealed that CD271 mRNA expression was increased in PSCs after coculture with pancreatic cancer cells. However, the level of CD271 mRNA expression subsequently decreased after the transient increase. Furthermore, CD271 mRNA expression was decreased in PSCs migrating toward pancreatic cancer cells through Matrigel. In the xenograft model, CD271⁺ PSCs were present at tumor margins/periphery and were absent in the tumor core. In conclusion, CD271 was expressed in PSCs around pancreatic tumors, but not in the center of the tumors, and expression decreased after long coculture with pancreatic cancer cells or after movement toward pancreatic cancer cells. These findings suggest that CD271⁺ PSCs appear at the early stage of pancreatic carcinogenesis and that CD271 expression is significantly correlated with a better prognosis in patients with PDAC.     

The glycosylation characteristic of hepatoma-associated antigen HAb18G/CD147 in human hepatoma cells

HAb18G/CD147 is a highly glycosylated transmembrane protein belonging to the immunoglobulin superfamily. Our previous studies have demonstrated that overexpressing HAb18G/CD147 enhances the metastatic potentials of human hepatoma cells. In the present study, to investigate the glycosylation characteristic of HAb18G/CD147 in human hepatoma cells, HAb18G/CD147 was first purified from human FHCC-98 hepatoma cells by immunoaffinity chromatography, and then introduced into human fibroblasts culture system for matrix metalloproteinases induction. As a result, the elevated levels of matrix metalloproteinases secreted by fibroblasts were detected by gelatin zymography. The lysates of human hepatoma FHCC-98 cell revealed two major forms of HAb18G/CD147 (43-66 and 35 kDa) by western blot assay. To elucidate whether the variation of molecule size were caused by different glycosylation, two different approaches were employed to accomplish this goal: deglycosylation with N-glycosylation inhibitor tunicamycin or endoglycosidases. A single deglycosylated core protein with molecular weight approximately 27 kDa was obtained from both methods. Furthermore, the results of endoglycosidases treatment also showed that two forms of HAb18G/CD147 contain different types of oligosaccharide chains, thus sensitive to different endoglycosidase. In conclusion, the present study demonstrated that purified native HAb18G/CD147 has the bioactivity of stimulating human fibroblasts to produce elevated levels of matrix metalloproteinases, and that the two different forms of HAb18G/CD147 are derived from the single core protein but differ in their degree and types of glycosylation.     

Pancreatic tumor cells influence the composition of the extracellular matrix

The malignant behavior of cancers depends on the microenvironmental context. We investigated compositional alterations of the extracellular matrix (ECM) in pancreatic cancer, with special emphasis on the proteoglycans decorin, lumican, and versican. Compared with normal controls (n=18), marked overexpression of these proteoglycans was observed in pancreatic cancer tissues (n=30) by quantitative RT-PCR (p<0.0001). Immunohistochemistry revealed abundance of proteoglycans in the ECM of pancreatic cancer specimens, whereas tumor cells themselves were devoid of either decorin, lumican or versican. RT-PCR confirmed pancreatic stellate cells (PSCs) as the major source of these proteins. Interestingly, TGFbeta1 and conditioned medium derived from pancreatic cancer cell lines synergistically suppressed the expression of known anti-tumor factors decorin and lumican, but stimulated the expression of pro-metastatic factor versican in cultured PSCs. These findings indicate that malignant cells can actively influence the composition of the ECM through TGFbeta1 and other soluble factors, altering their microenvironment in a tumor-favorable way.     

The Influence of Reactive Oxygen Species on the Adhesion of Pancreatic Carcinoma Cells to the Peritoneum

Postoperative peritoneal carcinomatosis is a significant clinical problem after “curative” resection of pancreatic carcinoma. Preoperative surgical trauma activates a cascade of peritoneal defense mechanisms responsible for postoperative intra-abdominal tumor recurrence. Reactive oxygen species (ROS) play a pivotal role in this postoperative inflammatory reaction. This study explores the influence of ROS on adhesion of human pancreatic carcinoma cells to human mesothelial cells. Furthermore this study explores the influence of ROS on the presentation of adhesion molecules on Panc-1 and mesothelial cells. ROS were produced using the enzymatic reaction of xanthine with xanthine oxidase (X/XO). A reproducible in vitro assay to study adhesion of human Panc-1 carcinoma tumor cells to a mesothelial cell monolayer of primary human mesothelial cells was used. Mesothelial monolayers were incubated with ROS produced prior to adhesion of the tumor cells. Incubation of the mesothelial cells with X/XO resulted in a significant increase (69.5%) in adhesion of Panc-1 in all patients. SOD/catalase, anti-oxidants, could reduce this increase by 56.7%. ROS significantly influenced the expression of the adhesion molecules ICAM-1, VCAM-1 and CD44h on mesothelial cells, but did not influence adhesion molecule expression on Panc-1. The ROS released during the post-operative inflammatory reaction may play an important role in the adhesion of pancreatic tumor cells to the mesothelium-possibly by influencing adhesion molecule expression on mesothelial cells. Therefore ROS can partly be responsible for the enhanced post-operative intra-abdominal tumor recurrence.Key words: reactive oxygen species, mesothelium, Panc-1     

Pancreatic cancer stimulates pancreatic stellate cell proliferation and TIMP-1 production through the MAP kinase pathway

Pancreatic adenocarcinoma is characterized by an intense desmoplastic reaction that surrounds the tumor. Pancreatic stellate cells (PSCs) are thought to be responsible for production of this extracellular matrix. When activated, PSCs have a myofibroblast phenotype and produce not only components of the extracellular matrix including collagen, fibronectin, and laminin, but also matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Since PSCs are found in the stroma surrounding human pancreatic adenocarcinoma, we postulate that pancreatic cancer could impact PSC proliferation and TIMP-1 production. Rat PSCs were isolated and cultured. Isolated PSCs were exposed to PANC-1 conditioned medium (CM) and proliferation, activation of the mitogen-activated protein (MAP) kinase pathway, and TIMP-1 gene induction were determined. Exposure to PANC-1 CM increased PSC DNA synthesis, cell number, and TIMP-1 mRNA (real-time PCR) as well as activating the extracellular-regulated kinase (ERK) 1/2. Inhibition of ERK 1/2 phosphorylation (U0126) prevented the increases in growth and TIMP-1 expression. PANC-1 CM stimulates PSC proliferation and TIMP-1 through the MAP kinase (ERK 1/2) pathway.     

The Influence of Reactive Oxygen Species on the Adhesion of Pancreatic Carcinoma Cells to the Peritoneum

Postoperative peritoneal carcinomatosis is a significant clinical problem after “curative” resection of pancreatic carcinoma. Peroperative surgical trauma activates a cascade of peritoneal defense mechanisms responsible for postoperative intra-abdominal tumor recurrence. Reactive oxygen species (ROS) play a pivotal role in this postoperative inflammatory reaction. This study explores the influence of ROS on adhesion of human pancreatic carcinoma cells to human mesothelial cells. Furthermore this study explores the influence of ROS on the presentation of adhesion molecules on Panc-1 and mesothelial cells. ROS were produced using the enzymatic reaction of xanthine with xanthine oxidase (X/XO). A reproducible in-vitro assay to study adhesion of human Panc-1 carcinoma tumor cells to a mesothelial cell monolayer of primary human mesothelial cells was used. Mesothelial monolayers were incubated with ROS produced prior to adhesion of the tumor cells. Incubation of the mesothelial cells with X/XO resulted in a significant increase (69.5%) in adhesion of Panc-1 in all patients. SOD/catalase, anti-oxidants, could reduce this increase by 56.7%. ROS significantly influenced the expression of the adhesion molecules ICAM-1, VCAM-1 and CD44h on mesothelial cells, but did not influence adhesion molecule expression on Panc-1. The ROS released during the post-operative inflammatory reaction may play an important role in the adhesion of pancreatic tumor cells to the mesothelium. Possibly by influencing adhesion molecule expression on mesothelial cells. Therefore ROS can partly be responsible for the enhanced post-operative intra-abdominal tumor recurrence.