胰腺导管腺癌中胰腺星形细胞的研究进展

胰腺星形细胞(pancreatic stellate cells,PSC)是胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)肿瘤微环境中最重要的成分,在PDAC发生发展中具有非常关键的作用.目前大量研究关注于PSC与胰腺癌细胞(pancreatic cancer cells,PCC)之间的相互作用及PSC在PDAC微环境中发挥的作用.PSC在许多情况下发生活化,如乙醇、氧化应激和高血糖等.PDAC早期即可出现PSC的活化,PCC可以诱导刺激PSC发生活化,活化的PSC可以产生大量胶原纤维,形成适宜PCC生长的间质微环境,促进PCC的增殖,减少化疗药物对肿瘤细胞的杀伤作用.另外,PSC还可以与间质中各种细胞成分如内皮细胞和各种免疫细胞相互作用,在血管生成、免疫逃逸和神经侵犯等方面协助肿瘤进展.因此,阐明PSC与肿瘤细胞以及其他间质成分之间复杂的相互作用至关重要.     

Tenascin distribution in basal cell carcinomas

The distribution of tenascin, an extracellular matrix protein highly expressed in the stroma around sites of epithelial-mesenchymal interaction during morphogenesis and in malignant neoplasms, was assessed in cryostat sections of 17 basal cell carcinomas using a polyclonal antibody. There was marked staining of the vascularized stroma around neoplastic islands, usually as an intense, well-defined band, but being more widespread and diffuse in sclerosing, infiltrative areas. Apparently enhanced staining was seen in tumours showing retraction artefact, which may be related to the observation that tenascin interferes with the cell binding function of fibronectin. Reduced staining was seen in areas showing evidence of tumour regression. Tenascin is an important component of the epithelial-mesenchymal interactive process and further studies on its distribution in benign and malignant skin tumours are required.     

膜联蛋白A5与宫颈黏膜上皮细胞癌变的关系

目的：确定在宫颈癌细胞系Hela细胞和SiHa细胞中是否有膜联蛋白A5(anxA5)的表达,为研究anxA5的功能及其与宫颈癌的关系奠定基础。方法：培养Hela细胞和SiHa细胞。TRIzol法提取细胞总RNA,RT- PCR法检测anxA5在mRNA水平的表达并将其基因片段连接于T载体,测序仪测序;Western印迹法和免疫细胞化学ABC法检测anxA5的表达。结果：两种宫颈癌细胞的RT-PCR结果和Western印迹法均显示目的条带;免疫细胞化学染色可见胞质和核膜被染成棕黄色;测序并经NCBI的BLAST进行比对,显示为人anxA5。结论：anxA5在宫颈癌细胞系Hela细胞和SiHa细胞的mRNA水平和蛋白质水平均有表达。     

The expression of tenascin‐C and tenascin‐W in human ossicles

The ossicles of the middle ear (the malleus, incus and stapes) transmit forces resulting from vibrations of the tympanic membrane to the cochlea where they are coded as sound. Hearing loss can result from diseases such as rheumatoid arthritis (RA) that affect the joints between the ossicles or degenerative processes like otosclerosis that lead to ankylosis of the footplate of the stapes in the oval window of the cochlea. In this study, immunohistochemistry was used to determine if the extracellular matrix glycoproteins tenascin‐C or tenascin‐W are expressed in the incudomalleolar and incudostapedial joints of ossicles dissected from human cadavers. Tenascin‐C, which is expressed during inflammatory conditions including RA, was seen in the articular cartilage of the incudomalleolar joints and the head of the stapes. Tenascin‐W, in contrast, was enriched in the annular ligament that anchors the footplate of the stapes into the oval window of the cochlea.     

Extreme skewing of annexin II and S100A6 expression identified by proteomic analysis of peritoneal B-1 cells

B-1 cells differ phenotypically, biochemically and functionally from conventional B-2 cells. The origin of these differences remains uncertain. We hypothesized that unbiased analysis of differences in protein expression between B-1 and B-2 cells might provide information not otherwise available, and thus undertook 1-dimensional (1D) gel analysis combined with mass spectrometry. We identified annexin II and S100A6 in peritoneal B-1 cells (B-1P) but not in splenic B-2 cells (B-2S); these results were confirmed by western blot analysis and reflected in mRNA expression. Further analysis of mRNA indicated that elevated expression levels of annexin II and S100A6 were unique to B-1P among several naive lymphoid populations. However, expression of annexin II and S100A6 protein was up-regulated in mitogenically stimulated B-2S. In both naive B-1 cells and stimulated B-2 cells, annexin II and S100A6 formed Ca++-sensitive complexes. These results confirm that the emerging field of proteomics detects differentially expressed molecules independently of RNA screening methods. These results identify two proteins (annexin II and S100A6) that are unexpectedly differentially expressed in B-1 cells and, although members of larger families, may fulfill unique, subset-specific functions. These results also validate 1D GE/LC-MS/MS as a reliable screening tool in identifying final protein product expression differences between B-1P and B-2S.     

MMP16在胰腺癌组织中的表达及其临床意义

目的：检测胰腺癌组织中MMP16蛋白表达情况,探讨其在胰腺癌发生、发展中的作用、及其临床意义。方法：应用免疫印迹法检测8对胰腺癌/癌旁组织中MMP16蛋白的表达情况;应用免疫组化EnVision法检测49例胰腺癌组织中MMP16蛋白的表达情况,分析其与临床病理因素的关系。结果：免疫印迹法检测显示,胰腺癌组织MMP16蛋白的相对表达量均高于相应癌旁组织(8/8);免疫组化检测显示,在胰腺癌组织中MMP16蛋白的表达率为85.7%(42/49),而在癌旁正常组织中MMP16蛋白表达率为9.1%(3/33);MMP16蛋白在癌组织中的表达率明显高于癌旁正常组织,差异有统计学意义(P<0.05)。MMP16表达与胰腺癌的组织分化程度、临床分期、淋巴结转移有关;该三因素的分组间均有统计学差异(P<0.05)。结论：MMP16在胰腺癌组织中呈高表达,因此可能在胰腺癌的发生、发展中起重要作用。     

Modified methods for isolation of pancreatic stellate cells from human and rodent pancreas

Primary cultures of pancreatic stellate cells (PSCs) remain an important basis for in vitro study. However, effective methods for isolating abundant PSCs are currently lacking. We report on a novel approach to isolating PSCs from normal rat pancreases and human pancreatic ductal adenocarcinoma (PDAC) tissue. After anaesthesia and laparotomy of the rat, a blunt cannula was inserted into the pancreatic duct through the anti-mesentery side of the duodenum, and the pancreas was slowly infused with an enzyme solution until all lobules were fully dispersed. The pancreas was then pre-incubated, finely minced and incubated to procure a cell suspension. PSCs were obtained after the cell suspension was filtered, washed and subject to gradient centrifugation with Nycodenz solution. Fresh human PDAC tissue was finely minced into 1×1×1 mm3 cubes with sharp blades. Tissue blocks were placed at the bottom of a culture plate with fresh plasma (EDTA-anti-coagulated plasma from the same patient, mixed with CaCl 2) sprinkled around the sample. After culture for 5–10 days under appropriate conditions, activated PSCs were harvested. An intraductal perfusion of an enzyme solution simplified the procedure of isolation of rat PSCs, as compared with the multiple injections technique, and a modified outgrowth method significantly shortened the outgrowth time of the activated cells. Our modification in PSC isolation methods significantly increased the isolation efficiency and shortened the culture period, thus facilitating future PSC-related research.     

Spatial expression of two anti-inflammatory mediators, annexin 1 and galectin-1, in nasal polyposis

BACKGROUND: There is renewed interest in the role played by specific counter-regulatory mechanisms to control the inflammatory host response, poorly investigated in human pathology. Here, we monitored the expression of two anti-inflammatory mediators, annexin 1 and galectin-1, and assessed their potential link to glucocorticoids' (GCs) effective control of nasal polyposis (NP). METHODS: Total patterns of mRNA and protein expression were analysed by quantitative real-time PCR (qPCR) and Western blotting analyses, whereas ultrastructural immunocytochemistry was used for spatial localization and quantification of each mediator, focusing on mast cells, eosinophils and epithelial cells. RESULTS: Up-regulation of the annexin 1 gene, and down-regulation of galectin-1 gene, was detected in polypoid tissue compared with nasal mucosa. Patient treatment with betamethasone augmented galectin-1 protein expression in polyps. At the cellular level, control mast cells and eosinophils displayed higher annexin 1 expression, whereas marked galectin-1 immunolabelling was detected in the granule matrix of mast cells. Cells of glandular duct epithelium also displayed expression of both annexin 1 and galectin-1, augmented after treatment. CONCLUSION: Mast cells and epithelial cells appeared to be pivotal cell types involved in the expression of both annexin 1 and galectin-1. It is possible that annexin 1 and galectin-1 could be functionally associated with a specific mechanism in NP and that GC exert at least part of their beneficial effects on the airway mucosa by up-regulating, in a specific cell target fashion, these anti-inflammatory agonists.     

Tenascin C expression is upregulated in pancreatic cancer and correlates with differentiation

BACKGROUND: Tenascin C is a large, hexameric, extracellular matrix protein that is present during embryonic development but essentially absent in adult tissues. It is involved in remodelling processes, such as wound healing and tumour development. Tissue expression of tenascin C correlates with prognosis in colorectal, cervical, and breast cancer and in carcinoma of the papilla of Vater. AIM: To study the expression of tenascin C in pancreatic cancer and to compare the staining results with the patients' clinicopathological data. MATERIAL AND METHODS: Formalin fixed, paraffin wax embedded specimens from 146 patients with pancreatic adenocarcinoma were stained with an anti-tenascin C monoclonal antibody. RESULTS: Tenascin C immunoreactivity was seen in most samples of pancreatic carcinoma: staining was weak in 72 (49%), moderate in 52 (36%), strong in 10 (7%), and negative in 12 (8%) samples. Tenascin C expression correlated with age (< or = 66 v >66 years) and poor differentiation (grades 1-2 v 3). There was no correlation between tenascin C expression and survival, clinical stage, tumour size, nodal status, distant metastasis, tumour location, or sex. CONCLUSION: Tenascin C expression was increased in most pancreatic carcinomas, but contrary to the results in other cancers, it is not a prognostic factor in pancreatic cancer.     

Differential expression of annexin I in human mammary ductal epithelial cells in normal and benign and malignant breast tissues

Annexins are a family of structurally related, water-soluble proteins that have calcium- and phospholipid-binding domains. Annexin I is thought to be involved in cell proliferation and differentiation and has recently been shown to be expressed on the surfaces of lymphoma cells where it acts as an endothelial cell adhesion molecule. To evaluate the expression of annexin I in relation to human breast cancer development and progression we used breast biopsy tissues. Immunohistochemical analysis of annexin I in paraffin-embedded ductal epithelial cells of various human breast tissues indicated that this annexin was not demonstrable in the ductal luminal cells of normal breast tissues (n = 11) and benign tumors (n = 10) (except for one ductal adenoma) but was generally expressed in various types of breast cancers, including noninvasive ductal carcinoma in situ (DCIS), invasive and metastatic breast tumors (n = 33). The results suggest that annexin I expression might correlate with malignant breast cancer progression but it is most likely involved at an early stage of human breast cancer development.     

Decreased annexin I expression in prostatic adenocarcinoma and in high-grade prostatic intraepithelial neoplasia

AIMS: To analyse annexin I expression in prostatic carcinoma. Annexin I belongs to a family of structurally related calcium and phospholipid-binding proteins implicated in signal transduction, DNA replication, cell proliferation and apoptosis. The decreased expression of annexin I, II and VII proteins has been reported in different types of cancer. METHODS AND RESULTS: Using immunohistochemistry, we analysed annexin I expression in 77 cases of prostatic adenocarcinoma (Gleason score 6, N = 40; Gleason scores 7-8, N = 27; and Gleason scores 9-10, N = 10) and high-grade prostatic intraepithelial neoplasia (PIN, N = 50). Immunoreactivity of annexin I in tumour cells was evaluated as negative (< 5% of cells), focally positive (5-25% of cells) or positive (> 25% of cells). In contrast to positive staining in adjacent benign prostatic epithelium, annexin I expression was decreased (focally positive) in 76% of cases of high-grade PIN (P < 0.0001) and was decreased or absent in 81% of prostatic adenocarcinomas (P < 0.0001). Annexin I expression in all higher grade tumours (Gleason scores 7-10) was only focally positive or absent. CONCLUSIONS: Expression of annexin I inversely correlates with the increasing histological grade of prostatic adenocarcinoma. By showing a progressive loss of annexin I expression in high-grade PIN, intermediate-grade and high-grade cancer, our findings suggest that the loss of annexin I expression occurs early in prostatic tumorigenesis and becomes more prominent throughout tumour progression. The loss of expression of annexin I may serve as a useful marker of prostate cancer development and progression.     

Loss of expression of antigen-presenting molecules in human pancreatic cancer and pancreatic cancer cell lines

Tumours evade immune recognition and destruction through loss or down-regulation of expression of antigen processing and antigen-presenting molecules such as the human leucocyte antigen (HLA class I) and transporter for antigen presentation (TAP). This study examined the expression of HLA class I, class II and TAP in human pancreatic carcinoma tissue and 19 immortalized pancreatic cancer lines using a panel of antibodies directed against allele-specific as well as monomorphic determinants of these molecules. In tissue samples, reduction or loss of HLA class I and TAP was observed in 76% of cases, loss or down-regulation of TAP expression in 53%. In pancreatic cell lines down-regulation or loss of class I and TAP expression was also observed frequently. However, reductions in class I and TAP expression were reversible upon exposure to interferon-gamma in vitro, suggesting a regulatory rather than structural defect in these genes. De novo class II expression was observed in 26% of tumours and 42% of cell lines and may reflect the differentiation status of the cells. The high rate of class I and TAP loss has implications for immunotherapy strategies for pancreatic cancer, as such changes could facilitate a selective growth advantage for malignant cells. However, the reinduction of expression of these molecules with cytokines such as interferon-gamma may ultimately allow their cytotoxic T cell-mediated destruction.     

Pleomorphic adenoma and carcinoma ex pleomorphic adenoma: immunohistochemical demonstration of the association between tenascin expression and malignancy

AIMS: To investigate tenascin expression in salivary gland tumours. Tenascin is a matricellular protein that has been studied in several tumour types. Its expression has been correlated with tumour morphogenesis as well as with local invasiveness and tumour metastatic behaviour. METHODS AND RESULTS: The distribution pattern of tenascin in a series of 63 pleomorphic adenomas (PA) and 20 carcinomas ex- pleomorphic adenoma (Ca ex PA) was studied immunohistochemically. Ten normal adult salivary glands were used as controls. Tenascin surrounded the excretory ducts of normal adult salivary gland tissue. It was absent in the basement membrane compartment of both benign and malignant mixed tumours. In the interstitial compartment of the extracellular matrix, the fibro-hyaline type expressed tenascin in a statistically significantly (P < 0.001) lower number of PA cases (25%) in comparison with both malignant and benign areas of Ca ex PA (75% and 90%, respectively). In the Ca ex PA group, a statistically significantly difference (P < 0.001) was found in the frequency of tenascin deposits around aggregates of neoplastic cells between metastasizing (73%) and non-metastasizing neoplasms (0%). CONCLUSIONS: These findings strongly support the hypothesis that tenascin deposition is involved in the mechanisms of malignant transformation of pleomorphic adenomas into carcinomas as well as being associated with clinical disease progression.     

Angiogenesis-associated protein annexin II in breast cancer: selective expression in invasive breast cancer and contribution to tumor invasion and progression

Many advanced human tumors including breast cancer overproduce plasmin that is known to promote angiogenesis and metastasis. The mechanism of this effect is poorly understood. Here we report that annexin II, an endothelial co-receptor for tPA (tissue-type plasminogen activator) and plasminogen, was undetectable in normal and hyperplastic ductal epithelial cells and ductal complexes. By contrast, it was consistently expressed in invasive breast cancer and ductal carcinoma in situ (DCIS) indicating its involvement in breast cancer. Using the well established invasive/metastatic MDA-MB231 cell line and the noninvasive/nonmetastatic MCF-7 human breast cancer cell line, we investigated the mechanism by which annexin II regulates breast cancer progression and metastasis. Western and Northern blot analyses demonstrate selective expression of annexin II in MDA-MB231 cells but not in poorly invasive MCF-7 cells suggesting its participation in invasive breast cancer. Since annexin II is a receptor for plasminogen, we tested whether MDA-MB231 cells are capable of producing plasmin in vitro. MDA-MB231 cell membranes induced plasmin generation in a time-dependent manner while those from MCF-7 cells failed to convert plasminogen to plasmin. The generated plasmin is capable of degrading ECM consequently facilitating cell invasion and migration, biological functions required for angiogenesis and metastasis. Plasmin generation and its dependent invasion and migration can be blocked by a monoclonal antibody to annexin II or angiostatin, potent inhibitors of angiogenesis, breast cancer, and metastasis. Our findings indicate that annexin II-dependent localized plasmin generation by human breast cancer cells could contribute to angiogenesis and metastasis. These results suggest that annexin II may be an attractive target for new anti-angiogenic and anti-breast cancer therapies.     

Tenascin labelling in colorectal biopsies: a useful marker in the diagnosis of collagenous colitis

AIMS: This study was undertaken to clarify whether immunohistological detection of tenascin (TN), an extracellular matrix glycoprotein that is expressed during stromal remodelling, may allow a more precise diagnosis of collagenous colitis. METHODS AND RESULTS: We studied multiple colorectal biopsies specimens from 15 patients with clinically suspected collagenous colitis for TN expression by using a monoclonal antibody. Biopsies from further 15 patients without symptoms and signs of collagenous colitis served as controls. In seven of the 15 cases with clinically suspected collagenous colitis a prominent and selective subepithelial tenascin expression was identified. The TN expression pattern closely correlated with the conventional detection of a subepithelial collagen band diagnostic of collagenous colitis. The immunohistological labelling for TN allowed a quicker and more precise measurement of the thickness of the diagnostic collagen deposits than conventional staining. By this approach one further case could be reclassified as collagenous colitis. CONCLUSIONS: Our data show that immunohistological detection of TN allows a more correct and easy diagnosis of collagenous colitis.     

Modulation of c-kit expression in pancreatic adenocarcinoma: A novel stem cell marker responsible for the progression of the disease

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers because of late symptoms and resistance to chemotherapy and radiation therapy. We have investigated the appearance of c-kit, a stem cell marker, in both normal adult pancreatic tissue and in cancerous tissue. Apart from some very pale staining of islets of Langerhans, normal pancreas was devoid of staining with antibodies to c-kit. In contrast, in cancerous tissue that still preserves the overall integrity of the pancreatic tissue, there was a clear labeling in islets of Langerhans, which seemed to be co-localized with insulin containing β cells. In other cases, where the pancreatic tissue was completely deteriorated, intensive labeling was clearly evident in remnants of both the exocrine and the endocrine tissues. The duct cells of the adenocarcinoma were moderately but clearly labeled with antibodies to c-kit. In contrast, in metastasis of PDAC, very intensive labeling of c-kit was evident. The location of KRAS, which is strongly associated with PDAC, was also analyzed at the initial stages of the disease, when islets of Langerhans still preserve their integrity to a large extent. KRAS was found exclusively in islets of Langerhans and overlapped in its location with insulin and c-kit expressing cells. It is suggested that the modulation of the expression of c-kit, visualized by antibodies to the oncogene molecule, may play an important role in the formation and progression of PDAC. The absence of c-kit in normal pancreas and its appearance in PDAC is probably due to a mutational event, which probably allows conversion of the β cells into cancer stem cells (CSC). Co-expression of both c-kit and KRAS, typical markers for CSC with overlapping with insulin in islets of Langerhans, strongly support the notion that β-cells play a central role in the development of PDAC. The use of specific drugs that can attenuate the kinase activity of c-kit or target KRAS expressing cancer cells should be tested in order to attenuate the progression of this lethal disease.     

施万细胞的层黏连蛋白表达变化及两者关系的初步研究

目的:观察层黏连蛋白(laminin,LN)在坐骨神经发育过程中的表达情况,以及LN和施万细胞(Schwann cells,SCs)的相互关系。方法:取E14、E17、P1和成年SD大鼠坐骨神经,免疫荧光组织化学染色检测LN表达情况;体外培养大鼠来源的SCs,经过外源性LN处理后,免疫荧光细胞化学染色检测LN、nidogen、type IVcollagen等细胞外基质成分的表达,酸性磷酸酶法检测SCs的黏附能力。结果:E17大鼠坐骨神经SCs有LN阳性免疫反应;经过外源性LN处理后,SCs有LN、nidogen、type IV collagen阳性免疫反应,且黏附能力增加。结论:在E17大鼠中,SCs开始分泌LN;LN具有促进SCs合成细胞外基质成分,并在其黏附过程中发挥作用。     

Very-low-density lipoprotein receptor-enhanced lipid metabolism in pancreatic stellate cells promotes pancreatic fibrosis

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