核心蛋白聚糖mRNA及其蛋白在结直肠癌中的表达

目的：研究核心蛋白聚糖（DCN）mRNA及其蛋白在结直肠癌中的表达。方法：采用原位杂交及免疫组织化学方法检测DCN mRNA及其蛋白在30例结直肠癌及20例相对正常大肠组织中的表达。结果：30例结直肠癌患者,DCN mRNA表达阳性率为66.67％（20／30）; 20例相对正常大肠腺细胞中,DCN mRNA表达阳性率为85.00％（17／20）,两者比较差异无显著性（P>0.05）。在30例结直肠癌癌细胞及20例相对正常大肠腺细胞内未发现DCN蛋白表达。结论：DCN mRNA在结直肠癌癌细胞及相对正常大肠腺细胞中均有表达。     

XXIX Italian Society for the Study of Connective Tissues (SISC) Meeting,October 29–30, 2009, Alghero,Italy

Soluble substances in the bovine nuchal ligament were removed by preliminary extractions with a buffer and dilute alkali solution. The insoluble residue was then extracted with 6 M guanidine HCl and with 6 M guanidine HCl containing a reducing agent (20 mM dithiothreitol) successively. Each of the two extracts contained a glycoprotein; that in the first extract was designated Glycoprotein A and that in the second Glycoprotein B. They were purified by ion-exchange chromatography and gel filtration till essentially homogeneous. Both proteins had similar molecular weights of 35,000 in SDS-polyacrylamide gel electrophoresis and by gel filtration, and their chemical compositions resembled each other closely. It is suggested that Glycoprotein B was present in the native state as a disulfide-bonded, aggregated form of Glycoprotein A. The compounds also showed similarity with the microfibrillar glycoprotein(s) previously reported in bovine nuchal ligament extracts.     

Decorin‐expressing adenovirus decreases collagen synthesis and upregulates MMP expression in keloid fibroblasts and keloid spheroids

Decorin is a natural transforming growth factor‐β1 (TGF‐β1) antagonist. Reduced decorin synthesis is associated with dermal scarring, and increased decorin expression appears to reduce scar tissue formation. To investigate the therapeutic potential of decorin for keloids, human dermal fibroblasts (HDFs) and keloid‐derived fibroblasts (KFs) were transduced with decorin‐expressing adenovirus (dE1‐RGD/GFP/DCN), and we examined the therapeutic potential of decorin‐expressing Ad for treating pathologic skin fibrosis. Decorin expression was examined by immunofluorescence assay on keloid tissues. HDFs and KFs were transduced with dE1‐RGD/GFP/DCN or control virus, and protein levels of decorin, epidermal growth factor receptor (EGFR) and secreted TGF‐β1 were assessed by Western blotting and ELISA. And type I and III collagen, and matrix metalloproteinase‐1 (MMP‐1) and matrix metalloproteinase‐3 (MMP‐3) mRNA levels were measured by real‐time RT‐PCR. Additionally, we immunohistochemically investigated the expression levels of the major extracellular matrix (ECM) proteins in keloid spheroids transduced with dE1‐RGD/GFP/DCN. Lower decorin expression was observed in the keloid region compared to adjacent normal tissues. After treatment with dE1‐RGD/GFP/DCN, secreted TGF‐β1 and EGFR protein expressions were decreased in TGF‐β1‐treated HDFs and KFs. Also, type I and III collagen mRNA levels were decreased, and the expression of MMP‐1 and MMP‐3 mRNA was strongly upregulated. In addition, the expression of type I and III collagen, fibronectin and elastin was significantly reduced in dE1‐RGD/GFP/DCN‐transduced keloid spheroids. These results support the utility of decorin‐expressing adenovirus to reduce collagen synthesis in KFs and keloid spheroid, which may be highly beneficial in treating keloids.     

蛋白聚糖与糖尿病微血管病变

蛋白聚糖在糖尿病微血管病变的发生发展过程中起重要的作用 ,大量研究表明其在糖尿病微血管病变时出现质和量的变化。糖尿病肾病和糖尿病视网膜病变时硫酸软骨素、透明质酸、硫酸皮肤素等多种蛋白聚糖在纤维化细胞外基质组织局部表达明显升高 ,而硫酸类肝素蛋白聚糖无明显变化或表达下降 ,在糖尿病视网膜病变时表达明显下降。此外 ,由于硫酸软骨素蛋白聚糖中小分子核心蛋白聚糖decorin能结合并中和转化生长因子 β1的活性 ,为糖尿病肾病的治疗提供一种可能的方法     

核心蛋白聚糖在Ishikawa细胞中的表达及意义

目的观察表皮生长因子受体（EGFR）信号通路抑制剂RG-14260（RG）单用及RG联合mTOR信号通路抑制剂亚罗奠司（RA）体外对子宫内膜癌Ishikawa细胞增殖及核心蛋白聚糖（DCN）表达的影响。方法子宫内膜癌Ishikawa细胞经RG及RG联合RA作用后,采用免疫组织化学法检测细胞增殖细胞核抗原（PCNA）的表达,反转录酶-聚合酶链式反应（RT-PCR）及免疫印迹法分别检测细胞内DCN基因和蛋白表达的变化。结果RG单用及联合RA均可降低Ishikawa细胞PCNA阳性细胞表达率,促进细胞DCN基因和蛋白的表达,但二者合用效果更为显著。结论单独和联合EGFR及mTOR信号通路抑制剂均可在基因和蛋白水平上上调Ishikawa细胞内DCN的表达,而升高的DCN反过来又可增强信号通路抑制剂对细胞增殖的抑制作用。     

探讨核心蛋白多糖体外抑制兔晶状体上皮细胞的转分化

目的:探讨核心蛋白多糖(decorin)对体外培养的兔晶状体上皮细胞(lens epithelial cells,LEC)分泌转化生长因子β1(transforming growth factorβ1,TGF-β1)和表达α-平滑肌肌动蛋白(α-SMA)的影响。方法:体外原代培养兔LEC,当细胞接近融合时转板培养,分别设置对照组和实验组,对照组为不用药培养12d;实验组为培养6d后加入不同浓度的decorin(10,1.0,0.1mg/L3个浓度组)继续培养6d,用ELISA法检测各组培养液上清中TGF-β1的水平,免疫细胞化学和计算机图像分析统计各组细胞中α-SMA的表达水平。结果:实验组TGF-β1水平和α-SMA水平均明显低于对照组的水平(117.9±18.1→427.2±41.3,111.7±26.7→427.2±41,236.0±28.6→427.2±41.3;15.7±8.7→97.2±26.9,16.2±9.5→97.2±26.9,54.9±29.6→97.2±26.9,P<0.01),decorin表现出明显的抑制作用;α-SMA的表达量随着TGF-β1水平的下降而下降,两者高度正相关(r=0.995,P<0.01)。结论:Decorin抑制活性TGF-β1的水平,下调α-SMA的表达。     

Degradation of cartilage by macrophages in culture: Evidence for the involvement of an enzyme which is associated with the cell surface

A cell culture system is described in which purified mononuclear phagocytes may be cultured with a cartilage substrate which is radiolabelled in its proteoglycan. Resident mouse peritoneal macrophages degraded this substrate, and did so more avidly if cultured in direct contact with it. There was no evidence for complete intralysosomal degradation of the proteoglycan of the cartilage. Lysates were found to contain considerable activity at pH 7, which was inhibited by the presence of 10% serum, or by boiling the lysate.

Proximity of macrophages to the substrate did not induce selective release of the lysosomal marker enzyme hexosaminidase, and concentrated enzymes secreted from the macrophages after treatment with the lysosomotropic agent ammonium chloride were ineffective in degrading cartilage at neutral pH.

The active enzyme in macrophage lysates at neutral pH was found to be sedimentable by 100,000 × g centrifugation for 1 hour, in absence of lysosomal protective agents. There is evidence for a cell membrane-associated process in the degradation of cartilage by these cells, which may be a proteolytic, endoglycosidic or free radical-mediated event.     

Human decorin regulates proliferation and migration of human lung cancer A549 cells

Background Decorin is a small leucine-rich proteoglycan and it plays an important role in regulation of cell growth and migration in various tumor cell lines. Decorin was found down-regulated in non-small cell lung cancer tissue and may be involved in regulation of lung cancer development. Methods In this study, lentivirus-mediated RNA interference and over expression were employed to change the expression levels of decorJn Jn lung cancer A549 cells. We tested the cell cycle of A549 cells and the expression of transforming growth factor （TGF）-[31, cyclin D1, epidermal growth factor receptor （EGFR）, P53, and P21. Results We found that up-regulation of decorin could inhibit proliferation, block cell cycle at G1 and decrease invasive activity of A549 cells. Moreover, we also show that up-regulation of decorin induced significant decreases of TGF-131, cyclin D1 expression, phosphorylation of EGFR, and increases of P53 and P21 expression. Opposite results were observed in A549 cells with down-regulation of decorin. Conclusion Our results suggest that decorin is a key regulator involved in proliferation and migration ofA549 cells.