涎腺腺样囊性癌高、低转移细胞系基因表达谱及基质金属蛋白酶表达差异

目的筛选与涎腺腺样囊性癌转移相关的候选基因，并对其中的候选基因进行初步的验证。方法用限制片段差异显示PCR技术（restriction fragments differential display PCR, RFDD-PCR）建立涎腺腺样囊性癌高、低转移细胞株（ACC-M、ACC-2）的表达谱。对两个表达谱的片段进行比较，通过生物信息学的分析，初步筛选出候选基因。用半定量逆转录PCR技术对筛选出的基因进行初步验证。结果RFDD-PCR方法共获得5420个基因片段，其中包含12个基质金属蛋白酶（matrix metalloproteinase，MMP）基因。半定量逆转录PCR方法发现MMP2、MMP7、MMP9、MMP14、MMP15、MMP24在ACC-M和ACC-2中的表达存在明显差异。结论构建了ACC-M和ACC-2细胞株的表达谱，为寻找目的基因奠定了基础。发现MMP2、MMP7、MMP9和MMP15与涎腺腺样囊性癌的发生、发展、转移有关，不同肿瘤细胞的转移能力可能与不同的MMPs家族基因相关。     

Cholecalciferol (Vitamin D<Subscript>3</Subscript>) Inhibits Growth and Invasion by Up-regulating Nuclear Receptors and 25-Hydroxylase (CYP27A1) in Human Prostate Cancer Cells

Epidemiological evidence suggests an inverse relationship between prostate cancer and serum vitamin D levels. We examined the ability of cholecalciferol (vitamin D₃), a calcitriol precursor, to inhibit or reverse cellular changes associated with malignant transformation and invasion and explored its mechanisms of action. The RWPE2-W99 human prostate epithelial cell line, which forms slow-growing tumors in nude mice, was used because it mimics the behavior of the majority of primary human prostate cancers. Cholecalciferol, at physiological levels: (i) inhibited anchorage-dependent and -independent growth; (ii) induced differentiation by decreasing vimentin expression with a concomitant decrease in motility/chemotaxis; (iii) decreased MMP-9 and MMP-2 activity with concomitant decrease in invasion; and (iv) exerted its effects by up-regulating vitamin D receptor (VDR), retinoid-X receptor-α (RXR-α), and androgen receptor (AR) in a dose-dependent manner. Furthermore, we found that RWPE2-W99 prostate cancer cells, similar to RWPE-1 cells (Tokar and Webber. Clin Exp Metast 2005; 22: 265–73), constitutively express the enzyme 25-hydroxylase CYP27A1 which is markedly up-regulated by cholecalciferol. Cholecalciferol has effects similar to those of calcitriol on growth, MMP activity, and VDR. The ability of CYP27A1 to catalyze the conversion of cholecalciferol to 25(OH)D₃ and of 25(OH)D₃ to calcitriol has been reported. RWPE2-W99 cells, similar to RWPE-1 cells, appear to have the rare ability to locally convert cholecalciferol to the active hormone calcitriol. Because it can inhibit cellular changes associated with malignant transformation and invasion, we propose that cholecalciferol may be an effective agent for the treatment of prostate cancer.     

Endocrinology and paracrinology: Roles of growth factors during peri-implantation development

Several growth factor ligand and receptor gene products havebeen shown to play roles during preimplantation mammalian development.Genes for insulin-like growth factors (IGFs), transforming growthfactors (TGFs), fibroblast growth factor (FGF), platelet-derivedgrowth factor (PDGF) and receptors for insulin, IGF, PDGF, TGFand epidermal growth factor (EGF) are expressed by early embryosof several species including mouse, rat, cow and sheep. Rolesof growth factors during early development have been demonstratedby addition of purified growth factors to culture medium orby molecular genetic techniques that interfere with gene expression.In this way, it has been shown that successful development ofthe blastocyst is dependent on the action of epidermal growthfactor (EGF) and leukaemia inhibitory factor (LIF). Recent experimentsshow that both LIF and EGF stimulate secretion of urokinase-typeplasminogen activator (uPA) and gelatinase B/ matrix metalloproteinase-9(MMP-9) in day 7 mouse blastocyst outgrowths. At the same time,tissue inhibitors of MMPs (TIMPs) are also expressed by embryonic,decidual and uterine tissues during the implantation process.It appears that LIF may act directly or indirectly, by inducingthe expression of other cytokines, to regulate the temporaland spatial production and activity of proteases and proteaseinhibitors to create a favourable environment for implantation.     

凝血酶对大鼠脑微血管内皮细胞的影响

目的 :探讨凝血酶 (Thrombin ,TM )对脑微血管内皮的影响。方法 :将大鼠脑微血管内皮细胞进行培养 ,培养液中加入 10U的TM或10U的TM + 0 .4mU的组织蛋白酶G(CaspethsinG ,CATG ) ,相差显微镜动态观察内皮细胞形态的变化 ,免疫组织化学技术检测基质金属蛋白酶 2 (MatrixMetalloproteinase 2 ,MMP 2 )表达的改变。 结果 :TM使内皮细胞发生收缩 ,细胞收缩程度具有时间依赖性 ,使内皮细胞MMP 2表达水平明显增加。TM +CATG加入培养液后 ,细胞形态、MMP 2表达与对照组比较均无明显统计学差异 (P >0 .0 5 )。结论 :TM通过激活蛋白酶激活受体 1(proteaseactivatedreceptor 1,PAR 1) ,使内皮细胞发生收缩 ,促进MMP 2表达 ,是TM增加血脑屏障 (BloodBrainBarrier,BBB)通透性的可能机制。     

Sensitivity of prostate tumors to wild type and M protein mutant vesicular stomatitis viruses

Because of its potent ability to induce apoptosis, vesicular stomatitis virus (VSV) is an attractive candidate as an oncolytic virus for tumor therapy. Previous studies have suggested that VSV selectively infects tumor cells due to defects in their antiviral responses making them more susceptible to VSV infection than normal cells. We tested this hypothesis in the prostate tumor system by comparing LNCaP and PC-3 prostate tumor cells to benign human prostatic epithelial cells from patient prostatectomy specimens. We compared the cell killing ability of a recombinant virus containing a wild-type (wt) M protein (rwt) and an isogenic M protein mutant virus (rM51R-M) that induces interferon (IFN) in infected cells and should display a greater selectivity for tumor cells. Our results showed that in single-cycle infection experiments, LNCaP cells were sensitive to killing by both wt and mutant viruses, while PC-3 cells were highly resistant to VSV-induced cell killing. LNCaP and benign prostate cells were similarly susceptible to both viruses, indicating that normal prostate cells are not inherently resistant to killing by VSV. In each of the cell lines, the rM51R-M virus induced similar levels of apoptosis to rwt virus, showing that the M protein does not play a significant role in apoptosis induction by VSV in these cells. In multiple-cycle infection experiments, LNCaP cells were more sensitive than benign prostatic epithelial cells to virus-induced cell killing by rM51R-M virus, but not rwt virus. Both viruses were equally effective at reducing LNCaP tumor volume in vivo following intratumoral and intravenous inoculation in nude mice, while PC-3 tumors were resistant to VSV treatment. None of the mice treated with rM51R-M virus died as a result of virus infection, while 50-71% of mice treated with rwt virus succumbed to virus infection. Similarly, when inoculated by the more sensitive intranasal route, the rM51R-M virus was less pathogenic than the rwt virus from which it was derived. These results indicate that M protein mutant viruses are superior candidates as oncolytic viruses for therapies of prostate tumors, but future strategies for use of VSV will require testing individual tumors for their susceptibility to virus infection.     

Suppressive effect of leflunomide metabolite (A77 1726) on metalloproteinase production in IL-1beta stimulated rheumatoid synovial fibroblasts

Leflunomide, an isoxazol derivative structurally unrelated to other immunomodulatory drugs, has proven to be efficacious in the treatment of rheumatoid arthritis (RA). This study was conducted to elucidate the mechanism by which leflunomide mediated antirheumatic effects. We investigated the effects of A77 1726, leflunomide's active metabolite, on mitogen-activated protein kinase (MAPK) activation in IL-1beta-stimulated rheumatoid synovial fibroblasts. The effects of A77 1726 on the secretion of matrix metalloproteinases (MMPs) from rheumatoid synovial fibroblasts were also examined. A77 1726 partially suppressed IL-1beta-induced ERK1/2 and p38 kinase activation. In contrast, A77 1726 efficiently suppressed IL-1beta-stimulated JNK1/2 kinase activation. Although no suppressive effect was demonstrated on MMP-2, A77 1726 markedly inhibited MMP-1, 3, and 13 secretions from IL-1beta-stimulated rheumatoid synovial fibroblasts. Tissue inhibitor of metalloproteinases-1 (TIMP-1) was constitutively produced from rheumatoid synovial fibroblasts and the suppressive effects of A77 1726 on TIMP-1 production were minimal. Our results suggest that the suppression of the MAPK signalling pathway and MMP synthesis in rheumatoid synovial fibroblasts is a possible mechanism for the inhibitory activity of leflunomide against rheumatoid arthritis.     

Plasminogen modulation of IL-1-stimulated degradation in bovine and human articular cartilage explants. The role of the endogenous inhibitors: PAI-1,α 2-antiplasmin,α 1-PI,α 2-macroglobulin and TIMP

The studies described here examine the involvement of the fibrinolytic cascade and its endogenous inhibitors in the regulation of activity of matrix metalloproteinases and cartilage degradation related to non-inflammatory joint disease, like osteoarthritis. An interleukin-1-induced model of degradation using [³⁵S]-labeled bovine and human articular cartilage explants was utilized. One goal of these studies was to compare the responses of bovine and human articular cartilage. Degradation was not inhibited by ₁-PI, PAI-1, ₂-macroglobulin, ₂-antiplasmin or TIMP-2, when IL-1 alone was added. Addition of human plasminogen to bovine explants, at concentrations found in human synovial fluid, increased degradation by three to fourfold. Under these conditions, the degradation was inhibited effectively by all of the endogenous inhibitors tested, indicating the presence of a cascade where activated chondrocytes are a source of u-PA. Plasminogen activated by u-PA gives plasmin, which is known to further activate pro-stromelysin. Stromelysin is essential for activation of collegenase. Not only TIMP, but also inhibitors at earlier steps of activation like PAI-1, ₂-antiplasmin, ₁-PI and ₂-macroglobulin inhibited degradation, and could provide cartilage protection in vivo. An experiment with human articular cartilage explants showed that very little or no degradation occurred when human articular cartilage explants were stimulated with interleukin-1 alone. Addition of human plasminogen (at physiologically relevant concentrations) resulted in significant degradation, which was inhibited in the same manner as in bovine explants, by inhibitors of the fibrinolytic cascade and TIMP. TIMP is much more efficient in human explants, indicating the limited participation of human plasmin in the degradation of human cartilage. Although speculative, it is possible that in vivo, cartilage degradation could be promoted not only by TIMP/MMP imbalance, but also accelerated by decreased levels of certain serpins in synovial fluid (e.g. PAIs, ₂-antiplasmin and ₁-PI).accepted by W. B. van den BergWork supported by OsteoArthritis Science Inc., One Kendall Square, Bldg. 200, Cambridge, MA 01139, USA.     

Matrix protein mediated shutdown of host cell metabolism limits vesicular stomatitis virus-induced interferon-alpha responses to plasmacytoid dendritic cells

Upon infection with many different viruses, plasmacytoid dendritic cells (pDC) produce large amounts of type I interferon (IFN-/β). To address why upon vesicular stomatitis virus (VSV) infection pDC, but not conventional myeloid DC (mDC), are induced to produce IFN-, pDC and mDC were differentiated from bone marrow cells (BM-DC). Upon VSV infection BM-pDC produced IFN-, whereas BM-mDC did not. Notably, upon infection with VSV-M2, a VSV variant expressing a M51R mutant matrix (M) protein that showed a reduced sequestration of host cell metabolism, BM-pDC and BM-mDC mounted massive IFN- responses. Both DC subsets showed comparable RNA levels of retinoic acid inducible gene-I (RIG-I) and Toll-like receptor (TLR) 7 and were able to respond upon triggering with double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) analogs. Moreover, upon VSV-M2 infection IFN- production by both DC subsets was largely dependent on viral replication. Interestingly, upon virus infection BM-pDC, but not BM-mDC, up-regulated mRNA levels of nuclear export factors Nup96/98, probably reflecting cellular mechanisms to circumvent viral escape strategies. Collectively, these results indicated that cell types induced to produce IFN- upon viral infection are not primarily defined by cellular receptor configurations but rather by complex virus/host cell interactions.     

慢性丙型肝炎患者血清leptin含量与HA、PⅢP水平的相关分析

目的：探讨了慢性丙型肝炎患者血清leptin含量与HA、PⅢP水平的关系。方法：应用放射免疫分析对32例慢性丙型肝炎患者进行了血清leptin和HA、PⅢP水平检测，并与35名正常健康人作比较。结果：慢性丙型肝炎患者血清leptin和HA、PⅢP水平均非常显著地高于正常人组（P〈0．01），相关分析显示，血清leptin与HA、PⅢP水平呈明显的正相关（r=0．6178．0．5706，P〈0．01）。结论：慢性丙型肝炎患者血清leptin水平的升高与肝脏炎症病变严重程度有关，leptin测定可作为—个判断肝脏炎症严重程度的指标，具有重要的临床价值。     

Eutopic endometrium and peritoneal,ovarian and bowel endometriotic tissues express a different profile of matrix metalloproteinases-2, -3 and -11, and of tissue inhibitor metalloproteinases-1 and -2

Endometriosis is subsequent to the ability of endometrial glands to invade normal tissues. Matrix metalloproteinases (MMPs)—enzymes that mediate normal tissue turnover, including endometrial breakdown during menstruation—appear to be involved in this invasive process. Here, we examined the immunohistochemical expression of MMP-2, MMP-3, MMP-11, tissue inhibitor metalloproteinase (TIMP)-1 and TIMP-2 in endometrium from women with (n=9) or without endometriosis (n=18) in comparison with peritoneal (n=20), ovarian (n=20) and colorectal endometriosis (n=20). Women with endometriosis showed decreased endometrial MMP-2 expression compared with women without endometriosis (mean±SD positive cells: 24.3±28.3% and 69.3±12.1%), together with loss of MMP-3 expression (0 versus 17.5%±20.2). MMP-11, TIMP-1 and TIMP-2 expression was similar in the two groups. Endometrial MMP-2, -3 and -11 expression and TIMP-1 and -2 expression were similar in women with endometriosis and in those with peritoneal endometriosis. MMP-2, -3 and -11 expression was higher in colorectal endometriosis than in ovarian and peritoneal endometriosis. TIMP-2 expression was lower in colorectal endometriosis (P=0.0002) and ovarian endometriotic cysts (P=0.003) than in peritoneal endometriosis. TIMP-1 expression did not vary according to the location of endometriotic lesions. These results suggest that MMP-2 and -3 and TIMP-2 may be involved in the pathogenesis of endometriosis. Interestingly, MMP-2 and -3 overexpression was related to the infiltrative nature of endometriotic lesions, with possible sequential expression from peritoneal to colorectal endometriosis.