ADAM17-shRNA转染的骨髓间充质干细胞对裸鼠乳腺癌移植瘤生长的影响

目的:探讨转染解聚素-金属蛋白酶17-shRNA(a disintegrin and metalloprotease 17-shRNA,ADAM 17-shRNA)的骨髓间充质干细胞(bone marrow mesenehymal stem cells,BMMSC)对乳腺癌MCF-7细胞裸鼠移植瘤的抑制效果.方法:全骨髓贴壁法分离并培养3周雄性SD大鼠的BMMSC,利用慢病毒介导的ADAM17-shRNA转染BMMSC.30只裸鼠建立MCF-7乳腺癌移植瘤模型,种植肿瘤细胞14d后建模成功.按照数字表法随机分成对照组(注射等量PBS)、BMMSC组(注射l×106/mlBMMSC)和转染组(注射1×106/ml转染ADAM 17-shRNA的BMMSC),每组10只.在种植细胞第15天开始经尾静脉注射BMMSC进行抑瘤实验(0.1 ml/只,每3d给药1次,共计5次),观察裸小鼠移植瘤的生长情况;抑瘤实验16d后处死裸鼠.利用Real-time PCR法检测移植瘤组织ADAM17 mRNA表达,Westem blotting法检测移植瘤组织ADAM 17蛋白表达.结果:抑瘤实验16d时,对照组、BMMSC组移植瘤体积明显高于转染组[(787.15±25.95)、(767.02±28.98) vs (361.89±19.75)mm3,均P＜0.01];BMMSC组、转染组抑瘤率明显高于对照组(2.57％、53.89％ vs 0.00％,均P＜0.05).对照组、BMMSC组ADAM17 mRNA的表达水平明显高于转染组(1.00±0.01、0.97±0.08 vs 0.30±0.09,均P＜0.05);对照组、BMMSC组ADAM 17蛋白表达水平明显高于转染组(0.70±0.09、0.68±0.02 vs 0.45±0.05,均P＜0.05).结论:ADAM 17-shRNA通过BMMSC介导可将ADAM17靶向归巢至裸鼠乳腺癌移植瘤并发挥抑瘤作用.     

PROTEOLYTIC ENZYMES IN WOUND HEALING: THE ROLE OF ENZYMATIC DEBRIDEMENT

Proteolytic enzymes are a family of proteins that serve to degrade necrotic debris derived from cell breakdown. They are produced endogenously often as precursor proteins whose activation is precisely regulated. These activated enzymes serve many functions in normal as well as pathological situations. In particular they are involved in the regulation of cell maturation and multiplication; collagen synthesis and turnover; the development and removal of the perivascular fibrin cuffs found in venous insufficiency and leg ulceration as well as the removal of dead tissues following inflammation. As a limited number of enzymes perform all these functions, it is difficult to predict the effects of applying synthetic proteolytic enzymes to a wound. Many such enzymes are currently commercially available and being promoted as alternatives to surgical wound debridement. It is important for their use to be considered in the context of their interaction with endogenous proteases, their physiological role in tissue, their ability to reach a desired target and the stage of wound healing at the time they are applied.     

Effect of Zinc Binding on the Structure and Stability of Fibrolase,a Fibrinolytic Protein from Snake Venom

Fibrolase is a metalloprotease with potential use as a fibrinolytic agent. Loss of the intrinsic zinc atom leads to a rapid decrease in enzymatic activity. Circular dichroism measurements indicate that there is a partial unfolding of an -helical section of the protein concomitant with the loss of zinc. Removal of zinc can be affected by elevated temperatures, acidic pH values, and addition of chelating agents. At low molar concentrations, both ethylenediaminetet-raacetic acid (EDTA) and dithiothreitol (DTT) were found to remove zinc efficiently. Analysis of the sequence of fibrolase identified a segment which possessed a high degree of homology with the metal binding site of other zinc proteases, such as thermolysin and the collagenases. However, the putative zinc binding site in fibrolase lacks the additional glutamate ligand found in thermolysin and subtilisin. This sequence is also predicted to adopt an -helical conformation. Together, these data indicate that there is a well-defined metal binding site in fibrolase and that metal binding is the most important factor governing the stability of this protein.     

UV‐induced EGFR signal transactivation is dependent on proligand shedding by activated metalloproteases in skin cancer cell lines

Exposure to extensive ultraviolet (UV) rays is a major cause of skin cancer, which is thought to be initiated by DNA mutations. Members of the epidermal growth factor receptor (EGFR) family are important in various pathophysiologic processes like cancer and are shown to be phosphorylated upon UV exposure. Here we show that EGFR phosphorylation by modest UV doses is dependent on metalloprotease activity and resultant epidermal growth factor (EGF) family proligand shedding. This proligand cleavage releases the mature ligand, which then binds to and activates EGFR. We show that UV induced EGFR phosphorylation in transformed cell lines of melanocyte and keratinocyte origin, which was reduced upon preincubation with a broad‐spectrum metalloprotease inhibitor, BB94. UV also activated EGFR downstream signaling via Erk and Akt pathways in a BB94‐sensitive manner. Furthermore, using neutralizing antibodies we found that proligand amphiregulin was required for UV‐induced EGFR activation in SCC‐9 cells. Using RNAi this EGFR activation was further shown to depend on the metalloproteases ADAM9 and ADAM17 in SCC‐9 cells. cDNA array hybridization and RT‐PCR analysis showed overexpression of a Disintegrin and a Metalloproteases (ADAMs) and EGF family proligands in melanoma cell lines. Additionally, blocking EGFR signal transactivation by BB94 led to increased apoptosis in UV‐irradiated cells. EGFR signal transactivation also led to increased stability of the DNA repair protein, PARP, under UV stress. Thus, both antiapoptotic and DNA repair pathways are activated simultaneously by EGFR signal transactivation. Together, our data provide novel insights into the mechanism of UV‐induced EGFR activation, suggesting broad relevance of the UV‐ADAM‐proligand‐EGFR‐Erk/Akt pathway and its significance in skin cancer. © 2008 Wiley‐Liss, Inc.     

Distinct progression‐associated expression of tumor and stromal MMPs in HaCaT skin SCCs correlates with onset of invasion

Matrix metalloproteinases (MMPs) are critically involved in tumor invasion and metastasis. However, failure of broad spectrum MMP inhibitors in clinical trials emphasizes the need for detailed analyses of the specific role of different MMPs in tumor malignancy. Using HaCaT‐keratinocyte clones representing distinct stages in skin squamous cell carcinoma (SCC) progression, we demonstrate the expression of specific tumor and stroma‐derived MMPs with the onset and maintenance of tumor invasion. Although MMP‐9‐positive leukocytes are present in benign and malignant tumor transplants at the onset of stromal activation and angiogenesis, mRNA expression of stroma‐derived MMP‐9 as well as MMP‐2, −13 and −14 is exclusively found in enhanced malignant tumor transplants. Their expression initiates with the onset of invasion, whereas being absent in early noninvasive stages of malignant transplants. In addition, a high expression of tumor‐derived MMP‐1, −2 and −14 contributes to malignant and invasive tumor growth. However, stroma‐derived MMP‐3 is exclusively restricted to very late‐stage invasive and malignant transplants. The functional contribution of these proteases to invasive growth is supported by the gelatinolytic activity in the tumor transplants that again initiates with the onset of invasive growth suggesting a crucial role of MMP‐2, −9, −13 and −14 for the establishment of a reactive stroma that promotes tumor invasion. These data demonstrate a complex cooperation of distinct tumor and stroma‐derived MMPs in the establishment of malignant tumors and provide the basis for a more specific use of highly selective MMP inhibitors during distinct stages of tumor progression. © 2009 UICC     

Involvement of caspases and transmembrane metalloproteases in sulphur mustard-induced microvesication in adult human skin in organ culture: Directions for therapy

While skin is a major target for sulphur mustard (HD), a therapy to limit HD-induced vesication is currently not available. Since it is supposed that apoptotic cell death and proteolytic digestion of extracellular matrix proteins by metalloproteases are initiating factors for blister formation, we have explored whether inhibition of these processes could prevent HD-induced epidermal–dermal separation using adult human skin in organ culture. Involvement of the caspase and the metalloprotease families was confirmed by the observation that their respective broad spectrum inhibitors, Z-VAD-fmk and GM6001, each suppressed HD-induced microvesication. The lowest effective concentrations were 10 and 100 μM, respectively. Using specific inhibitors for caspase-8 (≥10 μM) and caspase-9 (≥10 μM) we learned that HD-induced apoptosis is initiated by the death receptor pathway as well as by the mitochondrial pathway. Remarkably, blocking caspase-8 activity resulted in morphologically better conserved cells than blocking caspase-9 activity.     

培哚普利对急性冠脉综合征患者血基质金属蛋白酶2和9含量的影响

目的：探讨急性冠脉综合征（ACS）患者血基质金属蛋白酶（MMP）2和9含量的变化及其培哚普利的的干预影响。方法：选择60例健康人做对照，60例ACS患者随机被分为常规治疗组（n=30例）和培哚普利治疗组（n=30例），治疗3周，治疗前、后测量血清MMP-2和MMP-9含量的变化。结果：ACS患者血清MMP-2，9含量显著高于健康对照组（P〈0．01）；培哚普利组血清MMP-9含量显著低于常规治疗组（P〈0.05）。结论：培哚普利能够抑制基质金属蛋白酶9的分泌。     

CTGF和TIMP1双基因体外联合转染恒河猴腰椎间盘细胞对II型胶原和蛋白多糖的影响

目的 建立恒河猴腰椎间盘细胞体外培养模型,研究腺相关病毒（adeno-associated virus,AAV）介导的结缔组织生长因子（connective tissue growth factor,CTGF）和基质金属蛋白酶组织抑制因子1(tissue inhibitor of metalloproteinases 1,TIMP1) 双基因体外联合转染恒河猴椎间盘细胞较单基因对II型胶原和蛋白多糖合成的影响的变化,以探讨体外延缓椎间盘细胞退变的方法。方法 应用酶消化法培养恒河猴腰椎间盘髓核细胞,以感染复数（MOI） 为106的rAAV2-CTGF-IRES-TIMP1及rAAV2-CTGF、rAAV2-TIMP1分别感染髓核细胞,应用Western blot鉴定和RT-PCR检测II型胶原及蛋白多糖mRNA的表达,35S整合法性行蛋白多糖合成率的测定,SP-ABC免疫组化法检测Ⅱ型胶原含量。结果 rAAV2-CTGF-IRES-TIMP1双基因及rAAV-CTGF、rAAV-TIMP1单基因能够转染恒河猴椎间盘髓核细胞并在其内表达细胞因子。与对照组相比,CTGF可以促进II型胶原及蛋白多糖的合成,TIMP1可以促进蛋白多糖的合成,对II型胶原的合成未见到明显作用;双基因联合感染可以显著促进髓核细胞蛋白多糖和II型胶原的合成。结论 CTGF和TIMP1 单基因转染均可以促进蛋白多糖的合成,CTGF 对 II型胶原的合成亦有促进作用;双基因联合感染可以明显促进蛋白多糖和II型胶原的合成,其效果优于单基因,为多基因治疗椎间盘退变奠定了良好的基础。