纤黏连蛋白重组多肽CH50抑制黑色素瘤MMP-2和MMP-9表达、激活的研究

目的研究纤黏连蛋白重组多肽CH50对黑色素瘤B16细胞侵袭能力的影响,探讨CH50多肽抑制肿瘤生长、侵袭的机制。方法体外培养小鼠黑色素瘤B16细胞、小鼠腿部皮下注射B16细胞建立肿瘤动物模型。采用明胶电泳法检测B16细胞和黑色素瘤组织中基质金属蛋白酶MMP-2和MMP-9的表达和激活;以CH50多肽体外处理B16细胞或体内表达CH50,观察CH50下调MMPs表达以及对肿瘤细胞侵袭能力的抑制作用。结果B16细胞在体外培养条件下主要表达MMP-2,而在肿瘤微环境中则同时表达MMP-2和MMP-9。肿瘤组织中MMPs的表达明显高于体外培养B16细胞。CH50多肽对体外培养B16细胞的MMPs表达和激活无明显抑制作用,但处理后的B16细胞进入体内后表达MMPs的能力受到明显抑制。体内转染表达的CH50多肽亦可明显抑制肿瘤表达MMPs、并抑制肿瘤侵袭能力。结论纤黏连蛋白重组多肽CH50可以抑制肿瘤微环境中基质金属蛋白酶MMP-2和MMP-9的表达和激活,从而抑制黑色素瘤生长及侵袭能力。     

重组纤维连接蛋白多肽CH50对黑色素瘤B16细胞侵袭能力的抑制作用及机制

目的研究重组纤维连接蛋白多肽CH50对黑色素瘤B16细胞侵袭能力抑制作用及其机制。方法将小鼠黑色素瘤B16细胞用CFSE标记,经尾静脉注射接种小鼠,分别在6 h和24 h取肺组织作冰冻切片,观察肿瘤细胞在肺部聚集和对肺组织侵袭情况;接种15 d后解剖小鼠取肺,观察B16细胞在肺表面形成转移结节数量差异;观察CH50多肽对B16细胞结合纤维连接蛋白和纤维蛋白原的影响;RT-PCR法检测B16细胞中转移相关基因的表达水平;zymography法检测MMP-9水平。结果CH50多肽短时间作用于B16细胞,注射细胞6 h后肺组织荧光结节数显著减少;而作用较长时间,注射细胞24 h后肺组织荧光结节数减少更为明显。CH50多肽处理过的B16细胞在肺部形成转移结节明显减少。CH50多肽能够显著抑制B16细胞结合纤维连接蛋白及结合后的细胞铺展能力及B16细胞结合纤维蛋白原的能力,能够显著下调B16细胞cdc2、αv、β3、MMP-9等基因的表达与MMP-9分泌。结论CH50多肽能够抑制黑色素瘤B16细胞的侵袭能力,抑制瘤细胞与黏附分子结合,抑制转移相关基因的表达,从而使肿瘤细胞的生物学特征发生改变。     

人白细胞介素2基因转移对小鼠B16黑色素瘤细胞生长转移特性…

为开展人白细胞介素２基因转移瘤苗地抗肿瘤作用的研究。须首先获得能较稳定分泌ＩＬ的转基因肿瘤细胞，交了解其生长转移特性。应用ＸＭ６逆转录病毒载体将人ＩＬ２ｃＤＮＡ转入小鼠Ｂ１６黑色素瘤细胞。Ｓｏｕｔｈｅｒｎ杂交证实获得ＩＬ２基因转移的Ｂ１６细胞。     

输血对小鼠B16黑色素瘤肺转移及NK细胞活性的影响

<正> 癌症患者常因贫血和手术需要而输血。有报道同种输血可降低受者的免疫功能而对肿瘤病人的预后产生不良影响,但实验结果也有相互矛盾的情况,确切机制未明。本文比较同种输注血液不同组分对小鼠NK活性以及肿瘤肺转移的影响。     

蛇毒半胱氨酸蛋白酶抑制剂的表达对黑色素瘤B16细胞侵袭与转移的作用

目的探讨蛇毒半胱氨酸蛋白酶抑制剂cystatin（sv-cystatin）在黑色素瘤细胞侵袭与转移中的作用。方法构建真核表达质粒pcDNA3．1／sv-cystatin,采用脂质体法将重组质粒导人小鼠黑色素瘤B16FI细胞。G418筛选抗性克隆,Western blotting法和RT-PCR鉴定sv—cystatin在黑色素瘤细胞中的表达,四甲基偶氮唑盐（MTT）法检测肿瘤细胞体外生长和黏附能力的变化,体外侵袭、运动实验和小鼠实验性肺转移模型分析sv-cystatin表达对黑色素瘤细胞体内、外侵袭力的影响。结果sv-cystatin基因稳定转染后B16F1细胞体外侵袭与运动能力明显降低,其穿膜的细胞数显著低于B16F1／pcDNA3．1空载体组和未转染细胞组（P〈0．01）;sv—cystatin的表达可抑制C57BL／6小鼠肺转移瘤灶的形成;其体外增殖及黏附能力未见明显改变。结论sv—cystatin基因转染可抑制黑色素瘤B16细胞的体内、外侵袭与转移能力。     

诱导肺泡巨噬细胞分泌细胞毒因子抑制小鼠黑色素瘤肺转移

以腹腔注射卡介苗或康赛宁，再雾化吸入ｒＩＬ－２，来抑制小鼠黑色素瘤Ｂ１６－ＢＬ６细胞的肺转移。结果表明，小鼠肺转移被显著抑制，抑制率了高达７８．９５％，小鼠生存期最多延长了４８．２０％；腹腔注射Ｎ－单甲基－Ｌ－精氨酸（ＮＭＡ）使抑制作用显著减弱，平均下降了６９．００％。体外实验证明，Ｂ１６－ＢＬ６细胞对ＮＯ的细胞毒作用敏感，对ＴＮＦ不敏感。结果显示，肺转移的抑制作用可能主要是活化的肺泡世噬细胞分泌     

重组FN多肽CH50对巨噬细胞抗肿瘤功能的促进作用

重组ＦＮ多肽ＣＨ５０可以显著促进巨噬细胞产生ＮＯ的能力、增强其细胞毒作用。ＣＨ５０多肽可与ＩＦＮγ协同作用，进一步促进巨噬细胞产生ＮＯ的能力。体内实验亦表明，ＣＨ５０多肽可以抑制肿瘤细胞在体内生长，与ＩＦＮγ协同作用则可获得更佳效果     

可调控型反义基质金属蛋白酶9表达抑制人黑色素瘤细胞侵袭转移表型的研究

目的 探讨基质金属蛋白酶(MMP)-9与肿瘤转移的相关性。方法 利用基因重组技术构建反义MMP—9 cDNA四环素可调控型表达载体,用脂质体法转染反义MMP—9至转移性人黑色素瘤细胞株WM451(高表达MMP—9)。检测转染后细胞MMP—9表达水平的改变以及体外生长、侵袭、裸鼠体内成瘤及自发转移能力的变化。结果 转染反义基因后,WM451细胞MMP—9的表达及活性明显下降,同时MMP—2的表达也受到一定抑制,细胞生长速度、体外侵袭能力及棵鼠体内成瘤性及自发转移能力均受到一定程度抑制;运用四环素可以抑制四环素负调控逆转录病毒载体上的外源基因的表达。结论 反义MMP—9基因下调MMP—9的表达,可使人黑色素细胞转移能力受到一定程度的抑制,说明MMP—9在人黑色素瘤细胞转移过程中起重要作用。同时,四环素负调控逆转录病毒载体可以调控外源基因的表达。     

重组FN多肽CH50对巨噬细胞抗肿瘤细胞的促进作用

重组ＦＮ多肽ＣＨ５０可以显著促进巨噬细胞产生ＮＯ能力，增强其细胞毒作用，ＣＨ５０多肽可与ＩＦＮ－γ协同作用，进一步促进巨噬细胞产生ＮＯ的能力。体内实验亦表面，ＣＨ５０多肽可以抑制肿瘤细胞在体内生长，与ＩＦＮ－γ协同作用则可获得更佳效果。     

重组PAI-2基因抑制纤维肉瘤细胞侵袭转移的超微结构研究

目的：研究重组ＰＡＩ－２（纤溶酶原激活剂抑制剂－２）基因在裸鼠皮下纤维肉瘤移植瘤细胞中的表达及其对超微结构的影响。方法：应用常规诊断电镜和免疫电镜技术，观察肿瘤细胞的结构变化，定位研究ＰＡＩ－２蛋白的表达。结果：重组ＰＡＩ－２基因转染的纤维肉瘤细胞，其移植瘤具有ＰＡＩ－２蛋白的表达，以活性形式存在于细胞表面，同时细胞超微结构的恶性特征减弱，分化趋向成熟，侵袭转移能力下降。结论：ＰＡＩ－２基因的表达在肿瘤细胞侵袭转移机制中起负调控作用     

TNF Impairs In Vivo and In Vitro Natural Killer (NK) Susceptibility of B16 Melanoma Cells

Tumour necrosis factor alpha (TNF) is a multipotent cytokine which affects many biological properties of both normal and neoplastic cells. Here we show that treatment with TNF reduces B16-A melanoma cell susceptibility to normal and in vivo- and in vitro-activated NK cell-mediated killing. This resistance is associated with an enhancement of B16-A metastatic potential in normal syngeneic mice, but not in anti-asialo GM1-treated animals, further supporting the NK dependence of TNF-induced enhancement of metastatic ability. A significant increase of MHC class I expression on B16-A murine melanoma cells is observed after TNF treatment. In all these effects TNF interacts positively with interferon gamma (IFN gamma). Taken together, these results indicate that TNF treatment negatively affects the susceptibility of B16-A murine melanoma to NK effectors in vivo and in vitro. This decreased susceptibility may be related, at least in part, to enhanced expression of MHC class I antigens on tumour cells.     

In Vivo Effects of Human Bone Marrow Mesenchymal Stromal Cells on the Development of Experimental B16 Melanoma in Mice

Bulletin of Experimental Biology and Medicine - Experiments on F1(CBA×C57BL/6) mice with experimental metastatic melanoma B16 F10 showed that single intravenous injection of xenogeneic bone...     

Hydroxyurea regulates the development and survival of B16 Melanoma Cells by upregulating MiR-7013-3p

miRNAs are a family of short, noncoding RNAs that are involved in many processes in melanoma cells. MITF acts as a master regulator of melanocyte function, development and survival by modulating various genes. Hydroxyurea (HU) is used to treat melanoma, and miRNA expression is altered after HU treatment in B16 melanoma cells. In this study, we screened for miRNAs that were upregulated after HU treatment and that targeted the MITF gene. We found that miR-7013-3p exhibited increased expression after HU treatment and could bind to MITF. miR-7013-3p inhibited melanin production, proliferation, and migration and promoted apoptosis in B16 melanoma cells. The results may provide more information on the roles of miR-7013-3p in B16 melanoma cells.     

MRI Detection of Early Bone Metastases in B16 Mouse Melanoma Models

Bone metastasis causes significant morbidity in cancer patients, including bone pain, pathologic fractures, nerve compression syndrome, and hypercalcemia. Animal models are utilized to study the pathogenesis of skeletal metastases and to evaluate potential therapeutic agents. Previously published methods for imaging bone metastasis in rodent models have focused on identifying advanced stage metastasis using simple X-rays. Here we report MRI as a method for detecting early bone metastases in mouse models in vivo. B16 mouse melanoma cells were injected into the left cardiac ventricle of C57BL/6 mice and magnetic resonance (MR) images were obtained of the left leg following the development of metastatic disease, when tumor associated bone destruction was histologically present but not visible by X-ray. T1 and T2 relaxation times of bone marrow were measured in healthy control mice and B16 melanoma tumor-bearing mice. Mean T2 values for normal marrow were 28 ms (SD 5) and for diseased bone marrow were 41 ms (SD 3). T2 relaxation time of diseased bone marrow is significantly longer than that of normal bone marrow (P < 0.0001) and can be used as a marker of early bone metastases. These studies demonstrate that MR imaging can detect bone marrow metastases in small animals prior to development of cortical bone loss identified by X-ray.     

S-100蛋白和RANTES在B16黑色素瘤荷瘤鼠中的表达

目的 检测荷瘤鼠体内S-100蛋白及RANTES（T细胞特异性趋化因子）的表达,探讨其与肿瘤发生和转移的关系。方法 将培养的B16黑色素瘤细胞于实验组c57小鼠背部皮下注射,待肿瘤形成后,取局部淋巴结和肿瘤病变中心区及交界区皮肤,作免疫组化染色。结果 肿瘤早期组S-100蛋白和RANTES阳性反应物的面密度和数密度在淋巴结和肿瘤组织、癌周皮肤明显高于正常对照组和肿瘤晚期组（P〈0．05）。结论 S-100蛋白和IRANTES的表达程度与肿瘤的侵袭和进程呈负相关。     

Genetically Modified Murine Adipose-Derived Mesenchymal Stem Cells Producing Interleukin-2 Favor B16F10 Melanoma Cell Proliferation

Adipose-derived mesenchymal stem cells (ADSCs) are attractive tools for cancer gene therapy due to their intrinsic tropism to the tumor environment. Interleukin-2 (IL2) is recognized as a key regulatory molecule, which enhances the activity and growth of the immune effector cell function. High-Dose IL2 Therapy is an option for treatment of malignant melanoma but has frequent, often serious and sometimes life-threatening side effects. Here we investigated the effect of genetically modified ADSCs (GM-ADSCs) expressing IL2 in immunocompetent mouse models of subcutaneous and lung metastatic melanoma. Prior to in vivo studies, we demonstrated that IL2 produced by GM-ADSCs may act as a growth factor for melanoma cells due to the increased viability and reduced apoptosis of melanoma cells after in vitro treatment. Subcutaneous co-injection of IL2-expressing ADSCs with melanoma cells significantly enhanced the melanoma tumor growth. Furthermore, histological analysis of subcutaneous tumors for IL2 and Melan-A (a melanocytic differentiation marker) confirmed that most of cells in melanoma/IL2-ADSC co-injected tumors are melanoma cells, not IL2-ADSCs. In pulmonary metastases model, melanoma cells were injected intravenously and 10 days later mice were treated by systematical injection of GM-ADSCs. Intravenously injected IL2-ADSCs engrafted into melanoma lung tumors but were unable to reduce melanoma lung metastases. Besides, administered IL2-ADSCs significantly reduced systemic CD4+ cells and did not impact the total survival of lung metastases melanoma bearing mice. In conclusion, this study showed that IL2-producing ADSCs can favor B16F10 melanoma cell proliferation. Therefore, therapies utilizing IL2 have to be taken into careful consideration.     

Recombinant human prothrombin kringle-2 inhibits B16F10 melanoma metastasis through inhibition of neovascularization and reduction of matrix metalloproteinase expression

Angiogenesis, a multi-step process which involves endothelial cell proliferation, adhesion, migration, and basement membrane (BM) degradation, is essential for tumor metastasis. Here we show that recombinant human prothrombin kringle-2 (rk-2) inhibited bovine capillary endothelial cell migration with an IC₅₀ (concentration for half maximal inhibition) of 38 nM and inhibited adhesion to extracellular matrix (ECM) proteins. Because tumor metastasis requires angiogenesis, we examined whether rk-2 could inhibit metastases induced by injection of B16F10 melanoma cells into mice. The results revealed that the metastatic tumors in mouse lung were markedly decreased in a dose-dependent manner and acute lung injury induced by B16F10 melanoma metastasis was diminished by systemic rk-2 treatment. In immunohistochemical analysis, rk-2 reduced expression of vascular endothelial growth factor, which is a potent angiogenic activator and neovascularization in the mouse lung. Also, rk-2 diminished the expression of matrix metalloproteinase-2 and -9 in the mouse lung which induces tumor metastasis and angiogenesis. These data suggest that inhibition of B16F10 melanoma metastasis by rk-2 was caused by inhibition of neovascularization and reduction of matrix metalloproteinase expression.