阿托伐他汀通过ERK1/2通路抑制内皮微粒诱导的人脐静脉内皮细胞ICAM-1的表达

目的探讨阿托伐他汀对内皮细胞微粒(EMPs)诱导人脐静脉内皮细胞(HUVECs)细胞间黏附分子-1(ICAM-1)表达的影响及其与ERKl/2信号通路的关系。方法将HUVECs分为不同浓度EMPs作用组与阿托伐他汀干预组。应用Western印迹检测磷酸化ERK1/2和ICAM-1蛋白的表达,实时荧光定量PCR(qRT-PCR)检测ICAM-1 mRNA的表达。结果 EMPs可诱导HUVECs ICAM-1 mRNA和蛋白及磷酸化ERK1/2蛋白表达增加,且具有浓度和时间依赖关系(均P<0.01);阿托伐他汀及ERK1/2特异性抑制剂PD98059显著抑制EMPs诱导的HUVECs ICAM-1 mRNA和蛋白及磷酸化ERK1/2蛋白的表达(均P<0.01)。结论阿托伐他汀通过ERK1/2信号通路抑制EMPs诱导HUVECs ICAM-1表达。     

细胞外信号调节激酶1/2在血管内皮细胞衰老中的表达变化及作用

目的观察细胞外信号调节激酶1/2(ERK1/2)在血管紧张素Ⅱ(AngⅡ)诱导的内皮细胞中不同时点的表达变化。方法制备AngⅡRPMI1640培养液(10-6 mol/L)培养人脐静脉内皮细胞,采用MTT测定内皮细胞存活率,β-gal染色、细胞周期分析鉴定细胞衰老,透射电子显微镜观察衰老细胞超微结构。细胞免疫化学染色法分析Bcl-2、Bax蛋白表达变化,Western印迹测定磷酸化ERK1/2水平。结果 AngⅡ诱导组存活的细胞数为对照组的〔(81.9±0.04)%,P<0.01)〕;约80%的细胞呈现β-gal阳性染色,流式细胞仪检测细胞周期停滞于G0～G1,证实细胞衰老;透射电子显微镜可见AngⅡ诱导组衰老的细胞体积较大,核形不规则,细胞核染色质浓缩和凝聚。与对照组相比,Bcl-2 mRNA表达呈持续性降低,Bcl-2/Bax比值下降,ERK1/2磷酸化水平于12 h明显增加,24 h达到高峰(P<0.01),36 h下降至稳定,总ERK1/2蛋白水平无明显变化。结论ERK1/2信号转导途径参与AngⅡ诱导内皮细胞衰老的发生、发展过程,并可能通过调控内皮细胞Bcl-2/Bax比值来实现。     

TIMP-2、MMP-2和MT1-MMP在活化肝星状细胞中的表达及对细胞外基质合成分泌的影响

目的 研究金属蛋白酶组织抑制剂-2(TIMP-2)、基质金属蛋白酶-2(MMP-2)和膜型基质金属蛋白酶-1(MT1-MMP)在活化肝星状细胞(HSCs)中的表达,观察其对细胞外基质(ECM)合成分泌的影响.方法 原代分离培养大鼠HSCs活化后,分别给予40～160 pmol化学合成经修饰抗TIMP-2 siRNA进行干预,检测培养细胞上清液透明质酸(HA)、Ⅲ型前胶原(PCⅢ)和羟脯氨酸(Hyp)的含量,采用荧光实时定量PCR法检测TIMP-2、MMP-2、MT1-MMP、MMP-13、COL Ⅰ和COL Ⅲ mRNA的表达,western印迹检测TIMP-2、MT1-MMP和MMP-13蛋白表达及明胶酶谱法检测MMP-2蛋白表达.结果 应用化学合成经修饰抗TIMP-2 siRNA后,TIMP-2、MMP-2、MT1-MMP、COL Ⅰ和COL Ⅲ的表达明显降低,而MMP-13的表达则明显增加,培养细胞上清液中HA、PCⅢ和Hyp的含量也明显减少.结论 TIMP-2通过MT1-MMP介导MMP-2的活化,抑制TIMP-2的表达,MT1-MMP和MMP-2的表达随之降低,而HSCs合成分泌ECM也相应减少.     

Functional analysis of α1β1 integrin in human natural killer cells

Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the α1β1 and α2β1 integrins and down-regulate the expression of α6β1. By employing α1β1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the α1β1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-α2β1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-β1 LIA 1/2 mAb, but was unaffected by α1 and α2-specific mAb; as α3β1 and α6β1 were undetectable, the data indicate that the α1β1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-α1 HP-2B6 or its F(ab')₂ fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca²⁺ and Mg²⁺); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-α1 HP-2B6 enhanced TNF-α production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-α1 HP-2B6 mAb. Our data show that ligation of the α1β1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.     

uPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells

Malignant melanoma is a highly aggressive skin cancer characterized by an elevated grade of tumor cell plasticity. Such plasticity allows adaptation of melanoma cells to different hostile conditions and guarantees tumor survival and disease progression, including aggressive features such as drug resistance. Indeed, almost 50% of melanoma rapidly develop resistance to the BRAFV600E inhibitor vemurafenib, with fast tumor dissemination, a devastating consequence for patients’ outcomes. Vasculogenic mimicry (VM), the ability of cancer cells to organize themselves in perfused vascular-like channels, might sustain tumor spread by providing vemurafenibresistant cancer cells with supplementary ways to enter into circulation and disseminate. Thus, this research aims to determine if vemurafenib resistance goes with the acquisition of VM ability by aggressive melanoma cells, and identify a driving molecule for both vemurafenib resistance and VM. We used two independent experimental models of drug-resistant melanoma cells, the first one represented by a chronic adaptation of melanoma cells to extracellular acidosis, known to drive a particularly aggressive and vemurafenib-resistant phenotype, the second one generated with chronic vemurafenib exposure. By performing in vitro tube formation assay and evaluating the expression levels of the VM markers EphA2 and VE-cadherin by Western blotting and flow cytometer analyses, we demonstrated that vemurafenib-resistant cells obtained by both models are characterized by an increased ability to perform VM. Moreover, by exploiting the CRISPR-Cas9 technique and using the urokinase plasminogen activator receptor (uPAR) inhibitor M25, we identified uPAR as a driver of VM expressed by vemurafenib-resistant melanoma cells. Thus, uPAR targeting may be successfully leveraged as a new complementary therapy to inhibit VM in drug-resistant melanoma patients, to counteract the rapid progression and dissemination of the disease.     

Circulating MicroRNAs and Extracellular Vesicle–Containing MicroRNAs as Response Biomarkers of Anti–programmed Cell Death Protein 1 or Programmed Death-Ligand 1 Therapy in NSCLC

IntroductionAnti–programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) antibody therapy is a standard treatment for advanced NSCLC, and PD-L1 immunohistochemistry is used as a predictive biomarker for therapeutic response. However, because not all patients with NSCLC with high PD-L1 respond, and some patients with low PD-L1 expression exhibit durable benefit, more accurate predictive biomarkers are needed. Circulating microRNA (miRNA) and miRNA packaged in extracellular vesicles (EVs) are believed to play a role in intercellular communication among immune cells and between immune cells and tumor cells and may represent a good source of mechanism-related biomarkers.MethodsPretreatment plasma of patients with advanced NSCLC treated with single-agent anti–PD-1 or anti–PD-L1 antibody was used in this study. Plasma EVs were isolated using size-exclusion chromatography. Whole plasma and EV-containing RNAs were extracted. The miRNA profile was analyzed with a next-generation sequencing platform.ResultsSamples from 14 responders (patients who exhibited partial response or stable disease ≥6 mo) and 15 nonresponders (patients who exhibited progressive disease as per Response Evaluation Criteria in Solid Tumors) were analyzed. In total, 32 miRNAs (p = 0.0030–0.0495) from whole plasma and seven EV-associated miRNAs (p = 0.041–0.0457) exhibited significant concentration differences between responders and nonresponders. The results of some of these circulating miRNAs were validated in a separate cohort with eight responders and 13 nonresponders. The tumor PD-L1 level was also assessed using immunohistochemistry for patients involved in both cohorts.ConclusionsSpecific circulating miRNAs in whole plasma and plasma EVs are differentially expressed between responders and nonresponders and have potential as predictive biomarkers for anti–PD-1/PD-L1 treatment response.     

氨基葡萄糖对体外培养人软骨和滑膜细胞软骨寡聚基质蛋白分泌影响的比较

目的 比较氨基葡萄糖对体外培养软骨和滑膜细胞软骨寡聚基质蛋白(COMP)分泌的影响.方法 软骨细胞和滑膜细胞采自骨关节炎(OA)患者,采用分阶段酶消化法体外培养人软骨细胞和滑膜细胞,给予实验兔以氨基葡萄糖临床等效剂量灌胃后,抽取含药血清培养细胞.采用酶联免疫吸附试验(ELISA)检测药物对体外培养的人软骨细胞和滑膜细胞培养上清液中COMP的浓度.结果 体外培养滑膜细胞分泌的COMP浓度[(41.6±0.4)ng/ml]显著高于软骨细胞[(5.5±3.0)ng/ml](19＜0.05).氨基葡萄糖可显著增加体外培养软骨细胞的COMP分泌[(20.3±3.6)与(5.5±3.0)ng/ml,P＜0.05];而对体外培养滑膜细胞的作用较弱,可轻度减少COMP分泌[(36.6±1.3)与(41.6±0.4)ng/ml].结论 氨基葡萄糖含药血清可以促进体外培养软骨细胞分泌COMP,而对体外培养滑膜细胞无明显作用.     

基质金属蛋白酶抑制剂和肝纤维化关系的研究进展

基质金属蛋白酶抑制剂（TIMPs）是基质金属蛋白酶（MMPs）的特异性抑制剂,两者比例失常将导致细胞外基质合成和降解失去平衡,促进肝纤维化的形成。TIMPs表达的异常在肝纤维化形成过程中具有非常重要的作用,本文对TIMPs在肝纤维化形成中的变化及其对肝纤维化诊断、治疗等方面的作用作一综述。     

血管紧张素Ⅱ通过核因子κB上调巨噬细胞基质金属蛋白酶诱导因子的表达

目的 观察血管紧张素Ⅱ对巨噬细胞表达基质金属蛋白酶诱导因子的影响及其相关通路.方法 血管紧张素Ⅱ体外刺激人巨噬细胞,应用逆转录聚合酶链反应和Western blot测定基质金属蛋白酶诱导因子基因和蛋白表达;RNA干扰技术沉默核转录因子核因子κB p65亚基,评价核因子κB通路在血管紧张素Ⅱ上调巨噬细胞细胞基质金属蛋白酶诱导因子的表达中的作用.结果 血管紧张素Ⅱ刺激巨噬细胞后,基质金属蛋白酶诱导因子mRNA和蛋白表达均显著增加,核因子κB p65亚基沉默后,血管紧张素Ⅱ对基质金属蛋白酶诱导因子mRNA和蛋白表达无上调作用.结论 血管紧张素Ⅱ上调基质金属蛋白酶诱导因子的表达,此调控过程有核因子κB通路的参与.