过表达Tg737基因对肝癌细胞周期和凋亡的影响

目的:探讨Tg737基因过表达对人肝癌细胞株细胞周期和凋亡的影响及可能的机制。方法:将肝癌HepG2和MHCC97-H细胞分别用脂质体法转染pcDNA3.1-Tg737质粒(Tg737过表达组)或pcDNA3.1空载体(空载体组),或单纯脂质体孵育(脂质体组),以各自未处理的细胞为空白对照。48h后分别用流式细胞仪检测细胞周期与凋亡,Hoechst33342染料法检测细胞核形态,Westernblot法检测cyclinA、Bax、Bcl-2表达。结果:与各自的空白对照组比较,Tg737过表达组HepG2和MHCC97-H细胞的S期细胞数量与细胞凋亡率均明显增加(均P0.05),且细胞核均出现明显凋亡形态学改变,同时伴随cyclinA、Bax的表达上调和Bcl-2的下调(均P0.05);空载体组、脂质体组HepG2和MHCC97-H细胞镜下未见明显的凋亡形态学变化,且上述指标差异均无统计学意义(均P0.05)。结论:Tg737基因过表达可以抑制肝癌细胞周期进程、促进其凋亡,机制可能与Tg737参与调节cyclinA、Bax、Bcl-2有关的信号通路有关。     

短发卡状RNA抑制AKT2对肝癌细胞增殖及侵袭的影响

目的 探讨短发卡状RNA(siRNA)抑制AKT2对肝癌细胞增殖、凋亡和侵袭转移的影响.方法 设计并合成特异性靶向AKT2的siRNA片段并构建SMMC7721AKT2 siRNA表达质粒,将其转染SMMC7721细胞,通过G418筛选出稳定株.MTT法检测肝癌SMMC7721细胞的生存率变化;流式细胞术检测细胞周期;Western- blot检测P27、CyclinD1;Transwell实验和划痕实验分析细胞侵袭、转移能力的改变.结果 MTT检测显示AKT2干扰可抑制SMMC7721细胞的生长,与其他组比较差异有统计学意义(P＜0.05).流式细胞术显示AKT2干扰组细胞周期阻滞于G1期,G1期细胞比例上升,S期细胞比例下降.Western- blot检测显示CyclinD1表达下降,P27的表达上升.Transwell试验和划痕试验显示AKT2干扰组的侵袭和转移能力受到抑制.结论 AKT2基因沉默可明显抑制肝癌SMMC7721细胞的生长并阻滞细胞周期.AKT2基因沉默可抑制肝癌SMMC7721细胞的侵袭和转移能力.     

阿司匹林对肝癌细胞生长的抑制作用及其机制

目的 观察阿司匹林(AS)对肝癌细胞SMMC-7721生长的作用,并探讨其作用机制.方法 采用噻唑蓝(MTT)比色法检测不同浓度阿司匹林对肝癌细胞细胞生长的抑制作用以及流式细胞仪检测不同浓度阿司匹林对肝癌细胞细胞周期的影响;免疫组织化学方法检测阿司匹林对肝癌细胞细胞增殖核抗原(PCNA)、环氧合酶-2(COX-2)表达的影响;并用逆转录-聚合酶链反应(RT-PCR)测定COX-2基因的表达.结果 肝癌细胞经不同浓度的阿司匹林作用后,其生长抑制率明显升高(P<0.05),且与阿司匹林浓度有关和作用时间相关.细胞周期检测结果显示,经不同浓度阿司匹林作用后的SMMC-7721细胞,细胞周期发生明显变改.表现为随着阿司匹林浓度的增加,S期细胞比例下降明显,G0/G1期及G2/M期细胞比例则上升(10-2、10-3moL/L AS组与对照组比较P<0.05).免疫细胞化学染色显示,阿司匹林能下调肝癌细胞COX-2、PCNA的表达.RT-PCR检测表明,COX-2基因表达电泳条带与对照组比较辉度明显降低,其中10 mmol/L阿司匹林组最低,说明随着阿司匹林浓度增加,COX-2表达逐渐减少,阿司匹林能下调SMMC-7721细胞表达COX-2基因.结论 阿司匹林能有效抑制肝癌细胞SMMC-7721的生长,这种作用可能是其通过抑制细胞增殖,与抑制COX-2、PCNA的表达有关.     

下调CD133表达对肝癌细胞恶性生物学行为的影响

目的:探讨下调CDl33基因的表达对肝癌细胞生物学行为的影响。方法:将合成的CDl33小干扰核糖核酸分子(si RNA)转染至肝癌SMMC7721细胞并检测转染效率;分别以无转染与转染随机si RNA序列的SMMC7721细胞为空白对照和阴性对照,观察CDl33 si RNA转染后CDl33基因沉默效果,以及SMMC7721细胞主要生物学行为的变化。结果:转染24 h后,转染效率可达到(80.8±9.1)%;与空白对照组比较,CDl33 si RNA转染后的SMMC7721细胞CD133 m RNA及蛋白表达量分别降至空白对照组的10%与35%、细胞增殖活性明显降低、细胞凋亡率明显增加(41.3%vs.25.3%)并出现明显的S期阻滞,集落形成能力明显降低(均P0.05)。阴性对照组与空白对照组各指标无统计学差异(均P0.05)。结论:CDl33在肝癌细胞中可能起了癌基因作用,下调其表达能抑制肝癌的恶性生物学行为。     

甘露糖敏感型绿脓杆菌制剂对肝癌细胞周期的作用机制研究

目的 探讨甘露糖敏感性绿脓杆菌制剂(pseudomonasaeruginosa mannose sensitive haemagglutination vaccine,PA-MSHA)对肝细胞癌(hepatocellular carcinoma,HCC)细胞周期的作用及其机制.方法 培养人肝癌细胞株MHCC97L及HepG2,以不同剂量的PA-MSHA作用于肝癌细胞.MTT实验检测PA-MSHA对细胞增殖的影响.流式细胞技术检测PA-MSHA对细胞周期的作用.Western blot检测PA-MSHA作用前后细胞周期蛋白Cyclin D1、细胞周期蛋白依赖性激酶2(CDK2)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)以及细胞周期蛋白激酶抑制剂p21和p27的表达情况.结果 与对照组相比,PA-MSHA显著抑制MHCC97L和HepG2细胞的增殖,其作用呈剂量及时间依赖性(P＜0.05).PA-MSHA显著诱导肝癌细胞周期阻滞,PA-MSHA作用后G0-G1期和G2-M期细胞比例显著增高,而S期细胞比例则显著下降(P＜0.05).PA-MSHA显著抑制Cyclin D1、CDK2、PCNA蛋白的表达,而促进p21和p27的表达.外源性甘露糖可显著抑制PA-MSHA对肝癌细胞增殖和周期的作用(P＜0.05).结论 PA-MSHA通过调控细胞周期相关蛋白来诱导HCC细胞周期阻滞,抑制细胞增殖.这一作用是通过肝癌细胞表面的甘露糖的介导实现的.     

siRNA封闭胰岛素样生长因子Ⅰ类受体对肝癌细胞侵袭能力的影响及其机制

目的 探讨人胰岛素样生长因子1类受体(IGFIR)的短发夹环RNA质粒(pSUPER-siRNA-IGFIR)能否有效抑制人肝癌细胞株MHCC97H侵袭能力并探讨其相关机制.方法 设计并合成靶向IGFIR的siRNA片段并构建Psuper-siRNA-IGFIR表达质粒,将其转入MHCC97H、SMMC7721细胞,通过G418筛选出稳定株.通过黏附实验、Boyden小室实验、软琼脂克隆形成实验观察各细胞株侵袭能力的变化并采用明胶酶法测定肝癌细胞的金属蛋白酶(MMP)-2、MMP-9的含量,Western blot检测增殖相关基因的变化.并设空白对照组、阳性对照组.结果 MHCC97H-IG-FIR-siRNA、SMMC7721-IGFIR-siRNA黏附能力比对照组下降约5倍(MHCC97H)和6倍左右(SMMC7721),细胞迁移能力明显下降,下降各约3倍,细胞克隆形成率明显低于对照组约6倍和8倍,其MMP-2、MMP-9、ICAM-1、LNR、E-cadherin表达比对照组低.结论 构建的pSUPER-siRNA-IGFIR具有RNA干扰作用,可抑制肝癌细胞株转移能力.     

HDAC抑制剂SAHA对人肝癌细胞分化诱导作用的研究

目的 探讨组蛋白去乙酰化酶(histone deacetylases,HDAC)抑制剂SAHA(suberoylanilide hydroxamic acid)对人肝癌SMMC-7721细胞的分化诱导作用.方法 倒置显微镜观察SAHA对SMMC-7721细胞形态的影响;MTT比色法测定SAHA对SMMC-7721细胞增殖的抑制情况;免疫细胞化学检测SAHA对SMMC-7721细胞中甲胎蛋白(AFP)和增殖细胞核抗原(PCNA)表达的影响;流式细胞术(FCM)分析细胞周期;RT-PCR方法榆测处理前后SMMC-7721细胞p21WAF1基因mRNA的表达变化.结果 实验组细胞增殖速度显著减慢,与正常细胞形念变化相似;MTT比色法测定结果显示不同浓度SAHA对SMMC-7721细胞的增殖均有抑制作用,并有明显的剂量依赖和时间依赖关系;免疫细胞化学检测显示SAHA能显著降低PCNA和AFP在SMMC-7721细胞中的表达;流式细胞仪检测结果显示,SMMC-7721细胞经SAHA处理后,G0/G1期细胞明显增加,S期细胞则明显减少,细胞被阻滞于G0/G1期;RT-PCR检测结果表明,实验组细胞中p21WAF1 mRNA的表达明显增加.结论 SAHA对人肝癌细胞具有显著的诱导分化作用,诱导肝癌细胞分化的机理可能与抑制HDAC的活性,上调p21 WAF1 mRNA表达,及阻滞肝癌细胞G0/G1期有关.     

乙型肝炎病毒X基因在体内外对肝癌细胞增殖活性的影响

目的 探讨乙型肝炎病毒x(HBx)基因对肝癌细胞增殖活性的影响。方法将携带HBx基因的表达质粒pHA-HBx转染HepG2肝癌细胞,G418筛选阳性细胞克隆,RT-PCR鉴定HBx基因的整合及表达;通过细胞计数,观察HBx基因对肝癌细胞生长曲线和倍增时间的影响;流式细胞术检测细胞周期的变化;^3H-TdR掺入法检测细胞增殖活性;裸鼠接种观察HBx基因在体内对肝癌细胞增殖的影响。结果HBx基因在体内外对HepG2细胞的增殖活性均有明显影响,细胞生长曲线左移,倍增时间缩短;G0／G1。期细胞减少,S期和G2／M期细胞增多;转染后的细胞^3H-TdR掺入率较对照组增高;转HBx基因的裸鼠移植瘤的生长速度较对照组细胞明显加快。结论HBx基因在体内外均可提高肝癌细胞的增殖活性,增加肝癌细胞的恶性表型,加速肿瘤生长。     

细胞周期素D1反义cDNA治疗肝癌的研究

目的 通过基因反义封闭技术抑制细胞周期素D1（CydinD1）的表达，研究其对肝癌细胞增殖以及成瘤性的影响。方法 以肝癌HepG2细胞株为研究对象，通过转染可表达CyclinD1反义互补脱氧核苷酸（AScDNA）的质粒后，观察CyclinD1反义cDNA对肝癌细胞CyclinD1基因表达、体外增殖活性及裸鼠体内成瘤性的影响。结果 噻唑蓝（MTT）法检测细胞增殖活性显示转染表达反义CydinD1的质粒后，HepG2细胞的增殖受到抑制．抑制作用在48h左右最强；逆转录-聚合酶链反应（RT-PCR）检测显示CyclinD1 mRNA基因的表达明显被抑制；间接免疫荧光检测结果显示CyclinD1蛋白表达显著降低；流式细胞仪检测结果显示G0／G1期的细胞比例增高，G2＋M和S期的细胞比例下降，HepG2细胞周期在G1期被阻滞；裸鼠成瘤试验显示肝癌HepG2细胞的成瘤性受到明显抑制。结论 CyclinD1反义cDNA可以特异性的抑制肝癌HepG2细胞株CyclinD1蛋白的表达，从而调控细胞周期，抑制肝癌细胞增殖及体内成瘤性。CyclinD1反义cDNA对于肝细胞癌的生物治疗具有一定的应用前景。     

过表达核心蛋白聚糖基因对胆管癌细胞周期及凋亡的影响

目的 观察过表达核心蛋白聚糖(DCN)基因对胆管癌细胞周期及凋亡的影响.方法 脂质体介导真核表达质粒pEGFP-DCN和空载体pEGFP-N1转入人胆管癌细胞株QBC939,Westernblot检测DCN蛋白的表达,实时定量反转录-聚合酶链反应(RT-qPCR)检测DCN mRNA的表达,克隆形成实验计算克隆形成率,噻唑蓝(MTT)法检测细胞增殖活力,流式细胞术检测细胞周期及凋亡.结果 RT-qPCR示过表达DCN相对空载体上调32.34倍;Western blot结果示过表达组DCN上调2.00倍.pEGFP-DCN转染组细胞克隆形成数[(210.9±19.3)个]显著低于空质粒转染组[(608.7±56.3)个],差异有统计学意义(P＜0.05).转染pEGFP-DCN细胞与空载体比较,细胞增殖能力显著降低,差异有统计学意义(P＜0.05).流式细胞周期检测显示,胆管癌细胞转染pEGFP-DCN后G1～ Go期细胞比例明显升高,而G2～S期细胞减少,细胞周期被阻滞在G1～Go期,凋亡分析转染pEGFP-DCN的QBC939细胞较转染空载体细胞凋亡显著增加,半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3明显增高,而B细胞淋巴瘤/白血病-2(bcl-2)的表达水平明显降低(0.787±0.068比1.276±0.157).结论 转染DCN能够显著抑制胆管癌细胞的增殖,明显促进胆管癌细胞凋亡,而DCN促进胆管细胞凋亡与上调Caspase-3和下调bcl-2蛋白相关.     

Prognostic value of PD-1 and PD-L1 expression in patients with metastatic clear cell renal cell carcinoma

Objective

In renal cell carcinoma (RCC), several prognostic biomarkers have been identified and are under investigation. Several reports have shown that the expression of programmed death 1 (PD-1) and its ligand PD-L1 is associated with poor outcome for patients with RCC. The present study is aimed at evaluating the expression of PD-1 and PD-L1 and to investigate their clinical and prognostic significance in patients with clear cell RCC (CCRCC) having received molecular targeted therapies. In addition, we also evaluated the relationship between the expression of PD-1 and PD-L1 and intratumoral tumor infiltrating lymphocytes (TILs).

丝氨酸/苏氨酸蛋白激酶对肝癌细胞凋亡的促进作用

目的评价丝氨酸/苏氨酸蛋白激酶(HIPK1)促进肿瘤坏死因子(TNF)-α诱导的JNK/p38激活及其对肝癌细胞凋亡的影响。方法Hepa1-6细胞,分别感染重组腺病毒rAd-Lacz-V5(107PFU/孔)和rAd-myc-HIPK1(107PFU/孔)48h,荧光显微镜检测Lacz和HIPK1的蛋白表达,TNF-α处理细胞后Western blot检测JNK/p38激活情况;HIPK1的RNA干扰检测HIPK1在JNK/p38激活中的作用;TNF-α处理病毒感染的Hepa1-6细胞24h,DAPI染色荧光显微镜下了解细胞凋亡情况。结果荧光显微镜下见Lacz和HIPK1蛋白在Hepa1-6细胞中表达效率高;HIPK1组中TNF-α介导的磷酸化型JNK/p38的表达明显高于Lacz组,相反HIPK1的RNA干扰组中TNF-α介导的磷酸化型JNK/p38的表达明显低于对照组;细胞核形态学检测HIPK1组中细胞凋亡率为(30.72±5.67)%,而Lacz组为(9.82±3.41)%,两组间差异有统计学意义(P<0.01)。结论HIPK1促进TNF-α介导的JNK/p38的激活并诱导肝癌细胞Hepa1-6凋亡。     

Caveolin-1和HDAC1蛋白在肾细胞癌中的表达及意义

目的探讨Caveolin-1和HDAC1蛋白在肾细胞癌(renal cell carcinoma,RCC)及正常肾组织中的表达及临床意义,并探讨其与肿瘤生物学行为之间的关系。方法采用免疫组织化学方法检测51例RCC、15例正常肾组织及26例癌旁组织中Caveolin-1和HDAC1蛋白的表达,并分析其与患者临床病理特征的关系。51例RCC中组织学分级:高分化13例,中分化24例,低分化14例;临床分期:Ⅰ期16例,Ⅱ期20例,Ⅲ期10例,Ⅳ期5例。结果 Caveolin-1在正常肾组织、癌旁组织和RCC中的阳性表达率分别为33%(5/15)、42%(11/26)、65%(33/51),逐渐升高(P<0.05)。在51例RCC中,Caveolin-1的阳性表达率临床分期Ⅲ期和Ⅳ期高于Ⅰ期和Ⅱ期,低分化高于中、高分化,有淋巴结转移组高于无淋巴结转移组。Caveolin-1表达与病理学分期、分级和淋巴结转移均相关(P值分别为0.001、0.038和0.006,P均<0.05),随着肿瘤分期、分级的升高,Caveolin-1表达增强。RCC中Caveolin-1表达与年龄、性别、肿瘤所在部位均无关(P>0.05)。HDAC1在正常肾组织、癌旁组织和RCC中的阳性表达率分别为13%(2/15)、19%(5/26)、75%(38/51),呈现显著升高的趋势(P<0.05)。在51例RCC中,HDAC1的阳性表达率临床分期Ⅲ期和Ⅳ期高于Ⅰ期和Ⅱ期,低分化高于中、高分化,有淋巴结转移组高于无淋巴结转移组,肾癌组织中HDAC1表达率随肾癌TNM分期的增高和病理分级的增加而升高。RCC中HDAC1表达与年龄、性别、肿瘤所在部位均无关(P>0.05)。RCC中Caveolin-1的表达与HDAC1表达呈正相关。结论 Caveolin-1和HDAC1在RCC中高表达,可能是RCC发生、发展及浸润转移的重要因素之一,联合检测对判断RCC预后有指导意义。     

Cyclophilin a modulates dendritic cell differentiation from myeloblastic cell KG1

Introduction. Cyclophilin A (CypA) is a ubiquitously distributed intracellular protein as well as secreted protein and has recently been reported to be an immunomodulatory molecule. The objective of this study was to determine the effect of CypA on dendritic cell (DC) differentiation, activation, and functional maturation. Methods. KG1 cells (CD34⁺ human myeloblastic cell line) were treated with cytokines (GM-CSF+IL-4) and/or CypA and expression of cell-surface markers was analyzed by FACS analysis. The antigen uptake capacity of different DCs was determined by FITC-dextran uptake assay. Antigen presentation capacity of DCs was determined by allogeneic mixed lymphocyte reaction (MLR) by [³H]thymidine incorporation assay. To check the T cell polarization of KG1-derived DCs, various Th1 and Th2 cytokines secreted by allo-stimulated CD4⁺ and CD8⁺ T cells were determined by Bioplex cytokine assay. Role of p38 MAP kinase in DC functions was also investigated. Results. During the differentiation of KG1 cells to immature DCs, cell-surface expression of CD11b was increased by 30.6% for CypA alone; 55% for CypA plus cytokines; and 44% for cytokines alone. Similarly, CypA alone increased the cell-surface expression of CD11c by 59% as compared to CypA plus cytokines (68%) and cytokines alone (50%). CypA up regulated the antigen uptake capacity of the immature DCs to a greater extent (5 times) as compared to cytokines alone (2.5 times). Moreover, CypA augmented the capacity of DCs to present antigens to allogenic CD8⁺ T cells and also increased the secretion of Th1-type cytokines TNF-α and IFN-γ from the allogenic CD4⁺ T cells. Furthermore, CypA induced the phosphorylation and hence activation of MAP kinase p38. Pretreatment with SB-203580, a p38 inhibitor, significantly reduced MLR stimulatory capacity of CypA-induced DCs in both CD8⁺ T cells and CD4⁺ T cells (P < 0.05). Conclusions. CypA enhances DC differentiation and maturation by up regulating CD11b and CD11c expression. CypA can also augment DC antigen uptake and antigen presentation, which may be mediated by the P38 signaling pathway.     

Recruitment of osteoclast precursors by stromal cell derived factor-1 (SDF-1) in giant cell tumor of bone.

Giant cell tumor (GCT) of bone is a unique bone lesion that is characterized by an excessive number of multinucleated osteoclasts. GCT consists of neoplastic stromal cells, multinucleated osteoclasts and their precursors, thus serving as a naturally occurring human disease model for the study of osteoclastogenesis. It still remains unclear how stromal cells of GCT recruit osteoclast precursors. In the present study, we characterized the cellular components of GCT and confirmed the presence of CD14(+)-monocytes/CD68(+)-macrophages and CD34(+)-hematopoetic stem cells that express CXCR4, a specific receptor for SDF-1; SDF-1 gene expression and presence of SDF-1 protein were confirmed by real time RT-PCR, in situ hybridization, and immunohistochemistry in the GCT tissue and cultured cells. SDF-1 was present at 25-50 ng/ml in the conditioned media from the GCT cultures, which is in the range of physiological chemotactic concentration. Migration of osteoclast precursors was 2.5-fold higher in response to GCT conditioned media compared to the control media; and migration was inhibited by an average of 36% with anti-SDF-1 neutralizing antibody or competing recombinant SDF-1. These results suggest that SDF-1 is one of the significant chemoattractant factors involved in the recruitment of hematopoietic osteoclast precursor cells during tumor-induced osteoclastogenesis.     

Profilin 1 overexpression in renal cell carcinoma

Objectives: To gain information about overexpressed antigens in renal cell carcinoma (RCC) by using a chemical proteomics approach. Methods: RCC cell line 769P was cultured and proteome analysis was subsequently carried out in the culture supernatants. By using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and tandem mass spectrometry (LC‐MS/MS), proteins in the culture supernatants were searched. A MEDLINE search to define the functions of the identified proteins was carried out. Results: Four differentially regulated proteins (profilin 1, amyloid beta A4 protein [APP], proprotein convertase subtilisin/kexin type 1 inhibitor [ProSAAS], galectin‐3‐binding protein [LGALS3BP]) were selected. These were not overexpressed in normal kidney tissue or reported in RCC. Their levels were measured through western blotting of normal kidney and RCC tissues. No differences were observed in the expression levels of APP, ProSAAS or LGALS3BP between RCC and normal kidney tissues. Profilin 1 was overexpressed in RCC tissue. On the basis of this observation, an immunohistochemical analysis of profilin 1 in normal kidney and RCC tissues was carried out. In normal tissues, tubules that were sources of RCC stained positive for profilin 1. In RCC tissue, in contrast, the stromal cells in the tumors stained positive. Conclusions: Profilin 1 can be a key element in the pathological processes of RCC, such as tumorigenesis and/or tumor growth. Thus, it has the potential to serve as a diagnostic or progression biomarker and therapeutic target in RCC.     

Lipopolysaccharide transiently activates THP-1 cell adhesion

Lipopolysaccharide stimulation of adherent THP-1 cells induces morphological changes that are associated with the reorganization of the actin cytoskeleton. We hypothesized that LPS would also increase THP-1 cell adhesion and sought to determine the signaling mechanisms regulating this response. We show that LPS significantly increases THP-1 cell attachment after 1 h, supporting the idea that LPS can stimulate integrin function. By 4 h however, the number of adherent cells returned to control levels. Importantly, detached cells were determined to be viable by propidium iodide staining, indicating that the increase in cell adhesion was transient. LPS-induced adhesion to fibrinogen- but not fibronectin-coated wells was also transient, suggesting that adhesion reflected beta2 integrin activation. This idea was supported by the fact that LPS-induced adhesion could be blocked by a function-blocking anti-beta2 integrin antibody. Interestingly, the protein tyrosine phosphatase (PTP) inhibitor, phenylarsine oxide, prevented cell detachment. Taken together, these data suggest that LPS-mediated integrin activation is transient and can be regulated by PTP-mediated signaling events.