Lipoxin A_4通过调节肿瘤相关巨噬细胞对肝癌细胞株HepG2 microRNA表达谱的影响

肿瘤与炎症密切相关,作者以往已证实内源性促炎症缓解介质脂氧素A4(Lipoxin A4,LXA4)能在整体和细胞水平发挥抗肝癌细胞增殖和转移的作用。为进一步探讨LXA4通过调节肿瘤相关巨噬细胞对肝癌细胞株HepG2 microRNAs(miRNAs)表达谱的影响,该文首先提取经脂多糖(LPS)或LPS+LXA4作用24 h的人巨噬细胞株U937培养上清液,分别称为ACM或LCM,以模拟肿瘤的炎症微环境,并用此上清液刺激HepG2细胞,24 h后提取细胞总RNA,采用microRNA芯片miRCURYTM LNA Array(V16.0)检测,计算各样本中的miRNAs标准值及比值。以两组间Hy3荧光标记信号强度的比值≤0.5或≥2为标准判定差异表达miRNA。Real-time PCR检测hsa-miR-623的相对含量以验证基因芯片的结果。结果发现,与对照组细胞相比,经过ACM作用24 h的HepG2细胞有35个miRNAs上调、130个miRNAs下调。LCM组与ACM组相比,HepG2细胞有185个miRNAs上调、71个miRNAs下调。Real-time PCR检测的结果证实,hsa-miR-623的变化与基因芯片趋势一致。综上所述,LXA4能通过肿瘤相关巨噬细胞而间接发挥其调节HepG2细胞miRNAs表达谱的作用。     

神经肽Y 对炎症及低氧环境下不同癌细胞活力的影响

目的:研究神经肽Y对炎症反应及其对在低氧培养条件下的不同癌细胞活力的影响,探讨应激影响癌症发展的机制.方法:不同浓度神经肽Y(0,10-12M,10-10M,10-8M)与100 ng/ml LPS共孵育巨噬细胞24h后检测NO的浓度及iNOS表达的变化;不同浓度神经肽Y(0,10-9M,10-8M)刺激低氧培养条件下的肝癌细胞株HepG2与乳腺癌细胞株MCF-7 36h,采用cck-8检测细胞活力变化;不同浓度神经肽Y(0,10-9M,10-8)与LPS共孵育巨噬细胞24 h后取上清液离心,采用上清液培养两种癌细胞,并置于低氧条件下36h,cck-8检测细胞活力.结果:实验结果显示,神经肽Y可以抑制巨噬细胞NO的释放(P＜0.05),并降低iNOS的表达(P＜0.05);单独的神经肽Y对低氧培养条件下两种癌细胞的活力没有明显影响(P＞0.05);但在低氧培养条件中,相比于LPS组的条件培养基,LPS加神经肽Y组的条件培养基可明显增强MCF-7的活力(P＜0.05,P＜0.01),而HepG 2的活力则没有统计学差异.结论:神经肽Y可能通过抑制炎症反应,从而增强乳腺癌细胞MCF-7在低氧环境下的活力.     

胃癌中显著下调的miR-433的作用靶点研究

目的为了筛选胃癌中miRNAs的表达标记,验证胃癌相关miRNAs的作用靶点,建立一种新的诊断和治疗胃癌的方法。方法运用基因芯片技术检测3个正常胃组织标本,24个胃癌组织标本,胃癌细胞SGC7901和正常胃黏膜细胞GES-1中328个miRNAs的表达情况。用以上方法检测出在胃癌组织和SGC7901中,miR-433的表达水平显著下调。为了确保结果的准确性,采用实时荧光定量PCR对其进行验证。并用基因克隆和Western印迹方法分析miR-433的作用靶点。结果共有26个miRNAs在胃癌标本（包括24个胃癌组织和SGC7901）中异常表达。其中19个miRNAs下调,7个miRNAs上调。实时荧光定量PCR检测出miR-433在胃癌标本中的表达水平显著下调,该结果和基因芯片检测结果一致。另外,在本实验中发现miR-433与Grb2（growth factor receptor—bound protein 2）的表达呈负相关。结论胃癌相关miRNAs已进行了初步筛选。其中,miR-433可能是胃癌中的标记性miRNAs之一,Grb2是其作用靶点。这为建立新的以miRNAs为基础的诊断和治疗胃癌的方法提供了相关信息。     

草木犀石油醚提取物对小鼠巨噬细胞促炎介质的影响

目的研究草木犀石油醚提取物在体外的抗炎作用。方法采用小鼠巨噬细胞系RAW264．7建立炎症细胞模型,加入10μg/L的LPS培养液和不同浓度的草木犀石油醚提取物进行干预。ELISA法检测上清液中TNF-α,IL-1β,IL-6和NO的分泌量;实时荧光定量RT-PCR检测TNF-α,iNOS和COX-2的mRNA表达;Western印迹法检测COX-2蛋白的表达。结果草木犀提取物干预后细胞所分泌的炎性介质（TNF—α,IL-1β,IL-6和NO）与模型组相比均显著降低（P〈0．01）,并存在剂量依赖关系;RT-PCR结果显示干预后细胞TNF-α,iNOS和COX-2的mRNA表达水平显著降低（P〈0．01）,也存在剂量依赖关系;Western印迹结果显示草木犀石油醚提取物及地塞米松干预后COX-2蛋白水平明显降低（P〈0．01）。结论草木犀的石油醚提取物通过下调LPS诱导的巨噬细胞表达炎性介质而发挥其体外抗炎作用,且其下调作用呈剂量依赖性。     

结直肠癌组织异常表达miRNA的鉴定

目的：筛选结直肠癌组织异常表达的miRNAs。方法：采用Agilem基因芯片（V12．0）分析结直肠癌组织及其配对正常粘膜组织间差异表达的miRNAs,MiRNAs错误发生率（FDR）〈0．05和微矩阵显著性分析（SAM）q值〈0．05为差异显著。结果：鉴定出结直肠癌中32个差异表达的miRNAs,显著上调和下调各16个。实时定量PCR（RT—qPCR）证实基因芯片中4个表达上调的miRNAs在结直肠癌组织中也显著上调。结论：MiRNA基因芯片鉴定出了结直肠癌组织一系列新的差异表达的miRNAs。     

血小板反应蛋白4通过诱导肿瘤相关巨噬细胞M2型极化促进肝癌细胞转移

血小板反应蛋白4 (thrombospondin 4, THBS4)属于THBS家族成员,是细胞外基质分泌的蛋白质,参与调控细胞增殖、黏附及血管生成等多种生理过程。近来研究表明,机体在炎症刺激下加速产生THBS4并诱导巨噬细胞粘附与积累。我们的前期研究证实,THBS4在肝癌(hepatocellular carcinoma, HCC)中发挥促癌作用,但THBS4对肝癌免疫微环境的影响尚不明确。本文旨在分析THBS4通过诱导肿瘤相关巨噬细胞M2型极化,促进肝癌细胞转移的作用。通过肝癌条件培养基(HCC conditioned medium, HCM)模拟肿瘤微环境,发现在HCM作用下巨噬细胞中THBS4表达呈时间依赖性升高(P<0.05);下调THBS4促使M1型巨噬细胞标志物IL-1β、CD86的表达升高(P<0.01),而M2型标志物IL-10和CD206表达降低(P<0.01)。进一步通过Transwell共培养实验检测THBS4诱导的M2型巨噬细胞对肝癌转移的影响。将下调THBS4的M2型巨噬细胞(M2-TAMs)与HepG2肝癌细胞进行共培养。结果显示,下调T...     

CD 36和胞外信号调节激酶通路在脂多糖诱导炎症因子中的作用研究

本研究以鼠源巨噬细胞RAW264．7为模型,研究CD36和胞外信号调节激酶（ERK ）通路对脂多糖（LPS）诱导巨噬细胞分泌炎症因子的影响。首先用100 ng／ml LPS刺激正常及小干扰RNA （siRNA ）技术沉默CD36表达的巨噬细胞16 h ,检测巨噬细胞的ERK活性及分泌炎症因子如肿瘤坏死因子α（TNF‐α）、白细胞介素6（IL‐6）和IL‐10的水平;继而以20 nmol／L ERK抑制剂处理细胞,再用LPS刺激,检测以上各项指标的变化,进一步明确ERK通路与LPS诱导巨噬细胞分泌炎症因子的相关性。结果显示,经LPS刺激,巨噬细胞的ERK活性显著增强,分泌的促炎因子 TNF‐α和 IL‐6显著增高,抑炎因子 IL‐10水平无明显变化;与CD36正常表达的巨噬细胞相比,CD36表达下降的巨噬细胞ERK活性及促炎因子TNF‐α、IL‐6水平显著下降,抑炎因子IL‐10显著增多。与未处理组相比,ERK抑制剂预处理的巨噬细胞中LPS诱导的ERK活性显著降低,促炎因子 TNF‐α和 IL‐6水平降低,抑炎因子 IL‐10水平升高。结果提示,LPS能通过其受体———CD36,激活巨噬细胞内ERK活性,进而促进巨噬细胞促炎因子的分泌。     

冬凌草甲素缓解脂多糖诱导的Caco-2肠上皮细胞屏障损伤和SIRT1/eIF2α信号通路活性的抑制

目的 探索冬凌草甲素对脂多糖（lipopolysaccharide, LPS）诱导的肠上皮屏障障碍的影响及其生物学机制。方法将Caco-2细胞进行单层培养21 d使其分化后，分为对照组、LPS组（2μg/mL LPS处理24 h）、LPS+低剂量冬凌草甲素组（10μg/mL冬凌草甲素预处理30 min,2μg/mL LPS处理24 h）和LPS+高剂量冬凌草甲素组（40μg/mL冬凌草甲素预处理30 min,2μg/mL LPS处理24 h）。采用跨上皮电阻法和FITC-dextran 4通量法评估细胞单层屏障功能；CCK-8法检测细胞活力；乳酸脱氢酶（LDH）法检测LDH释放活性；DCFH-DA探针法检测细胞内活性氧水平；ELISA法检测炎症因子IL-1β和TNF-α分泌水平；免疫荧光法检测细胞的屏障功能相关蛋白闭合蛋白（occludin）和闭锁小带蛋白1(zonula occludens protein 1, ZO-1)的表达分布情况；Western blot法检测SIRT1和p-eIF2α的相对表达水平。结果 与LPS组比较，冬凌草甲素能改善LPS诱导的肠上皮细胞单层屏障障碍，增...     

脂多糖诱导肝细胞HepG2释放HMGB-1的研究

观察小剂量LPS(lipopolysaccharide)刺激下非坏死HepG2细胞是否存在HMGB1(high mobility group box-1 protein)移位及释放.以终浓度为100μg/L的LPS作用HepG2和RAW264.7细胞0、4、8、12、16、20、24h.LPS作用16~24h,HepG2和RAW264.7细胞培养上清中均可以检测到明显的HMGB1,与对照组差别有显著性(P<0.05).而MTT法和上清中LDH(lactate dehydrogenase)含量测定显示,LPS处理24h的HepG2存活率仍然非常高.免疫荧光观察到HMGB1的释放伴随着其从细胞核向胞浆的移位.由此可见,经LPS诱导,非坏死状态的HepG2细胞可发生HMGB1的移位及释放.     

TLR4 对人骨骼肌细胞炎症因子表达及胰岛素抵抗的影响

目的:研究TLR4对脂多糖(LPS)及Polymymin B(PMB)作用下的人骨骼肌细胞的炎症因子表达的影响及其在细胞胰岛素抵抗中的作用。方法:通过脂多糖(LPS)及Polymymin B(PMB)干预骨骼肌细胞24h,再用胰岛素刺激1h后,Real-time PCR检测检测骨骼肌细胞TLR4、MyD88、TNF-αmRNA的表达;Western blot检测TLR4,Myd88和CRP的表达;葡萄糖氧化酶法(GOD-POD法)检测细胞培养液中葡萄糖浓度。结果:TLR4高表达可以使炎症因子的表达增高,细胞培养液中的葡萄糖浓度增高;TLR4低表达可使炎症因子的表达降低,细胞培养液中的葡萄糖浓度没有明显变化。结论:TLR4调控了炎症因子的表达,继而可以引起胰岛素敏感性的改变,影响了胰岛素抵抗的发生。     

Role of lipoxin A4 in the cell-to-cell interaction between all-trans retinoic acid-treated acute promyelocytic leukemic cells and alveolar macrophages

Differentiation therapy with all-trans retinoic acid (ATRA) has been used successfully to treat acute promyelocytic leukemia (APL), but such treatment also causes differentiation syndrome (DS) by inducing APL cell infiltration into alveolar spaces. The mechanism underlying the clearance of infiltrated APL cells has not been investigated in detail. Lipoxin A(4) (LXA(4)) is an important anti-inflammatory mediator during the resolution of inflammation. In this study, the role of LXA(4) in the cell-cell interaction between alveolar macrophages (AMφ; NR8383 cells) and APL NB4 cells was investigated and found that conditioned medium (CM) harvested from ATRA-treated NR8383 (ATRA-NR8383) cells was able to induce the transmigration of ATRA-NB4 cells. However, the pro-migratory activity of CM was attenuated progressively when ATRA-NR8383 cells were co-cultured with increased cell dosages of apoptotic NB4 cells. A significantly higher amount of LXA(4) was released into the CM by ATRA-NR8383 cells when they were co-cultured with apoptotic ATRA-NB4 cells. Expression of a receptor for LXA(4) (ALX/FPR2) was enhanced in both ATRA-NB4 cells and ATRA-NR8383 cells. Exogenous LXA(4) treatment was able to inhibit the transmigration of ATRA-NB4 cells and induce the phagocytic clearance of apoptotic cells by ATRA-NR8383 cells. The anti-migratory activity of exogenous LXA(4) was attenuated by pre-treating ATRA-NB4 cells with an ALX/FPR2 inhibitor. We conclude that AMφ-derived LXA(4) plays an important role in the interaction between AMφ and APL cells and that this contributes to clearance of apoptotic APL cells.     

幽门螺杆菌感染细胞模型的建立及感染相关microRNAs的筛选鉴定

目的：建立稳定的幽门螺杆菌（H.pylori）感染人胃上皮细胞模型;筛选并鉴定H.pylori感染相关microRNAs（miRNAs）的表达,为深入研究感染相关miRNAs的调控作用机制奠定基础。方法：将H.pylori标准株按MOI=100：1感染人胃上皮细胞,通过检测炎性细胞因子及炎症反应关键酶的表达综合评价感染模型;采用博奥公司miRNAs V3.0芯片分析细胞感染前后miRNAs表达谱变化,运用实时定量PCR技术和Northern杂交对表达显著差异的miRNAs进行分析鉴定。结果：H.pylori感染细胞24 h后,细胞分泌促炎细胞因子IL-8显著升高（P〈0.01）;启动炎症反应的关键酶COX-2的表达明显增加。芯片数据显示：H.pylori感染引起超过2倍显著差异表达的miRNAs包括：表达上调的PREDICTED-MIR191、miR-155、miR-92b、miR-30b、miR-146a、miR-16等,和表达降低的miR-181b、miR-324。实时定量PCR和Northern杂交结果显示感染相关miR-155和miR-146a在H.pylori感染细胞模型中表达均显著增加（P〈0.01）。结论：miR-155和miR-146a在感染细胞模型中的表达增加提示二者可能参与H.pylori感染的免疫调控过程。     

Lipoxins and aspirin-triggered lipoxins in neutrophil adhesion and signal transduction

Lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 (ATL) are emerging as endogenous braking signals for neutrophil-mediated tissue injury. LXA4 and ATL and their metabolically stable analogues display potent inhibitory actions in human isolated cells and blood, including attenuation of expression of adhesion molecules on leukocytes and endothelial cells, neutrophil adhesion to endothelial cells and platelets under shear, and IL-8 production, key events of the acute inflammatory response. The underlying molecular mechanisms include interference with MAPK signaling pathways, modulation of the oxidative chemistry of superoxide, NO and ONOO-, inhibition of activation of NF-kappaB and AP-1, and consequently the expression of interleukin-8 and likely other pro-inflammatory genes. Collectively, these results add to the profile of LXA4/ATL rapid actions that contribute to "stop signaling" involved in regulating neutrophil functions during acute inflammation and suggest that aspirin inhibits neutrophil accumulation through triggering the synthesis of 15-epi-LXA4.     

Mutations of serine 236-237 and tyrosine 302 residues in the human lipoxin A4 receptor intracellular domains result in sustained signaling

Lipoxin A(4) (LXA(4)) is a potent negative modulator of the inflammatory response. The antiinflammatory activities of LXA(4), such as inhibition of agonist-induced polymorphonuclear cell (PMN) chemotaxis and upregulation of beta-2 integrins, require the expression of a G-protein-coupled, high-affinity LXA(4) receptor (LXA(4)R). We now report that stimulation of PMN with proinflammatory agonist N-formyl peptides (FMLP), calcium ionophore A(23187), or phorbol mirystate acetate (PMA) is followed by marked downregulation of LXA(4) binding (B(max) decrease of approximately 45%) and decreased activation of phospholipases A(2) (PLA(2)) and D (PLD). Elucidation of the mechanisms underlying these effects was addressed by structure-function analyses of the intracellular domains of LXA(4)R. Mutant molecule, S236/S237 --> A/G (LXA(4)R(pk)) and Y302 --> F (LXA(4)R(tk)) were obtained by site-directed mutagenesis to yield receptors lacking the putative targets for serine/threonine kinase- or tyrosine kinase-dependent phosphorylation. Expression of wild-type and mutated LXA(4)R sequences in CHO and HL-60 cells was used to examine LXA(4) ligand-receptor interactions and signal transduction events. Results indicated that cells expressing LXA(4)R(pk) or LXA(4)R(tk) displayed sustained activation of PLA(2) and PLD in contrast to the transient ones obtained with LXA(4)R(wt) (peak activation at 2-3 min). Moreover, inhibition of LXA(4)-dependent PLA(2) activity by PMA in LXA(4)R(wt) transfected CHO cells was not observed in cells expressing LXA(4)R(pk). Phosphopeptide immunoblotting revealed that the functional differences between wild-type and mutant LXA(4) receptors are accompanied by distinct changes in the receptor protein phosphorylation pattern. Further characterization of these and related LXA(4)R intracellular domains will help to better understand specific events that regulate the antiinflammatory activities of LXA(4).     

脂氧素A_4对大鼠急性肝衰竭的保护作用

目的观察脂氧素A4(LXA4)对大鼠急性肝衰竭的保护作用并初步探讨其机制。方法采用D-氨基半乳糖(D-GalN)+脂多糖(LPS)建立大鼠急性肝衰竭动物模型。30只大鼠随机分为四组:正常组、模型组、脂氧素组、四氢化吡咯二硫代氨基甲酸酯(PDTC)组。肝组织病理切片观察肝组织损伤情况;自动生化仪检测肝功能;ELISA法检测血清TNF-α及IL-6水平;免疫组化法检测肝组织核因子κB(NF-κB)表达。结果大鼠急性肝衰竭时,肝组织炎症坏死明显,肝脏转氨酶明显升高,血清IL-6及TNF-α水平显著升高,肝组织NF-κB表达明显增强;脂氧素组及PDTC组组织学改变均明显好转,肝脏转氨酶、细胞因子及NF-κB表达明显下降(与模型组比较,P0.01,);脂氧素组及PDTC组比较,上述指标之间P0.01。结论 LXA4对大鼠急性肝衰竭有明显的保护作用,这种保护作用部分是通过阻断肝组织NF-κB活化,减少促炎细胞因子的释放来实现的。     

LXA4 stimulates ZO-1 expression and transepithelial electrical resistance in human airway epithelial (16HBE14o-) cells

Lipoxin A(4) (LXA(4)) is a biologically active eicosanoid produced in human airways that displays anti-inflammatory properties. In cystic fibrosis and severe asthma, LXA(4) production has been reported to be decreased, and, in such diseases, one of the consequences of airway inflammation is disruption of the tight junctions. In the present study, we investigated the possible role of LXA(4) on tight junction formation, using transepithelial electrical resistance (TER) measurements, Western blotting, and immunofluorescence. We observed that exposure to LXA(4) (100 nM) for 2 days significantly increased zonula occludens-1 (ZO-1), claudin-1, and occludin expression at the plasma membrane of confluent human bronchial epithelial 16HBE14o- cells. LXA(4) (100 nM) stimulated the daily increase of the 16HBE14o- cell monolayer TER, and this effect was inhibited by boc-2 (LXA(4) receptor antagonist). LXA(4) also had a rapid effect on ZO-1 immunofluorescence at the plasma membrane and increased TER within 10 min. In conclusion, our experiments provide evidence that LXA(4) plays certainly a new role for the regulation of tight junction formation and stimulation of the localization and expression of ZO-1 at the plasma membrane through a mechanism involving the LXA(4) receptor.     

Synthesis of lipoxins and other lipoxygenase products by macrophages from the rainbow trout, Oncorhynchus mykiss

Rainbow trout macrophages maintained in short term culture when incubated with either calcium ionophore, A23187, or opsonized zymosan synthesize a range of lipoxygenase products including lipoxins and leukotrienes. These cells are unusual in that they generate more lipoxin than leukotriene following such challenge. The main lipoxin synthesized was lipoxin (LX) A4. This compound was identified by cochromatography with authentic standard during reversephase high performance liquid chromatography, by ultra violet spectral analysis, radiolabeling following incorporation of [14C]arachidonic acid substrate into macrophage phospholipids, and gas chromatography electron impact mass spectrometry of the methyl ester, trimethylsilyl ether derivative. Other 4-series lipoxins synthesized by trout macrophages were identified as 11-trans-LXA4, 7-cis-11-trans-LXA4, and 6(S)-LXA4. These cells also produced 5-series lipoxins tentatively identified as LXA5, 11-trans-LXA5 and possibly 6(S)-LXA5. No LXB4 or LXB5 was, however, detected. The dynamics of leukotriene and lipoxin release were also determined. Lipoxin generation was slower than leukotriene generation the latter reaching a maximum after 30 min of exposure to ionophore (5 microM, 18 degrees C) compared with 45 min for the former.     

HA纳米载体介导转hGM-CSF基因的HepG2疫苗诱导的抗瘤效应研究

目的：观察HA纳米颗粒载体介导转染hGM-CSF基因的HepG2细胞疫苗体外抗肿瘤效应,为hGM-CSF基因修饰的HepG2细胞疫苗的临床应用提供依据。方法：HA纳米颗粒载体介导hGM-CSF基因转染HepG2细胞制备转GMCSF基因的HepG2细胞疫苗。密度梯度离心法分离人PBMC,体外诱导人PBMC。WST-1法测定PBMC的增殖活性及对HepG2细胞的杀伤效应,流式细胞术分析CD4＋和CD8＋的阳性表达率,ELISA法测定INF-γ的分泌。结果：WST-1结果显示,转基因HepG2组疫苗能诱导PBMC增殖,其增殖率优于野生型疫苗（p〈0.05）;其诱导的PBMC对HepG2的杀伤率高于各野生型疫苗组和各空白对照组（p〈0.05）。FCM结果显示,转基因HepG2疫苗组PBMC中CD4＋和CD8＋阳性表达率均高于各野生型疫苗组和各空白对照组（p〈0.05）。ELISA结果显示,转基因组PBMC培养上清中IFN-γ含量为1989.76±254.21pg/ml,高于各野生型疫苗组和各空白对照组（p〈0.05）。结论：HA纳米颗粒载体介导转染hGM-CSF基因能增加HepG2细胞疫苗的免疫原性,转hGM-CSF基因HepG2细胞疫苗可有效诱导PBMC增殖、分化,增加INF-γ的分泌,提高其对HepG2细胞的杀伤作用。     

Clotrimazole presents anticancer properties against a mouse melanoma model acting as a PI3K inhibitor and inducing repolarization of tumor-associated macrophages

The immune system is a key component of tumorigenesis, with the latter promoting the development of cancer, its progression and metastasis. In fact, abundant infiltration of tumor-associated macrophages (TAM), which are M2-like macrophages, has been associated with a poor outcome in most types of cancers. Here, we show that lactate produced by murine melanoma B16F10 cells induces an M2-like profile in cultured macrophages. Further, we demonstrate that clotrimazole (CTZ), an off-target anti-tumor drug, abolishes lactate effects on the activation of macrophages and induces the expression of M1-like markers. We show that clotrimazole has cytotoxic effects on tumor cells by negatively modulating PI3K, which inhibits glycolytic metabolism and leads to a diminishing lactate production by these cells. These effects are more pronounced in cancer cells exposed to conditioned media of M2-polarized macrophages. Moreover, clotrimazole inhibits tumor growth in a murine model of implanted melanoma, reduces lactate content in a tumor microenvironment and decreases vascular endothelial growth factor expression. Finally, clotrimazole drastically diminishes TAM infiltration in the tumors, thereby inducing M1 polarization. Collectively, these findings identify a new antitumor mechanism of clotrimazole by modulating the tumor microenvironment (TME), particularly the activation and viability of TAM.     

转hGM-CSF基因HepG2细胞疫苗侵袭性和生长活性初步检测

目的：探讨经60Co处理的转hGM-CSF基因的HepG2肝癌疫苗的侵袭性和生长活性变化。方法：体外培养三种肝癌细胞（①野生型HepG2肝癌细胞②转染hGM-CSF基因的HepG2肝癌细胞③60Co射线处理的转hGM-CSF基因的HepG2肝癌疫苗）采用MTT方法检测三种细胞在24h、48h、72h的OD值并绘出生长曲线;利用transwell小室进行体外侵袭实验来观察上述三种细胞侵袭性;用RT-PCR技术检测上述三种细胞基质金属蛋白酶2（MMP-2）在mRNA水平上表达的变化;结果：经60Co照射处理的转hGM-CSF基因的HepG2肝癌疫苗组OD值在相同培养时间点较其他两组明显变小且差异有显著性（P〈0.05）。三种细胞（上述①②③种细胞）transwell侵袭试验显示：③组穿过人造基底膜的细胞数量明显少于前两组;PT-PCR示：③组细胞的MMP-2的mRNA的表达明显低于①②。结论：经过60Co处理过的转hGM-CSF基因的HepG2肝癌疫苗的侵袭性和生长活性均明显降低。