肺癌脑转移型A549／GFP-2细胞的筛选及其条件液对脑微血管内皮细胞的作用

目的：建立肺癌脑转移模型,筛选脑转移倾向细胞A549／GFP-2,探讨A549和A549／GFP-2条件液对脑微血管内皮细胞的作用,揭示肺癌脑转移的机制。方法：利用胸腔原位注射法筛选出A549脑转移细胞亚型A549／GFP-2,原代培养大鼠脑微血管内皮细胞,观察A549和A549／GFP-2细胞条件液对脑微血管内皮细胞增殖的影响和细胞内HIF-1α和VEGF表达的改变。结果：胸腔内原位种植较好地反应了临床肺癌脑转移的过程。不同浓度的A549和A549／GFP-2细胞条件液对脑微血管内皮细胞增殖的影响不同,低浓度（〈30％）对脑微血管内皮细胞有促进的作用;高浓度（〉60％）对脑微血管内皮细胞的增殖有不同程度的抑制作用,且有随浓度增加抑制作用增强的趋势。A549和A549／GFP-2细胞条件液能提高脑微血管内皮细胞内HIF-1α和VEGF的表达。结论：胸腔内原位种植是建立肺癌脑转移的稳定模型。肺癌脑转移与肺癌细胞在生长过程中分泌的HIF-1α和VEGF等细胞因子破坏了脑微血管内皮细胞的结构有关。     

缺氧对大鼠脑微血管内皮细胞增殖和凋亡的影响及其机制研究

目的:探讨缺氧对大鼠脑微血管内皮细胞增殖和凋亡的影响及其可能的分子机制。方法:从2周龄的SD大鼠中提取脑微血管内皮细胞。体外模拟脑缺氧微环境,将体外培养的脑微血管内皮细胞分别置于常氧(21%O_2)和低氧环境(1%O_2)下处理6、12和24小时,采用四甲基偶氮唑盐(MTT)法观测不同时间点细胞增殖能力的变化;用AnnexinV-FITC/PI双标流式细胞术观察不同时间点细胞凋亡情况;Real time-PCR和Western blot法检测细胞中(Hypoxia-inducible factor-1α) HIF-1αm RNA和蛋白的表达。进一步使用HIF-1αsi RNA靶向沉默HIF-1α基因,再检测低氧环境下HIF-1αm RNA和蛋白表达、细胞增殖以及凋亡水平的变化。结果:随着低氧处理时间的延长,大鼠脑微血管内皮细胞的增殖能力被显著抑制,同时凋亡水平显著增加,HIF-1αm RNA和蛋白表达水平显著升高。使用HIF-1αsi RNA特异性阻断HIF-1α表达后,低氧环境下HIF-1αm RNA和蛋白表达明显降低,同时细胞活性增加,细胞凋亡率显著下降。结论:缺氧微环境能够通过上调HIF-1α的表达抑制大鼠脑微血管内皮细胞增殖并促进其凋亡。     

脑微血管内皮细胞氧糖剥夺条件培养液对PC12细胞活性的影响

目的:观察脑微血管内皮细胞氧糖剥夺后条件培养液对PC12细胞的影响.方法:原代培养大鼠脑皮质微血管内皮细胞,传至三代.制备其正常、氧糖剥夺、复糖复氧三种状态条件液,并观察内皮细胞在这三种状态下的形态改变;将这三种条件培养液作用于PC12细胞,分4组:正常对照组(Normal)、正常内皮细胞条件液组(N-CM)、氧糖剥夺内皮细胞条件液组(I-CM)、复糖复氧内皮细胞条件液组(R-CM).每组分别设6h、12 h、24 h、48 h、72 h、96 h、120 h、144 h 8个时间点,MTS/PMS法检测三种脑微血管内皮细胞条件液作用后的不同时间点PC12细胞活性的变化.结果:氧糖剥夺后脑微血管内皮细胞发生核固缩等明显病理改变,复糖复氧后这些改变有所恢复.N-CM组、R-CM组与相应时间点的Normal组PC12细胞活性相比差异均有统计学意义(P＜0.01).正常和复糖复氧内皮细胞条件液显著抑制了PC12细胞的增殖和活性.N-CM组、R-CM组与相应时间点的I-CM组PC12细胞活性相比差异均有统计学意义(P＜0.01).结论:正常脑微血管内皮细胞条件液抑制了PC12细胞的活性,氧糖剥夺后的脑微血管内皮细胞务件液抑制效应消失,复糖复氧后,这种抑制效应也同时恢复.     

沙利度胺抑制肿瘤细胞分泌VEGF/bFGF机制的探讨*

目的: 探讨Cereblon(CRBN)对沙利度胺抑制人肺癌A549细胞及人肝癌HepG2细胞分泌VEGF/bFGF的影响。方法: 采用慢病毒介导的短发夹RNA(shRNA)干扰技术建立稳定敲低CRBN的A549细胞系(A549_CRBN)及HepG2细胞系(HepG2_CRBN)并通过实时定量PCR(Real-time PCR)和蛋白质印记(Western blot)实验验证。将A549细胞分为阴性对照组(A549_luciferase)、CRBN低表达组(A549_CRBN);HepG2细胞分为阴性对照组(HepG2_luciferase)、CRBN低表达组(HepG2_CRBN),以上细胞按照 3×10⁵ cells/well接种到6孔板中,放入37℃,5%CO2的培养箱中培养24 h,分别加入1 ml含100 μmol/L沙利度胺(thalidomide组)和1 ml 1‰ DMSO(control组)的培养液,继续培养24 h再行后续实验,每组设计3个复孔。MTS法检测沙利度胺对细胞增殖的影响;Real-time PCR检测VEGF、bFGF、c-jun mRNA表达,ELISA法检测VEGF、bFGF蛋白表达。结果: 与对照组比较,沙利度胺在浓度为1、10、50、100 μmol/L 时对A549 及HepG2细胞的增殖能力无显著影响(P＞0.05)。与A549_CRBN或HepG2_CRBN组比较,A549_luciferase及HepG2_luciferase组分泌的VEGF及bFGF均显著降低(P＜0.05)。与A549_luciferase或HepG2_luciferase细胞的对照组比较,沙利度胺可抑制A549_luciferase和HepG2_luciferase细胞的VEGF和bFGF的表达(P＜0.05),而对A549_CRBN和HepG2_CRBN细胞中VEGF和bFGF的表达无显著抑制作用;与HepG2_luciferase细胞的对照组比较,沙利度胺可抑制HepG2_luciferase细胞的c-Jun表达(P＜0.01),而对HepG2_CRBN细胞的c-Jun表达无显著抑制作用。结论: 沙利度胺对A549和HepG2细胞VEGF和bFGF表达的抑制作用可能是通过CRBN介导的,而c-Jun可能是抑制作用的关键转录因子之一。     

RGD肽对肺癌A549 细胞增殖侵袭能力的影响及其机制研究

目的:研究RGD肽对肺癌A549细胞增殖凋亡及侵袭迁移的影响,并探讨其作用机制。方法:不同浓度RGD肽处理肺癌A549细胞后,MTT检测肺癌细胞的增殖能力,流式细胞仪检测肺癌细胞凋亡及周期分布,Transwell检测其迁移及侵袭能力的变化,Western blot检测RGD肽对肺癌A549细胞MMP2、MMP9的表达水平影响。结果:当RGD肽浓度增加至50 mg/L时,肺癌A549细胞增殖明显受到抑制,且这种抑制作用呈剂量依赖关系;RGD肽组A549细胞G0/G1期细胞比例增高,细胞凋亡率由(6.1±0.1)%增至(15.2±0.5)%;在迁移和侵袭试验中,RGD肽组A549细胞的穿膜细胞数分别由123±10和43±10降至45±5和18±5;RGD肽组A549细胞MMP2、MMP9表达水平显著降低。结论:RGD肽对肺癌A549细胞的增殖有明显抑制作用,并促进其凋亡,可能与RGD肽改变其周期分布有关,RGD肽可明显抑制A549细胞的迁移及侵袭,可能与其下调MMP2、MMP9的表达相关。     

姜黄素对A549肺癌细胞增殖、侵袭和迁移的抑制作用及与Keap1/Nrf2信号通路的关系

目的探究姜黄素对肺癌A549细胞增殖、侵袭和迁移的抑制作用及其机制。方法通过MTT法检测姜黄素对A549细胞增殖能力的影响;通过流式细胞术测定姜黄素对A549细胞凋亡的调节作用;通过Transwell试验,观察姜黄素对肺癌A549细胞的侵袭和迁移能力的影响;采用Western blot实验和RT-PCR实验,观察姜黄素对其Keap1/Nrf2信号通路的调节作用。结果增殖实验、凋亡实验和Transwell实验显示,姜黄素能够显著性抑制癌细胞的增殖、侵袭和迁移,并能够促进其凋亡。Western blot检测显示姜黄素能够显著抑制Keap1和Nrf2表达;RT-PCR实验结果显示姜黄素能够显著抑制Keap1mRNA和Nrf2 mRNA表达。结论姜黄素可能通过抑制Keap1/Nrf2信号通路蛋白的表达而抑制肺癌A549细胞的增殖、迁移和侵袭,并促进其凋亡。     

低氧处理不同时间对人肺腺癌A549细胞增殖的影响 及相应缺氧模型探讨*

目的:观察低氧处理不同时间对人肺腺癌A549细胞增殖的影响,探讨合理的人肺腺癌细胞株A549体外模拟缺氧时间。方法:将人肺腺癌细胞A549细胞株在低氧环境下分别培养12 h、24 h、48 h、72 h,设置常氧对照组,通过CCK8法测定A549细胞存活率,RT-PCR和免疫印迹分别检测细胞缺氧诱导因子-1α(hypoxia-inducible factor-1α, HIF-1α)和血管内皮生长因子(vascularendothelial growth factor, VEGF)mRNA及蛋白的表达。结果:低氧24 h组A549细胞存活率最高,低氧48 h、72 h组A549细胞存活率呈时间依赖性明显下降(P0.001)。自低氧12 h起,A549细胞HIF-1αmRNA和VEGFmRNA的表达开始随低氧时间延长而显著增加(P均0.001);HIF-1α和VEGF蛋白表达自24 h开始随低氧时间延长而显著增加(P均0.001)。结论:低氧诱导的A549细胞存活率呈时间依赖性降低,而HIF-1α、VEGF表达呈时间依赖性增高,人肺癌细胞株A549缺氧模型最适时间为24 h。     

通络救脑注射液对脑微血管内皮细胞活性影响的特征

目的：观察通络救脑注射液对培养的正常及缺血脑微血管内皮细胞的活性影响。揭示其通络作用的效应靶点与特征。方法：原代培养大鼠脑皮质微血管内皮细胞,传至第三代。分为正常及拟缺血组,采用培养基氧糖刺夺（OGD）法建立拟缺血模型。通过四甲基偶氮唑盐（MTT）比色分析法测定不同浓度的通络救脑注射液对正常及OGD内皮细胞的活性影响。结果：通络救脑注射液作用于正常脑微血管内皮细胞,与未加药组比较,小剂量药物抑制细胞活性趋势,大剂量促进细胞活性趋势,剂量总趋势呈反抛物线形;通络救脑注射液作用于OGD组脑微血管内皮细胞,小剂量范围促进内皮细胞的增殖活性,呈显著和极显著差异,而大剂量组则抑制细胞活性,剂量总趋势呈抛物线形。结论：通络救脑注射液对正常及缺血脑微血管内皮细胞具有双向调节作用,药物剂量与细胞增殖活性呈非线性关系。天剂量与小剂量可能是不同的作用机制,反映了中药复方药效的多维性。     

胸腔原位种植与经左心室注射建立肺癌脑转移动物模型的比较

目的为研究肺癌脑转移机制提供一种可靠的造模方法。方法 18只BALB/c nude裸鼠随机分为2组,分别经胸腔原位种植与经左心室注射的方法,接种处于对数期生长的人肺腺癌PC-9细胞(1×106/0.1 m L),接种后观察裸鼠状态,在裸鼠出现严重恶液质时处死。解剖裸鼠,观察肺、脑、肝、肾转移情况;病理取材、HE染色观察。结果胸腔原位种植组:3周后,第4、6、9号裸鼠可见胸壁瘤结凸起形成,渐增大;裸鼠于第4~6周开始出现体重减轻,并逐渐出现恶液质,分别于第5~7周处死。开胸后见:胸腔广泛灰白色肿瘤结节、团块形成,双侧肋骨、胸膜、脊柱多发种植灶,双肺被侵蚀压缩,颜色苍白,形态改变。HE染色见:肺表面广泛种植瘤形成,与正常肺组织分界清楚;仅6号裸鼠出现脑转移。经左心室注射组:裸鼠于第3周开始出现体重下降,并逐渐出现恶液质,全部裸鼠于第4周处死。开胸后:除11、18号裸鼠胸壁见2~3个散在瘤结分布(直径约1~3 mm),其余胸腔视野正常;肺组织轮廓清楚,未见瘤结生成。HE染色见:9只裸鼠均出现大小不一的多发脑转移灶。胸腔原位种植组:脑转移率为11.1%;经左心室注射组:脑转移率为100%。结论经左心室注射建立肺癌脑转移动物模型的方法,较胸腔原位种植的方法保证了更高的脑转移率。     

脑微血管内皮细胞条件培养液对星形胶质细胞的影响及通络中药的干预特征

目的:揭示脑微血管内皮细胞生理、病理及通络中药处理后不同状态的培养液对正常星形胶质细胞影响的特征,从细胞间相互作用角度探讨脑微血管内皮细胞与星形胶质细胞的生物学关系,为阐释脑微环境稳定的血脑屏障维护机制以及通络中药通过内皮细胞调节脑内微环境理论假说提供新的证据。方法:制备正常、拟缺血和拟缺血合并通络救脑注射液处理的大鼠脑微血管内皮细胞条件培养液,观察其对星形胶质细胞活性和凋亡率的影响。结果:与正常星形胶质细胞相比,正常内皮细胞条件培养液能够降低正常星形胶质细胞的活性,并促进星形胶质细胞的凋亡;而拟缺血处理的内皮细胞条件培养液能够提高正常星形胶质细胞的活性和凋亡率;拟缺血合并通络药物处理的内皮细胞条件培养液对正常星形胶质细胞的活性有提高作用,并显著降低其凋亡率。结论:三种不同处理方式的内皮细胞条件培养液对正常星形胶质细胞活性和凋亡产生不同的影响,提示不同状态的微血管内皮细胞对脑内微环境产生影响,通络救脑注射液可能通过调节微血管内皮细胞的分泌而对星形胶质细胞发挥作用。     

HIF-1-alpha激活的长链非编码RNA-ANRIL与肺腺癌的关系的研究

摘要目的：现已经证实缺氧诱导因子-1alpha（hypoxia inducible factor-1,HIF-1alpha）与肺腺癌（lung adenocarcinoma，LA）侵袭性有关。 INK4 基因座中反义非编码RNA（CDKN2B antisense RNA 1，ANRIL）是目前确认的能够促进肿瘤发生发展的一种长链非编码 RNA（long noncoding RNA，lncRNA）。而本研究即探究在肺腺癌中HIF-1琢与ANRIL之间是否存在一定的联系。方法：利用实时定 量PCR 技术（quantitative real-time polymerase chain reaction，qRT-PCR）检测人体肺腺癌组织和肺腺癌细胞系A549 中HIF-1-alpha和 ANRIL的表达水平，然后利用小干扰RNA（small interfering RNA，siRNA）和基因过表达技术使HIF-1alpha低量和过量表达，再检测 肺腺癌细胞系A549 中ANRIL表达水平的变化。结果：ANRIL和HIF-1alpha在人体肺腺癌组织和肺腺癌细胞系A549 中的表达水平 呈现正相关，而且ANRIL 和HIF-1alpha在癌组织中的表达水平均高于癌旁组织，并且通过HIF-1alpha的低量和过量表达，ANRIL亦呈 现相应的变化。结论：ANRIL 和HIF-1alpha在肺腺癌组织中的表达具有相关性，而且HIF-1琢能够刺激激活ANRIL的表达，因此 ANRIL-HIF-1alpha可能为肺腺癌的诊疗提供一个崭新的靶点。     

Curcumin reverses cis-platin resistance and promotes human lung adenocarcinoma A549/DDP cell apoptosis through HIF-1α and caspase-3 mechanisms

Curcumin, a yellow pigment derived from Curcuma longa Linn, has been favored by the Eastern as dietary ingredients for centuries. During the past decade, extensive investigations have revealed curcumin sensitized various chemotherapeutic agents in human breast, colon, pancreas, gastric, liver, brain and hematological malignant disorders in vivo and in vitro. Several pathways and specific targets including NF-κB, STAT3, COX-2, Akt and multidrug resistant protein have been identified to facilitate curcumin as a chemosensitizer. Recent studies suggest HIF-1α participated in the development of drug resistance in cancer cells and targeting HIF-1α either by RNAi or siRNA successfully overcame chemotherapeutic resistance. To investigate the mechanism basis of curcumin as a chemosensitizer in lung cancer, we examined curcumin's effects on HIF-1α in cis-platin (DDP) sensitive A549 and resistant A549/DDP cell lines by RT-PCR and Western blot. HIF-1α in A549/DDP cells was found to be overexpressed at both mRNA and protein levels together with a poor response to DDP. Results from transient transfection and flow cytometry showed the HIF-1α abnormality contributed to DDP resistance in A549/DDP lung cancer cells. Combined curcumin and DDP treatment markedly inhibited A549/DDP cells proliferation, reversed DDP resistance and triggered apoptotic death by promoting HIF-1α degradation and activating caspase-3, respectively. Expression of HIF-1α-dependent P-gp also seemed to decrease as response to curcumin in a dose-dependent manner. Our findings shed light on drug resistant reversing effect of curcumin in lung cancer cells by inhibiting HIF-1α expression and activating caspase-3.     

NF-kappaB inhibition by an adenovirus expressed aptamer sensitizes TNFalpha-induced apoptosis

Prolonged activation of NF-kappaB is involved in the pathogenesis of chronic inflammatory diseases and associated cancers. NF-kappaB activation is considered to be a main mechanism opposing TNFalpha-induced apoptosis. We investigated whether inhibition of NF-kappaB could sensitize tumor and endothelial cells to TNFalpha-induced apoptosis. As such, we developed a novel H1 RNA polymerase III promoter driven adenoviral vector to express an RNA aptamer, Ad-A-p50, which selectively inhibits NF-kappaB activation in the nucleus. This event sensitizes human lung adenocarcinoma cells (A549) and human endothelial cells (HUVEC) to TNFalpha-induced apoptosis through the multiple pathways regulated by NF-kappaB, including Bcl-XL, HIF-1alpha, and VEGF. Our findings also suggest a new mechanism of HIF-1alpha regulation by NF-kappaB in the normoxic environment. RNA aptamer inhibition of NF-kappaB offers exciting opportunities for sensitizing inflammatory and tumor cells to TNFalpha-induced apoptosis.     

Hyperbaric oxygen preconditioning promotes angiogenesis in rat liver after partial hepatectomy

Hyperbaric oxygen preconditioning (HBO-PC) increases the level of HIF-1alpha (hypoxia inducible factor-1alpha) and its target gene VEGF (vascular endothelial growth factor) which is involved in angiogenesis. Liver regeneration is an angiogenesis-dependent process. We hypothesized that HIF-1alpha and VEGF mediated the angiogenesis effect of HBO-PC on regenerating rat liver. Male Sprague Dawley rats received HBO-PC followed by 70% partial hepatectomy. Proliferation of hepatocytes and endothelial cells was evaluated by BrdU (bromodeoxyuridine) staining. Microvascular density was assessed by immunohistochemistry. mRNA expression of HIF-1alpha was assessed by quantitative RT-PCR and protein levels of HIF-1alpha and VEGF were assessed by western blot. HIF-1alpha DNA-binding activity was determined with an ELISA-based kit. HBO-PC increased the proliferation index of endothelial cells and microvascular density at 48 h after partial hepatectomy. The protein level and DNA-binding activity of HIF-1alpha and the protein level of VEGF were increased by HBO-PC before and after partial hepatectomy. Partial hepatectomy alone also increased proliferation index and the expressions of HIF-1alpha and VEGF. Our results indicated that the angiogenesis effect of HBO-PC on liver after partial hepatectomy could be achieved by increased HIF-1alpha activity and VEGF expression. However, the angiogenic effect of HBO-PC is moderate and HBO-PC failed to produce additional effect on the enhancement of HIF-1alpha and VEGF induced by partial hepatectomy alone.     

Damage-associated molecular patterns in the pathogenesis of osteoarthritis: potentially novel therapeutic targets

Albendazole (ABZ) has an anti-tumor ability and inhibits HIF-1α activity. HIF-1α is associated with glycolysis and vascular endothelial cell growth factor (VEGF) expression, which plays an important role in cancer progression. These clues indicate that ABZ exerts an anti-cancer effect by regulating glycolysis and VEGF expression. The aim of this study is to clarify the effects of ABZ on non-small cell lung cancer (NSCLC) cells and explore the underlying molecular mechanisms. The expression levels of HIF-1α and VEGF were detected using western blot analysis, and the effect of ABZ on glycolysis was evaluated by measuring the relative activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) and detecting the production of lactate in A549 and H1299 cells. The results showed that ABZ decreased the expression levels of HIF-1α and VEGF and suppressed glycolysis in under hypoxia, but not normoxic condition. Inhibiting HIF-1α also suppressed glycolysis and VEGF expression. Additionally, ABZ inhibited the volume and weight, decreased the relative activities of HK, PK, and LDH, and reduced the levels of HIF-1α and VEGF of A549 xenografts in mouse models. In conclusion, ABZ inhibited growth of NSCLC cells by suppressing HIF-1α-dependent glycolysis and VEGF expression.     

Astrocyte responses to injury: VEGF simultaneously modulates cell death and proliferation

Hypoxia is linked to changes in blood-brain barrier (BBB) permeability, and loss of BBB integrity is characteristic of many pathological brain diseases including stroke. In particular, astrocytes play a central role in brain homeostasis and BBB function. We investigated how hypoxia affects astrocyte survival and assessed whether VEGF release through hypoxia-inducible factor-1alpha (HIF-1alpha) induction plays a role in tolerance of these cells to insult. Thus primary astrocytes were subjected to normoxic (21% O(2)), hypoxic (1% O(2)), or near-anoxic (<0.1% O(2)) conditions in the presence or absence of glucose. Cell death was significantly initiated after combined oxygen glucose deprivation, and, surprisingly, astrocyte proliferation increased concomitantly. Near anoxic, but not hypoxic, conditions stabilized HIF-1alpha protein and provoked DNA binding activity, whereas oxygen and glucose deprivation accelerated HIF-1alpha accumulation. Unexpectedly, Hif-1alpha knockdown studies showed that elevated VEGF levels following increased insult was only partially due to HIF-1alpha induction, suggesting alternative mechanisms of VEGF regulation. Notably, endogenous VEGF signaling during insult was essential for cell fate since VEGF inhibition appreciably augmented cell death and reduced proliferation. These data suggest Hif-1 only partially contributes to VEGF-mediated astrocyte responses during chronic injury (as occurs in clinical hypoxic/ischemic insults) that may ultimately be responsible for disrupting BBB integrity.