YAP调控肺癌A549/DDP细胞顺铂耐药性的机制分析

目的探讨Yse相关蛋白(Yes-associated protein, YAP)对A549/DDP细胞顺铂耐药性的影响及其机制。 方法采用MTS检测顺铂对肺腺癌A549细胞以及肺腺癌耐顺铂A549/DDP细胞的50%抑制浓度(IC₅₀)。使用qPCR以及Western blot检测A549和A549/DDP细胞中YAP mRNA以及蛋白的表达。Western blot检测维替泊芬(Verteporfin, VP)处理A549/DDP后YAP蛋白表达变化。将A549/DDP细胞设置为DMSO对照组、顺铂组(DDP组)、维替泊芬组(VP组)、顺铂联合维替泊芬组(DDP+VP组),采用MTS检测0、24和48 h各处理组细胞活力的变化。采用qPCR检测VP处理A549/DDP后干细胞标志物ALDHA1、CD133、OCT4、NANOG、SOX2的mRNA表达变化。 结果顺铂对A549和A549/DDP细胞的IC50分别为(4.07±0.03)μg/ml、(23.44±0.98)μg/ml,耐药指数为5.76。A549/DDP细胞中YAP显著高于A549细胞(P<0.05)。VP处理A549/DDP细胞后YAP蛋白表达水平较DMSO组明显降低。DDP+VP组较单独DDP组,其细胞活力在24 h和48 h均明显降低(P<0.05)。qPCR检测发现A549/DDP细胞经VP处理后,干细胞标志物ALDHA1、CD133、OCT4、NANOG、SOX2的mRNA均较DMSO组明显降低(P<0.05)。 结论YAP可能参与了肺癌细胞的顺铂耐药。抑制YAP可通过抑制肿瘤干细胞特性,进而发挥逆转耐药的作用。     

人参皂苷增加顺铂抗肺癌A549细胞活性的机理研究

目的观察人参皂苷与顺铂联合作用肺癌A549细胞的细胞毒作用,探索相关作用机制。方法 MTT实验检测人参皂苷、顺铂对人肺癌A549细胞的细胞毒作用;提取药物处理细胞总蛋白,Western blot检测细胞survivin蛋白的表达变化;Annexin V/PI双染、流式细胞仪检测细胞凋亡的发生。结果人参皂苷和顺铂对肺癌A549细胞的半数抑制浓度(IC50)值分别为(43.26±3.27)和(2.34±0.15)μmol/L;20、40μmol/L人参皂苷与顺铂联合作用A549细胞,顺铂对A549细胞的IC50值减少为(1.42±0.33)、(0.61±0.04)μmol/L,P<0.01。Western blot检测显示人参皂苷剂量依赖性的降低肺癌A549细胞survivin蛋白的表达。流式细胞仪凋亡检测显示人参皂苷使顺铂诱导的细胞凋亡增加(P<0.01)。结论人参皂苷可以显著增加顺铂对肺癌A549细胞的致凋亡作用,下调survivin蛋白表达可能是其增敏的主要机制。     

全反式维甲酸联合维生素D3对肺癌原代细胞化疗敏感性的影响

目的探讨全反式维甲酸(ATRA)联用维生素D3(VD3)逆转肺癌细胞多药耐药、提高肺癌细胞化疗敏感性的机制。方法将ATRA、VD3联合作用于培养后的人肺癌原代细胞,观察肺癌原代细胞对顺铂化疗的敏感性及多药耐药相关蛋白(MRP)、肺耐药蛋白(LRP)的表达水平。结果人肺癌原代细胞MRP、LRP呈高表达;ATRA及VD3作用后肺腺癌原代细胞MRP、LRP表达量明显降低,肺鳞癌MRP、LRP的表达量无明显变化;顺铂化疗的敏感性提高。结论MRP、LRP在肺癌尤其非小细胞肺癌中高表达,可反映肿瘤细胞多药耐药性;ATRA或VD3可下调肺腺癌MRP、LRP基因的表达水平,提高肺腺癌的化疗敏感性;但对肺鳞癌MRP、LRP基因表达水平无明显影响;ATRA和VD3联用有协同作用。     

ERK通路对胃癌细胞顺铂敏感性的调节作用

目的:研究在人胃癌细胞中,细胞外信号调节蛋白激酶(extracellular signal-regulated kinase,ERK)通路对人胃癌细胞顺铂敏感性的影响.方法:采用MTT法测定顺铂对两种胃癌细胞MGC-803、BGC-823的增殖抑制影响,测定PD98059作用BGC-823细胞后顺铂对细胞的增殖抑制影响.Western blot方法检测MGC-803、BGC-823细胞中谷胱甘肽-S-转移酶π(glutathione S-transferases π,GGST-π)蛋白的表达;及PD98059作用BGC-823细胞24 h后BGC-823细胞中p-ERK、GsT-π蛋白的表达.结果:顺铂作用两种胃癌细胞7 2 h后.MGC-803,BGC-823细胞的IC50值分别为0.71和1.21 mg/L,且MGC-803细胞的增殖率明显低于BGC-823细胞的增殖率(P<0.05).MGC-803细胞顺铂的敏感性高于BGC-823细胞.MGC-803细胞不表达GST-π蛋白,BGC-823细胞强表达GST-兀蛋白.20 μmol/LPD98059作用BGC-823细胞24 h后,p-ERK蛋白表达明显下调,GST-π蛋白表达亦明显下调.20 μmol/L PD98059作用BGC-823细胞24 h后,加入顺铂培养48 h,BGC-823+PD98059组增殖率明显低于对照组,BGC-823细胞对顺铂的敏感性明显增强.结论:下调胃癌细胞GST-π蛋白的表达可增强其对顺铂的敏感性;抑制胃癌细胞中ERK通路的活化,可以降低其细胞内GST-π蛋白表达;ERK通路通过调节人胃癌细胞GST-π基因表达影响其对顺铂的敏感性.     

GRP78的表达非小细胞性肺癌细胞A549对顺铂的敏感性

目的探讨下调GRP78的表达后是否增强非小细胞性肺癌细胞对顺铂的敏感性。方法通过蛋白印记的方法检测内质网应激后GRP78的表达变化;用流式细胞仪检测用siRNAGRP 78抑制GRP 78的表达上调后凋亡细胞的数目;用A549肿瘤细胞对裸鼠进行了皮下荷瘤,在体内检验GRP78的表达水平对顺铂耐药的影响;运用免疫组织化学的方法检测肺癌患者的肿瘤组织中GRP78的表达水平。结果内质网应激能上调GRP78的表达;抑制内质网应激诱导的GRP78的表达上调后,细胞对顺铂的敏感性增加;将GRP78基因表达干扰后,荷瘤组织的生长明显受到顺铂的抑制;20例肺癌患者的肿瘤组织与癌旁组织相比,存在GRP78的高表达。结论 GRP78的表达水平上调降低非小细胞性肺癌细胞对顺铂的敏感性,具体的参与调控的信号通路需要进一步的机制研究。     

结肠癌细胞源exosomes的分离鉴定及其表面蛋白CD44v6的表达

目的 探索结肠癌colo205细胞系对exosomes的分泌功能,并分析热休克作用对表面蛋白CD44v6表达量的影响.方法 采用超速离心法分离colo205细胞分泌的exosomes和热休克处理后colo205细胞分泌的exosomes(Heat shocked exosomes,HS-Exo),经220 nm微孔滤膜过滤纯化后,在透射电镜下观察其形态,并用SDS-PAGE方法分析细胞与exosomes的蛋白组成,Western Blot检测表面CD44v6的表达情况.结果 经透射电镜观察,正常exosomes与HS-Exo形态基本相似,均为圆形或椭圆形膜性囊泡,直径大多在30～100 nm之间,且经热休克处理的colo205细胞及其分泌的HS-Exo,在CD44v6表达量上较正常colo205细胞及exosomes显著上调(P＜0.05).结论 结肠癌colo205细胞系可分泌exosomes,且超速离心结合滤膜过滤的分离纯化方法切实可行;热休克作用可使CD44v6表达上调,说明HS-Exo较exosomes可能在肿瘤免疫治疗方面有更重要的应用价值.     

核桃青皮提取物联合顺铂对Lewis肺癌细胞的诱导凋亡作用

目的观察不同浓度核桃青皮提取物对Lewis肺癌细胞的诱导凋亡作用。方法用0、1、2和4μg/L浓度核桃青皮提取物分别或联合顺铂作用于体外培养的大鼠Lewis肺癌细胞,采用MTT法检测药物对细胞的诱导凋亡作用。结果不同浓度核桃青皮提取物作用30 min、3、12、24 h,均能明显诱导Lewis肺癌细胞发生凋亡,作用呈现时间-剂量依赖性;联合顺铂后,可使细胞凋亡更加明显。结论低浓度的核桃青皮提取物可明显诱导Lewis肺癌细胞发生凋亡,与顺铂联合可产生协同作用。     

骨肉瘤细胞来源的exosomes制备和鉴定

目的探讨从骨肉瘤细胞培养上清提取exosomes及鉴定其表面标志。方法采用超滤离心联合蔗糖密度梯度超速离心的方法从骨肉瘤细胞培养上清液中分离exosomes,用透射电子显微镜观察exosomes的形态,Western印迹鉴定其表面CD81及组织相溶性复合体(MHC)-I类分子的表达。结果透射电镜下见骨肉瘤细胞分泌的exosomes为圆形或类圆形小体,直径约60¹00 nm,聚集成团或散在分布;Exosomes表达CD81及MHC-Ⅰ类分子,exosomes CD81及MHC-I类分子的表达水平较骨肉瘤细胞高。结论从骨肉瘤细胞培养上清液中成功分离出exosomes,其表达抗原提呈相关的免疫分子。     

微小RNA-21在顺铂联合5-氟尿嘧啶抑制肺癌细胞株作用中的表达变化

目的探讨顺铂和5-氟尿嘧啶在体外联合用药对肺癌细胞株A549细胞存活率、细胞周期的影响以及在这过程中微小RNA-21(microRNA-21,miR-21)表达水平的变化。方法顺铂和5-氟尿嘧啶单药或联合用药处理肺癌A549细胞后,采用CCK-8法检测细胞存活率;倒置显微镜观察细胞形态变化;流式细胞仪技术检测细胞周期;实时荧光定量PCR检测miR-21的表达变化。结果顺铂和5-氟尿嘧啶单药对A549细胞的抑制作用都具有浓度依赖性,两者联合用药48h后细胞的存活率明显低于单药组(P0.05),并且细胞形态改变更加明显,悬浮、变圆的细胞也更多;顺铂处理A549细胞后,将细胞阻滞于S期和G2期,5-氟尿嘧啶阻滞A549细胞于S期,联合用药后细胞阻滞于S期和G2期;随着顺铂和5-氟尿嘧啶作用浓度的增加,miR-21的表达水平上调,联合用药后miR-21的表达亦上调,但趋势没有顺铂组明显(P0.05)。结论顺铂在体外联合5-氟尿嘧啶对A549细胞存活率的抑制有协同作用,同时可以上调细胞内miR-21表达水平。     

PDCD5蛋白基于顺铂为基础的非小细胞肺癌化疗敏感性

目的探讨程序性细胞死亡因子(PDCD)5过表达后顺铂对A549细胞生物学行为和凋亡作用的影响,同时测定PDCD5在非小细胞肺癌血清中的表达,分析其与化疗后临床参数及疗效的相关性。方法采用四甲基偶氮唑盐(MTT)法、流式细胞仪研究PDCD5表达对肺癌细胞化疗敏感性的影响。采用酶联免疫吸附试验(ELISA)检测血清中PDCD5蛋白水平。结果 rhPDCD5增强了顺铂诱导A549细胞的增殖和凋亡。共检测40例非小细胞肺癌血清标本,PDCD5蛋白表达与患者年龄、性别无关,与吸烟、TNM分期、有无淋巴结转移及疗效有显著相关性。PDCD5蛋白的低表达与不良疗效呈正相关。结论 RhPDCD5对顺铂诱导的肺癌细胞毒性具有明显的敏感性。PDCD5蛋白高表达的患者可能更适合以顺铂为基础的化疗。PDCD5作为细胞凋亡的调控因子,可能在肿瘤的发病机制和发展过程中发挥重要作用。     

肺癌细胞裂解物负载对树突状细胞分泌的exosome诱导抗肿瘤作用的影响

目的为制备高效的胞外体（exosome）肿瘤疫苗提供理论依据。方法用细胞因子诱导培养树突状细胞（DC）,将肺癌细胞裂解物负载DC,提取exosome用exosome活化T细胞（负载组）,以未负载DC的exosome（未负载组）及肺癌细胞裂解物负载DC（DC组）活化的T细胞为对照,MTr法检测三组肺癌细胞的杀伤率。结果exosome中有HSP70、HLA及CEA表达。活化T细胞／肺癌细胞为25：1、10：1、5：1时负载组杀伤率均明显高于未负载组及DC组（P均〈0．05）。结论肺癌细胞裂解物负载能增强DC分泌的exosome诱导的抗肿瘤作用;本研究为制备高效的exosome肿瘤疫苗提供了理论依据。     

Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid.

Exosomes are 30-100 nm diameter vesicles formed by inward budding of endosomal compartments and are produced by several cell types, including T-cells, B-cells and dendritic cells (DC)s. Exosomes from DCs express major histocompatibility complexes (MHC) class I and II, and co-stimulatory molecules on their surface, and can induce antigen-specific activation of T-cells. The aims of the present study were to investigate for the presence of exosomes in bronchoalveolar lavage fluid (BALF) from healthy individuals, and to establish if these exosomes bear MHC and co-stimulatory molecules. The authors analysed BALF taken from seven healthy volunteers and used exosomes from monocyte-derived DC (MDDC) cultures as a reference. After ultracentrifugation, exosomes were bound to anti-MHC class II coated magnetic beads and analysed by flow cytometry and electron microscopy. The authors report for the first time that exosomes are present in BALF. These exosomes are similar to MDDC derived exosomes as they express MHC class I and II, CD54, CD63 and the co-stimulatory molecule CD86. The results demonstrate that exosomes are present in the lung, and since they contain both major histocompatibility complex and co-stimulatory molecules it is likely that they are derived from antigen presenting cells and might have a regulatory role in local immune defence.     

自体 CIK 细胞联合培美曲塞和顺铂治疗非小细胞肺癌的临床效果研究

目的：自体 CIK 细胞联合培美曲塞和顺铂治疗非小细胞肺癌的临床效果研究。方法选取2012年2月至2015年7月于我院诊治的非小细胞肺癌患者共87例进行研究,按照随机原则分为研究组(n =44)和对照组(n =43)。对照组实施培美曲塞和顺铂一线治疗,研究组采用自体 CIK细胞联合培美曲塞和顺铂治疗。比较2组患者治疗前后免疫功能变化情况,评价临床疗效、生活质量评分、药物毒性反应。结果研究组患者在治疗后的各项免疫功能指标(自然杀伤细胞、CD3+、CD4+、CD8+、CD4+/CD8+)均明显优于治疗前、对照组(P 值均<0.05)；研究组和对照组的疾病控制率分别为86.36%和65.12%,研究组均高于对照组(χ2=5.363,P <0.05)；研究组生活质量总稳定率(52.27%)高于对照组(18.60%)(χ2=10.752,P <0.05)；研究组患者的胃肠道反应发生率(Ⅰ~Ⅱ度)、中性粒细胞减少发生率(Ⅰ~Ⅱ度、Ⅲ~Ⅳ度)均低于对照组(χ2值分别为12.164、11.553、5.132,P 值均<0.05)。结论对非小细胞肺癌患者行自体 CIK 细胞联合培美曲塞和顺铂治疗,患者免疫功能、生活质量明显改善,疾病控制率高,药物毒性反应低,效果显著,值得推广。     

Novel method for extracting exosomes of hepatocellular carcinoma cells

AIM:To develop a novel method for the rapid and efficient extraction of exosomes secreted by tumor cells.METHODS:Unlike the traditional extraction method,the supernatants of cell cultures were concentrated,and the exosomes were isolated promptly and effectively using a novel nanomaterial called ExoQuick.Coomassie brilliant blue staining was used for protein quantification,and the morphology of the exosomes extracted by both methods was visualized by transmis-sion electron microscopy.Exosome marker proteins were detected by Western blot analysis.Two potential hepatoma-associated proteins,tissue transglutaminase2(TGM2)and annexin A2,were analyzed.RESULTS:The exosomes separated by the new extraction assay based on the nanomaterial were discshaped,intact vesicles with lipid bilayer membranes.They were approximately 30-100 nm in diameter,which is similar to the diameter of exosomes isolated by the traditional method.The protein concentration of exosomes extracted by the new method was approximately780μg/108 cells,and therefore,it was 19 times higher than that of exosomes extracted in the traditional manner.There were differences between the total proteins of Huh-7 cells and the exosomal proteins.Typical exosome proteins,such as the transmembrane protein CD63 and heat shock protein 70,were confirmed.Two potential hepatoma-associated proteins were also identified.TGM2 was first found to exist in the exosomes of human liver cancer cells,but annexin A2 was not secreted into exosomes.CONCLUSION:The new extraction method based on the nanomaterial is quick and efficient.The cancerassociated protein TGM2 can be secreted through an exosome-mediated non-classical secretion pathway,and it may be a valuable tumor marker.     

PI3K/Akt和MAPK/ERK信号通路在胃癌细胞来源的Exosome促进同源肿瘤细胞增殖中的作用

目的:研究胃癌细胞来源的Exosome对同源肿瘤细胞增殖的影响,初步探讨PI3K/Akt和MAPK/ERK信号转导通路在此过程中的作用.方法:采用离心超滤和蔗糖密度梯度离心的方法从胃癌MGC803细胞的上清液中分离出胃癌细胞来源的Exosome.应用透射电子显微镜观察Exosome的形态,MTT检测细胞活力,Weste...     

巨噬细胞外泌体抑制HBV DNA复制

目的探讨巨噬细胞(macrophage,M?)外泌体抑制HBV DNA复制的机制及与慢性乙型肝炎(chronic viral hepatitis B,CHB)肝损伤的相关性。方法体外分离鉴定M?外泌体,并将其与Hep G2.2.15细胞共培养,利用实时定量PCR检测共培养后HBV DNA复制的变化,分别分析M?及其上清外泌体中微小RNA(micro RNA,mi RNA)-638的表达;同时入组CHB免疫活化(immune activation,IA)期、低(非)复制(inactive carrier,IC)期患者,并以同期健康者作为对照组,通过实时定量PCR检测血清外泌体中mi RNA-638的表达,并与肝损伤、HBV DNA载量进行相关性分析。结果 M?外泌体可以抑制HBV DNA复制;M?及其上清外泌体中mi RNA-638高表达;CHB患者IA期mi RNA-638的表达水平低于IC期;CHB患者血清外泌体中mi RNA-638的表达水平与ALT、AST和HBV DNA水平呈负相关。结论 M?外泌体可以抑制HBV DNA复制,外泌体中mi RNA-638的表达水平与肝脏炎性损伤和病毒复制密切相关,M?外泌体相关mi RNA可能是潜在的抑制HBV复制、减轻肝脏炎症的靶点。     

Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment

The identification of lung tumor-initiating cells and associated markers may be useful for optimization of therapeutic approaches and for predictive and prognostic information in lung cancer patients. CD133, a surface glycoprotein linked to organ-specific stem cells, was described as a marker of cancer-initiating cells in different tumor types. Here, we report that a CD133⁺, epithelial-specific antigen-positive (CD133⁺ESA⁺) population is increased in primary nonsmall cell lung cancer (NSCLC) compared with normal lung tissue and has higher tumorigenic potential in SCID mice and expression of genes involved in stemness, adhesion, motility, and drug efflux than the CD133⁻ counterpart. Cisplatin treatment of lung cancer cells in vitro resulted in enrichment of CD133⁺ fraction both after acute cytotoxic exposure and in cells with stable cisplatin-resistant phenotype. Subpopulations of CD133⁺ABCG2⁺ and CD133⁺CXCR4⁺ cells were spared by in vivo cisplatin treatment of lung tumor xenografts established from primary tumors. A tendency toward shorter progression-free survival was observed in CD133⁺ NSCLC patients treated with platinum-containing regimens. Our results indicate that chemoresistant populations with highly tumorigenic and stem-like features are present in lung tumors. The molecular features of these cells may provide the rationale for more specific therapeutic targeting and the definition of predictive factors in clinical management of this lethal disease.     

Effect of PI3K-mediated autophagy in human osteosarcoma MG63 cells on sensitivity to the chemotherapy drug cisplatin

Objective: To investigate the influence of autophagy on sensitivity to the chemotherapy drug cisplatin(DDP) and the role of PI3 K in autophagy. Methods: MTT methods and l ow cytometer, with rapamycin up-regulating the autophagy and 3-MA down-regulating the autophagy, were employed to measure the proliferation inhibition rate on DDP-treated osteosarcoma cells and the change in cell cycle. The expression of intracellular protein was detected by Western blot. The autophagy of MG63 cell was observed using l uorescence microscope and transmission electron microscope. Results: Western blot showed that basic autophagy level of MG63 cell was significantly lower than that of h FOB cell. MTT test revealed that the cell proliferation inhibition rate in the group treated with rapamycin and DDP, group treated with 3-MA and DDP, and group only treated with DDP was signii cantly dif erent. It was demonstrated by the l ow cytometry that in group treated with DDP, inhibition on autophagy can increase the cell numbers in G1 phase and reduce the cell numbers in S phase of cell cycle. Increase of autophagosome in MG63 cytoplasm was observed under l uorescence microscope. Conclusions: Up-regulating the autophagy signii cantly reduced the sensitivity of MG63 cell to chemotherapy with DDP. DDP induced autophagy of MG63 cell and blocked the cell cycle at G1 phase.     

Effect of 5-Aza-2’-deoxycytidine on immune-associated proteins in exosomes from hepatoma

AIM: To study the effect of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on heat shock protein 70 (HSP70), human leucocyte antigen-Ⅰ (HLA-Ⅰ) and NY-ESO-1 proteins in exosomes produced by hepatoma cells, HepG2 and Hep3B. METHODS: Exosomes derived from HepG2 and Hep3B cells treated with or without 5-aza-CdR were isolated and purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under electron microscope. Concentration of proteins in exosomes was measured...