阿霉素对不同转移能力肝癌细胞及肝癌干细胞相关基因表达的影响

目的 探讨阿霉素（adriamycin, ADM）对不同转移能力肝癌细胞及肝癌干细胞相关基因表达的影响。方法　利用MTT法检测ADM对人肝癌细胞系MHCC97-L、HCCLM3的抑制作用,计算各自的半数致死浓度（median lethal dose, LD50）;Western blot法检测ADM作用下人肝癌细胞系MHCC97-L、HCCLM3中干细胞基因Nanog、Oct-4、Sox2、ARID1A、Wnt5b的表达,并分析干细胞基因在两种细胞中表达变化的差异。结果 ADM浓度越高,对人肝癌细胞的抑制作用越明显,其对MHCC97-L和HCCLM3的半数致死浓度分别为0.4212 μmol/L和0.5249 μmol/L;随着ADM（LD50）作用时间的延长,MHCC97-L和HCCLM3中Wnt5b的表达水平先升高后降低,Nanog蛋白的表达先降低后升高,Sox2在HCCLM3中表达水平逐渐升高。Wnt5b和Nanog 4 h内在低转移能力肝癌细胞系MHCC97-L中的表达变化均值均小于其在高转移能力肝癌细胞系HCCLM3时的表达变化均值,差异有统计学意义（P<0.05）。结论 ADM可促进MHCC97-L和HCCLM3细胞的凋亡,且对MHCC97-L的作用强于HCCLM3;干细胞相关基因Wnt5b与肝癌细胞的恶性程度呈负相关;Nanog和Sox2均与肿瘤的转移密切相关,且Nanog和Sox2可能与肝癌耐药密切相关。     

TEY1基因在不同转移潜能肝癌细胞中表达的定量分析

背景和目的:我们先前的研究提示,肝癌转移抑制基因可能在人染色体8p上。TEY1基因(TEnni-Yokusei1,转移抑制的日文)是最近从人染色体8p发现的前列腺癌转移抑制基因。本研究通过对不同转移潜能肝癌细胞株中TEY1基因的表达水平进行定量分析,探讨TEY1基因作为肝癌转移抑制基因的可能性。方法:提取肝癌细胞株MHCC97-H、MHCC97-L、HCCLM3和正常肝细胞系L02的RNA,反转录合成第一链cDNA,对反转录产物进行荧光定量PCR。用看家基因GAPDH的cDNA起始浓度作为参照标化TEY1的cDNA起始浓度,对标化结果进行单因素方差分析。结果:MHCC97-H、MHCC97-L、HCCLM3和L02标化后的TEY1cDNA起始浓度分别为(3.57±2.62)×10-4、(5.02±1.95)×10-4、(2.82±0.78)×10-5、(15.20±0.58)×10-4。肝癌细胞的TEY1基因表达水平和正常细胞之间存在显著性差异(P<0.001);HCCLM3的TEY1基因表达水平与MHCC97-H和MHCC97-L相比差异有显著性(P<0.05);MHCC97-L的TEY1基因表达水平是MHCC97-H的1.4倍,但两者无显著性差异(P>0.05)。结论:TEY1基因表达水平与肝癌细胞的转移潜能呈负相关,可以推测TEY1基因可能是肝癌转移的抑制基因。     

杂交瘤单抗4F11识别CD90+肝癌干细胞并抑制其侵袭【院士点评：靶向CD90+肿瘤干细胞可能治疗肝癌转移】

目的:筛选识别肝癌干细胞的单克隆抗体并研究其体外的抗肿瘤作用,为肝癌干细胞靶向治疗提供候选抗体药物。方法:无血清悬浮培养及PKH26染色分析人肝癌细胞株MHCC97H中是否存在肝癌干细胞。流式细胞术检测MH-CC97H细胞及其成球细胞中7种肿瘤干细胞标志物的表达,以及MHCC97H细胞中肝癌干细胞标志物CD90和不同杂交瘤单抗3G7、4F11、11C9、15B7、15D2识别抗原的共表达情况。无血清悬浮培养法和CCK8法检测单抗4F11对MHCC97H细胞及其成球细胞自我更新和增殖的影响,Transwell实验检测单抗4F11对MHCC97H细胞体外侵袭和迁移的影响。结果:PKH26染色实验显示,MHCC97H细胞球体由单个肝癌干细胞增殖分化形成。MHCC97H细胞球中CD90+MHCC97H细胞比例较亲本MHCC97H细胞显著增加[(18.0±7.5)%vs(2.3±1.0)%,P<0.05]。杂交瘤单抗4F11、3G7、11C9、15B7、15D2均能识别MHCC97H细胞中CD90+MHCC97H细胞,其中单抗4F11对CD90+MHCC97H细胞的识别比例为(47.2±4.4)%,其对MHCC97H成球细胞增殖的抑制率远大于对亲本MHCC97H细胞增殖的抑制率[(29.4±3.8)%vs(12.0±2.2)%,P<0.05]。单抗4F11抑制MHCC97H细胞成球,抑制率达(58.0±20.8)%。单抗4F11能显著抑制MHCC97H细胞体外侵袭和迁移,抑制率分别为(48.6±5.1)%和(47.6±3.6)%。结论:杂交瘤单抗4F11能特异性识别CD90+肝癌干细胞,抑制肝癌细胞的侵袭和迁移,可作为肝癌干细胞靶向治疗的候选抗体药物。     

上调miR-200a表达对人肝癌细胞MHCC97中边缘群细胞干细胞特性的影响

目的:探讨转染miR-200a mimics对人肝癌细胞系MHCC97中边缘群细胞(SP)干细胞特性的影响。方法:利用流式细胞技术从MHCC97细胞中分离出SP细胞。转染组为应用脂质体介导转染miR-200a mimics的SP细胞,对照组为未接受转染的SP细胞。采用实时荧光定量RT-PCR检测两组SP细胞miR-200a的表达情况。四甲基偶氮唑盐(MTT)和软琼脂克隆形成实验检测细胞增殖情况。Transwell小室检测细胞迁移能力。裸鼠成瘤实验检测细胞体外成瘤能力。结果:转染miR-200a mimics可明显上调SP细胞miR-200a的表达。MTT结果显示转染组SP细胞的增殖能力明显低于对照组(P<0.05),转染组软琼脂中克隆形成率(24.70±5.54)%明显低于对照组(51.63±7.11)%（P<0.05)。Transwell实验显示转染组细胞穿膜数为(18.52±4.27)个,明显少于对照组(63.34±7.41)个(P<0.05)。裸鼠成瘤实验中转染组成瘤数(1/8)也低于对照组(8/8)（P<0.05)。结论:上调人肝癌细胞系MHCC97中SP细胞miR-200a的表达,能够抑制SP细胞的增殖和迁移,降低体外成瘤能力,即抑制SP细胞的干细胞特性。     

吉西他滨联合YH-16对肝癌干细胞样细胞的影响

目的：探讨吉西他滨联合重组人血管内皮抑制素YH-16对裸鼠CD133+MHCC97H肝癌移植瘤中肝癌干细胞样细胞的影响。方法：用免疫磁珠分选MHCC97H中肝癌干细胞样细胞,将其接种到20只裸鼠皮下,建立移植瘤模型。治疗组给予吉西他滨和YH-16,对照组给予等量生理盐水,比较两组移植瘤大小变化及原代培养细胞球形成情况,流式细胞术检测原代培养中CD133+细胞百分率。结果：治疗组的移植瘤明显缩小,治疗组和对照组细胞球形成数目分别为(7.5±2.4)个 vs (16.0±3.5)个,细胞球直径分别为(178.30±19.21) μm vs (131.06±20.96) μm（P＜0.05）;治疗组和对照组原代培养中CD133+细胞百分数分别为（6.24±0.96）% vs（18.26±1.76）%（P＜0.05）。结论：低剂量吉西他滨联合YH-16可抑制血管内皮细胞破坏肝癌干细胞样细胞的血管巢,进而选择性清除肝癌干细胞样细胞。     

LASS2基因抑制HCCLM3肝癌细胞的转移

目的：探讨LASS2基因对HCCLM3肝癌细胞转移的影响。方法：Northern blot分析高转移潜能肝癌细胞系HCCLM3和低转移潜能肝癌细胞系MHCC97-L中LASS2基因的mRNA水平。构建包含LASS2基因编码框的真核表达载体pCMV—HA2-LASS2，通过脂质体转染HCCLM3细胞，经G418筛选，建立稳定表达LASS2基因的HCCLM3细胞株。通过分析LASS2在HCCLM3中过表达后，HCCLM3细胞在迁移、侵袭等转移能力上的改变，Western blot、明胶酶谱及原位酶谱分析MMP-2合成、分泌、激活的变化。结果：Northern blot显示，HCCLM3细胞中LASS2基因的表达水平低于MHCC97-L细胞。当LASS2基因过表达后，HCCLM3细胞的迁移及侵袭能力受到显著抑制（P〈0．001），虽然细胞内MMP-2的合成未改变，但细胞MMP-2的分泌及MMP-2的激活均受抑制。结论：LASS2基因可下调MMP-2的分泌及激活，可使肝癌细胞HCCLM3的转移能力受到明显抑制，提示LASS2基因对肝癌细胞HCCLM3的转移具有抑制作用。     

肝癌Huh7干细胞样细胞的富集培养及鉴定

目的：建立一种体外有效富集、培养和鉴定具有肝癌干细胞特征的细胞亚群的方法。方法：采用成球培养法利用肿瘤干细胞样细胞（cancer stem cell, CSC）分化培养基对肝癌Huh7细胞进行富集培养,获得的干细胞样细胞于体外进一步扩增获得肝癌干细胞球。流式细胞术检测Huh7干细胞样细胞表面肿瘤干细胞标志物EpCAM、CD90和CD133的表达,平板克隆集落形成实验和裸鼠成瘤实验分别检测Huh7细胞和Huh7干细胞样细胞的克隆集落形成能力、体内成瘤能力。结果：Huh7细胞成球培养3~7 d后即可形成肝癌干细胞样细胞球,获得的干细胞样细胞具有自我更新和增殖能力,其EpCAM阳性细胞比例较Huh7细胞明显增加\[（99.6%±0.31）% vs（0.12%±0.05）%,P<0.01\],但两种细胞CD90\[(0.11%±0.06)% vs (0.09%±0.07)%, P>0.05\]和CD133\[(0.17%±0.08)% vs (0.15%±0.05)%, P>0.05]表达差异无统计学意义。Huh7干细胞样细胞克隆集落形成数量明显多于Huh7细胞\[（188.67±12.5）vs （79±16.7）个,P<0.01\];当接种量为5×104个细胞时,与接种Huh7细胞的对照裸鼠相比,接种Huh7干细胞样细胞的裸鼠成瘤时间更短（11 vs 30 d）,成瘤率更高（100% vs 16.67%）;接种5×105数量级的细胞时,实验组成瘤体积\[（171.90±10.94）vs（86.39±11.21）mm3, P<0.01\]和瘤体质量\[（2.98±0.82）vs（0.32±0.17）g, P<0.01\]均明显大于对照组。结论：利用成球培养法能够从Huh7肝癌细胞系中富集培养获得Huh7肝癌干细胞,其具有比Huh7细胞更强的成瘤能力。     

高转移潜能人肝癌细胞特异性结合肽对肝癌侵袭和转移的影响

目的 研究高转移潜能人肝癌细胞特异性结合肽(AWYPLPP肽)对肝癌侵袭和转移的影响.方法 采用Matrigel侵袭实验、迁移实验、二苯基溴化四氮唑蓝(MTT)实验以及黏附实验,探讨AWYPLPP肽对高转移潜能人肝癌细胞HCCLM3侵袭表型的影响.裸鼠皮下接种HCCLM3细胞,建立肿瘤肺转移模型,观察AWYPLPP肽对HCCLM3肺转移的影响.结果 侵袭实验结果显示,在AWYPLPP肽浓度为0.1～100 μmol/L时,能显著促进HCCLM3细胞的侵袭能力,呈剂量效应关系.迁移实验、MTT实验和黏附实验表明,AWYPLPP肽对HCCLM3细胞的迁移、增殖和黏附能力无影响.HCCLM3细胞皮下接种30 d后处死裸鼠,AWYPLPP肽组出现明显的肺转移,肺转移率为88.9%(8/9),与PBS组比较,差异有统计学意义(P<0.05),但对皮下肿瘤的生长无明显影响.结论 AWYPLPP肽能促进高转移潜能人肝癌细胞体外侵袭能力以及肿瘤肺转移;进一步寻找肿瘤细胞表面与AWYPLPP肽结合的受体,可能为深入研究肝癌侵袭转移的机制和设计干预治疗的靶点提供新思路.     

卵巢癌SKOV3细胞中侧群细胞的生物学特性

目的 分选人卵巢癌细胞系SKOV3中的侧群（side population, SP）细胞,并探讨其是否具有肿瘤干细胞的生物学特性。方法 流式细胞仪检测、分选经DNA染料Hoechst 33342染色后SKOV3中的SP细胞,并对侧群细胞（SP）与非侧群细胞（non-SP）作细胞生物学鉴定,包括增殖能力、克隆形成能力、侵袭及迁移能力、自我更新能力及细胞周期。结果 SKOV3细胞系中SP细胞比例为（1.12±0.104）%,SP细胞增殖速度快于non-SP细胞,差异有统计学意义（P<0.05）。接种相同数量的细胞,SP细胞克隆形成率高于non-SP细胞（P<0.05）。Transwell实验显示,SP细胞侵袭与迁移的细胞数与non-SP相比均明显增多（P<0.05）。SP细胞体外能分化为Non-SP细胞,具有自我更新能力。SP细胞大多处于细胞周期的G0/G1期。结论 卵巢癌细胞系SKOV3中的SP细胞具有肿瘤干细胞的生物学特性,SP细胞表型可考虑作为富集卵巢癌干细胞样细胞的有效方法。     

人胆囊癌SP细胞的分离及其干细胞标记物的表达*

目的:探索在人胆囊癌GBC-SD 细胞系中是否存在具有干细胞特性的SP细胞（side population cells）,以及SP细胞、非SP细胞和GBC-SD 细胞系表达常见干细胞标记物ABCG2、Oct- 4、CD34的情况。方法:采用荧光激活细胞分选技术（FACS）分选出人胆囊癌SP细胞和非SP细胞,通过RT-PCR、Western blot、流式细胞术以及免疫荧光化学技术检测SP细胞、非SP细胞以及GBC-SD 细胞系表达ABCG2、Oct- 4 和CD34的情况。结果:人胆囊癌GBC-SD 细胞系中存在着少量的具有干细胞潜能的SP细胞,其所占的比例为（0.64± 0.08）% ,SP细胞相对于非SP细胞和GBC-SD 细胞,具有更强的体外侵袭力（P<0.05）;并且ABCG2 在胆囊癌SP细胞中呈现出高表达的状态（89.56±3.86）% ,在非SP细胞中几乎不表达（1.32± 0.49）% ,在未分选胆囊癌细胞系中ABCG2 呈弱表达（12.37± 1.61）% ,P=0.001;Oct- 4 在三种细胞中的表达分别为:（94.87± 1.40）% 、（88.16± 2.34）% 、（90.17± 1.61）% ,P>0.05;CD34在mRNA水平高表达于非SP细胞和GBC-SD 细胞,不表达于SP细胞;在蛋白水平几乎均不表达于这三种细胞,其含量依次为:（1.78± 0.51）% 、（0.63± 0.21）% 、（0.96± 0.38）% ,P>0.05。结论:人胆囊癌GBC-SD 细胞系中存在具有干细胞特性的SP细胞,并且具有ABCG2+Oct- 4+CD34-的表型特征。      

Biomarkers in clear cell renal cell carcinoma

The treatment of advanced renal cell carcinoma (RCC) has evolved significantly following the identification of the von Hippel–Lindau (VHL) gene and the function of its protein, and subsequent development of antiangiogenic therapies. A series of clinical trials resulted in the approval of three new agents with significant activity in this disease. Additional studies are now underway to identify subsets of patients most likely to benefit. This article reviews the current therapy for advanced RCC and the development of biomarkers in RCC. This requires the identification of disease characteristics at a clinical, genetic and molecular level associated with response and/or surrogate measures of clinical benefit. Currently, a variety of prognostic factors (lactate dehydrogenase, performance status, disease-free interval, hemoglobin and calcium levels) are utilized to predict the survival of RCC patients. The use of validated biomarkers in either serum/plasma, urine or tissue could enhance this process, as well as define at the molecular and genetic levels, factors associated with response to therapy and/or the development of resistance. Examples include plasma VEGF levels, VHL gene mutation status and carbonic anhydrase IX levels in tumor tissue, among others. Validation of such biomarkers is crucial in order for them to be clinically useful.     

Biomarkers in clear cell renal cell carcinoma

The treatment of advanced renal cell carcinoma (RCC) has evolved significantly following the identification of the von Hippel-Lindau (VHL) gene and the function of its protein, and subsequent development of antiangiogenic therapies. A series of clinical trials resulted in the approval of three new agents with significant activity in this disease. Additional studies are now underway to identify subsets of patients most likely to benefit. This article reviews the current therapy for advanced RCC and the development of biomarkers in RCC. This requires the identification of disease characteristics at a clinical, genetic and molecular level associated with response and/or surrogate measures of clinical benefit. Currently, a variety of prognostic factors (lactate dehydrogenase, performance status, disease-free interval, hemoglobin and calcium levels) are utilized to predict the survival of RCC patients. The use of validated biomarkers in either serum/plasma, urine or tissue could enhance this process, as well as define at the molecular and genetic levels, factors associated with response to therapy and/or the development of resistance. Examples include plasma VEGF levels, VHL gene mutation status and carbonic anhydrase IX levels in tumor tissue, among others. Validation of such biomarkers is crucial in order for them to be clinically useful.     

Non-small and small cell lung carcinoma cell lines exhibit cell type-specific sensitivity to edelfosine-induced cell death and different cell line-specific responses to edelfosine treatment

The unique signal transduction pathways that distinguish non-small cell lung carcinoma (NSCLC) from small cell lung carcinoma (SCLC) are poorly understood. We investigated the ability of edelfosine, an inhibitor of phosphatidylinositol-specific phospholipase C (PLC) to inhibit cell viability among four NSCLC cell lines and four SCLC cell lines. The differential sensitivity of cells to edelfosine's cytostatic and cytotoxic effects has been attributed to edelfosine-induced changes in the activities of many enzymes, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), p38 kinase, and poly(ADP-ribose) polymerase (PARP). To investigate the role of these enzymes in edelfosine-induced cytotoxicity, we correlated edelfosine-induced changes in enzyme activity and cell viability among the different NSCLC and SCLC cell lines. We found that NSCLC cells are much more susceptible to the cytotoxic effects of this drug than are SCLC cells. Three out of the four edelfosine-sensitive NSCLC cell lines (NCI-H157, NCI-H520, NCI-H522) exhibit G2/M arrest, significant apoptosis and some degree of JNK activation in response to drug treatment. In contrast, none of the SCLC cell lines exhibit edelfosine-induced G2/M arrest or significant apoptosis. A comparison of the edelfosine-induced effects among the sensitive and resistant lung cancer lines indicates that there is little correlation between edelfosine-induced cytotoxicity and altered activities of JNK, ERK, p38, or cleavage of PARP. These results demonstrate that edelfosine-induced changes in JNK, ERK, p38, or PARP are not good predictors of cell susceptibility to edelfosine-induced cytotoxicity. Thus, edelfosine-induced inactivation of PLC may disrupt signaling cascades downstream of PLC that are unique to individual cellular environments. These findings also identify edelfosine as one of the few potential chemotherapeutic agents that has a greater cytotoxic effect against NSCLC cells than SCLC cells.     

桥接整合因子1通过AKT-mTOR通路诱导非小细胞肺癌H1975细胞周期阻滞

目的：研究桥接整合因子1（bridging intergrator 1,Bin1）基因过表达后对非小细胞肺癌细胞株H1975细胞周期的影响及其作用机制。方法：构建携带Bin1基因的CMV-MCS-GFP-SV40-Neomycin-Bin1质粒,并转染H1975细胞（Bin1+组）,另设置空白质粒转染组（Bin1-组）及空白对照组（Ctrl组）,利用RT-PCR和Western blotting分别检测3组细胞中Bin1在mRNA和蛋白质水平的表达情况。流式细胞术检测不同处理组H1975细胞周期的变化,Western boltting分别检测各组中AKT、mTOR磷酸化水平及细胞周期相关蛋白（周期蛋白D1、CDK4、Rb）的表达情况。结果：与Bin1-组、Ctrl组比较,Bin1+组H1975细胞中Bin1在mRNA、蛋白水平表达明显上调（均P<0.05）; H1975细胞阻滞在G1期\[（60.53±1.89）% vs（46.14±1.56）%、（47.33±2.07）%,均P<0.05\]; Bin1+组H1975细胞内p-AKT、p-mTOR表达下调（均P<0.05）,AKT、mTOR表达变化无统计学差异（P>0.05）;周期蛋白D1、CDK4的表达量均明显下调(P<0.05),Rb表达量明显增加（P<0.05）。结论：Bin1基因在H1975细胞株过表达后明显诱导细胞周期阻滞,其机制可能是通过抑制AKT-mTOR通路及其细胞周期相关蛋白实现的。     

A human squamous cell carcinoma cell line.

An epithelial cell line COLO 16 has been established from a human squamous carcinoma, characterized and maintained for over two years. The cells produce a parathyroid-like hormone and carcinoembryonic antigen. The line is definitely not a "HeLa contaminant." The cell line is available to other investigators.