一株胃癌干细胞功能性单克隆抗体的鉴定及功能研究

[目的]从抗人胃癌干细胞单克隆抗体杂交瘤库中筛选鉴定识别胃癌干细胞的单克隆抗体,并证明其具有抑制胃癌干细胞自我更新能力和侵袭功能的功能性单抗,为靶向人胃癌干细胞治疗胃癌提供有治疗潜能的单抗候选药物。[方法]采用无血清培养、PKH26染色及流式细胞术等方法确定人胃癌细胞系SNU-5中存在肿瘤干细胞,可作为研究抗人胃癌干细胞单抗的细胞模型。应用双色免疫荧光、流式细胞分选等技术分析鉴定单克隆抗体25G5是识别胃癌干细胞的单克隆抗体。用肿瘤干细胞的成球生长实验、Transwell侵袭实验及耐药性实验研究分析25G5单抗对胃癌干细胞功能的影响。[结果]SNU-5细胞在无血清培养基中存活并增殖成球形生长,SNU-5球体细胞中表达胃癌干细胞标志物CD44~+和CD90~+的细胞比例分别为72.4%和8.55%,较亲本细胞分别提高了21.3倍和2.4倍,表明SNU-5中存在有CD44~+、CD90~+的胃癌干细胞。细胞免疫荧光结果显示25G5单抗识别的抗原分子与CD44、CD90胃癌干细胞标志物共染表达于胃癌干细胞膜上。流式细胞术分选的25G5~+细胞的成球率为(21.4±0.3)%,显著高于25G5-细胞(12.3±0.7)%和亲本细胞(16.1±1.0)%。Transwell侵袭实验、细胞耐药性实验和动物体内致瘤实验结果显示25G5~+细胞的侵袭能力和耐药能力也显著高于25G5-细胞和亲本细胞,表明25G5单抗识别的细胞是具有自我更新、高侵袭、高耐药性特征的肿瘤干细胞。体外功能研究发现,单抗25G5能显著抑制SNU-5胃癌细胞在无血清培养液中的成球生长,抑制率可达46.4%,也能显著地抑制SNU-5成球细胞(Sphere细胞)的侵袭,抑制率达58.4%。单抗25G5是一株能显著抑制SNU-5中肿瘤干细胞自我更新和侵袭能力的功能性抗人胃癌干细胞单抗。[结论]单克隆抗体25G5是一株抗胃癌干细胞的功能性单抗,为胃癌干细胞靶向治疗的候选抗体药物。     

人胃癌CD90+干细胞可能影响胃癌的转移和患者的预后

目的： 分离并鉴定人胃癌细胞系SNU-5中的肿瘤干细胞,探讨人胃癌CD90+干细胞对胃癌转移和预后的影响。 方法： 无血清悬浮培养及PKH26染色确定SNU-5细胞系中是否存在肿瘤干细胞,流式细胞术分析SNU-5亲本及球体细胞中肿瘤干细胞标志物的表达,分选CD90+SNU-5细胞并进行体外生物学特征研究及SCID鼠致瘤实验。收集肿瘤医院腹部外科95例胃癌患者肿瘤病理标本,免疫组化方法检测胃癌组织中CD90的表达。 结果： SNU-5细胞无血清悬浮培养11 d后形成的细胞球体中存在单个PKH26阳性细胞。无血清悬浮培养可将CD90+SNU-5细胞富集6.1倍,且CD90可在球体细胞中与标示干细胞的PKH26共染。CD90+SNU-5细胞较CD90- SNU-5细胞和亲本SNU-5细胞具有更高的自我更新能力\[成球率（7.7±1.1）% vs (1.3±0.4)%、(1.8±0.3)%,均P<0.01\]和侵袭能力\[侵袭细胞数(283.3±30.2) vs (48.0±7.5)、(156.7±72)个,均P<0.01\]。CD90+SNU-5细胞在重症联合免疫缺陷（severe combined immune deficency,SCID）小鼠皮下接种2×102个细胞6周即可致瘤（1/6）,而接种2×104个CD90- SNU-5细胞10周才能致瘤（1/6）。95例胃癌患者组织中CD90的表达与胃癌的远处转移显著相关（P<0.01）,且CD90阳性胃癌患者的生存期明显短于CD90阴性的患者（P<0.01）。 结论： 人胃癌细胞系SNU-5中存在具有更强自我更新及侵袭能力的CD90+干细胞,人胃癌组织中CD90+干细胞数量与肿瘤的转移与患者生存期明显相关。     

高转移胃癌细胞株的建立及其肿瘤干细胞的生物学特征

目的:建立高转移胃癌细胞株并研究其肿瘤干细胞生物学特征.方法:将胃癌细胞株SNU-5接种至裸鼠皮下成瘤后,取肺部转移灶,通过机械分离法并反复接种裸鼠,获取细胞株SNU-5-V12,采用无血清悬浮培养及PKH26染色确定SNU-5-V12细胞中存在肿瘤干细胞.流式细胞术分析SNU-5和SNU-5-V12细胞中CD44肿瘤干细胞标志物表达情况,分选SNU-5和SNU-5-V12细胞中CD44+细胞并进行体外生物学特征研究及裸鼠致瘤实验.结果:建立高转移SNU-5-V12细胞株,SNU-5-V12细胞无血清悬浮培养11d后形成的细胞球体中存在单个PKH26阳性细胞.流式分析显示高转移SNU-5-V12细胞中干细胞标志物CD44比例显著高于SNU-5细胞[(72.9±1.5)％ vs (8.96±1.2)％],且SNU-5-V12的CD44+细胞比SNU-5的CD44+细胞具有更强的自我更新能力[成球率(27.8±1.7)％ vs (20.4±1.0)％,P＜0.01],转移细胞数增加1.64倍[(329.5±7.5) vs (200±2.0)个,P＜0.01],耐药能力也显著增强[IC50:(0.286 vs 0.196)μ∥ml,P＜0.01],裸鼠皮下接种2×102个CD44+ SNU-5-V12细胞2个月2/6裸鼠可致瘤,而CD44+ SNU-5细胞需要接种2×104个细胞2个月时仅1/6裸鼠致瘤.结论:获得高转移胃癌细胞株SNU-5-V12,其CD44+细胞体内外功能显著增强,为胃癌干细胞的靶向治疗提供有价值的细胞模型.     

靶向胃癌干细胞单克隆抗体2C12的鉴定及功能研究

目的:研究胃癌干细胞的功能性单克隆抗体2C12体外功能实验,为胃癌干细胞的靶向治疗提供候选单抗药物。方法:以胃癌细胞系SNU-5为细胞模型,流式细胞术检测其亲本细胞和通过无血清悬浮培养的球体细胞中2C12+细胞的表达水平,双色细胞免疫荧光检测单抗2C12和CD90识别的抗原在SNU-5细胞中的表达情况。流式细胞术分选SNU-5细胞中2C12+和2C12-细胞,采用无血清悬浮培养成球实验和Transwell小室法分别检测其自我更新能力和侵袭能力。用单抗2C12处理SNU-5的球体细胞后,检测其对SNU-5的球体细胞自我更新能力和侵袭能力的影响。结果:2C12+细胞在SNU-5的球体细胞中的表达水平明显高于亲本细胞。免疫荧光结果显示单抗2C12识别的抗原分子能与CD90在SNU-5细胞上共定位。流式细胞术分选结果表明SNU-5细胞中2C12+细胞成球数明显高于2C12-细胞（103.3±9.07 vs 50.67±5.86）（P<0.01）,侵袭能力也明显较高（209.3±12.9 vs 99.0±3.61）（P<0.01）。对2C12单抗的体外功能研究发现,不同浓度的单抗2C12（0 μg/mL、20 μg/mL、40 μg/mL）能显著抑制SNU-5的球体细胞的成球能力（122.0±5.66、83.0±5.66、59.0±4.24）,抑制率达到31.97%和51.64%,同时侵袭能力也显著降低（178.0±8.49、106.5±5.0、78.0±2.83）,抑制率达到40.17%和56.18%。结论:单抗2C12是一株抗胃癌干细胞的功能性单抗,能够显著抑制胃癌干细胞的干性相关特征,为胃癌干细胞靶向治疗的候选抗体药物。     

丹参酮ⅡA磺酸钠联合顺铂腹腔化疗对胃癌生长的影响

目的 探讨丹参酮ⅡA磺酸钠联合顺铂腹腔化疗对大鼠种植性胃肿瘤生长的影响.方法 利用Walker-256细胞株建立鼠种植性胃肿瘤模型,将40只Wistar雄性胃肿瘤大鼠随机分为4组:生理盐水(NS)组、顺铂(CDDP)组、丹参酮ⅡA磺酸钠(STS)组、顺铂+丹参酮ⅡA磺酸钠(CDDP+STS)组.接种后第7天,生理盐水组每只腹腔注入NS 2 mL/kg;顺铂组每只腹腔注入顺铂5 mg/kg;丹参酮ⅡA磺酸钠组每只腹腔注射丹参酮ⅡA磺酸钠25 mg/kg;顺铂+丹参酮ⅡA磺酸钠组每只腹腔注入顺铂5 mg/kg,并加用丹参酮ⅡA磺酸钠25 mg/kg.每周2次,连用6周.6周后处死动物,取完整肿瘤组织,测量肿瘤体积并计算肿瘤体积抑制率,采用TdT介导的DNA末端原位标记染色(TUNEL)法检测胃癌细胞凋亡指数(AI).结果 生理盐水组、顺铂组、丹参酮ⅡA磺酸钠组及顺铂+丹参酮ⅡA磺酸钠组肿瘤体积分别为(1.69±0.51)cm3、(1.06±0.38)cm3、(1.12±0.41)cm3和(0.67±0.13)cm3,顺铂组、丹参酮ⅡA磺酸钠组及顺铂+丹参酮ⅡA磺酸钠组肿瘤体积较NS组明显减小(P<0.05,P<0.01),抑瘤率分别为37.28%、33.73%、60.36%(P<0.05),顺铂+丹参酮ⅡA磺酸钠组对小鼠胃肿瘤的抑制作用优于各治疗组.生理盐水组、顺铂组、丹参酮ⅡA磺酸钠组及顺铂+丹参酮ⅡA磺酸钠组的凋亡指数分别为(5.75±1.34)%、(24.15±4.80)%、(23.85±3.23)%和(45.10.4±3.75)%,顺铂组、丹参酮ⅡA磺酸钠组及顺铂+丹参酮ⅡA磺酸钠组显著高于生理盐水组(P<0.01);而顺铂+丹参酮ⅡA磺酸钠组较顺铂组及丹参酮ⅡA磺酸钠组明显升高(P<0.01),但顺铂组和丹参酮ⅡA磺酸钠组之间差异无统计学意义(P0.05).结论 丹参酮ⅡA磺酸钠能抑制胃肿瘤生长,诱导胃癌细胞凋亡,并且能够协同传统细胞毒药物顺铂的抗肿瘤作用.     

抗肝癌干细胞单克隆抗体的鉴定及功能

目的:鉴定可靶向肝癌干细胞的功能性单克隆抗体,为肝癌干细胞靶向治疗提供抗体候选药物.方法:以肝癌细胞系Bel7402-V3为模型,采用流式细胞术分选ESA+细胞后检测其耐药能力及致瘤能力.双色细胞免疫荧光检测单抗3G7和ESA识别的抗原蛋白在Bel7402-V3细胞中的表达情况,同时采用此法检测sphere中PKH26与3G7的共染情况.流式细胞术分选3G7+细胞后检测其自我更新能力,并采用CCK-8法检测其耐药能力;甲基纤维素成球实验,检测单抗3G7对细胞自我更新能力的影响;CCK-8法检测单抗3G7对细胞增殖和耐药能力的影响.结果:流式细胞术分选ESA+细胞的耐药性明显高于ESA-细胞,其IC50值分别为2.30 μmol/L、0.49 μmol/L(P<0.01),ESA+细胞的致瘤性较ESA-细胞高至少40倍.细胞免疫荧光结果显示单抗3G7识别的抗原分子能与ESA在Bel7402-V3细胞上共定位,并能与标识干细胞的PKH26染料共染.流式细胞术分选3G7+细胞体外成球率明显高于3G7-细胞[(30.4±3.4)% vs (8.8±1.8)%](P<0.01),耐药性也明显较高(IC50值:1.014 μmol/L vs 0.365 μmol/L).抗体体外功能研究发现,单抗3G7能显著抑制Bel7402-V3的甲基纤维素成球,抑制率达到37.2%;同时,单抗3G7能抑制sphere细胞的增殖,抑制率为41.7%(P<0.01);经单抗3G7处理过细胞的耐药能力显著降低,实验组与对照组的IC50分别为0.56 μg/ml和0.68 μg/ml.结论:单克隆抗体3G7是一株抗肝癌干细胞的功能性单抗,为肝癌干细胞靶向治疗的候选抗体药物.     

PD-1单抗联合顺铂或吉西他滨化疗对KRAS突变非小细胞肺癌A549 细胞移植瘤小鼠模型的治疗作用

目的：探讨程序性死亡受体-1(PD-1)单抗联合顺铂或吉西他滨在KRAS基因突变非小细胞肺癌（NSCLC）A549细胞移植瘤小鼠模型治疗中的作用。方法：构建免疫系统-肿瘤双人源化A549细胞小鼠移植瘤模型,将60只小鼠按随机数字表法分成6组（10只/组）,分别为对照组（200μL/kg PBS）、PD-1单抗组（20 mg/kg PD-1单抗）、顺铂组（3 mg/kg顺铂）、PD-1单抗+顺铂组（20 mg/kg PD-1单抗+3 mg/kg顺铂）、吉西他滨组（30 mg/kg吉西他滨）和PD-1单抗+吉西他滨组（20 mg/kg PD-1单抗+30 mg/kg吉西他滨）。TUNEL和DAPI双染色法检测移植瘤组织中细胞凋亡水平,测量移植瘤体积和质量并计算肿瘤生长抑制率,免疫组化法检测移植瘤微血管密度（MVD）。结果：成功构建免疫系统-肿瘤双人源化NSCLC A549细胞小鼠移植瘤模型,PD-1单抗+顺铂组移植瘤的细胞凋亡率、肿瘤生长抑制率均最高,移植瘤体积、质量和MVD均最小,与其他5组小鼠比较差异均有统计学意义（均P<0.05）。结论：顺铂与PD-1单抗具有协同活性,而吉西他...     

ENO1在胃癌干细胞中表达及其与胃癌侵袭转移的相关性研究

摘 要：［目的］ 探讨ENO1在胃癌干细胞中的表达及其对胃癌侵袭转移的影响。［方法］ 以胃癌SNU-5模型,实时荧光定量PCR和双色细胞免疫荧光检测ENO1在其分选的CD44+细胞中的表达情况。运用慢病毒载体稳定干扰SUN-5中ENO1的表达,并用实时荧光定量PCR和Western blot检验干扰效率。流式细胞术分选稳定干扰ENO1的SNU-5中CD44+细胞后（shRNA-ENO1）,实时荧光定量PCR检测其Oct-4、Sox 2以及干性相关基因Nanog的表达,同时分别进行细胞成球实验、体外侵袭与体内致瘤的胃癌干细胞生物学特性研究,Western blot检测ENO1对胃癌干细胞侵袭转移影响的相关分子机制。［结果］ SNU-5的CD44+细胞中ENO1基因和蛋白表达明显高于CD44-细胞,并建立稳定干扰ENO1的SUN-5。shRNA-ENO1细胞中干性基因Oct-4、Sox 2和Nanog中mRNA的表达显著低于PLV-Ctr细胞（P<0.05）。与PLV-Ctr细胞相比较,shRNA-ENO1细胞的自我更新能力、侵袭能力和肿瘤重量显著降低。Western blot检测shRNA-ENO1细胞中Vimentin、Snail和N-cadherin下调表达,同时E-cadherin上调表达,并伴随AKT和PI3K的磷酸化水平降低,提示ENO1的作用可能通过PI3K/AKT信号通路激活。［结论］ ENO1在胃癌干细胞中高表达,其在调控胃癌侵袭转移能力中发挥重要作用。     

抗胃癌干细胞单克隆抗体的筛选鉴定及功能研究

目的:筛选鉴定靶向胃癌干细胞的功能性单克隆抗体,为胃癌干细胞靶向治疗提供抗体候选药物。方法:以胃癌细胞系BGC-823为模型,采用流式细胞术分选CD44+细胞后检测其自我更新和致瘤能力。双色细胞免疫荧光检测单抗11H5和CD44在BGC-823细胞中的表达情况。流式细胞术分选11H5+细胞后检测其自我更新能力、细胞增殖和耐药能力。结果:流式细胞术分选CD44+细胞成球率明显高于CD44-细胞［（27.2±1.6）% vs （12.9±1.2）%］（P<0.01）,CD44+细胞的致瘤性比CD44-细胞至少高100倍。细胞免疫荧光结果显示单抗11H5识别的抗原能与CD44在BGC-823细胞上共定位。流式细胞术分选11H5+细胞体外成球率明显高于11H5-细胞［（21.4±2.0）% vs （6.2±1.0）%］（P<0.01）,耐药性也明显高于11H5-细胞(IC50值:0.986 μmol/L vs 0.315 μmol/L)。抗体体外功能研究发现,单抗11H5能显著抑制BGC-823细胞成球,抑制率达到47.9%。单抗11H5能抑制其sphere细胞的增殖,抑制率为44.9%（P<0.05）。经单抗11H5作用的耐药能力显著降低(IC50值:0.52 μg/ml vs 0.64 μg/ml)。结论:单克隆抗体11H5是一株抗胃癌干细胞的功能性单抗,为胃癌干细胞靶向治疗的候选抗体药物。     

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。     

胃癌SP表型细胞侵袭转移相关性研究

目的：观察人胃癌SNU5肿瘤细胞系及其高侵袭细胞亚系中是否含有SP细胞,并验证该表型细胞与侵袭转移的关系。方法：采用Transwell小室,体外筛选出高侵袭细胞;经Hoechst33342染色,应用流式检测并分选出SP表型以及非SP表型细胞;应用无血清培养方法检测SNU5亲本细胞、SP表型以及非SP表型细胞在无血清培养中的成球能力。将分选出的SP表型以及非SP表型细胞进行侵袭实验、划痕迁移实验。裸鼠皮下接种,检测SP表型以及非SP表型细胞的自发性肺转移能力。基因芯片的方法分析SP表型以及非SP表型细胞的基因表达谱差异。结果：经过三轮筛选建立了稳定的高侵袭亚细胞群SNU5-P3。SP分选检测发现,SNU5-P3的SP细胞比例为1.6%;无血清培养发现,SNU5-P3-SP细胞成球数为104±19,显著强于SNU5以及SNU5-P3-non-SP细胞。侵袭实验检测发现,SNU5-P3-SP细胞的侵袭能力比SNU5、SNU5-P3-non-SP细胞增强近2.5倍。划痕迁移检测发现,SNU5为-P3-SP运动能力显著增强,划痕24h愈合。体内移植瘤转移实验发现SNU5-P3-SP细胞自发性肺转移率为100%,SNU5为50%,而SNU5-P3-non-SP仅有16.7%。基因芯片分析,获得733个差异基因,其中上调基因450个,36%的基因与侵袭转移有关。结论：胃癌SP表型细胞具有干细胞的特征,在胃癌侵袭、转移中起到关键作用。     

Epigenetic regulation of Delta-Like1 controls Notch1 activation in gastric cancer

The Notch signaling pathway drives proliferation, differentiation, apoptosis, cell fate, and maintenance of stem cells in several tissues. Aberrant activation of Notch signaling has been described in several tumours and in gastric cancer (GC), activated Notch1 has been associated with de-differentiation of lineage-committed stomach cells into stem progenitors and GC progression. However, the specific role of the Notch1 ligand DLL1 in GC has not yet been elucidated. To assess the role of DLL1 in GC cancer, the expression of Notch1 and its ligands DLL1 and Jagged1, was analyzed in 8 gastric cancer cell lines (KATOIII, SNU601, SNU719, AGS, SNU16, MKN1, MKN45, TMK1). DLL1 expression was absent in KATOIII, SNU601, SNU719 and AGS. The lack of DLL1 expression in these cells was associated with promoter hypermethylation and 5-aza-2'dC caused up-regulation of DLL1. The increase in DLL1 expression was associated with activation of Notch1 signalling, with an increase in cleaved Notch1 intracellular domain (NICD) and Hes1, and down-regulation in Hath1. Concordantly, Notch1 signalling was activated with the overexpression of DLL1. Moreover, Notch1 signalling together with DLL1 methylation were evaluated in samples from 52 GC patients and 21 healthy control as well as in INS-GAS mice infected with H. pylori and randomly treated with eradication therapy. In GC patients, we found a correlation between DLL1 and Hes1 expression, while DLL1 methylation and Hath1 expression were associated with the diffuse and mixed type of gastric cancer. Finally, none of the samples from INS-GAS mice infected with H. pylori, a model of intestinal-type gastric tumorigenesis, showed promoter methylation of DLL1. This study shows that Notch1 activity in gastric cancer is controlled by the epigenetic silencing of the ligand DLL1, and that Notch1 inhibition is associated with the diffuse type of gastric cancer.     

肝癌高转移细胞株的肿瘤干细胞生物学特性研究

目的:建立高转移肝癌细胞株,研究其肿瘤干细胞生物学特征,为靶向人肝癌干细胞治疗提供有价值的细胞模型。方法:肝癌细胞系Bel7402接种裸鼠皮下,成瘤后,取小鼠肺部转移灶,通过机械分离法,获取肺部肝转移细胞,体外扩大培养后,再次接种裸鼠。如此反复接种裸鼠,获得稳定肺转移肝癌细胞株Bel7402-V13。采用无血清悬浮培养及PKH26染色确定Bel7402-V13中肿瘤干细胞的存在。流式细胞术分析亲本Bel7402和Bel7402-V13中肿瘤干细胞标志物ESA的表达情况,分选Bel7402和Bel7402-V13中ESA+细胞并进行体外生物学特征研究及裸鼠致瘤实验。结果:建立高转移Bel7402-V13细胞株,Bel7402-V13细胞无血清悬浮培养7d后形成的细胞球体中存在单个PKH26阳性细胞。与Bel7402中ESA+细胞相比,流式分析显示干细胞标志物ESA比例高转移Bel7402-V13显著提高5.67倍（17.6±0.4 vs 3.1±1.5）。Bel7402-V13中ESA+细胞具有更强的自我更新能力［成球率:（94.8±7.5）% vs （52.3±6.9）%,P<0.01］,侵袭能力提高1.51倍（397.7±79.4 vs 262.0±40.1,P<0.01）,耐药能力也显著增强（IC50:0.286 vs 0.196,P<0.01）,ESA+细胞Bel7402-V13在裸鼠皮下接种2×102个细胞3月即可致瘤（3/6）,而接种2×102个ESA+细胞Bel7402细胞3月才能致瘤（1/6）。结论:获得高转移肝癌细胞株Bel7402-V13,伴随ESA+细胞增加,其体内外功能显著增强,为肝癌干细胞的靶向治疗提供有价值的细胞模型。     

Sunitinib synergizes the antitumor effect of cisplatin via modulation of ERCC1 expression in models of gastric cancer

We evaluated the effects of sunitinib monotherapy and in combination with cisplatin in human gastric cancer cell lines. Sunitinib showed antiproliferative effect in gastric cancer cells line with high PDGFRA expression. Knockdown of PDGFRA showed that sunitinib sensitivity was correlated with the basal expression of PDGFRA. Synergistic growth inhibitory activity in combination with cisplatin was identified. We further explored how sunitinib potentiated the activity of cisplatin. We found that sunitinib treatment resulted in the down-regulation of ERCC1 expression via the modulation of PDGFRA expression in gastric cancer cells. The effect was verified via SNU484 xenograft model. Our data support the rationale of clinical trial using sunitinib in combination of cisplatin in gastric cancer.     

Upregulated microRNA‐193a‐3p is responsible for cisplatin resistance in CD44(+) gastric cancer cells

Cisplatin is a well‐known anticancer drug used to treat various cancers. However, development of cisplatin resistance has hindered the efficiency of this drug in cancer treatment. Development of chemoresistance is known to involve many signaling pathways. Recent attention has focused on microRNAs (miRNAs) as potentially important upstream regulators in the development of chemoresistance. CD44 is one of the gastric cancer stem cell markers and plays a role in regulating self‐renewal, tumor initiation, metastasis and chemoresistance. The purpose of the present study was to examine the mechanism of miRNA‐mediated chemoresistance to cisplatin in CD44‐positive gastric cancer stem cells. We sorted gastric cancer cells according to level of CD44 expression by FACS and analyzed their miRNA expression profiles by microarray analysis. We found that miR‐193a‐3p was significantly upregulated in CD44(+) cells compared with CD44(?) cells. Moreover, SRSF2 of miR‐193a‐3p target gene was downregulated in CD44(+) cells. We studied the modulation of Bcl‐X and caspase 9 mRNA splicing by SRSF2 and found that more pro‐apoptotic variants of these genes were generated. We also found that downstream anti‐apoptotic genes such as Bcl‐2 were upregulated, whereas pro‐apoptotic genes such as Bax and cytochrome C were downregulated in CD44(+) cells compared to CD44(?) cells. In addition, we found that an elevated level of miR‐193a‐3p triggered the development of cisplatin resistance in CD44(+) cells. Inhibition of miR‐193a‐3p in CD44(+) cells increased SRSF2 expression and also altered the levels of multiple apoptotic genes. Furthermore, inhibition of miR‐193a‐3p reduced cell viability and increased the number of apoptotic cells. Therefore, miR‐193a‐3p may be implicated in the development of cisplatin resistance through regulation of the mitochondrial apoptosis pathway. miR‐193a‐3p could be a promising target for cancer therapy in cisplatin‐resistant gastric cancer.     

Lipid raft-dependent death receptor 5 (DR5) expression and activation are critical for ursodeoxycholic acid-induced apoptosis in gastric cancer cells

Ursodeoxycholic acid (UDCA) is known as a suppressor of cholestatic liver diseases and colorectal cancer development. Here, we demonstrate that UDCA induces apoptosis without necrotic features in SNU601, SNU638, SNU1 and SNU216 human gastric cancer cells, implying its possible use as an effective chemotherapeutic agent in treatment of gastric cancer. UDCA-induced apoptosis was dominantly mediated by an extrinsic pathway dependent on caspase-8, -6 and -3. UDCA increased expression of death receptor 5 [(DR5), also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2], and this DR appeared to be responsible for UDCA-induced apoptosis, as evidenced by DR5 knockdown. UDCA triggered formation of lipid rafts that played crucial roles in UDCA-induced apoptotic actions. Lipid rafts were required not only for provision of a proper site for DR5 action but also for mediation of DR5 expression. In addition, reactive oxygen species (ROS) and protein kinase C (PKC) δ appeared to be implicated in UDCA-induced raft-dependent DR5 expression. Our results indicate that UDCA-induced apoptosis is mediated by DR5 expression, which is regulated by the raft formation/ROS production/PKCδ activation pathway and DR5 localization into lipid rafts in gastric cancer cells. Tumor-suppressive activity of UDCA was confirmed in an in vivo system: UDCA (120 mg/kg/day) significantly decreased tumor growth in gastric cancer xenograft mice. Taken together, our results demonstrate that UDCA can be used as a potent chemotherapeutic agent for treatment of gastric cancer.     

双功能抗体介导胃癌杀伤效应的研究

目的 以化学方法将抗CD3及抗三硝基苯（TNP）的单克隆抗体（McAb)交联为双功能抗体。利用此双功能抗体介导人外周血淋巴细胞（PBLS）对于不同抗胃癌McAb所针对的胃癌细胞进行体内外杀伤试验。方法 利用化学交联法制备双功能抗体,以酶联免疫吸附试验(ELISA）测定McAbs活性,以ELISA桥联法、琼脂糖免疫双扩散及十二烷基磺酸钠－降丙烯酰胺凝胶电泳（SDS－PAGE）对于双功能抗体进行鉴定,以细胞杀伤试验及肿瘤生长抑制实验检测双功能抗体作用。结果 将抗CD3及抗TNP McAb交联为双功能抗体,此双功能抗体可介导PBLS对于不同抗胃癌McAb所针对的胃癌细胞进行体内外杀伤试验。结论 此双功能抗体在肿瘤生物免疫治疗中具有应用前景。     

胃癌单克隆抗体偶合物导向化疗的实验研究

将制备的鼠抗人胃癌单克隆抗体纯化后，以Ｉｏｄｏｇｅｎ法标记＾１２５Ｉ，Ｍａｎａｄｅ法偶平阳霉素。间接免疫荧光试验和＾３Ｈ－ＴｄＲ掺入法（抑制率８１．８５％）证明标记物免疫活性保持良好，５只人胃癌大鼠模型尾静脉注射＾１２５Ｉ－ＭｃＡｂ，放射免疫自显影有４只移植瘤体外显像（８０％），Ｔ／ＮＴ值为３．１２，显著高于对照组（Ｐ〈０．０１），ＰＰＭ导向治疗动物试验，４只荷瘤大鼠注射ＰＰＭ５次，肿瘤抑制率为５     

程序性细胞死亡因子4在胃癌组织中的表达及临床病理学意义

目的:探讨胃癌组织中程序性细胞死亡因子4(pro-grammed cell death4,PDCD4)的表达及其临床病理学意义。方法:应用免疫组织化学和Western印迹杂交研究方法检测61例胃癌组织中PDCD4的表达,观察其与胃癌临床病理学参数之间的关系。结果:免疫组化染色显示,在正常胃组织中,阳性细胞比例均在80%以上。61例胃癌中,PD-CD4低表达者(阳性细胞比例<30%)占65·6%(40/61),高表达(阳性细胞比例介于30%~80%)者占34·4%(21/61)。Western印迹分析结果显示,同癌旁正常组织相比,所有胃癌组织PDCD4蛋白表达均明显下调。相关分析表明,PDCD4表达下调或缺失与肿瘤的不良分化相关,P<0·01,而与性别、年龄及肿瘤的TNM分期无关。结论:PDCD4蛋白在胃癌中多呈低表达,并与胃癌的分化程度有关,在胃癌的发生、发展过程中起重要作用。