一株靶向CD90+MHCC97-L细胞单克隆抗体治疗肝癌的实验研究

目的:研究抗人肝癌干细胞单抗28C10体内外功能,为肝癌干细胞的靶向治疗提供有应用价值的候选治疗剂。方法:采用无血清成球实验、侵袭实验和CCK-8方法等检测分析28C10单抗对MHCC97-L sphere的细胞自我更新、侵袭和耐药的影响。裸鼠体内治疗实验研究单抗28C10联合顺铂对MHCC97-L移植瘤生长的作用。Western-Blot方法鉴定该单抗识别抗原的分子量。结果:流式细胞检测结果显示单抗28C10能够识别MHCC97-L中CD90阳性细胞的比例为2.75%。体外功能实验结果显示单抗28C10能显著抑制MHCC97-L sphere细胞的无血清成球能力和侵袭能力,抑制率分别达33.33%和53.8%。28C10能显著抑制MHCC97-L sphere的顺铂耐药能力,其IC50为0.74 μg/ml,而对照组的IC50为1.42 μg/ml。抗体体内治疗实验结果显示,低、高剂量抗体28C10均能抑制肝癌移植瘤的生长,抑制率分别达到了24.0%和67.7%,单独顺铂组肝癌移植瘤的抑制率为35.9%,而顺铂联合高剂量抗体28C10组对肝癌移植瘤的抑制率达到70.1%。Western-Blot结果显示单抗28C10识别的抗原蛋白分子量约100 kD。结论:筛选获得了1株抗肝癌干细胞的功能性单抗,为靶向肝癌干细胞治疗肝癌奠定了重要的基础。     

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。     

不同乳腺癌干细胞亚群的自我更新和致瘤能力差异

目的 分析原代培养乳腺癌细胞中不同乳腺癌干细胞亚群的自我更新、致瘤能力差异。方法 采用流式细胞术和免疫荧光实验,从乳腺癌原代培养细胞中分选、鉴定CD44⁺CD24^-/low、ALDH1⁺和ALDH1⁺CD44⁺CD24^-/low细胞亚群,分析各分选细胞占总肿瘤细胞的比例;通过克隆形成实验观察各组细胞的自我更新能力;通过裸鼠致瘤实验观察各组细胞的致瘤能力。结果 CD44+CD24^-/low细胞占总细胞比例的7.2%,ALDH1⁺细胞所占比例为4.6%,ALDH1⁺CD44⁺CD24^-/low细胞所占比例为1.5%。三组细胞自我更新和致瘤能力由强至弱依次为ALDH1⁺CD44⁺CD24^-/low、ALDH1⁺、CD44⁺CD24^-/low细胞,各组细胞间比较差异均存在统计学意义（P<0.05）。 结论 乳腺癌组织中CD44+CD24-/low、ALDH1⁺和ALDH1⁺CD44⁺CD24^-/low三组乳腺癌干细胞亚群之间的自我更新和致瘤能力有明显差异,ALDH1⁺CD44⁺CD24^-/low亚群细胞具有最强的自我更新和致瘤能力,提示ALDH1⁺CD44⁺CD24^-/low可能是更具有特异性的乳腺癌干细胞标志物。     

抗HSP90抗体通过内化eHSP90抑制人胃癌干细胞并提高顺铂的抗癌效果

目的：探索抗HSP90单克隆抗体28C10通过靶向肿瘤干细胞促进顺铂(cisplatin,DDP)对人胃癌细胞PAMC82恶性生物学行为的抑制效果及其可能的作用机制。方法:28C10单独或与DDP联合处理人胃癌细胞PAMC82,采用不同实验方法检测该细胞的无血清成球能力、迁移和侵袭能力与克隆形成能力,CCK-8法检测28C10对PAMC82细胞恶性生物学行为和协同DDP抗癌能力的影响。采用细胞免疫荧光及流式细胞术检测PAMC82细胞中HSP90及eHSP90(extracellular HSP90)的表达、定位、eHSP90⁺亚群比例,以及28C10处理后对ALDH⁺、CD44⁺、eHSP90⁺细胞亚群的影响。采用WB实验检测28C10作用后PAMC82细胞中HSP90、干性相关蛋白以及PI3K/AKT/mTOR信号通路蛋白表达的变化。结果：胃癌细胞PAMC82膜表面表达eHSP90,具有2%～3%的eHSP90⁺细胞亚群,且eHSP90⁺细胞多为与ALD...     

肝癌干细胞抗体靶向治疗的实验

目的研究抗人肝癌干细胞鼠单抗15B7的生物学特征、体内外功能,探讨靶向肝癌干细胞是否能够有效抑制肝癌移植瘤复发、自发性肺转移以及延长荷瘤小鼠的生存期。方法采用双色免疫荧光、双色流式细胞技术、皮下成瘤实验,检测、鉴定15B7单克隆抗体能够识别肝癌干细胞(hepatocellular carcinomacancer stem cells, HCC-CSC)。从人肝癌细胞系BEL7402中以流式细胞仪分选具有CD133+或ESA+表型的细胞。在此基础上采用CCK-8细胞增殖实验、侵袭实验、迁移实验等检测分析15B7单抗对CD133+表型的细胞增殖、侵袭、迁移的作用以及对细胞周期的影响。裸鼠体内治疗实验研究15B7单抗对BEL7402移植瘤生长的抑制作用。以Western blot方法鉴定该功能性单抗的抗原。结果双色免疫荧光和双色流式检测显示15B7单抗能与HCC-CSC的标志物ESA、CD133共染;流式分选15B7+或ESA+或CD133+的细胞在体外无血清培养条件下有良好的成球生长能力;流式分选的15B7+细胞裸鼠皮下接种1×104个/只,2月可形成肿瘤,以上实验证明15B7单抗是抗肝癌干细胞的单抗。体外功能实验结果显示15B7单抗能够抑制CD133+细胞的增殖、迁移、侵袭,抑制率分别达13.8%、157%和30.9%,同时还能诱导CD133+细胞发生G1期阻滞。体内治疗实验研究结果表明15B7单抗能明显抑制裸鼠肝癌移植瘤生长,抑制率可达60.5%。Western blot显示15B7单抗识别HCC-CSC表达的抗原蛋白相对分子质量约50 kD。结论15B7单抗能明显抑制裸鼠体内人肝移植瘤的生长,为肝癌干细胞的靶向治疗提供有重要应用价值的候选抗体药物。     

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.     

载脂蛋白C3 转基因小鼠脾脏免疫细胞表型及其活性分析

目的：分析载脂蛋白C3（Apo C3）转基因小鼠脾脏免疫细胞的表型及活性。方法：以12只ICR背景Apo C3转基因小鼠为研究模型,选取鼠龄/性别匹配的野生型小鼠作为对照,采集小鼠球后静脉丛血液,以酶反应显色比色法联合酶标仪测定血浆甘油三酯水平,选取血浆甘油三酯含量≥ 1 000 mg/dL （11.3 mmol/L）的小鼠用于后续实验。取小鼠脾脏制备成单细胞悬液,采用细胞膜表面分子、胞内分子及核内分子染色法,通过流式细胞术检测转基因小鼠和野生型小鼠脾脏免疫细胞频率和表型。再将转基因小鼠或野生型小鼠脾脏细胞与结肠癌细胞MC38分别以1：1、1：3、3：1效靶比共培养72 h后,通过流式细胞术检测CD8⁺ T细胞CD107a的表达。结果：Apo C3转基因小鼠血浆甘油三酯含量较野生型小鼠显著升高（P<0.01）,而体质量差异无统计学意义（P>0.05）。与野生型小鼠相比,Apo C3转基因小鼠脾脏CD3⁺CD8⁺、CD3⁺CD8⁺NKG2D⁺细胞频率明显增多,髓系抑制性Gr-1⁺CD11B⁺细胞频率增加,脾脏NK1.1⁺NK、NK1.1⁺NKG2D⁺细胞的频率明显减少（P均>0.05）;脾脏CD3⁺CD4⁺NKG2D⁺、CD3⁺CD4⁺IL-17⁺细胞频率以及调节性T细胞,NKT细胞,γδT细胞,B细胞和巨噬细胞频率均无明显变化（P均>0.05）。Apo C3转基因小鼠CD8⁺CD62L^-CD44⁺、CD8⁺CD44⁺KLGR1⁺频率升高,以效应性T细胞为主,且CD8⁺ T细胞分泌IFN-γ活性增强;转基因小鼠中CD8⁺ T淋巴细胞CD107a的表达增加差异均具有统计学意义（P均<0.05）。结论：Apo C3转基因小鼠呈现高水平血浆甘油三酯,其免疫细胞表型及活性具有显著变化,其中CD8⁺ T细胞频率及生物活性上调,髓系抑制性细胞频率升高,NK细胞频率降低;并且转基因小鼠中CD8⁺T淋巴细胞对靶细胞杀伤能力增强。     

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。     

SKP2基因表达水平对肝癌细胞放射敏感性的影响

目的 研究人肝癌组织中S期激酶相关蛋白2（SKP2）表达与预后的关系以及肝癌细胞中SKP2表达对人肝癌细胞放射敏感性的影响。方法 使用TCGA数据库中肝癌组织和正常对照组织的测序结果,分析SKP2基因在肝癌组织中的表达情况及其与患者预后的关系。使用Western blot检测肝癌细胞放射照射前后SKP2蛋白水平表达变化。利用CRISPR/Cas9技术敲除肝癌细胞SKP2基因的启动子和第1外显子,建立SKP2基因敲除的PLC/PRF/5和Hep3B肝癌细胞系模型,并进一步进行放射照射。用克隆形成实验检测SKP2基因敲除细胞的放射敏感性变化情况。结果 对TCGA数据库分析结果显示,与正常组织比较,SKP2基因在肝癌组织中表达升高。对SKP2高、低水平肝癌患者总体生存率进行K‐M法分析,结果显示SKP2高表达肝癌患者预后相对较差,其5年生存率为34.6%,SKP2低表达肝癌患者则为50.6%（HR=2.18,95%CI为1.46~3.27,P<0.001）。体外细胞实验显示肝癌细胞受到放射照射后SKP2表达水平明显升高。在CRISPR/Cas9敲除SKP2^‐/‐肝癌细胞中,克隆形成实验结果显示SKP2^‐/‐肝癌细胞的放射敏感性较SKP2^+/+明显增加。结论 SKP2基因在肝癌组织中表达升高,且高表达者较低表达者预后差。放射可上调PLC肝癌细胞SKP2的表达水平,而敲除SKP2后其放射敏感性明显增加。     

乳腺癌细胞系中肿瘤干细胞相关亚群的初步研究

目的 通过乳腺癌细胞系及其干细胞的培养,化疗药干预和流式细胞仪筛选鉴定,探讨不同乳腺癌细胞系中CD44⁺CD24^-/low细胞比例,及富集乳腺癌干细胞相关亚群的方法。方法 通过细胞培养乳腺癌细胞系MCF-7、MDA-MB-231,观察生长曲线,比较化疗药物干预下生长情况;利用流式细胞仪检测两种乳腺癌细胞系中CD44⁺CD24^-/low细胞比例;无血清悬浮培养,化疗药(多西紫杉醇、表阿霉素)干预这两种乳腺癌细胞系,观察其是否形成细胞球。结果 (1)MDA-MB-231细胞系倍增时间短,生长速率高于MCF-7细胞系;(2)MCF-7细胞系中可能存在较大比例肿瘤干细胞,其对化疗抵制,能自我更新;(3)化疗敏感性用两独立样本t检验,MCF-7细胞,差异没有统计学意义;MDA-MB-231细胞,差异有统计学意义,提示MDA-MB-231细胞系对该方案化疗较敏感;(4)无血清悬浮培养,MDA-MB-231细胞系未发现明显细胞球;MCF-7细胞系初次无血清培养约6天出现细胞球。加入化疗药筛选后两种细胞,见大部分肿瘤细胞逐渐死亡,未发现明显细胞球;(5)流式细胞仪检测,MCF-7、MDA-MB-231两种细胞系中主要是CD44⁺CD24⁺亚群和CD44^-CD24⁺亚群,CD44⁺CD24^-/low细胞比例分别2.07%和0.20%。结论 (1)MDA-MB-231细胞系增值较快,恶性度相对较高,其对TA联合化疗药物较敏感;MCF-7细胞系中可能存在少量肿瘤干细胞,对化疗抵制,能自我更新;(2)无血清培养液培养MCF-7细胞系能形成悬浮细胞球;流式细胞仪检测两种细胞系中CD44⁺CD24^-/low细胞比例小;(3)CD44⁺CD24^-/low表型可能不是乳腺癌干细胞唯一特异性的表面标志。     

高转移肺癌细胞株的建立及其肿瘤干细胞生物学特性

 目的 研究高转移肺腺癌细胞株的肿瘤干细胞生物学特征。方法 肺腺癌细胞系A549接种裸鼠皮下成瘤后,取肺的转移灶,通过机械分离法获取肺转移细胞,体外扩大培养后,再次接种裸鼠。如此反复接种裸鼠,获得稳定肺转移的细胞株A549-V13。采用无血清悬浮培养及PKH26染色确定A549-V13存在肿瘤干细胞。流式细胞术（FACS）检测和分选A549和A549-V13中肿瘤干细胞标志物CD133的表达并进行体内外生物学特征实验。结果 建立稳定高转移A549-V13细胞株。无血清悬浮培养A549-V13,7天后形成的球形细胞团中存在单个PKH26阳性细胞。FACS检测显示,与A549中CD133⁺细胞相比,高转移A549-V13细胞株中CD133⁺细胞的表达比例显著提高4.85倍。A549-V13中CD133⁺细胞具有更强的自我更新能力,侵袭能力提高1.42倍,耐药能力也显著增强,其IC₅₀提高1.26倍,A549-V13的CD133⁺细胞在裸鼠皮下接种2×102个细胞3月致瘤4/6,而接种2×10²个A549的CD133⁺细胞3月才致瘤2/6。结论 建立高转移肺癌细胞株A549-V13,伴随CD133⁺细胞表达比例的增加,其体内外生物学功能显著增强。     

T-Cell Membrane CD45RA (2H4) and CD45RO (UCHL1) Determinants: I, Diverse Patterns of Expression in Mature (Post-Thymic) T-Cell Proliferations

By simultaneous two- and three-colour flow cytometry, this study analysed the expression of membrane CD45RA (2H4) and CD45RO (UCHL1) determinants by normal thymocytes (n = 5) and peripheral blood lymphocyte subpopulations (CD4⁺, n = 21; CD8⁺, n = 12; CD8^dim+, n = 12) and compared these patterns with those of T-cells from representative CD4⁺CD8^- (n = 8), CD4⁺CD8⁺ (n = 2), CD4^-CD8⁺ (n = 10) and CD4^-CD8^- (n = 1) proliferations. These comprised cases of prolymphocytic leukaemia (T-PLL, n = 5), adult T-cell leukaemia-lymphoma (ATLL, n = 2), Sezary Syndrome (SS, n = 4), chronic lymphocytic leukaemia (T-CLL, n = 4), and lymphoproliferative disease of granular lymphocytes (LDGL, n = 5). Normal thymocyte fractions, of which a mean of 85% cells co-expressed membrane CD4 and CD8, were predominantly (mean 89%) 2H4^-UCHL1⁺ with the remaining cells consisting of 2H4^intUCHL1⁺ and 2H4⁺UCHL1^- components. Further analysis showed that virtually all CDla⁺ thymocytes were UCHL1⁺ whereas the CD1a^- fraction comprised similar proportions of both UCHL1^- and UCHL1⁺ subpopulations. Similarly, normal blood CD4⁺, CD8⁺ and CD8^dim+ lymphocytes showed reciprocal CD45RA/CD45RO expression and could be phenotypically grouped into 2H4⁺UCHL1^- 2H4^intUCHL1⁺ and 2H4^-UCHL1⁺ subpopulations. Mean proportions of 48% and 68%, for CD4⁺ and CD8⁺ lymphocytes respectively, showed a composite 2H4⁺UCHL1^- phenotype, whereas the percentage of NK-associated CD8^dim+ cells with this phenotypic pattern was considerably higher (mean, 85%). Normal lymphocyte subpopulations lacking both determinants (2H4^-UCHL1^-) were only rarely noted. Comparing normal patterns of CD45RA/CD45RO expression with those of the T-cell proliferations revealed diverse and abnormal patterns of staining for 3/6 of the CD4⁺CD8^- SS and ATLL, and for 5/5 of the T-PLL (CD4⁺CD8^-, n = 2; CD4⁺CD8⁺, n = 2; and CD4^-CD8⁺, n = 1) cases studied. In contrast, the nine cases of CD4^-CD8⁺ T-CLL and LDGL all showed CD45RA/CD45RO staining patterns similar to that of normal CD8⁺/CD8^dim+ blood lymphocytes (i.e. a predominance of 2H4⁺UCHL1^- cells). Although the variant CD45RA/CD45RO pattern types of the CD4⁺ proliferations did not appear to be related to either the diagnostic category or other phenotypic characteristics, the high proportion of abnormal patterns within this case group suggests that recognition of these abnormalities may be potentially relevant to the differentiation of benign and malignant CD4⁺ proliferations and, in addition, may be of aetiological importance with respect to the diverse acquired defects in immunity commonly seen in patients with such disorders.     

Enhanced antitumor effects by the coculture of allotumor RNA-pulsed dendritic cells with autologous cytokine-induced killer cells on hormone-refractory prostate cancer

In this study, we evaluated antitumor effects of allotumour RNA-transfected dendritic cells (DCs) cocultured with autologous cytokine-induced killer cells (CIKs) on hormone-refractory prostate cancer. The cocultured cells enhanced prostate cancer cytolysis from 26% (CIKs-induced cytolysis) to 80.8%. They also increased the productions of CD4⁺ Th1 (IFN-γ⁺IL-4^-, 55.52%) and CD8⁺ T (IFN-γ⁺, 69.59%) cells determined by intracellular cytokines IFN-γ /IL-4 staining and reduced the rate of CD4⁺ CD25⁺ cells from 18.72% (in CIKs) to 9.72%. The cocultured cells significantly inhibited tumor growth in SCID mouse and induced cancer cells necrosis and apoptosis. Our study indicates that tumor RNA-pulsed DCs cocultured with autologous CIKs significantly enhance antitumor immunity, which can be induced by increased CD4⁺ Th1 and CD8⁺ T cells and decreased CD4⁺CD25⁺ regulatory T (T_reg) cells. This provides a potential immunotherapy strategy for HRPC.     

The clonal hierachy in multiple myeloma

In this report we evaluated the number and phenotype of blood circulating B-cell subsets at different stages of differentiation in 26 patients with newly diagnosed multiple myeloma (MM). In all patients, plasma cells and/or plasma blasts could be identified by flow cytometry with a mean frequency of 1.20% and 0.07%, respectively. In 76.9% of the patients these cells showed aberrant expression mainly of CD56, CD28 and CD117, none of these markers were found on the earlier B-lymphocytes. Clonal B-cells preceding the plasma blast stage were identified by patient specific IgH RT-PCR on sorted B-cell subsets. The clonal cells included the less differentiated CD38⁺/CD19⁺ and CD38^-/CD19⁺ subsets, illustrating that the clonal cells are part of an ongoing differentiation process. Further, the presence of CD38^-/CD19⁺     

T-Cell Membrane CD45RA (2H4) and CD45RO (UCHL1) Determinants: II, Aberrant HLA-ABC Expression by CD45RA and CD45RO Cell Subpopulations of Mature CD4+ T-Cell Leukaemias

Six thymocyte suspensions, 10 normal blood CD4⁺ CD8^- lymphocyte-enriched fractions and leukaemic cells from 24 patients with CD4⁺ mature T-cell lymphoid malignancy (five Sezary Syndrome, six adult T-cell leukaemia-lymphoma and 13 cases of T-cell prolymphocytic leukaemia) were examined in this study for the expression of membrane HLA-ABC by CD45RA (2H4) and CD45RO (UCHL1) subpopulations. These analyses showed that the main increase in HLA-ABC expression by normal CD4⁺ CD8^- blood lymphocytes (mean 490 to 760 FITC units) paralleled the loss of membrane 2H4 whilst the acquisition of UCHL1 was not associated with any significant change in HLA-ABC staining intensity. The sequence of 2H4 differentiation by normal thymocytes, based on the observed increasing levels of HLA-ABC staining intensity appeared to be (a) CD1a ⁺ 2H4^- UCHL1⁺ (25 HLA-ABC fluorescent units), (b)CD1a^-2H4^intUCHL1⁺ (134 units), and (c) CD 1a^- 2H4 ⁺ UCHL1 ^- (197 units). Quantitative estimates of membrane HLA-ABC expression by leukaemic T-cells revealed marked heterogeneity between individual cases irrespective of diagnostic subgroup. Based on the lower observed limits for normal CD4⁺ 2H4⁺ (318 units) and CD4 ⁺ 2H4^- (478 units) fractions, 14% and 38% respectively of the leukaemic 2H4⁺ and 2H4^- components examined showed reduced HLA-ABC expression. Two cases showed very low membrane HLA-ABC levels that were within the range observed for normal CD1a^- thymocytes. In contrast, HLA-ABC staining intensities exceeding that of corresponding normal CD4⁺ 2H4⁺ (710 units) and CD4⁺ 2H4^- (1286 units) subpopulations were seen in a high proportion (65%) of leukaemic 2H4 ⁺ components, with only 14% of 2H4^- fractions showing raised levels and, in two cases, these staining intensities exceeded three times the normal observed limits. In addition to the quantitative differences in HLA-ABC expression, a remarkably consistent (81% of evaluable cases) feature of the leukaemic T-cells was that the 2H4^-UCHL1⁺ subpopulation in CD4⁺ malignancies had a lower HLA-ABC level than the 2H4⁺UCHL1 subpopulation. This was in marked contrast to normal post-thymic T-cells where increasing HLA-ABC expression was seen with increasing UCHL1 (or decreasing 2H4) staining. These results suggest that leukaemic T-cells have an aberrant intra-thymic and post-thymic sequence of 2H4/UCHL1 expression which has become 'uncoupled' from CD1a/HLA-ABC expression.     

CD44+/CD24+表型的宫颈癌Siha细胞对顺铂的耐药及机制研究

背景与目的：肿瘤干细胞的存在是肿瘤细胞抗拒化疗的原因之一。该研究探讨CD44⁺/CD24⁺宫颈癌Siha细胞对顺铂的耐药性及相关机制。方法：体外培养Siha细胞,流式荧光激活细胞分选仪分选CD44⁺/CD24⁺Siha细胞,噻唑蓝(thiazolyl blue,MTT)法检测不同浓度顺铂对细胞的体外抑制情况,采用流式细胞仪检测10 μg/mL顺铂作用于CD44⁺/CD24⁺ Siha细胞24、48和72 h时的细胞凋亡率,实时定量聚合酶链式反应(quantitutive real-time polymerase chain reaction,qRT-PCR)和蛋白［质］印迹法(Western blot)检测CD44⁺/CD24⁺ Siha细胞中Oct-4、ABCG2、Bcl-2的表达,同时设立亲代Siha细胞作为对照。结果：不同浓度顺铂(0.1、1、5、10、15和20 μg/mL)对CD44⁺/CD24⁺ Siha细胞的增殖抑制作用较亲代Siha细胞小［(88.4±1.5)% vs (92.9±1.5)%,(79.9±1.0)% vs (84.7±1.1)%,(69. 8±0.8)% vs (75.1±2.9)%,(59.0±0.7)% vs (65.8±2.7)%,(49.6±0.9)% vs(52.1±0.5)%,(45.1±0.7)% vs (48.8±1.0)%,P<0.05］;与亲代细胞相比,当10 μg/mL顺铂作用于CD44⁺/CD24⁺Siha细胞时,发现在24、48和72 h时的细胞凋亡率均较小［(3.05±0.16)% vs (5.17±0.27)%,(17.94±2.02)%vs (32.60±4.28)%和(40.14±3.01)% vs (56.62±5.32)%,P<0.05］。qRT-PCR和Western blot实验均显示CD44⁺/CD24⁺ Siha细胞高表达Oct-4、ABCG2和Bcl-2,与亲代Siha细胞比较差异有统计学意义(P=0.015)。结论：CD44⁺/CD24⁺ Siha细胞可以抵抗顺铂诱导的细胞凋亡,具有化疗抵抗性,并且高表达肿瘤干细胞表面标志物如Oct-4和ABCG2,该结果在宫颈癌肿瘤干细胞的有效分选以及肿瘤靶向治疗方面有深远意义。