β-榄香烯对MCF-7／ADM细胞株中乳腺癌干细胞作用的初步研究

目的探讨乳腺癌耐阿霉素细胞株MCF-7／ADM与阿霉素敏感细胞株MCF-7／S中乳腺癌干细胞（BCSCs）含量及乳腺癌耐药蛋白（BCRP）和P-糖蛋白（P-gp）表达的差异,观察中药β-榄香烯（β-ELE）对BCSCs及BCRP和P-gp表达的影响。方法 应用无血清细胞培养法培养MCF-7／ADM和MCF-7／S细胞株,形态学观察不同细胞株无血清细胞球培养的成球率,RT PCR检测两种细胞株中BCRP和P-gp的mRNA水平,流式细胞仪检测BCRP和P-gp的阳性表达率及CD44+CD24^-／low细胞比例。应用15μg/ml β-ELE作用MCF-7/ADM细胞株48h后,检测细胞成球率及BCRP、P-gp基因与蛋白表达的变化。结果 与MCF-7／S细胞比较,MCF-7／ADM细胞的成球率及BCRP、P-gp mRNA水平较高。MCF-7／ADM细胞中BCRP和P-gp蛋白的阳性表达率分别为（77.78±9.55）％和（32.33±5.12）％,CD44+CD24-／low细胞比例为（64.79±11.78）％,均高于MCF-7／S细胞的（3.97±1.51）％、（14.26±2.51）％和（18.79±3.28）％,差异均有统计学意义（P＜0.01）。β-ELE能明显抑制MCF-7／ADM细胞的成球率及BCRP、P-gp基因与蛋白的表达（P＜0.01）。结论 MCF-7／ADM是BCSCs相对富集的一种耐药细胞株且高表达BCRP和P gp,β-ELE能够抑制MCF-7／ADM细胞中BCSCs比例及其成球率,并降低耐药蛋白的表达。     

乳腺癌干细胞富集与鉴定的初步研究

目的:通过从MCF-7、ZR-75-1、MDA-MB-231乳腺癌细胞系中培养富集及鉴定乳腺癌干细胞(breast cancer stem cell,BCSC),寻找培养与富集乳腺癌干细胞的方法。方法:贴壁培养MCF-7、ZR-75-1、MDA-MB-231细胞系,倒置显微镜观察各细胞形态;流式细胞仪分别分选收集CD44-CD24-、CD44-CD24+、CD44+CD24-及 CD44+CD24+ 细胞,其中CD44+CD24-为乳腺癌干细胞,其余三类为对照组;MTT法计数细胞,绘制MCF-7、ZR-75-1、MDA-MB-231细胞系生长曲线;MCF-7细胞系进行无血清悬浮培养1个周期,流式细胞仪检测分子表面标记物CD44+CD24-含量,贴壁培养的CD44+CD24-乳腺癌干细胞为对照组;将分选的MCF-7（CD44+CD24-）和分选的其余MCF-7细胞（非CD44+CD24-）进行干性成球实验,鉴定CD44+CD24-干性表达。结果:MCF-7、MDA-MB-231细胞系富含表面标志物CD44-CD24-的乳腺癌细胞;ZR-75-1细胞系富含分子表面标志物CD44+CD24+的乳腺癌细胞;生长曲线显示MCF-7、ZR-75-1、MDA-MB-231均呈持续增长,MDA-MB-231细胞生长较MCF-7、ZR-75-1细胞快;通过无血清悬浮培养CD44+CD24-乳腺癌干细胞由19.4%富集到88.9%;成球实验中CD44+CD24-表型细胞成球数量较分选的其余MCF-7细胞（非CD44+CD24-表型）明显增多,成球率分别为（36.5±1.7）%,（1.1±0.5）%。结论:流式细胞仪可成功分选出分子表面标志物为CD44+CD24-的乳腺癌干细胞;CD44+CD24-可能不是乳腺癌干细胞唯一的表面标志物;MDA-MB-231细胞系较MCF-7、ZR-75-1细胞系生长快;无血清悬浮培养法可简便、高效地富集乳腺癌干细胞;CD44+CD24-乳腺癌干细胞干性表达较强。     

PHⅡ-7对人乳腺癌多药耐药细胞MCF-7/ADR体外杀伤活性和逆转耐药作用

背景与目的:多药耐药(multidrug resistance,MDR)是恶性乳腺癌化疗失败的主要原因,而ABC转运蛋白家族的P-glycoprotein(P-gp)高表达是MDR的主要机制之一.因此研制能抑制P-gp表达及其功能的新型抗癌药是目前研究的热点.本研究旨在研究PH Ⅱ-7对人乳腺癌多药耐药细胞MCF-7/ADR体外抗瘤活性及逆转耐药的作用.方法:采用MTT法检测PHⅡ-7、多柔比星(adriamycin,ADR)及二者联合应用对人乳腺癌细胞MCF-7和耐药细胞株MCF-7/ADR的IC50值;采用流式细胞仪测定PH Ⅱ-7对MCF-7及MCF-7/ADR细胞凋亡的诱导作用;采用逆转录PCR和实时荧光定量PCR方法检测PHⅡ-7对多药耐药基因1(multidrug resistance gene 1,mdr1)表达水平的影响;通过流式细胞仪观察PHⅡ-7处理前后,MCF-7/ADR细胞内Rhodamine123浓度的改变,分析PHⅡ-7对p-gp外排功能的影响.结果:PH Ⅱ-7对MCF-7和MCF-7/ADR细胞均有生长抑制作用,IC50值分别为(6.07±0.85)和(5.51±1.22)μmol/L;低浓度PHⅡ-7与ADR联合应用能增强其细胞毒作用,二者具有协同作用.PHⅡ-7可诱导MCF-7和MCF-7/ADR细胞凋亡;在诱导凋亡过程中,PHⅡ-7可降低MCF-7/ADR细胞内mdrl基因的表达,抑制p-gp外排功能.结论:PHⅡ-7可诱导MCF-7/ADR凋亡,并具有对MCF-7相同的体外杀伤活性.PHⅡ-7可通过抑制P-gp的表达和功能逆转MCF-7/ADR耐药性.     

Anxa2和P-gp蛋白相互作用对多药耐药乳腺癌细胞迁移与侵袭影响的研究

目的:探讨Anxa2和P-gp蛋白相互作用对多药耐药乳腺癌细胞迁移与侵袭的影响.方法:采用小干扰RNA技术下调人多药耐药乳腺癌细胞MCF-7/ADR中P-gp的表达,并筛选稳定的单克隆细胞株;利用噻唑蓝(MTT)比色法和Transwell实验研究P-gp的表达对MCF-7及其耐药细胞MCF-7/ADR的增殖、迁移和侵袭能力的影响;利用免疫沉淀和免疫荧光分析多药耐药细胞中Anxa2和P-gp的相互关系.结果:蛋白印迹法检测发现,Anxa2和P-gp在耐药乳腺癌细胞中均高表达;MCF-7/ADR与其亲本细胞MCF-7相比,迁移和侵袭能力明显增强;MCF-7/ADR细胞中P-gp的表达被抑制后,其迁移和侵袭能力显著下降,并且差异有统计学意义,P＜0.05.免疫沉淀证实,Anxa2和P-gp之间存在相互作用;免疫荧光发现,Anxa2和P-gp在细胞膜上存在很好的共定位.结论:降低P-gp的表达可以明显抑制耐药乳腺癌细胞MCF-7/ADR的迁移和侵袭能力,Anxa2和P-gp之间存在较好的共定位和相互作用.提示Anxa2和P-gp的相互作用有可能对增强多药耐药乳腺癌细胞的侵袭转移能力起到一个重要的作用.     

乙醛脱氢酶阳性乳腺癌干细胞的分离培养及其生物学特性分析

 目的　证实人类乳腺癌MCF-7细胞系中存在ALDH+乳腺癌干细胞,并研究ALDH+乳腺癌干细胞体外增殖、侵袭能力等生物学特性。方法　应用流式细胞术从MCF-7细胞中分离并培养ALDH+ 乳腺癌干／祖细胞,通过划痕试验、MTT法生长曲线测定、以及Transwell法等检测ALDH+乳腺癌干细胞的生物学特性。结果　MCF-7细胞系中,CD-／low24 CD+44 细胞比例约为1.4 %,ALDH+ CD-／low24 CD+44 细胞比例约为1.2 %; ALDH+乳腺癌干细胞与CD-／low24 CD+44 细胞两者的生长曲线基本一致;CD-／low24 CD+44 ,ALDH+ CD-／low24 CD+44 组细胞划痕区细胞间距离明显缩短,其迁移能力明显强于对照组,且两群干细胞之间存在差异;Transwell实验结果,与对照组相比,CD-／low24 CD+44 、ALDH+ CD-／low24 CD+44 两组细胞有大量细胞过膜,三组MTT检测吸光度值分别为1.05±0.098、1.56±0.075、1.67±0.032。结论　MCF-7细胞系中存在CD-／low24 CD+44 和ALDH+ CD-／low24 CD+44 乳腺癌干细胞,ALDH可以作为鉴定乳腺癌干细胞的分子标志之一。     

内分泌及化疗对ER+乳腺癌CD44+CD24-/low细胞亚群的实验与临床研究

目的:探讨内分泌治疗及化疗对雌激素受体(estrogen receptor,ER)阳性的乳腺癌CD44+CD24-/low细胞亚群的影响.方法:用3个浓度的雌二醇(estradiol,E2)和E2+他莫西芬(tamoxifen,TAM)处理乳腺癌MCF-7细胞,用工作浓度的化疗药物(多西他赛、紫杉醇、表柔比星和5-氟尿嘧啶)处理MCF-7细胞,FCM法检测各组细胞中CD44+CD24-/low的细胞比例,并检测ER+的转移性乳腺癌(metastatic breast cancer,MBC)患者化疗前、后外周静脉血中CD45-CD44+CD24-/low的细胞比例.结果:不同浓度E2培养MCF-7细胞10和20 d后,CD44+ CD24-/low细胞比例均高于对照组,E2+TAM组低于相应浓度E2组.各化疗药物作用MCF-7细胞24 h后,CD44+CD24-/low细胞比例均上升;而继续培养至20 d后,CD44+ CD24-/low细胞比例降低.部分缓解和疾病稳定的MBC患者,化疗后外周血CD45-CD44+CD24-/low细胞比例降低;而疾病进展者化疗后外周血CD45-CD44+CD24-/low细胞比例上升.结论:E2促进MCF-7细胞中CD44+CD24-/low细胞亚群的生长,TAM可抑制MCF-7细胞中CD44+CD24-/low细胞亚群的生长;MCF-7细胞中CD44+CD24-/low细胞亚群对多西他赛、紫杉醇、表柔比星和5-氟尿嘧啶均有抵抗能力;化疗有效患者外周血中CD45-CD44+CD24-/low细胞比例降低.     

化疗对三阴性乳腺癌CD44+CD24-/low细胞亚群的影响

目的:观察化疗对三阴性乳腺癌CD44+CD24-/low细胞亚群的影响。方法:用0.2、1、5倍药物血浆峰浓度(peak plasma concentration,PPC)的紫杉醇、多西紫杉醇、氟尿嘧啶、表柔比星、长春瑞滨和吉西他滨分别作用人三阴性乳腺癌MDA-MB-231细胞24、48和72h后,MTT法检测细胞生长情况;FCM法检测1倍PPC的各药物作用48h以及细胞复苏后CD44+CD24-/low细胞含量的变化;另外,FCM法检测10例转移性三阴性乳腺癌患者在化疗前后,外周血中CD44+CD24-/low细胞的含量变化。结果:6种药物均可抑制MDA-MB-231细胞增殖。1倍PPC的各药作用后,CD44+CD24-/low细胞含量未明显升高,以氟尿嘧啶组含量降低最为明显(P<0.05)。转移性三阴性乳腺癌患者在化疗后,外周血中CD44+CD24-/low细胞含量降低(P<0.05)。结论:化疗药物紫杉醇、多西紫杉醇、氟尿嘧啶和表柔比星可降低MDA-MB-231细胞株中CD44+CD24-/low细胞亚群的含量。化疗可使转移性三阴性乳腺癌患者外周血中CD44+CD24-/low细胞含量降低。     

乳腺癌耐阿霉素细胞MCF-7/ADR外泌体传递tTG参与MCF-7细胞耐药的研究

目的:研究乳腺癌耐阿霉素细胞MCF-7/ADR通过外泌体传递tTG参与敏感细胞MCF-7耐药的过程。方法:本实验首先培养MCF-7、MCF-7/ADR细胞,然后检测MCF-7、MCF-7/ADR细胞中外泌体的含量,并将外泌体提取后备用。MCF-7/ADR细胞外泌体(EXO/ADR)与MCF-7细胞共培养建立MCF-7的外泌体耐药细胞(MCF-7+EXO/ADR)。使用Western blot法检测MCF-7、MCF-7/ADR、MCF-7+EXO/ADR细胞中的tTG蛋白的表达水平,使用流式细胞术分别检测敏感细胞株外泌体(EXO/S)、耐药细胞株外泌体(EXO/ADR)中tTG的表达水平。使用CCK-8法分别检测MCF-7、MCF-7/ADR、MCF-7+EXO/ADR细胞对阿霉素的敏感性情况。结果:实验结果显示,在EXO/S、EXO/ADR中均可以表达CD63、TSG101,但是较低表达Calnexin。经过流式细胞术检测发现,EXO/S、EXO/ADR中的tTG表达水平分别为(18.3±3.63)%、(46.07±8.31)%,二者tTG的表达有明显差异。经过Western blo...     

低毫安(10mA)电化学治疗对人乳腺癌细胞耐药性的逆转作用

目的:研究低毫安(10mA)电化学治疗对人乳腺癌细胞阿霉素耐药株(MCF-7/ADR)多药耐药(MDR)性的逆转作用及其机制.方法:用MTT法测定电化学治疗对细胞的生长抑制作用,荧光分光光度法检测细胞内阿霉素(ADR)的浓度,流式细胞术检测细胞p-糖蛋白(P-gp)的表达.结果:低毫安电化学治疗(0.1C-2C)能不同程度降低ADR对MCF-7/ADR细胞的IC5o;2C的电化学治疗能显著提高ADR在MCF-7/ADR细胞内的浓度,降低MCF-7/ADR细胞P-gp的表达.结论:电化学治疗能逆转MCF-7/ADR细胞的MDR,其机理可能是抑制了P-gp的功能表达,增加了细胞内ADR的积累.     

Enhanced myeloid specificity of CD117 compared with CD13 and CD33

The c-kit proto-oncogene encodes a 145 kd tyrosine kinase transmembrane receptor, which plays a key role in haemopoiesis. The c-kit has been classified as CD117 and is especially useful in the differential diagnosis of acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL). We analysed 104 consecutive cases (55 AML, 23 B-cell lineage ALL, three T-cell ALL, 11 blast crisis of chronic myeloproliferative disorders and 12 cases of myelodysplastic syndromes with more than 10% of blasts) referred to our Hospital for immunophenotypic diagnosis and compared the expression pattern of CD13, CD33 and CD117 using the same fluorochrome (phycoerythrin-PE). The recommendations of the EGIL group were followed in order to establish lineage involvement of the blastic population. The threshold used to assign positivity for CD117 was 10%. Bcr/abl, TEL/AML-1 and MLL rearrangements were assessed by molecular methods. CD117 expression was detected in 91% of AML and MDS. All the negative cases corresponded to acute monocytic leukemias. The calculated specificity for myeloid involvement was 0.86 for CD117, 0.36 for CD13 and 0.44 for CD33 (P < 0.005). CD117 was also positive in four cases of ALL. None of these cases showed bcr/abl or MLL rearrangements. In the light of these findings, CD117 expression should yield a higher score, at least one point, in the system currently applied for the diagnosis of biphenotypic acute leukemias (BAL) as its myeloid specificity is greater than that of CD13 and CD33. Moreover, its absence in AML could identify two subgroups of M5b cases. The coexpression of CD117 with cytoplasmic CD79a is often associated with CD7 reactivity, suggesting a stem cell disorder. CD117 should be included on a routine basis for the immunophenotypic diagnosis of acute leukemias.     

CD5, CD10, and CD23 expression in Waldenstrom's macroglobulinemia

CD5, CD10, and CD23 are cell surface antigens used to distinguish B-cell disorders. The expression of these antigens and their clinical significance in Waldenstrom's macroglobulinemia (WM), an uncommon B-cell disorder, remains to be clarified. We therefore determined expression of CD5, CD10, and CD23 by flow cytometric analysis on bone marrow lymphoplasmacytic cells (CD19+ k/l light chain restricted) for 171 serially biopsied patients with findings of the consensus panel definition of WM. Importantly, we also correlated laboratory and clinical data, as well as existence of a familial history of a B-cell disorder in view of reports suggesting familial predisposition in WM. These studies demonstrated tumor cell expression of CD5, CD10, and CD23 in 15 of 171 patients (9%), 11 of 161 patients (7%), and 37 of 105 patients (35%), respectively. Coexpression of CD23 with CD5 or CD10 was common. Tumor Lymphoplasmacytic from 10 of 15 (66%) and 3 of 11 (27%) patients with WM that expressed CD5 and CD10, respectively, also showed expression of CD23 (P = 0.01 and P = 0.08, respectively). Among patients with CD23 expression, increased serum immunoglobulin (Ig) M levels were observed compared with patients without CD23 expression (P = 0.05). No differences in age at diagnosis; presence of adenopathy and/or splenomegaly; bone marrow involvement; serum IgA, IgB, and b2 macroglobulin levels; hematocrit; platelet count; or familial history of WM or a related B-cell disorder were observed among patients with and without CD5, CD10, and CD23 expression. These studies demonstrate that CD5, CD10, and CD23 are commonly found in WM and that their expression should not exclude the diagnosis of WM. Moreover, expression of CD23 may define a clinically distinct subset of patients with WM.     

人CD46、CD55和CD59与肿瘤免疫逃逸及免疫治疗

膜结合补体调节蛋白CD46、CD55和CD59在肿瘤细胞膜上表达或过表达,保护肿瘤细胞免受免疫系统的攻击,成为肿瘤细胞免疫逃逸的途径之一.如何下调肿瘤细胞表面三者表达或抑制其功能以增强其对补体依赖的细胞毒作用的敏感性备受关注.     

CD46 CD55 CD59在结肠癌组织中的表达及意义

目的:检测CD46、CD55、CD59在结肠癌组织中的表达情况,分析其与结肠癌临床病理参数间的相关性及意义。方法:选取有详细性别、年龄、组织分化、病理分期、肿瘤部位、组织类型资料的组织芯片标本,包括121 例结肠癌和121 例癌旁结肠组织,均为2004年10月至2006年6 月第四军医大学西京消化病医院胃肠外科手术切除标本。应用免疫组织化学改良二步法分别检测CD46、CD55、CD59的表达情况。结果:CD46、CD55及CD59在结肠癌组织中的阳性表达率均显著高于对应的癌旁组织（P < 0.001）。 CD46表达水平与性别、年龄、组织分化、TNM 病理分期、肿瘤位置、病理组织类型均无关（P > 0.05）。 CD55、CD59的表达水平与性别、年龄、肿瘤位置、病理类型无关（P > 0.05）,而与组织分化、TNM 病理分期有关（P < 0.05）。 其表达强度阳性率中低分化组明显高于高分化组（P < 0.05）。 TNM 分期中Ⅲ、Ⅳ期患者肿瘤病理组织强阳性表达率高于Ⅰ、Ⅱ期阳性表达率,两者比较有统计学意义（P < 0.05）。 结论:CD46、CD55及CD59在结肠癌组织中高表达,特别是CD55、CD59的表达与肿瘤分化、病理分期相关,提示三者表达水平与结肠癌生物学行为密切相关。      

Poor prognosis of mediastinal non-Hodgkin's lymphoma with an immature phenotype of CD2+, CD7 (or CD5)+, CD3-, CD4-, and CD8-

Nine children with mediastinal non-Hodgkin's lymphoma (NHL) were treated according to our new regimen which is characterized by intensified therapy with high-dose cytosine arabinoside (HDCA). After induction therapy with a combination of five drugs, such as vincristine, doxorubicin, cyclophosphamide, 1-asparaginase, and prednisolone, intermediate dosages of methotrexate (MTX) (1 g/m2) and HDCA (1.5 g/m2 x 12 doses) were administered. All but one patient (88.9%) achieved complete remission and then received this intensified therapy. With a median follow-up period of 25.5 months, five patients are still in complete remission, but three patients have relapsed. From the phenotypic point of view, these relapsed patients showed only very immature T-cell differentiation antigens such as CD2 and CD7 (or CD5). These results suggest that HDCA as intensified therapy for children with mediastinal NHL seems to be effective. However, for patients with an immature phenotype of T-lineage cells, more sophisticated regimens should be prepared.     

Agranular CD4+/CD56+ cutaneous neoplasm

CD4+ CD56+ cutaneous neoplasm with hematological relapse is a rare malignant disease and has been described recently in the literature as blastic or agranular NK-cell leukemia/lymphoma. The origin of this neoplasm is uncertain. We describe a 75-year-old patient with a primary cutaneous neoplasm CD4+ CD56+ who evolved to leukemic phase despite standard lymphoma chemotherapy. Morphologically, the cells were undifferentiated without granules in the cytoplasm. The immunophenotype showed the expression of CD4, CD56, CD68, CD33, CD7, CD2, CD45RA, and CD38. Histological analysis revealed a cell infiltration mainly located in the dermis. T-cell receptor and immunoglobulin heavy chain genes were in germline configuration. Cytogenetic study showed complex structural abnormalities with a deletion of the chromosome 5 del(5q). The clinical course was aggressive with an early hematological relapse.     

The CD11/CD18 leukocyte glycoprotein deficiency

CD11/CD18 leukocyte glycoprotein deficiency is a rare, inherited disorder of leukocyte function, manifested by recurrent severe bacterial infections. A deficiency in the expression of a family of leukocyte membrane glycoproteins (the CD11/CD18 glycoproteins) represents the molecular basis for this disease.