重组反义c-myc腺病毒抑制K562细胞生长、诱导凋亡的作用

目的：研究重组反义cmyc腺病毒（Ad—AS-c-myc）对人慢性粒细胞K562细胞系抑制生长，诱导凋亡作用及分子机制，探讨Ad-As-c—myc用于慢性粒细胞白血病基因治疗的可能性。方法：采用重组腺病毒Lac—Z（Ad—LacZ）及Ad—AS-c—myc联合多聚季胺转染K562细胞，Xgal染色判断转染效率。采用形态学、MTT试验、RT—PCR、DNA凝胶电泳、流式细胞仪及免疫细胞化学等方法进行研究。结果：多聚阳离子可提高腺病毒载体对K562细胞的转染，Ad—LacZ＋多聚季胺转染率达86％。AdAS—c—myc能抑制K562细胞c—myc基因在转录水平的表达，K562细胞生长受抑（抑制率38％）；Ad—AS-c—myc可使K562细胞G2、G2期阻滞，增殖相关基因PCNA表达降低，Apo2．7蛋白阳性细胞率增高，DNA电泳出现DNA梯形条带。结论：Ad—AS-c-myc对K562细胞具有抑制生长及诱导凋亡作用，其生物学作用与K562细胞c-myc及PCNA的表达降低有关。Ad-AS-c-myc在慢性粒细胞白血病基因治疗中具有潜在的临床价值。     

As2S2诱导K562细胞凋亡的分子机制初步研究

目的　探索As2 S2 对K5 6 2细胞的诱导凋亡作用及其机制。方法　细胞凋亡的检测用流式细胞分析、基因组DNA电泳、细胞形态学观察等方法 ,Westernblot方法用于蛋白表达的检测 ,基因表达的变化用半定量RT PCR方法。结果　 3～ 5 μmol L的As2 S2 作用 4 8～ 72h即可诱导K5 6 2细胞凋亡。 3μmol LAs2 S2 作用 72h时 ,细胞凋亡率达 (34.4± 3.3) % ;5 μmol LAs2 S2 作用 4 8,72h时 ,细胞凋亡率分别为 (2 1.8± 3.6 ) %和 (4 6 .0± 5 .2 ) %。 5 μmol LAs2 S2 作用下 ,As2 S2 可降低Bcr Abl和JAK2的蛋白含量。As2 S2 从基因水平上调bax表达 ,下调c myc表达。As2 S2 作用后 ,Caspase 3被激活。As2 S2 也能诱导慢性粒细胞性白血病 (CML)患者单个核细胞凋亡。结论　As2 S2 可诱导CML细胞凋亡 ,Bcr Abl含量的降低可能起了很重要的作用。bax表达的增加、c myc表达的减少、JAK2蛋白含量的降低以及Caspase 3激活也可能参与了该细胞凋亡机制。     

原位杂交技术在髓系微小残留白血病检测中的初步应用

应用原位杂交法以~(32)P 标记 c—myc 为探针,分析了5例急性髓细胞白血病完全缓解期单个骨髓细胞中 c—myc 基因的转录水平,并与正常对照进行比较。结果表明,正常骨髓中仅有 c—myc 基因低表达细胞,而5例患者中有3例可检出 c—myc 基因过高表达的异常细胞(白血病细胞)。该技术为髓系微小残留白血病的检测提供了新途径。     

c-myc反义寡核苷酸对表达FHIT基因的结肠癌细胞增殖及凋亡作用的影响

目的：观察脂质体介导的c—myc反义寡核苷酸对表达FHIT基因的结肠癌细胞增殖及凋亡作用的影响。方法：用脂质体法将重组FHIT基因的PRC／CMV质粒和空载体质转染到人结肠细胞株SW480，随后分别转染c—myc反义寡核苷酸。Western blot法检测细胞FHIT和c—myc的表达；MTF法检测细胞的增殖；AO／EB染色法和流式细胞分析技术检测细胞的凋亡。结果：转染FHIT基因后，SW480细胞有明显的FHIT蛋白表达而转染空载体的细胞未检测到FHIT蛋白表达。C—myc反义寡核苷酸转染后，对SW480细胞c—myc的表达有明显的抑制作用（P〈0．01）；对FHIT＋／-SW480细胞的增殖均有明显的抑制作用（P〈0．01），且对FHIT＋SW480细胞的抑制作用明显强于FHIT—SW480细胞（P〈0．05）。同时，c-myc反义寡核苷酸对FHIT＋／-SW480细胞的凋亡均有明显的促进作用，对FHIT＋SW480细胞凋亡的促进作用明显强于FHIT—SW480细胞（P〈0．05）。结论：FHIT基因的表达和癌基因c—myc的表达抑制共同作用可以发挥较强的抗肿瘤细胞的效果，为多基因联合干预治疗肿瘤奠定了理论基础。     

原位杂交技术在髓系微小残留白血病检测中的初步应用

我们应用原位杂交法,以~(32)P标记c—myc为探针,分析了5例急性髓细胞白血病完全缓解期单个骨髓细胞中c—myc基因的转录水平,并与正常对照进行比较。结果表明,正常骨髓中仅有c—myc基因低表达细胞,而5例患者中有3例可检出c—myc基因过高表达的异常细胞(白血病细胞)。该技术为髓系微小残留白血病的检测提供了新途径。     

白血病细胞凋亡与bcl-2和bax-alpha表达的研究

目的　研究白血病细胞细胞凋亡率与 bcl- 2和bax- alpha阳性率及表达水平关系。方法　选择难治或复发的化疗耐药白血病患者和初治化疗敏感白血病患者各 10例 ,分离白血病细胞 ,柔红霉素 (5 0 0 ng/ ml)诱导凋亡 ,应用流式细胞术比较分析耐药组和敏感组白血病细胞的凋亡率与 bcl- 2和 bax- alpha阳性率和表达水平的关系。结果　化疗耐药组和化疗敏感组对柔红霉素诱导的凋亡率有显著差异 ,敏感组 (2 3.2 6 %± 12 .94% )显著高于耐药组 (2 .2 6 %± 1.44 % ) ,(P=0 .0 0 1) ;耐药组白血病细胞凋亡率与 bcl- 2阳性率呈负相关 (r=- 0 .6 47,P=0 .0 43) ,与 bax- alpha阳性率和相对荧光强度呈正相关 (r=0 .883,P=0 .0 0 1;r=0 .814,P=0 .0 0 4)及 bcl- 2 :bax- alpha比率呈负相关 (r=-0 .893,P=0 .0 0 1) ;敏感组凋亡率 bax- alpha的阳性率和荧光强度呈正相关 (r=0 .6 5 3,P=0 .0 41,r=0 .76 2 ,P=0 .0 10 ) ,与 bcl- 2 :bax- alpha比率呈负相关 (r=- 0 .741,P=0 .0 14)。结论　白血病细胞耐药性与细胞的凋亡抗性有关 ,这种凋亡抗性与 bcl- 2和 bax- alpha的表达比例增高有关 ,特别是 bax- alpha的表达水平     

拓扑替康抑制HL-60细胞增生和诱导凋亡机制与c-myc的关系

目的探讨拓扑替康（TPT）抑制白血病细胞的作用机制。方法采用反转录-聚合酶链反应（RT-PCR）和免疫组织化学方法,检测TPT作用后c—myc mRNA及其蛋白的表达情况。结果0．15μmol／L TPT作用HL-60细胞12h后,HL-60细胞c—myc mRNA表达为0．17±0．03,与空白对照组的1．11±0．25相比明显降低,差异具有统计学意义（P〈0．05）。HL-60细胞c—myc蛋白表达阳性细胞百分率为19．67％,较空白对照组的68．33％明显降低,差异具有统计学意义（P〈0．05）。结论拓扑替康抑制HL-60细胞增生和诱导凋亡可能是通过c—myc途径起作用的。     

丹参酮诱导HL—60细胞分化及凋亡的流式细胞术分析

应用体外细胞培养及流式细胞仪技术，分析了丹参酮作用前后HL－60细胞的细胞周期分布、细胞增殖指数、p53、c－myc、c－fos及bc1－2基因产物的表达变化，并以全反式维甲酸为对照。结果表明：0．5μg．ml－1的丹参酮作用后，HL－60细胞趋向粒细胞系统分化，有部分细胞发生凋亡（11．8％），丹参酮通过阻止HL－60细胞进入S期而抑制其DNA合成，其作用的分子机制与c－fos基因的表达增高、c－myc基因及bc1－2基因的表达降低有关，诱导分化与细胞凋亡的关系尚有待进一步探讨。     

人膀胱移行细胞癌HPV16/18型感染与c-erbB_2、H-ras、c-myc表达的关系

目的　研究人膀胱移行细胞癌 (TCC)中HPV 16 / 18型感染与c erbB2 、H ras、c myc蛋白产物表达的相互关系。方法应用免疫组织化学法检测经聚合酶链反应证实的 34例HPV 16 / 18感染阳性、2 0例HPV 16 / 18感染阴性的TCC组织和 7例正常膀胱组织中c erbB2 、H ras、c myc蛋白产物的表达 ,并经统计学处理。 结果　HPV 16 / 18感染阳性组c erbB2 、H ras、c myc蛋白产物表达阳性率分别为 5 5 .9% (19/ 34)、5 8.8% (2 0 / 34)、6 1.8% (2 1/ 34) ;HPV 16 / 18感染阴性组分别为 5 5 .0 % (11/ 2 0 )、6 5 .0 % (13/2 0 )、6 5 .0 % (13/ 2 0 ) ;正常膀胱粘膜上述 3种蛋白产物表达阳性例数依次为 0、1、0例。HPV 16 / 18感染与c erbB2 、H ras、c myc蛋白产物表达无关 (P >0 .0 5 )。c erbB2 、H ras、c myc蛋白产物阳性表达率在癌组织和正常膀胱粘膜之间有显著性差异 (P <0 .0 5 ) ,且其阳性表达率与TCC的病理分级相关 (P <0 .0 5 )。癌组织内c erbB2 与c myc蛋白产物表达呈正相关 (P <0 .0 1)。 结论　在TCC的发生、发展过程中 ,HPV 16 / 18可能不是主要通过c erbB2 、H ras、c myc蛋白产物的改变来发挥作用。c erbB2 、H ras、c myc蛋白产物的改变有可能为TCC发生的晚期事件 ,提示应注意其与临床预后的关系     

急性白血病细胞C-myc基因表达的研究

目的：研究C—myc基因在急性白血病(AL)细胞中的表达及其与AL的分型、临床特征、疗效及预后因素的关系。方法：采用免疫组化方法检测54例初治AL骨髓细胞C—myc蛋白表达情况，分析其与FAB分型、临床特征、疗效及预后因素的关系。结果：AL骨髓细胞中C—myc基因的表达显著高于正常对照组(尸＜0．05)。C—myc基因表达在急性髓系白血病(AML)与急性淋巴细胞白血病(ALL)无显著差异；在AML各亚型中，也无显著性差异(P＞0．05)。C—myc的表达与初诊时的白细胞数呈正相关(r＝0．50，P＜0．05)。结论：C—myc基因的紊乱在AL的发病中起着重要作用，与白血病的某些临床特征、疗效及预后因素密切相关。     

造血干细胞移植治疗成人急性白血病

造血干细胞移植治疗成人急性白血病5例，包括3例第一次完全缓解（CR1）急性粒细胞白血病，1例CR1急性淋巴细胞自血病，1例急性粒细胞白血病复发。其中异基因骨髓移植（alloBMT）4例，同基因外周血造血干细胞移植（Syn－PBSCT）1例。所有病例均采用TBI＋CY＋CCNU作预处理。所有病例均移植成功。2例发生急性移植物抗宿主病（aGVHD），其中1例又发生慢性GVHD。1例未缓解患者移植后完全缓解，但于 69天死于间质性肺炎；3例已分别无病生存22、10和3个月；余1例（急淋）移植后12个月死于复发。结果尚提示外周血干细胞移植造血重建更快。     

Molecular Analysis of Rearranged VH Genes during B Cell Chronic Lymphocytic Leukemia: Intraclonal Stability is Frequent but not Constant

Several genetic mechanisms have been shown to diversify the expressed antibody repertoire of commited B lymphocytes. These include V gene replacement, ongoing gene rearrangement and somatic hypermutation. These mechanisms may be operational at discrete points in the B cell differentiation pathway and generate idiotype diversity in various malignant B cell tumors. In particular, V region mutations have been established as a major mechanism of tumor escape from anti-idiotype immunotherapy in some lymphoma. On the other hand, previous studies on a few selected cases have shown that this mutation process does not affect the B cell clone during chronic lymphocytic leukemia. However, to what extent this intraclonal stability is a general phenomenon during B cell CLL is not clear. Therefore, we randomly selected 6 patients suffering from classical B cell CLL (sIgM (+), CD5 (+), CD19 (+)) at different stages of the disease and analysed the intraclonal variability of the expressed variable region of the heavy chain (VH). After PCR amplification of the cDNA corresponding to the rearranged VDJ regions, the products were cloned and sequenced. In five cases, multiple clone analysis did not show any intraclonal variability whatever the stage of the disease. Furthermore, in a single case, this intraclonal stability was confirmed during a three year period of time when the disease progressed. The sixth case behaved differently since we found multiple nucleotide substitutions, apparently accumulating as the malignant clone expanded. Besides the theoretical difficulties that these changes can induce during immunotherapy, two findings merit further discussion: 1) the distribution of the ongoing mutations affecting the VH region was not suggestive of an antigen driven selection, 2) this intraclonal variability was specific for the VH region, since the VL region showed no intraclonal variation.     

Familial Hairy Cell Leukemia

Familial hairy cell leukemia (HCL) occurs rarely, and HCL occurring in association with other hematologic malignancies is even rarer. We describe two cases of familial HCL syndromes: a mother and son with HCL, and a HCL patient whose aunt developed Hodgkin's Disease (HD). This is the first reported familial association of HCL with HD.     

RNAi沉默CDX2基因对急性髓细胞白血病细胞增殖影响的实验研究

目的:探讨RNA干扰技术沉默CDX2基因对人急性髓细胞白血病细胞系THP1增殖影响的意义.方法:针对CDX2mRNA序列设计合成2对编码小干扰RNA(siRNA)的DNA模板,构建pGenSi1-CDX2siRNA重组质粒,转染THP1细胞.采用蛋白质印迹法检测重组质粒对CDX2蛋白表达的影响,MTT法观察THP1细胞增殖情况,Annexin-V-PI双染法流式细胞术检测转染重组质粒后细胞凋亡状况.结果:成功构建pGenSil-CDX2 siRNA重组质粒,成功转染THP1细胞,并能特异地抑制CDX2基因的表达;转染重组质粒后,THP1的活力分别降低为(39.8±5.0)%,与空质粒组组间差异有统计学意义,P=0.016.重组质粒组的THP1细胞凋亡率为37.3%～42.1%.结论:pGenSil-CDX2 siRNA重组质粒明显下调XDX2在急性髓细胞白血病细胞中的表达,并抑制肿瘤细胞生长,促进其凋亡.     

Malignant Cell Detection in Burkitt's Lymphoma Using Third-complementarity-determining Region (CDRIII), Clone-specific Probe Developed by Sequencing DNA from Stored Slides

The DNA sequence of the third-complementarity-determining region (CDRIII) of the immunoglobulin heavy chain (IgH) gene in a case of Burkitt's lymphoma was determined hy polymerase chain reaction (PCR) using template DNA extracted from a smear stored at room temperature for more than one year. The DNA sequence obtained from the stored slide was compared with that of DNA from a frozen lymph node biopsied at the initial presentation. The sequences were shown to he identical, implying that DNA from a smear on a stored slide can he used as a source of DNA for PCR amplification, sequencing, and development of a clone-specific probe. Using oligonucleotides generated from one of the CDRIII sequences of the IgH gene as molecular probes, a retrospective study for the malignant clone on the smears was carried out. Malignant ceils were detectable in the peripheral blood at an early stage of hone marrow relapse but not in the peripheral blood or bone marrow at the initial presentation. No malignant clone was detected in the bone marrow when testicular infiltration was diagnosed by examination of a pathological specimen. Thus, the technique permits molecular analysis of hematologic malignancies of B-cell lineage in cases where fresh or frozen specimens are not available.     

Effects of CT-Xp Gene Knock down in Melanoma Cell Lines

Cancer/testis (CT) genes are encoded by genes that are normally expressed only in the human germ line but which are activated in various malignancies. CT proteins are frequently immunogenic in cancer patients and their expression is highly restricted to tumors. They are thus important targets for anticancer immunotherapy. In several different tumor types, the expression of CT-X genes is associated with advanced disease and poor outcome, indicating that their expression might contribute to tumorigenesis. CT-X genes encoding members of the MAGE protein family on Xq28 have been shown to potentially influence the tumorigenic phenotype. We used small interfering RNA (siRNA) to investigate whether CT-X mapping to the short arm of the X-chromosome might also have tumorigenic properties and therefore be potentially targeted by functional inhibitors in a therapeutic setting. siRNAs specific to GAGE, SSX and XAGE1 were used in cell proliferation, migration and cell survival assays using cell lines derived from melanoma, a tumor type known to present high frequencies of expression of CT antigens. We found that of these, those specific to GAGE and XAGE1 most significantly impeded melanoma cell migration and invasion and those specific to SSX4 and XAGE1 decreased the clonogenic survival of melanoma cells. Our results suggest that GAGE, XAGE1 and SSX4 might each have a role in tumor progression and are possible therapeutic targets for the treatment of melanoma and other malignancies.     

Novel Mutations in a Lethal Case of Lymphomatous Adult T Cell Lymphoma with Cryptic Myocardial Involvement

The autopsy of a 65-year-old diabetic African American male revealed significant left myocardial involvement by adult T-cell leukemia/lymphoma (ATLL) despite normal pre-mortem fluorodeoxyglucose (FDG) uptake by positron emission tomography/computed tomography (PET/CT). Due to pre-existing diabetic cardiomyopathy with reduced ejection fraction (EF) and compatible imaging studies, cardiac lymphomatous involvement was not suspected. While peripheral blood was negative for leukemia, next-generation sequencing of a lymph node revealed at least eight novel mutations (AXIN1, R712Q, BARD1 R749K, CTNNB1 I315V, CUX1 P102T, DNMT3A S199R, FGFR2 S431L, LRP1B Y2560C and STAG2 I771M). These findings underscore a diagnostic pitfall in a rare lymphomatous variant of ATLL infiltrating myocardium and contribute to its molecular characterization.     

Allogeneic Hemopietic Stem Cell Transplants for the Treatment of B Cell Acute Lymphocytic Leukemia

Objective: Explore the feasibility of allo- hemopietic stem cell transplants in treating patients with B cell acute lymphocytic leukemia. Methods: Between september 2006 and February 2011, fifteen patients with B cell acute lymphocytic leukemia (ALL) were treated by allo-hemopietic stem cell transplants (HSCT). Stem cell sources were peripheral blood. Six patients were conditioned by busulfan (BU) and cyclophosphamide (CY) and nine patients were conditioned with TBI and cyclophosphamide (CY). Graft versus host disease (GVHD) prophylaxis regimen consisted of cyclosporine A (CSA), methotrex ate (MTX) and mycophenolatemofetil (MMF). Results: Patients received a median of 7.98×108·kg-1 (5.36-12.30×108·kg-1) mononuclear cells (MNC). The median time ofANC> 0.5×109/L was day 12 (10-15), and PLT> 20. 0×109/L was day 13 (11-16). Extensive acute GVHD occurred in 6 (40.0%) patients, and extensive chronic GVHD was recorded in 6 (40.0%) patients. Nine patients were alive after 2.5-65 months follow-up. Conclusion: Allogeneic stem cell transplant could be effective in treating patients with B cell acute lymphocytic leukemia.     

水杨酸钠对HL-60/VCR细胞多药耐药的部分逆转

目的 :研究水杨酸钠 (Na Sal)对HL 6 0 VCR细胞多药耐药的部分逆转作用。方法 :采用噻唑蓝(MTT)法、流式细胞术等方法 ,观察非甾体抗炎药Na Sal对HL 6 0 VCR细胞多药耐药的部分逆转作用。结果 :Na Sal对HL 6 0 VCR细胞的生长具有抑制作用并表现出剂量依赖性 ,1mmol L的非细胞毒剂量的Na Sal和化疗药物联合应用可以提高多种化疗药物的细胞毒性作用 ,逆转倍数为 1 0 1～ 3 2 6 ,相对逆转效率为 0 5 2 %～ 73 6 2 %。流式细胞仪测定细胞内柔红霉素 (DNR)浓度发现 ,Na Sal并不增加HL 6 0 VCR细胞内DNR浓度。结论 :Na Sal能有效地部分逆转HL 6 0 VCR细胞的多药耐药性