端粒酶逆转录酶基因反义寡核苷酸抑制肺癌细胞端粒酶活性和诱导细胞凋亡

目的 研究端粒酶逆转录酶（hTERT)基因反义寡核苷酸（ASODN）对肺癌细胞端粒酶活性和细胞凋亡的影响。方法 实验分为ASODN组、正义寡核苷酸（SODN）组和空白对照组,所用ASODN和SODN的浓度分别为10μmol/L,脂质体为16mg/L,采用端粒重复扩增法、逆转录－聚合酶链反应、Western Blot及流式细胞术分别观察各组端粒酶活性、hTERP mRNA和蛋白质表达以及细胞凋亡。结果 ASODN组显著下调或抑制肺癌细胞端粒酶活性和hTERT表达,但直到第21天才出现细胞凋亡增多。结论 端粒酶活性与hTERT表达密切相关。     

Bcl-2反义寡核苷酸对环已亚胺诱导U251细胞凋亡的影响

目的 探讨Bcl-2反义寡核苷酸对环已亚胺（CHX）诱导胶质瘤细胞U251凋亡的影响。方法在光镜下观察Bcl-2反义寡核苷酸、CHX及二者联合应用对U251凋亡的形态学改变;应用MTT法检测各组U251细胞的抑制率;通过DNA电泳检测CHX诱导U251细胞凋亡的情况。结果 Bcl-2反义寡核苷酸可明显促进CHX诱导的U251凋亡,对照组、SODN组与CHX组、AODN组、SODN＋CHX组及AODN＋CHX组的细胞抑制率相比P均〈0．01,AODN组、SODN＋CHX组和AODN＋CHX组的细胞抑制率相比P均〈0．01;Bcl-2反义寡核苷酸可促进细胞凋亡,呈现出特征性的DNA梯状带。结论Bcl-2反义寡核苷酸和CHX均可诱导胶质瘤细胞U251发生凋亡,且Bcl-2反义寡核苷酸可明显促进CHX诱导的胶质瘤细胞U251凋亡,提高胶质瘤细胞对CHX的敏感性。     

Ku70反义寡核苷酸增强甲状腺癌细胞的辐射敏感性

目的 研究DNA修复基因Ku70反义寡核苷酸(ASODN)对甲状腺癌细胞辐射敏感性的影响.方法 设计合成特异性靶向Ku70的ASODN,以不同浓度转染人甲状腺髓样癌细胞(TT),并设脂质体和正义寡核苷酸(SODN)对照组进行比较.采用Western印迹技术检测各组细胞中Ku70蛋白表达水平.利用不同剂量60 Co γ射线照射细胞后,细胞克隆形成实验检测细胞存活分数.CCK-8法、Annexin-V/PI染色法分析ASODN对细胞活力和细胞凋亡的影响.彗星电泳法比较γ射线照射后DNA双链断裂的修复效率.结果 转染ASODN各组细胞Ku70蛋白表达均较脂质体对照组、SODN组明显降低,并呈浓度依赖性;照射后细胞存活率降低(P＜0.01),凋亡率显著增加(P＜0.01),DNA双链断裂的修复效率降低(P＜0.01).结论 ASODN能够特异性地下调甲状腺癌细胞Ku70的表达,抑制γ射线照射后细胞存活和DNA双链断裂修复,提高肿瘤细胞的辐射敏感性.     

CRKL基因对K 562细胞的作用研究

目的：通过研究CRKL基因反义寡核苷酸（AODN）对K562细胞增殖及凋亡的作用。探讨CRKL基因对慢性粒细胞性白血病（CML）发病的影响。方法：应用CRKL基因反义寡核苷酸作用于K562细胞，通过^3H-TdR掺入，流式细胞术，Rt-PCR等方法观察K562细胞形态学，细胞周期，细胞动力学及基因表达等变化。结果：CRKL基因ASODN对K562细胞的增殖有抑制作用。并可诱导K562细胞的凋亡。结论：CRKL基因在CML的发病中可能有重要作用。     

存活素与血管内皮生长因子反义寡核苷酸对人甲状腺髓样癌裸鼠模型的作用

目的探讨存活素与血管内皮生长因子（VEGF）反义寡核苷酸（ASODN）对人甲状腺髓样癌（MTC）裸鼠模型的作用。方法构建的28只人甲状腺髓样癌裸鼠模型，随机分配到对照组和治疗组。对照组包括未处理组，正义寡核苷酸（SODN）组（VEGF SODN组和存活素SODN组）和SODN联合组；治疗组包括ASODN组（VEGF ASODN组和存活素ASODN组）和ASODN联合组。各组每日一次瘤内注射相应处理液，连续7d。治疗结束后l周末，杀死全部瘤鼠，切取瘤体，测算瘤重和抑瘤率以评价肿瘤生长情况，瘤组织石腊切片分别行HE、免疫组化和DNA末端原位标记法（TUNEL法）染色以评价组织病理构造、VEGF与存活素蛋白表达和细胞凋亡的变化。结果HE染色显示，治疗组血管生成明显降低。此外，相对于对照组，治疗组具有显著的VEGF和存活素低表达、高凋亡、低瘤重和高抑瘤率特点（均P〈0．01），并且ASODN联合组较ASODN组更为显著（均P〈0．05），而对照组间和SODN组间未见上述各特点的统计学差异。结论在裸鼠MTC模型，人甲状腺髓样癌存活素和VEGF的单或双靶向基因沉默治疗能够达到明显抑瘤效果。并且联合治疗更为突出。这为发展甲状腺髓样癌的基因治疗提供了实验依据。     

存活素反义寡核苷酸对甲状腺癌细胞生长的抑制作用

目的 探讨利用凋亡抑制基因存活素反义寡核苷酸（ASODN）诱导甲状腺癌细胞凋亡的效应。方法 设计合成特异性靶向存活素的ASODN，以不同浓度和时间对人甲状腺髓样癌细胞进行转染，并设空白、正义（SODN）对照组进行比较。采用RT-PCR、Western印迹、免疫细胞化学技术检测各组细胞中存活素mRNA、蛋白表达水平，DNA裂解分析、吖啶橙／溴化乙锭（AO／EB）染色法、流式细胞仪检测细胞凋亡水平和形态变化，MTT法检测细胞生长抑制情况。结果 转染ASODN各组细胞存活素mRNA和蛋白表达均较空白对照组、SODN组明显减弱；细胞凋亡率显著增加，细胞生长相应受抑，上述效应在ASODN转染24h最为明显，并呈浓度依赖性；而各时段SODN组与空白对照组间各项指标差异均无统计学意义。结论 ASODN能够特异性地下调甲状腺癌细胞中存活素基因表达，进而诱导肿瘤细胞凋亡，并抑制其增殖。     

MDM2反义寡核苷酸的化疗增敏作用研究

目的:通过MDM2反义寡核苷酸(MDM2-AS)抑制白血病细胞系K562和HL-60中MDM2的表达,研究其化疗增敏作用。方法:细胞分为对照组、AS组、顺铂组和联合处理组,分别单独或联合MDM2-AS转染细胞,以RT-PCR和WESTERNBLOT检测MDM2的表达;以单细胞凝胶电泳检测细胞DNA损伤;以流式细胞仪检测处理后各组细胞的凋亡率。结果:经MDM2-AS转染后细胞的MDM2表达明显受抑制(P<0.05),而经过MDM2-AS与顺铂联合处理细胞,K562和HL60细胞凋亡率明显增加(P<0.01),与顺铂处理组相比亦明显增加(P<0.05)。结论:以MDM2-AS反义封闭抑制MDM2的表达,可以增加白血病细胞的化疗敏感性,提示这一方法在白血病治疗中的广泛应用前景。     

Chk1/2反义寡核苷酸对DDP诱导白血病原代细胞凋亡影响的研究

目的:观察白血病原代细胞在顺铂作用下细胞周期变化和转染Chk1/2反义寡核苷酸对顺铂诱导原代细胞凋亡的影响。方法:用RT-PCR、westernblot及免疫组化检测白血病骨髓单个核细胞(BMMNC)中Chk1和Chk2的表达,用流式细胞仪检测顺铂作用下白血病BMMNC细胞周期变化。以脂质体作为载体,转染Chk1/2反义寡核苷酸于BMMNC,用流式细胞仪检测转染Chk1/2反义寡核苷酸后顺铂作用下BMMNC细胞凋亡率。结果:在白血病BMMNC中,Chk1和Chk2mRNA水平均存阳性表达,Chk1蛋白表达强,而Chk2蛋白表达弱。10μmol/L顺铂作用下BMMNC出现S期阻滞,转染Chk1反义寡核苷酸可明显增加顺铂诱导下BMMNC凋亡率,转染Chk2反义寡核苷酸不能增加顺铂诱导下BMMNC凋亡率。结论:Chk1可作为白血病增敏治疗的有效靶点。     

血管内皮生长因子反义寡核苷酸抑制甲状腺癌血管生成的效应

目的探讨反义寡核苷酸(ASODN)抑制甲状腺癌细胞血管内皮生长因子(VEGF)表达及内皮细胞生长的效应。方法设计合成靶向VEGF的ASODN转染人髓状甲状腺癌细胞系(TT)细胞,并制备相应条件培养基作用内皮细胞ECV304,设正义寡核苷酸(SODN)和空白对照组进行比较。观察细胞生长状态,RT PCR、免疫细胞化学法检测TT细胞VEGFmRNA和蛋白表达,四氮唑蓝法检测TT和ECV304细胞生长抑制率(IR),流式细胞仪、吖啶橙/溴化乙锭染色法检测ECV304细胞凋亡状态。结果ASODN组TT细胞VEGFmRNA和蛋白表达显著低于SODN和对照组(P<0.01),但IR差异无统计学意义(P>0.05);各转染组ECV304细胞IR差异亦无统计学意义(P>0.05);而经各ASODN组TT细胞条件培养基作用的ECV304细胞生长明显受抑,IR(分别为0.21±0.03、0.31±0.01、0.42±0.22)显著高于SODN组(0.05±0.03,P<0.01),并伴明显细胞凋亡,上述效应呈浓度依赖性。结论ASODN可通过特异性封闭甲状腺癌细胞VEGF表达,抑制内皮细胞生长,干扰肿瘤血管生成。     

survivin反义寡核苷酸对SMMC-7721细胞增殖和凋亡的影响

目的 观察survivin反义寡核苷酸(ASODN)对人肝癌细胞株SMMC-7721增殖和凋亡的影响.方法 人工合成survivin基因ASODN和正义ODN(SODN),并行硫代磷酸化修饰,通过脂质体途径转染SMMC-7721;分别用RT-PCR和Western blot检测survivin mRNA和蛋白表达;用MTT法检测ASODN对SMMC-7721增殖的影响;流式细胞仪检测细胞周期变化及细胞凋亡率;倒置显微镜观察细胞形态变化.结果 SMMC-7721可强表达survivin mRNA和蛋白;ASODN呈浓度依赖性抑制survivin mRNA和蛋白表达及SMMC-7721增殖,诱导细胞凋亡,使细胞阻滞于G2/M期.SODN对survivin mRNA和蛋白及SMMC-7721的增殖、细胞周期无明显抑制作用.结论 脂质体介导转染survivin ASODN可抑制细胞增殖、使细胞阻滞于G2/M期,从而促进细胞凋亡.     

髓系白血病中EVI1基因的表达及其临床意义

目的研究EVI1基因在成人急性髓系白血病(AML)和慢性粒细胞白血病(CML)中的表达及其临床意义。方法采用多重逆转录-聚合酶链反应(多重RT-PCR)技术分析2002年9月至2005年3月华中科技大学同济医学院附属同济医院收治的45例AML和43例CML骨髓单个核细胞中EVI1基因mRNA的表达,分析EVI1mRNA阳性白血病患者的临床特征及疗效。结果45例AML患者中有8例(17.8%)EVI1阳性,其中M11例、M23例、M54例。43例CML患者中有8例(18.6%)EVI1阳性,其中慢性期2例,占5.7%(2/35),加速期与急变期各3例,均占75.0%(3/4),加速期与急变期组的EVI1基因表达率高于慢性期组(P<0.01)。EVI1阳性的AML患者早期病死率高;EVI1基因阳性表达与CML白血病临床分期及预后相关。结论EVI1基因的高表达在髓系白血病的发生中,特别是在慢性粒细胞白血病慢性期向急性期转化中起重要作用。     

Myelodysplastic syndromes and acute leukemia developing after imatinib mesylate therapy for chronic myeloid leukemia

During therapy with imatinib, some patients with chronic myeloid leukemia (CML) develop chromosomal abnormalities in Philadelphia chromosome (Ph)-negative cells. These abnormalities are frequently transient and their clinical consequence is unclear. Although some reports have suggested that the abnormalities might be associated with secondary myelodysplastic syndrome (MDS), the diagnosis has not always been established using standard criteria. We report 3 cases of patients treated with imatinib for CML who were subsequently found to have chromosomal abnormalities in Ph-negative cells. One of them developed acute myelogenous leukemia (AML) and the other 2 developed high-risk MDS that rapidly transformed to AML. These cases were identified in a total study group of 1701 patients. Although these occurrences are rare, the findings highlight the need for close monitoring of patients with CML treated with imatinib.     

Homozygous deletions of the p16 tumor-suppressor gene are associated with lymphoid transformation of chronic myeloid leukemia

The p16 gene, also referred to as MTS1, INK4, CDK4I, or CDKN2, at chromosome 9p21 has recently been described as a tumor suppressor that may be involved in a wide range of tumors. We have used a semiquantitative multiplex polymerase chain reaction assay to search for deletions of the p16 gene in 34 patients with chronic myeloid leukemia in blast crisis (CML BC), 19 patients with acute lymphoblastic leukemia (ALL), and 25 patients with acute myeloid leukemia (AML). Homozygous deletions of p16 exons were found in 5 of 10 (50%) patients with CML in lymphoid BC and in 5 (26%) ALL patients, but in only 1 (2%) case with AML. No deletions were found in CML BC of nonlymphoid phenotype. Comparison of chronic phase DNA or remission DNA with acute leukemia DNA in 5 individuals showed that the p16 deletions were acquired and not inherited, directly implicating these lesions in the pathogenesis of the disease. We conclude that functional elimination of the p16 gene, or a closely mapping gene, is involved in a significant number of patients with CML in lymphoid transformation.     

Rare occurrence of N-ras point mutations in Philadelphia chromosome positive chronic myeloid leukemia

Point mutations of the N-ras oncogene are relatively common in acute myelogenous leukemia (AML) cells, occurring in some 25% to 50% of patient samples. We used a technique involving the direct nucleotide sequencing of in vitro amplified N-ras genomic fragments to determine the frequency of N-ras point mutations in chronic myeloid leukemia (CML) cells at various stages of the disease. This approach will detect N-ras point mutations in a mixed population of cells if the mutation is present in 25% or more of the cells. We could not demonstrate any point mutation at N-ras codons 12,13 or 59-63 in any of the 44 CML cases analyzed, which included 21 blast crisis samples. In contrast with AML N-ras point mutations are exceedingly rare in CML.     

The incidence of submicroscopic deletions in reciprocal translocations is similar in acute myeloid leukemia, BCR-ABL positive acute lymphoblastic leukemia, and chronic myeloid leukemia

We compared the incidence of submicroscopic deletions accompanying balanced translocations using interphase fluorescence in situ hybridization (FISH) in 245 patients with chronic myeloid leukemia (CML), 79 patients with acute lymphoblastic leukemia (ALL) and BCR-ABL (n=70) or MLL rearrangements (n=29), and 412 patients with acute myeloid leukemia (AML) with CBFB-MYH11 (n=122), PML-RARalpha (n=108), AML1-ETO (n=112), or MLL rearrangements (n=98). The incidence of submicroscopic deletions was 2-9% depending on the entity.     

Intracardiac mass in chronic myeloid leukemia

Chronic myeloid leukaemia (CML) is a neoplastic disorder of myeloid cell lines and is a less aggressive disease compared to acute myeloid leukemia (AML). Although cardiovascular complications are not uncommon, intracardiac thrombosis in CML is rarely reported. Herein, we report a case of CML presenting with an intracardiac thrombus attached to the posterior mitral leaflet, and subsequently resulting in peripheral embolization.     

Characterization of chronic myeloid leukemia stem cells

Although tyrosine kinase inhibitors have redefined the care of chronic myeloid leukemia (CML), these agents have not proved curative, likely due to resistance of the leukemia stem cells (LSC). While a number of potential therapeutic targets have emerged in CML, their expression in the LSC remains largely unknown. We therefore isolated subsets of CD34(+) stem/progenitor cells from normal donors and from patients with chronic phase or blast crisis CML. These cell subsets were then characterized based on ability to engraft immunodeficient mice and expression of candidate therapeutic targets. The CD34(+)CD38(-) CML cell population with high aldehyde dehydrogenase (ALDH) activity was the most enriched for immunodeficient mouse engrafting capacity. The putative targets: PROTEINASE 3, SURVIVIN, and hTERT were expressed only at relatively low levels by the CD34(+)CD38(-)ALDH(high) CML cells, similar to the normal CD34(+)CD38(-)ALDH(high) cells and less than in the total CML CD34(+) cells. In fact, the highest expression of these antigens was in normal, unfractionated CD34(+) cells. In contrast, PRAME and WT1 were more highly expressed by all CML CD34(+) subsets than their normal counterparts. Thus, ALDH activity appears to enrich for CML stem cells, which display an expression profile that is distinct from normal stem/progenitor cells and even the CML progenitors. Indeed, expression of a putative target by the total CD34(+) population in CML does not guarantee expression by the LSC. These expression patterns suggest that PROTEINASE 3, SURVIVIN, and hTERT are not optimal therapeutic targets in CML stem cells; whereas PRAME and WT1 seem promising.