去甲斑蝥素联合IL-12、IL-15处理增强PBMC对Raja细胞的杀伤效应【创新点评：中药抗癌药物去甲斑蝥素增强PBMC对淋巴瘤的杀伤－郑胡镛】

目的:探讨细胞因子IL-2、IL-15激活的人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)对去甲斑蝥素(norcantharidin,NCTD)处理后的人Burkitt淋巴瘤Raji细胞的杀伤效应及其可能机制.方法:锥虫蓝拒染法检测NCTD对Raji细胞和PBMC细胞增殖的影响;LDH释放法检测IL-2、IL-15激活后的PBMC对K562细胞和Raji细胞的杀伤;流式细胞术(flow cytometry,FCM)检测IL-2、IL-15诱导后PBMC表面NKG2D的表达,以及NCTD作用前后Raji细胞表面NKG2D配体(MICA、MICB、ULBP1、ULBP2、ULBP3)的表达.结果:NCTD抑制Raji细胞的增殖,具有剂量、时间依赖性(P＜0.05),但对PBMC增殖无影响(P＞0.05).在效靶比为10∶1、20∶1时,IL-2、IL-15激活的PBMC对K562细胞的杀伤率较未激活的PBMC明显增高[(52.42±3.89)％vs (15.82±5.12)％,(79.55±9.22)％vs(27.67±3.66)％,P＜0.05];PBMC对NCTD处理后Raji细胞杀伤率较未经NCTD处理的Raji细胞明显增高[(23.63±6.20)％vs (5.04±1.25)％,(41.80±4.09)％ vs (8.59±2.19)％;P＜0.05],且IL-2、IL-15激活的PBMC对NCTD处理后Raji细胞的杀伤率较NCTD处理前明显提高[(38.97±2.76)％ vs(13.19±3.67)％,(63.09 ±7.30)％ vs(19.89 ±4.15)％;P＜0.05].激活后PBMC表面NKG2D表达率升高[(44.91±5.85)％vs (25.28±7.69)％,P＜0.05].NCTD作用后Raji细胞表面NKG2D的配体ULBP2表达显著升高[(12.69±3.99)％vs(1.03 ±0.42)％,P＜0.05],而其他配体MICA、MICB、ULBP1、ULBP3的表达无明显变化(P＞0.05).结论:NCTD联合细胞因子IL-2、IL-15处理能增强PBMC对Raji细胞的杀伤效应,其机制与NCTD上调Raji细胞表面ULBP2表达和细胞因子上调PBMC表面NKG2D表达有关.     

miR-15a诱导人乳腺癌细胞MCF-7凋亡的作用机制

目的 探讨miR-15a在诱导乳腺癌细胞凋亡中的作用及机制.方法 采用定量聚合酶链反应检测人乳腺上皮细胞株MCF-10A和乳腺癌细胞株MCF-7中miR-15a的表达水平.采用软件预测miR-15a的靶点,并通过荧光素酶报告基因系统验证.将miR-15a经脂质体法转染MCF-7细胞,采用Western blot法检测Bcl-2蛋白的表达,并采用流式细胞术检测MCF-7细胞的凋亡率.结果 miR-15a在乳腺癌细胞株MCF-7中的表达明显低于乳腺上皮细胞株MCF-10A (0.253∶1,P＜0.0001).miR-15a可显著抑制抗凋亡基因Bcl-2 3′-UTR荧光素酶报告基因的表达(P＜0.05).与未转染组(对照组)相比,miR-15a转染组MCF-7细胞中Bcl-2的表达水平显著下降,凋亡明显增加(P＜0.05).结论 miR-15a 作为一个潜在的抑癌基因,可通过靶向于Bcl-2诱导乳腺癌细胞MCF-7发生凋亡,其可为乳腺癌的临床治疗提供新的靶点和理论依据.     

紫杉醇对宫颈癌HeLa细胞增殖和荷瘤裸小鼠肿瘤生长抑制作用

目的研究紫杉醇对人宫颈癌HeLa细胞增殖和对荷瘤裸小鼠肿瘤生长的抑制作用。方法采用噻唑蓝(MTT)法观察紫杉醇对HeLa细胞增殖的抑制作用;并在裸小鼠人宫颈癌模型上观察紫杉醇对荷瘤裸小鼠肿瘤生长的抑制作用。结果紫杉醇对HeLa细胞具有明显的抑制作用并有剂量时间依赖关系;荷瘤裸小鼠动物实验表明,紫杉醇对裸小鼠的肿瘤生长也具有明显的抑制作用,治疗组Ⅰ和治疗组Ⅱ的抑瘤率为分别26.38%和39.04%。结论紫杉醇对HeLa细胞的增殖和荷瘤裸小鼠肿瘤的生长具有明显的抑制作用。     

MicroRNA-206 expression levels correlate with clinical behaviour of rhabdomyosarcomas

Background:

Rhabdomyosarcomas (RMSs) are primarily paediatric sarcomas that resemble developing skeletal muscle. Our aim was to determine the effects of microRNAs (miRNA) that have been implicated in muscle development on the clinical behaviour of RMSs.

Methods:

Expression levels of miR-1, miR-206, miR-133a and miR-133b were quantified by RT–PCR in 163 primary paediatric RMSs, plus control tissues, and correlated with clinico-pathological features. Correlations with parallel gene expression profiling data for 84 samples were used to identify pathways associated with miR-206. Synthetic miR-206 was transfected into RMS cell lines and phenotypic responses assessed.

Results:

Muscle-specific miRNAs levels were lower in RMSs compared with skeletal muscle but generally higher than in other normal tissues. Low miR-206 expression correlated with poor overall survival and was an independent predictor of shorter survival in metastatic embryonal and alveolar cases without PAX3/7-FOXO1 fusion genes. Low miR-206 expression also significantly correlated with high SIOP stage and the presence of metastases at diagnosis. High miR-206 expression strongly correlated with genes linked to muscle differentiation and low expression was associated with genes linked to MAPkinase and NFKappaB pathway activation. Increasing miR-206 expression in cell lines inhibited cell growth and migration and induced apoptosis that was associated with myogenic differentiation in some, but not all, cell lines.

Conclusion:

miR-206 contributes to the clinical behaviour of RMSs and the pleiotropic effects of miR-206 supports therapeutic potential.     

Growth of Human Lymphoma Cells in SCID Mice

Two EBV-negative human lymphoma cell lines raised in this laboratory and peripheral blood cells of a patient with large cell lymphoma in leukemic phase were injected intravenously or intraperitoneally into C.B.17 SCID mice. One line (OCI-LY18) was derived from the pleural fluid of a patient with a large cell, immunoblastic malignant lymphoma. Cells of this line are of B cell origin and characterized by multiple rearrangements of the JH locus. The second line (OCI-LY17) was grown from the peripheral blood of a patient with a large cell lymphoma of T-cell phenotype which has characteristic rearrangement of the T-cell receptor beta chain. The cells directly obtained from a patient with large cell non-cleaved malignant lymphoma were of B-cell origin.

Animals carrying OCI-LY18 developed large tumor masses within 6-8 weeks of inoculation. The tumors were detected in the intestine, mesentery, retroperitoneum, lymph nodes, spleen, lung and kidney. The masses resembled the primary tumor with respect to histological appearance and immunological phenotype. It was possible to generate secondary cell lines from the tumors found in the inoculated SCID mice. Injection of one of these secondary cell lines into SCID mice resulted in the rapid development of lymphoma and as few as 10 cells were sufficient to establish the disease in the inoculated animals. In contrast cells of OCI-LY17 produced small tumor aggregates that did not appear to progress over time and did not cause death of the animals. The tumors were identified by the same phenotypic profile as that seen in the primary cell line and sections of the patient derived lymph node biopsy.

The sample of peripheral blood cells directly derived from the patient gave rise to tumor nodules in multiple locations. Their morphology and immunological phenotype was consistent with the original disease seen in the patient.     

miR-15a寡核苷酸抑制Raji细胞生长及增强阿糖胞苷诱导Raji细胞凋亡的研究(英文)

Objective: The aim of the research was to study whether microRNA-15a (miR-15a) oligonucleotide could inhibitcell growth and enhance cytarabine (Ara-C)-induced apoptosis in Raji cells. Methods: Transfecting miR-15a oligonucleotideinto Raji cells with LipofectamineTM 2000, and then combined with Ara-C. IC50 value and cell proliferation were detected byCCK8 assay; the expression levels of Bcl-2 mRNA and protein were evaluated by RT-PCR and indirect immuno-fluorescence.The apoptotic cells were observed by Hoechst Dyeing; AnnexinV/PI double dyeing method was used to detect the cellapoptotic rate by Flow Cytometry (FCM). Results: After Raji cells were transfected with miR-15a oligonucleotide for 48 h, Bcl-2 protein expression levels obviously decreased, however, there was no difference in Bcl-2 mRNA levels, as compared withthe control group and blank group (P < 0.05). CCK8 assay showed that miR-15a oligonucleotide decreased the cell growth at24, 48 and 72 h, moreover, miR-15a oligonucleotides combined with Ara-C obviously decreased the cell growth than miR-15agroup, Ara-C group and scrambled oligonucleotides (SODN) + Ara-C group. Meanwhile, miR-15a oligonucleotides combinedwith Ara-C significantly decreased IC50 of Ara-C (10.41 μg/mL), which were obviously lower than those of Ara-C group (15.43μg/mL) and SODN plus Ara-C group (14.92 μg/mL). Plenty of apoptotic cells could be seen with Hoechst dyeing. AnnexinV/PIdouble dying assays by FCM indicated that the cell apoptotic rates in earlier period and late period of miR-15a + Ara-C groupwere 20.93% and 25.27%, respectively, which were obviously higher than those of miR-15a group, Ara-C group and SODNplus Ara-C group. Conclusion: miR-15a oligonucleotides can inhibit cell growth and enhance Ara-C-induced apoptosis inRaji cells.     

Growth Inhibition and Apoptosis Induction of HumanUmbilical Vein Endothelial Cells by Apogossypolone

Aims and Background: Prostate cancer is one of the most common malignant tumors in the male reproductivesystem, which causes the second most cancer deaths of males, and control of angiogenesis in prostate lesions is ofobvious importance. This study assessed the effect of apogossypolone (ApoG2) on proliferation and apoptosis ofhuman umbilical vein endothelial cells (HUVECs). Subjects and Methods: HUVECs were treated with differentconcentrations of ApoG2. The survival rate of HUVECs were determined by MTT assay. Utrastructural changesof HUVECs were assessed with transmission electron microscopy. Apoptosis in HUVECs was analyzed by flowcytometry and cell migration by Boyden chamber assay. Matrigel assays were used to quantify the development oftube-like networks. Results: ApoG2 significantly inhibited HUVEC growth even at 24 h (P<0.05). The inhibitoryeffect of ApoG2 is more obvious as the concentration and the culture time increased (P<0.05). These resultsindicate that ApoG2 inhibits the proliferation of HUVECs in a time- and concentration-dependent manner withincrease of the apoptosis rate. Besides, ApoG2 reduced the formation of total pseudotubule length and networkbranches of HUVECs. Conclusions: The results suggest that ApoG2 inhibits angiogenesis of HUVECs by growthinhibition and apoptosis induction.     

hTERT反义酸对淋巴白血病细胞端粒酶活性的抑制(英文)

目的 研究和探索hTFRT反义核酸对淋巴白血病细胞端粒酶活性的影响。方法 用PCR ELISA试剂合测定端粒酶的活性;免疫组织化学和流式细胞仪技术测定hTERT蛋白的含量;用RT-PCR和电泳技术测定hTERT mRNA水平。结果 实验结果显示,10μmol/L AS PS-ODN作用72 h以后,hTERT mRNA和蛋白的水平显著下降,端粒酶的活性明显受到抑制。结论 hTERT反义核酸是淋巴白血病细胞端粒酶活性的良好的抑制剂。     

hTERT反义酸对淋巴白血病细胞端粒酶活性的抑制(英文)

目的研究和探索 hTERT 反义核酸对淋巴白血病细胞端粒酶活性的影响。方法用 PCR ELISA 试剂合测定端粒酶的活性;免疫组织化学和流式细胞仪技术测定 hTERT 蛋白的含量;用 RT-PCR 和电泳技术测定 hTERT mRNA 水平。结果实验结果显示,10 μmol/L AS PS-ODN 作用72 h 以后,hTERT mRNA 和蛋白的水平显著下降,端粒酶的活性明显受到抑制。结论 hTERT 反义核酸是淋巴白血病细胞端粒酶活性的良好的抑制剂。     

单价抗CD20抗体诱导人B细胞淋巴瘤Raji细胞的凋亡

背景与目的:抗CD20抗体和片段已应用于非霍奇金淋巴瘤的临床治疗,但仍需要开发新的抗CD20抗体和片段(未修饰的或放射性标记的),以治疗对美罗华(利妥昔单抗)无反应的患者。鼠源性抗CD20抗体HI47的嵌合抗体片段Fab和F(ab)'2已被构建。本研究目的是观察HI47(鼠抗-CD20抗体)和其嵌合抗CD20抗体片段抑制肿瘤细胞生长和诱导肿瘤细胞凋亡的作用。方法:用免疫荧光法测定抗CD20抗体与CD20+人B细胞淋巴瘤Raji细胞的结合能力;MTT法测定抗CD20抗体片段对Raji细胞生长的影响;用膜联蛋白Ⅴ染色和DNA琼脂糖凝胶电泳检测和验证抗CD20抗体片段诱导Raji细胞凋亡。结果:HI47和其嵌合的抗CD20抗体片段均可与CD20+Raji细胞结合,结合率可达90%以上;HI47不能与美罗华竞争结合Raji细胞;HI47和其嵌合的抗CD20抗体片段浓度为100μg/ml对Raji细胞的抑制率分别为:(57.0±1.5)%、(65.2±2.5)%、(77.2±3.2)%;单价的抗CD20抗体片段Fab(20μg/ml)能够诱导Raji细胞的凋亡,早期凋亡率为17%。结论:HI47的嵌合抗体片段对Raji细胞有抑制作用,能诱导Raji细胞的凋亡。     

siRNA抑制DNA-PKcs表达及对HeLa细胞增殖的影响

背景与目的：建立抑制DNA-PKcs表达的细胞模型,以此探讨DNA-PKcs的功能。材料与方法：构建DNA-PKcs的siRNA抑制表达载体,利用Lipofectamine介导,转染HeLa细胞,筛选稳定表达的转化克隆。Western blot检测DNA-PKcs表达。通过细胞生长速度检测细胞辐射敏感性变化。结果：设计了作用于DNA-PKcs不同位点的3条siRNA,并构建表达质粒,转染HeLa细胞,获得了3个稳定转化克隆,Western blot分析表明其DNA-PKcs表达受到明显抑制,细胞对了射线和紫外线的敏感性增加,接种裸鼠后的肿瘤生长速度减慢。结论：成功建立了DNA-PKcs表达抑制细胞模型,并且发现DNA-PKcs表达抑制后除影响细胞的辐射敏感性外,还可能与肿瘤细胞增殖有关。     

环氧合酶-2对多发性骨髓瘤细胞血管内皮生长因子表达的调节作用

目的探讨多发性骨髓瘤（MM）细胞中环氧合酶-2（COX-2）对血管内皮生长因子（VEGF）表达的调控作用及其临床意义。方法应用人淋巴细胞分离液提取43例多发性骨髓瘤患者骨髓中的单个核细胞,Western blotting（WB）方法检测COX-2及VEGF的蛋白表达,分析其与MM的国际分期系统（ISS）分期、预后的相关性;RT-PCR方法检测多发性骨髓瘤U266细胞COX-2及VEGF mRNA的表达;MTT方法检测COX-2特异性抑制剂对多发性骨髓瘤U266细胞的生长抑制效应。结果多发性骨髓瘤细胞患者COX-2及VEGF过度表达,与ISS分期及预后密切相关;年龄、β2-微球蛋白（β2-MG）、血钙（Ca）及骨髓中浆细胞百分比（BMPC）等预后指标与COX-2的相关系数分别为0.692,0.791,0.571及0.856,与VEGF的相关系数分别为0.731,0.645,0.511及0.823;应用选择性COX-2抑制剂塞来昔布作用于U266细胞48 h,VEGF mRNA及蛋白的表达明显下调,与未处理组相比,统计学差异显著（P〈0.01）。结论 COX-2与VEGF高表达于多发性骨髓瘤患者,与疾病的ISS分期及预后密切相关,干预COX-2可部分下调VEGF的表达,COX-2可望成为多发性骨髓瘤治疗的靶点。     

PI3K/AKt通路参与苦参碱抑制raji细胞增殖与凋亡过程的机制研究

[目的]探讨苦参碱体外诱导非霍奇金淋巴瘤raji细胞增殖与凋亡的机制是否与PI3K/AKt信号通路有关.[方法]M TT法检测苦参碱作用后raji细胞的增殖抑制率;流式细胞仪检测苦参碱作用后raii细胞的细胞周期的变化;RT-PCR检测苦参碱作用后PTEN、AKt、caspase3等mRNA水平的变化;Western blot检测PTEN、pBad、Bad、pAKt、AKt、caspase3等蛋白的表达.[结果]苦参碱可抑制raji细胞的增殖,随着药物浓度的提高和作用时问的延长,细胞的增殖抑制率逐渐升高,与对照组比较,差异均有显著(P＜0.001);苦参碱诱导raii细胞周期停滞于G1期(P＜0.05);苦参碱可通过磷酸化AKt来上调PTEN的表达,引起Bad、p21、p27等的变化,与对照组分别比较表达水平差异均有统计学意义(P＜0.05),最终诱导caspase家族级联反应,引发肿瘤细胞的凋亡.[结论]苦参碱可通过PI3K/AKt信号通路抑制非霍奇金淋巴瘤raii细胞的增殖,诱导raji细胞凋亡.     

白血病细胞bcl-2基因表达与药物敏感性的关系

目的:探讨白血病细胞bcl-2基因表达与药物敏感性的关系.方法:首先用Western印迹法检测HL60、U937细胞以及3例急性髓系白血病患者(AML)的骨髓单个核细胞的Bcl-2基因蛋白表达,继而用MTT法检测8种化疗药物对上述细胞的抑制率.结果:HL60细胞Bcl-2蛋白表达水平高于U937细胞,柔红霉素(DNR)对 HL60的抑制率低于U937细胞,AML原代白血病细胞Bcl-2蛋白的高表达伴随低药敏.结论:白血病细胞Bcl-2蛋白表达水平与其对化疗药物的敏感程度呈负相关性.     

Bcl-2 Gene and Prognosis of B-cell Lymphoma

Both the polymerase chain reaction (PCR) and Southern blot analysis were used to detect bcl-2 gene rearrangement in B-cell lymphoma. Recent molecular studies have shown that the translocation of karyotypic abnormalities t(14;18) results in the juxtaposition of the candidate proto-oncogene bcl-2 on chromosome 18 with the immunoglobulin heavy-chain locus on chromosome 14. We detected the bcl-2 rearrangement in three of six follicular lymphomas (50%), two of five follicular and diffuse lymphomas (40%), one of 13 diffuse medium-sized cell lymphomas (7.7%) and two of 33 diffuse large cell lymphomas (6.0%) through Southern blot analysis. With PCR, the rearrangement was demonstrated in five of eight follicular (63%), three of five follicular and diffuse (60%), seven of 36 diffuse large cell lymphomas (19%) and two of 13 diffuse medium-sized lymphomas (15%). Diffuse large cell lymphomas with bcl-2 rearrangement detected by PCR have a good prognosis as do cases of follicular lymphoma The bcl-2 gene is closely related to follicular lymphoma as described in previous reports and has general prognostic importance in diffuse large cell lymphoma of B cell type.     

The Heterogeneity of Target Recognition by Lymphokine-activated Killer Precursor Cells

Lymphokine-activated killer (LAK) cells were generated from peripheral blood lymphocytes (PBL) that were depleted of mature cytotoxic natural killer (NK) cells. PBL NK activity was abolished by pretreatment of effector cells with the toxic lysosomotropic agent l -leucine methyl ester (LME) or by depletion of effector cells by K562 monolayer absorption (MA). Both treatments markedly reduced the proportion of cells expressing NK-associated markers such as CD 16 (Leu 11b, B73.1), Leu 7, and NKH-1 (Leu 19), whereas these treatments had minimal effects on cells expressing T cell markers (CD 3, CD 4, and CD 8). LME and MA also drastically decreased the proportion of K562 target-binding lymphocytes. LAK activity against NK-sensitive and NK-resistant targets can be generated from the NK cell-depleted PBL by incubation with interleukin-2. Peak LAK activity generated from MA-treated PBL was later than the peak of LAK activity generated from either untreated or LME-treated PBL. Although MA of PBL on NK-resistant S4 sarcoma targets had little effect on NK activity, LAK activity against both K562 and S4 targets was reduced. These results suggest that there are at least three LAK precursor subpopulations in PBL: mature NK cells that can bind and kill K562 targets (LME-sensitive and MA-sensitive); "pre-NK"cells that can bind but cannot kill (LME-resistant and MA-sensitive); and non-NK cells that cannot bind and cannot kill K562 targets (MA-resistant).