抗CD47抗体联合阿糖胞苷靶向治疗NOD/SCID小鼠单核细胞白血病的研究

目的：探讨CD47在急性髓细胞白血病NOD/SCID小鼠模型中的预后意义及靶向治疗的最佳策略。方法：将分选的CD34+CD38-白血病干细胞（leukemia stem cells, LSCs）移植入NOD/SCID小鼠体内,建立小鼠急性单核细胞白血病模型;抗人CD47单克隆抗体单独或联合阿糖胞苷治疗白血病小鼠7~14 d,并进行疗效分析。将LSCs与小鼠巨噬细胞在含抗CD47单克隆抗体的培养液中共培养,观察CD47对巨噬细胞吞噬LSCs能力的影响。结果：THP-1细胞中存在CD34+CD38- LSCs,比例约为0.12%±0.06%,将分选后的CD34+CD38- LSCs（比例高达97.0%±1.7%）移植入NOD/SCID小鼠后成功建立白血病模型。体内实验表明,阿糖胞苷（7 d）联合抗CD47单克隆抗体（14 d）治疗后,白血病小鼠外周血和骨髓中CD33+CD45+白血病细胞下降最明显（P<0.01）,生存时间明显长于其它各组。体外共培养2 h后,抗CD47单克隆抗体组吞噬指数（76.9%±12.2%）明显高于抗CD45单克隆抗体组（7.60%±2.4%,P<0.01）。结论：CD47高表达是急性髓细胞白血病的预后不良因素。阿糖胞苷联合抗CD47单克隆抗体可有效杀灭普通白血病细胞和白血病干细胞,对彻底治愈急性髓细胞白血病具有重要临床意义。     

糖皮质激素对急性淋巴细胞白血病Puma表达的影响

目的：糖皮质激素可通过调节某些基因的表达而诱导细胞凋亡,但在治疗白血病过程中对促凋亡蛋白Pum a表达的影响尚不清楚,该实验试探讨糖皮质激素在诱导体外培养的急性淋巴细胞白血病细胞凋亡过程中对Pum a表达的影响。方法：利用人急性淋巴细胞白血病细胞移植到免疫缺陷小鼠体内建立的细胞系,地塞米松处理后MTT比色法进行药物敏感性实验、W estern印迹及实时荧光定量PCR检测Pum a蛋白及mRNA的表达。结果：来源于临床疗效好的病例样本,其体外培养的白血病细胞对地塞米松敏感,来源于临床疗效差的病例样本,其体外培养的白血病细胞对地塞米松耐药,耐药组与敏感组白血病细胞间对地塞米松的体外敏感性有显著差异(P<0.05)。地塞米松处理后耐药组及敏感组均未见Pum a蛋白及mRNA的表达上调。结论：糖皮质激素在诱导体外培养的白血病细胞凋亡过程中,不能引起Pum a表达的上调。     

卡介苗体外对急性淋巴细胞性白血病患儿细胞毒性T淋巴细胞杀伤HL-60细胞效应的影响

目的：探讨卡介苗（BCG）体外对急性淋巴细胞性白血病(ALL)患儿外周血细胞毒性T淋巴细胞（CTL）杀伤HL-60细胞效应的影响。方法：应用Ficoll-Hypaque法分离ALL患儿（白血病组）和健康儿童（对照组）的外周血单个核细胞,在白介素-2（IL-2）、植物血凝素（PHA）和BCG作用下诱导成CTL细胞;应用流式细胞仪测定CD3、CD3+CD4+、CD3+CD8+比例变化;应用MTT法检测CTL对HL-60细胞的杀伤力。结果：培养前2 d,对照组和白血病组CTL细胞数量及体积均无明显变化,第3天开始两组细胞均开始增殖,体积变大,6～10 d达高峰;培养至第10天时,加入BCG的白血病组和对照组细胞数量分别较无BCG的白血病组和对照组增多;白血病组CD3+CD8+比例明显高于对照组,经BCG作用后两组CD3+CD8+比例均明显升高;白血病组CTL对HL-60细胞的杀伤力明显低于对照组。结论：BCG可协同IL-2、PHA体外促进CTL细胞增殖并提高其对HL-60细胞的杀伤力。     

脐血血清对急性白血病化疗后骨髓抑制期CFU-GM生长的影响

探讨脐血血清对急性白血病化疗后骨髓抑制期造血细胞增殖的促进作用。采用体外CFU-GM培养法观察正常成人血清和脐血血清对正常造血细胞和急性白血病化疗后骨髓抑制期造血细胞增殖的影响。结果显示：脐血血清能明显促进正常CFU－GM和急性白血病化疗后骨髓抑制期CFU－GM生长。因此：脐血在体外能够刺激正常骨髓和急性白血病化疗后骨髓抑制期骨髓造血祖细胞增殖,提示急性白血病强化疗和干细胞移植时输注脐血可促进骨髓造血功能恢复。     

脐血血清对急性白血病化疗后骨髓抑制期CFU—GM生长的影响

探讨脐血血清对急性白血病化疗后骨髓抑制期造血细胞增殖的促进作用，采用体外ＣＦＵ－ＧＭ培养法观察正常成人血清和脐血血清对正常造血细胞和急性白血病化疗后骨髓抑制期造血细胞增殖的影响。结果显示：脐血血清能明显促进正常ＣＦＵ－ＧＭ和急性白血病化疗后骨髓抑制期ＣＦＵ－ＧＭ生长，因此：脐血在体外能够刺激正常骨髓和急性白血病化疗后骨髓抑制期骨髓造血祖细胞增殖，提示急性白血病强化疗和干细胞移植时输注脐血可促进骨髓     

CHFR基因和PARP-1基因对裸鼠Raji淋巴瘤细胞增殖和凋亡调控的研究

目的 CHFR基因是一种抑癌基因,低表达或表达缺失可能促淋巴瘤细胞增殖,PARP-1基因参与其对淋巴瘤细胞增殖的调控,本研究旨在通过过表达CHFR基因及分别沉默CHFR、PARP-1基因,观察其对裸鼠移植瘤Raji细胞的影响,探讨CHFR基因和PARP-1基因调控裸鼠Raji淋巴瘤细胞的增殖和凋亡机制。方法 在非霍奇金B细胞裸鼠Burkitt淋巴瘤移植瘤Raji细胞中,用5-氮杂-2′-脱氧胞苷(5-Aza-dC)过表达CHFR基因,及用慢病毒介导的shRNA分别沉默CHFR、PARP-1基因,测定各组裸鼠移植瘤的肿瘤的大小及重量;采用实时荧光定量PCR技术测定各组CHFR及PARP-1基因mRNA含量;应用MSP方法检测各组CHFR的甲基化状态;分别通过TUNEL试剂盒和免疫组化监测凋亡指数(AI)和微血管密度(MVD)。结果 5-Aza-dC组的裸鼠在非霍奇金B细胞Burkitt淋巴瘤移植瘤Raji细胞能增强CHFR基因mRNA的表达,降低PARP-1基因mRNA的表达;在体内环境中,5-Aza-dC能够促进移植瘤Raji细胞的凋亡,抑制移植瘤的生长(P<0.05),这些结果...     

永生化骨骺软骨细胞的生物学性状研究

目的比较永生化骨骺软骨细胞与原代骨骺软骨细胞的生物学性状,为细胞替代疗法治疗小儿身材矮小提供理论依据。方法利用SV40LTag诱导骨骺软骨细胞永生化,经G418稳定筛选后,观察细胞形态,应用MTT、血清依赖性实验、软琼脂克隆形成试验、裸鼠致瘤试验分析其生物学性状。结果原代软骨细胞体外传代至第6代左右即出现衰老现象,而转化软骨细胞可连续传代,未出现衰老迹象。转化软骨细胞更趋向于长梭形。两者的生长曲线比较相似,但转化软骨细胞的饱和密度明显高于正常软骨细胞。两者都对血清有依赖性,都为二倍体核型,均不能在软琼脂上形成克隆。经8周观察两者都不能使裸鼠致瘤。结论永生化骨骺软骨细胞为良性转化,为转基因细胞移植治疗身材矮小等疾病的研究提供了安全的细胞来源。     

羟基磷灰石纳米粒子体外抗白血病作用的研究

目的探讨羟基磷灰石(HAP)纳米粒子体外抗白血病作用。方法采用MTT法检测单用HAP和联合化疗药物对K562细胞的增殖抑制及化疗增敏作用,并用倒置显微镜观察药物对K562细胞形态学的影响。结果①HAP和抗癌药物对K562细胞增殖均可产生影响,在倒置显微镜下,可见全部测定孔细胞增殖均较对照孔低下。②K562细胞生长抑制率与HAP的浓度和作用时间呈明显正相关。③HAP与化疗药物合用具有更良好的抑癌效果。结论HAP纳米粒子明显抑制K562细胞生长,具有体外抗白血病作用和化疗增敏作用;HAP纳米粒子对白血病及肿瘤的治疗将具有良好的开发应用前景。     

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目的探讨B细胞淋巴瘤/白血病-2基因(bcl-2)在儿童急性B淋巴细胞白血病(B-ALL)的发生、发展及预后中的意义,分析反义寡核苷酸技术(ASODN)在儿童B-ALL的临床应用前景。方法选取2006年10月至2010年10月青岛大学医学院附属医院儿科初诊B-ALL患儿36例,收集骨髓标本,获取骨髓单个核细胞(BMMCs),采用实时荧光定量PCR方法检测BMMCs中bcl-2 mRNA的表达;将bcl-2-ASODN用脂质体介导转染细胞后检测儿童B-ALL白血病细胞增殖和凋亡改变;联合bcl-2-ASODN和VDLP(长春新碱、柔红霉素、门冬酰胺酶和地塞米松)化疗药物,四甲基偶氮唑盐法(MTT法)检测bcl-2-ASODN对儿童B-ALL白血病细胞体外化疗药物敏感性的影响。结果 (1)bcl-2 mRNA在B-ALL患儿中表达升高,与对照组比较差异有统计学意义(P<0.05),化疗敏感组患儿治疗后bcl-2 mRNA表达明显减低,较化疗耐药组差异有统计学意义(P<0.05);(2)bcl-2-ASODN用脂质体介导转染后白血病细胞增殖减弱、凋亡增加,较对照组差异有统计学意义(P<0.05);(3)体外联合bc...     

三氧化二砷抑制人胃癌裸鼠皮下移植瘤生长及机制的研究

目的探讨三氧化二砷(As2O3)抗人胃癌裸鼠皮下移植瘤的作用及其机制。方法实验用30只裸鼠建立人胃癌裸鼠移植瘤模型,随机分为3组,即生理盐人组、低剂量As2O3组和高剂量As2O3组,比较各组的抑瘤作用,并对标本分别进行光镜和电镜观察及原位末端标记(TUNEL)检测。结果As2O3能显著抑制胃癌裸鼠移植瘤的增长,并能诱导肿瘤细胞凋亡,未见As2O3引起肝、肾系统损害。结论As2O3对人胃癌细胞裸鼠移植瘤具有显著的抗癌作用,此作用与诱导癌细胞凋亡密切相关。     

Effect of rat atrial natriuretic factor on in vivo and in vitro aldosterone and corticosterone secretions in the rat during the perinatal period.

The effect of rat atrial natriuretic factor (rANF) on aldosterone and corticosterone secretion was investigated in vivo in 21-day-old rat fetuses injected intravenously through the umbilical vein and in vitro on isolated adrenal cells from 17-, 19- and 21-day-old fetuses and 1-, 2-, 3- and 4-week-old rats. In vivo, rANF (50 pmol/50 microliters/fetus) inhibited both basal levels and secretion of aldosterone stimulated by adrenocorticotropin hormone (ACTH(1-24), 0.25 pmol/50 microliters/fetus), but not corticosterone secretion. In vitro, the addition of graded concentrations of rANF (0.001, 0.01 and 10 nmol/l) to the incubation medium did not affect the basal aldosterone and corticosterone secretions of fetal and neonatal adrenal cells. ACTH(1-24) (0.1 nmol/l) stimulated productions of both corticosterone and aldosterone by the adrenal cells at all stages studied. The addition of graded concentrations of rANF to the incubation medium containing ACTH(1-24) (0.1 nmol/l) induced a dose-dependent inhibition of aldosterone secretion by the adrenal cells from 21-day-old fetuses and newborn rats. In contrast, no effect was observed on cells from 17- and 19-day-old fetuses. At all stages investigated, the three doses of rANF were unable to affect ACTH-induced corticosterone secretion in vitro. In isolated adrenal cells from 2-week-old rats, rANF (10 nmol/l) inhibited the secretion of aldosterone induced by ACTH(1-24) (0.1 nmol/l), and by different steroids of the aldosterone synthetic pathway (progesterone, 11-deoxycorticosterone, corticosterone, 1 mumol/l for each steroid). These results suggest that rANF is a specific inhibitor of aldosterone synthesis in the perinatal period of the rat and that the inhibitory effect of rANF occurs both during the early and late pathways of aldosteroidogenesis.     

Interferon synthesis by human colostral leucocytes.

The antiviral potential of human colostral leucocytes was assessed by their capacity to produce interferon. Leucocytes cultured from colostrum were stimulated by mitogens or Newcastle disease virus (NDV) to produce interferon which, by metabolic and physicochemical criteria, corresponded to normal human leucocyte interferon. Prepartum cells produced higher levels than postpartum cells. Colostral cells were less efficient producers than blood leucocytes.     

Indomethacin and ibuprofen effect on IL-1ra production by mononuclear cells of preterm newborns and adults

The in vitro effect of indomethacin (IM) and ibuprofen (IB) on the production of the interleukin-1 receptor antagonist (IL-1ra) by cord blood mononuclear cells (CBMC) from preterm newborns was compared to that of peripheral blood mononuclear cells (PBMC) from adults. Mononuclear cells (MC) were incubated with lipopolysaccharide (LPS) in the absence or presence of various concentrations of IM and IB. The level of IL-1ra in the supernatants was tested by ELISA. The results showed a lower ability of MC from preterm newborns to produce IL-1ra as compared with adult cells, supporting the assumption of neonatal immune cell immaturity. IM at pharmacological concentrations caused inhibition of IL-1ra secretion by PBMC from adults whereas IB suppressed the secretion of IL-1ra at higher concentrations only. At the same concentrations neither drug had an in vitro effect on the production of IL-1ra by CBMC of preterm newborns. In conclusion, the lower ability of CBMC of preterm newborns to produce IL-1ra in response to LPS and the absence of an IM and IB effect on the secretion of this cytokine by these cells as compared with PBMC of adults, suggest an underdevelopment of the immune response in preterm newborns.     

A new hamster fibrosarcoma model for in vitro/in vivo evaluation of cancer chemotherapeutic agents.

A hamster fetal cell clone has been developed for in vitro chemotherapeutic studies as well as in an in vivo fibrosarcoma model system. Highly reproducible quantitative in vitro chemotherapeutic data can be obtained with this cell line within 5 days, and as few as 10(2) cells produce rapidly growing fibrosarcomas when injected subcutaneously into adult hamsters. We found using these cells in vitro that 1-beta-D-arabinofuranosylcytosine (ara-C) can antagonize the effect of 5-azacytidine (aza-C) if given simultaneously or if aza-C treatment is preceded by a 2-hr exposure to ara-c. Using the same cell line as in vivo model for chemotherapy it was also shown that ara-C and cyclocytidine significantly inhibited tumor growth. This hamster cell line may be quite useful as an in vitro/in vivo model system for the study of cancer chemotherapeutic agents.     

Effect of vitamin K on neonatal erythrocytes.

This study investigated the possible oxidative effect of vitamin K3 (menadione) and Vitamin K1 (Konakion) on neonatal erythrocytes by controlled in vitro exposure. Menadione caused only mild morphological changes and did not decrease ATP levels. However, it oxidized intracellular hemoglobin to methemoglobin in neonatal cells more than in adult cells. Reduced glutathione contents were higher in neonatal cells, but less available for antioxidant protection. Konakion did not increase methemoglobin levels in newborn infants after a prophylactic injection. In vitro exposure to Konakion did not affect reduced glutathione and ATP levels, nor did it oxidize hemoglobin. However, extensive morphological changes were observed, attributed to the effect of its solvent. Therefore, it seems that menadione, which is no longer administered to newborns, causes oxidative stress in neonatal cells whereas Konakion, the current vitamin K1, does not, either in in vitro studies or by therapeutic administration.     

地高辛对非霍奇金淋巴瘤细胞体外药物敏感性的研究

目的了解强心苷类药物地高辛对非霍奇金淋巴瘤组织细胞的体外药物敏感度及与阿糖胞苷联合作用效果。方法体外培养非霍奇金淋巴瘤细胞,予不同浓度地高辛、阿糖胞苷及两药联合进行干预,应用MTS比色法检测细胞的生长及凋亡状况。结果(1)地高辛对非霍奇金淋巴瘤细胞的体外药物敏感度:①各浓度组地高辛分别作用于12组非霍奇金淋巴瘤细胞24 h、48 h和72 h后,各组抑制率均不同程度增高;②不同浓度地高辛对非霍奇金淋巴瘤组织细胞的作用敏感率随时间变化差异有显著性。(2)地高辛对阿糖胞苷化疗效果的协同作用:①析因设计方差分析显示:不同浓度的地高辛对非霍奇金淋巴瘤细胞的作用有显著性差异(F_(地高辛)=20.567,P0.01),不同浓度的阿糖胞苷作用组亦有显著性差异(F_(阿糖胞苷)=57.182,P0.01)。且两者具有一定协同作用。②单独效应方差结果分析显示:各浓度组地高辛对非霍奇金淋巴瘤细胞的作用仅在阿糖胞苷浓度为100μmol/L时,差异具有显著性(F=24.390,P0.01)。结论(1)地高辛在体外可对非霍奇金淋巴瘤细胞有一定抑制作用。(2)地高辛对阿糖胞苷的化疗效果有一定增强,在阿糖胞苷高浓度时协同作用效果更为明显。     

小鼠胚胎干细胞定向诱导为胰岛样细胞过程中印记基因变化

目的：探讨体外诱导对小鼠胚胎干细胞株SF1-G印记基因Kcnq1、Cdkn1c的影响。方法：体外诱导小鼠胚胎干细胞SF1-G向胰岛样细胞分化,RT-PCR和细胞免疫荧光检测胰岛细胞标志基因表达;聚合酶链式反应-限制性内切酶片段长度多态性 (PCR-RFLP)检测印记基因Kcnq1、Cdkn1c在分化各阶段细胞中的亲本表达情况。结果：胰岛细胞标志性基因Insulin、Glucagon、Somatostatin、IAPP、Glut2在诱导分化后的细胞都有表达;分化终末细胞能表达胰岛细胞特异性蛋白胰岛素、C肽及Somastatin;PCR-RFLP检测分析显示,体外诱导分化后细胞的印记基因Kcnq1、Cdkn1c呈双等位基因表达。结论：胚胎干细胞经体外诱导培养可定向分化为胰岛样细胞;用此种分化方法可导致胚胎干细胞印记基因表达的改变,出现印记丢失。［中国当代儿科杂志,2010,12(12)：954-958］     

A new hamster fibrosarcoma model for in vitro/in vivo evaluation of cancer chemotherapeutic agents

A hamster fetal cell clone has been developed for in vitro chemotherapeutic studies as well as in an in vivo fibrosarcoma model system. Highly reproducible quantitative in vitro chemotherapeutic data can be obtained with this cell line within 5 days, and as few as 10² cells produce rapidly growing fibrosarcomas when injected subcutaneously into adult hamsters. We found using these cells in vitro that 1-β-D-arabinofuranosylcytosine (ara-C) can antagonize the effect of 5-azacytidine (aza-C) if given simultaneously or if aza-C treatment is preceded by a 2-hr exposure to ara-c. Using the same cell line as in vivo model for chemotherapy it was also shown that ara-C and cyclocytidine significantly inhibited tumor growth. This hamster cell line may be quite useful as an in vitro/in vivo model system for the study of cancer chemotherapeutic agents.     

Effect of indomethacin on IL-1beta, IL-6 and TNFalpha production by mononuclear cells of preterm newborns and adults

The in vitro effect of indomethacin (IM) on the production of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)alpha by cord blood mononuclear cells (CBMC) from preterm newborns was compared to that of peripheral blood MC of adults (PBMC). MC isolated from peripheral blood of adults (PBMC) and cord blood of preterm newborns (CBMC) were incubated without or with lipopolysaccharide (LPS) in the absence or presence of various concentrations of IM. The levels of IL-1beta, IL-6 and TNFalpha in the supernatants were tested by ELISA. MC isolated from preterm newborns were less sensitive to the in vitro effect of IM on IL-1beta and TNFalpha secretion than adult cells. While the spontaneous secretion of IL-1beta and TNFalpha and the production of TNFalpha induced by LPS were significantly increased following incubation of adult PBMC with IM, only the spontaneous synthesis of TNFalpha by CBMC of preterm newborns was affected by this drug. The in vitro production of IL-6 by MC in the two groups was not affected by the addition of IM. It is suggested that IM may affect the preterm's immune response. However, the role of the drug in the frequency and severity of infections in the neonatal intensive care unit needs further investigation.