转白血病抑制因子基因人胚肺成纤维细胞对脐血CD34~＋细胞体外扩增的影响

背景：单份脐血的造血细胞数量有限,难以满足成人的需要,如何有效地扩增脐血造血干/祖细胞是目前研究的热点。目的：构建人白血病抑制因子基因修饰的人胚肺成纤维细胞,观察转基因细胞对脐血CD34＋造血干/祖细胞体外扩增的影响。方法：建立转人白血病抑制因子基因的饲养层细胞,用RT-PCR法和ELISA法鉴定目的基因的表达;采用免疫磁珠法分离脐血CD34＋造血干/祖细胞,流式细胞术检测纯度;将CD34＋造血干/祖细胞与饲养层细胞共培养,流式细胞术检测各组增殖效果;扩增后的造血干/祖细胞用跨膜迁移实验检测自发迁移率和基质细胞衍生因子1诱导迁移试验以鉴定体外扩增的造血干/祖细胞的归巢能力。结果与结论：成功建立转基因饲养层细胞,RT-PCR法和ELISA法证实有目的基因表达,与人白血病抑制因子转基因饲养层细胞共培养7d后CD34＋造血干/祖细胞可大量扩增,同时表面黏附分子的表达量仍较高。体外迁移实验显示与转基因饲养层细胞共培养的造血细胞的诱导迁移率明显高于对照组,可以较好地保持其归巢能力。因此转人白血病抑制因子基因的饲养层细胞可有效扩增脐血CD34＋造血干/祖细胞,延缓其分化,并且体外扩增后仍保持较高的归巢能力。     

食管癌组织和外周血miRNA检测技术及其应用的研究进展

食管癌是人类常见的八大肿瘤之一,全世界每年约有30万人死于食管癌,其中70%发生在我国。食管癌起病隐匿,大部分患者就诊时已是中晚期,此时实施外科手术治疗的5年存活率仅为20%～30%。如何提高食管癌的早期诊断,尽早治疗对提高食管癌患者生存率具有重要意义。目前用于食管癌筛查的首选方法是胃镜组织活检,但是组织活检可能会导致肿瘤扩散,且胃镜检查患者痛苦,不能被无症状者接受,     

C-kit、NPM1、FLT3基因突变在656例中国急性髓系白血病患者中的分布及其对预后的影响

目的 探讨急性髓系白血病(AML)患者中C-kit、NPM1、FLT3基因突变的发生率和分布情况,并分析其对预后的影响.方法 应用基因测序方法分别检测656例AML患者C-kit基因8、17号外显子,NPM1基因12号外显子,FLT3基因20(酪氨酸激酶区,TKD)、14、15号外显子(内部串联重复,ITD)基因突变情况,并随访患者的预后.结果 656例AML患者中检出C-kit基因8号外显子突变6例(0.9％),17号外显子突变33例(5.0％),NPM1基因突变169例(25.8％),FLT3-TKD突变46例(7.1％),FLT3-ITD突变178例(27.1％).至少有1个突变的患者341例(50.3％).C-kit的8号外显子检测到6种突变类型;C-kit的17号外显子检测到8种突变类型;NPM1检测到15种突变类型,其中10种类型已有报道(A、B、C、D、Nm、I*、J、J+、S、13),5种类型未见报道;TKD检测到11种突变类型.t(8;21)/M2患者常伴有C-kit的17号外显子突变;inv(16)/M4患者常伴有C-kit的8号外显子突变;M3患者中除FLT3基因突变外未检测到其他突变.NPM1和ITD基因突变多见于正常核型患者,在形态学亚型中多见于M5和M1,阳性病例多伴随高白细胞计数、骨髓原始细胞增多、CD34低表达和CD33高表达.随着发病年龄的增长,突变数量增多,白细胞计数平均值增高,高白细胞计数的患者比例升高.在正常核型的群体中突变阳性的比例较其他群体增高,2种、3种突变的比例增多,差异均有统计学意义(P值均＜0.01).FLT3-ITD突变阳性病例中位生存时间(10.0±1.2)个月,与阴性群体中位生存时间(17.0±2.4)个月相比差异有统计学意义(P =0.004),单独NPM1突变与否对总生存率没有显著影响,但NPM1+/ITD-患者的总生存率最高.结论 C-kit、NPM1、FLT3基因突变类型及数量在MICM分型中都有特殊的分布,并与白细胞计数、骨髓原始细胞数和预后均有相关性.对染色体核型正常的患者,基因突变可作为判断预后新的分子标志.     

喹硫平与利培酮治疗老年精神分裂症对照研究

目的探讨喹硫平对老年首发精神分裂症的临床疗效及安全性。方法6例首发老年精神分裂症患者随机分成两组,分别给与喹硫平与利培酮治疗8周,用阳性和阴性症状量表（PANSS）和副反应量表（TESS）评定疗效和不良反应。结果两组疗效相当（P〉0．05）,喹硫平不良反应显著少于利培酮（P〈0．05）。结论喹硫平对老年精神分裂症安全有效。     

IL-24基因修饰增强CIK细胞对HL-60细胞的细胞毒作用

目的:研究IL-24基因修饰的CIK细胞与同源树突状细胞共培养后对白血病细胞的杀伤作用及其机制.方法:从健康人外周血单个核细胞中常规诱导DC和CIK 细胞,电穿孔法将IL-24基因导入CIK细胞中(获得细胞为CIK-IL24),RT-PCR 和ELISA法检测CIK细胞中IL-24基因的表达,FCM和ELISA法检测转基因前后CIK表型及分泌细胞因子能力的变化,将CIK 细胞和同源DC共培养,FCM法检测共培养的DC-CIK细胞对HL-60细胞细胞毒活性的变化.结果:通过电穿孔法成功将IL-24基因导入CIK细胞,与对照组相比,转IL-24基因后CIK细胞中CD3~+、CD3~+CD56~+细胞的比例无明显改变,CD4~+CD25~+细胞比例显著下降.IL-24可上调CD3+CD56+细胞表面粘附分子CD54、CXCR4的表达,转染IL-24基因后CIK分泌TNF-α和IFN-γ的能力显著增强,与DC共同作用HL-60细胞时转染IL-24基因后的CIK细胞细胞毒活性明显增强.结论:通过IL-24基因修饰,明显增强了CIK细胞对HL-60细胞的杀伤能力,其机制与IL-24促进CIK分泌TNF-α、IFN-γ,上调CIK细胞表面粘附分子的表达,减少CD4~+CD25~+调节性T细胞比例等密切相关.     

CD34~＋造血干/祖细胞在转hLIF基因腺病毒载体饲养层细胞中的扩增及其移植SCID小鼠的实验研究

目的:建立转hLIF基因腺病毒载体的饲养层细胞,观察对CD34+造血干/祖细胞的扩增作用,并研究移植辐射损伤模型SCID小鼠的效果.方法:建立转hLIF基因腺病毒载体的饲养层细胞,并用RT-PCR法鉴定目的基因;采用免疫磁珠法分离脐带血CD34+造血干/祖细胞,流式细胞术检测纯度;将CD34+造血干/祖细胞与饲养层细胞共培养,流式细胞术检测各组增殖效果,建立辐射损伤模型SCID小鼠,将扩增后的CD34+造血干/祖细胞经CFDA SE荧光标记后移植入SCID小鼠体内,通过RT-PCR和观察荧光标记细胞来检测小鼠内的人源细胞.结果:建立的转基因饲养层细胞均有绿色荧光,RT-PCR法证实有目的基因表达,免疫磁珠法分离的CD34+造血干/祖细胞纯度可达(95.6±2.58)%,与饲养层细胞共培养后CD34+造血干/祖细胞可扩增13.2倍,表面粘附分子CXCR4和CD54表达量仍较高,移植入SCID小鼠四周后,仍可见带有荧光标记的人源细胞,RT-PCR证明人源基因Alu的存在.结论:建立的转hLIF基因腺病毒载体饲养层细胞可以有效地扩增CD34+造血干/祖细胞,延缓其分化,并且有较高的移植效率和造血活性.     

细胞因子联合对4.5Gyγ射线照射比格犬造血损伤的治疗作用

目的 观察细胞因子组合对4.5 Gy γ射线照射比格犬造血系统损伤的治疗效果,为极重度骨髓型急性放射病的临床救治提供实验依据。方法 16只比格犬均给予4.5 Gy ⁶⁰Co γ射线全身照射,随机分为照射对照、综合对症和细胞因子3组。细胞因子组动物在综合对症支持治疗的基础上应用rhG-CSF、rhIL-11和rhIL-2联合治疗。2 d检测1次外周血象,分别于照射前4 d,照射后1和45 d收集骨髓和外周血进行造血细胞集落培养,制备胸骨病理切片观察组织形态学改变。结果 照射后各组动物外周血各类细胞数急剧下降,细胞因子联合治疗可提高白细胞最低值(1.04×10⁹/L,而照射对照组和综合对症组分别为0.28×10⁹/L和0.68×10⁹/L),缩短血小板减少持续时间(细胞因子组24 d,综合对症组33 d),使红细胞维持在正常值范围;照射后1 d骨髓及外周血中造血干细胞集落形成率明显下降,照射后45 d 2个治疗组造血干细胞集落数均恢复为照射前水平;照射对照组动物骨髓造血细胞完全消失,细胞因子治疗使得骨髓造血功能完全恢复,与照射前水平比较差异无统计学意义。结论 rhG-CSF、rhIL-11和rhIL-2联合应用可提高极期时外周血白细胞最低值,加速白细胞、血小板和红细胞恢复,促进4.5 Gy γ射线照射犬体内残留造血干/祖细胞的增殖、分化和成熟,从而加速造血功能的重建。rhG-CSF、 rhIL-11和rhIL-2不失为治疗极重度骨髓型急性放射病的有效措施。     

Experimental therapeutic effect of combined cytokines on beagle dogs exposed to 4.5 Gy γ-rays

Objective To evaluate the effects of combined administration of recombinant human interleukin-11(rhIL-11),recombinant human G-CSF(rhG-CSF)and recombinant human interleukin-2 (rhIL-2)on acute radiation sickness(ARS)beagles.Methods Sixteen beagle were irradiated with 4.5 Gy60 Co γ-rays to establish ARS models,and were divided into irradiation control group,supportive care group and combined cytokines treatment group.After irradiation irradiation control group was given no treatment,the dogs in supportive care group received purely symptomatic treatment,while combined cytokines treatment group received rhIL-11 50μg/(kg·d)and rbG-CSF 10μg/(kg·d)subcutaneously(0-14 d)and rhIL-2 1×1 06 U/d(29-43 d)besides symptomatic treatment.Manifestation and characteristics of ARS beagles were observed,and the survival time were recorded.At last,post-mortem examination and histological examination were performed.Results All animals underwent nausea,diarrhea and fever.After irradiation,all animals in irradiation control group died in two weeks,and the mean survival time was 12.7 d,while only one died at 33 d in supportive care group.All dogs in combined cytokine group survived at 45 day after exposure,and their haematopoiesis and gastrointestinal tract were recovered.Conclusions Combination of rhIL-11 + rhG-CSF + rhIL-2 treatment could be significantly effective on ARS beagles irradiated by 4.5 Gy60 Co γ-rays,which could accelerate injured haemotopoiesis and intestinal tract recovery,increase the survival rate and improve the life quality of animals.     

Initial research of screening for the differentially expressed proteins in beagles irradiated with 4.5 Gy60 CO γ-rays by two-dimensional gel electrophoresis and mass spectrometry

Objective To explore the mechanisms of cytokines on acute radiation disease in irradiated beagles.Methods The sera of beagles irradiated with 4.5 Gy γ-rays with cytokines treatment was collected at different time points post irradiation.The two-dimensional gel electrophoresis(2-DE)was used to isolate and compare the differentially expressed proteins in sera.HD-MS was used to analyze the differentially expressed proteins with significance,and the amino acid sequences should be determined. Results High resolution 2-DE gel map was obtained.There were six differentially expressed proteins in sera of irradiated beagles at different time points.Four protein spots were successfully identified by MS.A significant spot was identified as serum amyloid A(SAA)by HD-MS,with relative molecular mass of 13 077 and isoelectfie point of 6.26.Expression of SAA was not found 1 d pre-irradiation and 36 d postirradiation,but increased slightly 1 d(0.2166)and significantly 14 d post-irradiation(0.4577). Conclusions The expression of serum amyloid A was consistent with the process of acute radiation injury,which might indicate the turnover of the disease.     

Clotting mechanism in beagles irradiated by 4.5 Gy γ-rays

Objective To explore the clotting mechanism in beagles irradiated by 4.5 Gy γ-rays after treatment with supportive care,or supportive care and combined cytokines.Methods Sixteen beagles were divided into irradiation control group,Supportive care group and combined cytokines treatment group.Platelet aggregation test,thrombelagtography (TEG) and the time measurement were analyzed in vitro.Results In irradiation group and supportive care group,the platelet aggregation rates in beagles were decreased markedly and the k value of TEG was increased 7 d post-irradiation,while those indexes in combined cytokines treatment group changed little.At 14 d post-irradiation,each parameter of TEG in irradiated group changed obviously.The values of r,k,r+k and M were elevated significantly,clotting time and the maximum coagulation time of thrombus delayed,the Ma value was decreased markedly,and the maximum elasticity amplitude of thrombus was diminished.All parameters in combined cytokines treatment group were better than those in supportive care group.The thrombin time was prolonged obviously in irradiated group 14 d post-irradiation,while the thrombin time was the longest at 2-3 weeks post irradiation in supportive care group and combined cytokines treatment group(P>0.05).Conclusions Cytokines could improve the platelet aggregation and the blood clotting functions of beagles suffering from acute radiation sickness.