人脐血和外周血单个核细胞增生活性及紫外线B照射抑制作用的研究

目的：比较人脐血（ＣＢ）和外周血（ＰＢ）单个核细胞（ＭＣｓ）的增生活性及其紫外线Ｂ照射（ＵＶＢ）在增生活性上的抑制作用。方法：应用ＰＨＡ诱导细胞增生以＾３Ｈ－ＴｄＲ结合检测增生活性（ｃｐｍ）；ＵＶＢ照射后的ＰＨＡ刺激和ＭＬＣ抑制作用，以未照射细胞的活性面分数表示。结果：人ＣＢＭＣｓ和ＰＢＭＣｓ ｃｐｍ平均值无统计学差异，但单个实验的标准偏差均显示人ＣＢＭＣｓ的ｃｐｍ高于ＰＢＭＣｓ，二者对小剂量ＵＶ     

MHC半相合脾加骨髓细胞诱发H22荷瘤鼠的抗肿瘤效应

目的：观察MHC半相合脾加骨髓细胞移植抗小鼠H22实体瘤的效果。方法：以皮下接种H22肝癌细胞的BALB/c×C57BL/6杂交F1代雌性小鼠为受鼠,以健康雌性F1、雄性C57BL/6、雄性C3H小鼠为MHC全相合、半相合、不相合供鼠,观察移植后的抑瘤情况；观察供鼠细胞经~(60)Co照射的MHC半相合移植对WBC、生化和嵌合体的影响；比较供鼠细胞经与不经~(60)Co照射的MHC半相合移植的GVHD情况。结果：供鼠细胞经/不经~(60)Co照射的MHC半相合移植小鼠的肿瘤明显较小,与单纯化疗未进行移植者比较,差异具有统计学意义(P＜0．05)；但受鼠未经化疗预处理的MHC半相合细胞输注没有出现抗肿瘤效应；供鼠细胞经7．5 Gy ~(60)Co照射的MHC半相合移植能明显降低GVHD反应,且对外周血白细胞、生化无不良影响。结论：经7．5 Gy~(60)Co照射的MHC半相合脾加骨髓细胞移植能对H22肝癌细胞产生移植物抗肿瘤效应并降低GVHD反应。     

吲哚胺2，3-二氧化酶基因修饰的DCs对小鼠移植物抗宿主病的抑制作用

目的:探讨小鼠树突状细胞(dendritic cells,DCs)转染吲哚胺2,3-二氧化酶(indolamine 2,3-dioxygenase,IDO)基因后对移植物抗宿主病(graft versus host disease,GVHD)的抑制作用。方法:用携带IDO基因的重组腺病毒感染BALB/c小鼠来源的树突状细胞,后者与C57BL/6小鼠骨髓移植物共培养,将经过处理的骨髓移植给BALB/c小鼠(C57BL/6→BALB/c小鼠GVHD模型,H-2~h→H-2~d),观察、比较各组GVHD表现(包括GVHD评分、生存期、病理学改变),并行嵌合体检测及观察混合淋巴细胞反应(mixed lymphocyte reaction,MLR)。结果:致死剂量照射的受体小鼠接受经IDO处理的骨髓移植(bone marrow transplantation,BMT)后,未出现明显的GVHD反应,生存期显著延长,3个月时生存率大于80%;对照组均在移植后2周左右出现明显的GVHD表现,生存期未超过1个月。IDO-DC治疗组未出现明显的组织病理学损害;3个月时IDO-DC治疗组仍然保持比较高的嵌合;IDO-DC组的T细胞对C57BL/6、BALB/c淋巴细胞的反应性与对C3H淋巴细胞反应性相比显著降低(P<0.05)。结论:经IDO基因修饰的DCs可以选择性去除骨髓移植物中异基因反应性T细胞,从而特异性地抑制GVHD并能及早地免疫重建。     

树突状细胞诱导的半相合CTL灭活后对小鼠移植肺癌的抑制作用

目的:探讨60Co灭活的树突状细胞(dendritic cell,DC)诱导的MHC半相合细胞毒性T淋巴细胞(cytotoxicity T lymphocyte,CTL)回输的抗小鼠移植肺癌作用。方法:体外培养CB6F1小鼠来源的DC,流式细胞术检测DC表型,用CB6F1-DC诱导针对C57BL/6小鼠来源的转移性肺癌细胞(Lewis lung cancer,LLC)的细胞毒性T细胞(cytotoxicity T lymphocyte,CTL),经60CO灭活的CTL回输给荷LLC瘤C57BL/6小鼠。细胞毒实验检测CTL细胞的抗肺癌效应,病理检测荷瘤小鼠肝、脾、小肠、皮肤变化,观察移植物抗宿主病(graft-versus-host-disease,GVHD)的发生及肺部肿瘤转移情况,观察荷瘤小鼠的存活时间。结果:流式细胞检测证实培养出成熟DC。DC诱导的CTL灭活后回输治疗LLC肺转移瘤小鼠后,小鼠肺质量显著降低[(0.27±0.06)vs(0.52±0.07)g,P<0.05],平均生存期显著延长[(78.10±16.50)vs(49.30±6.45)d,P<0.05]。荷瘤小鼠脾淋巴细胞对LLC细胞的杀伤活性随着效靶比的增加逐渐增强,最大杀伤率可达(32.7±1.64)%(效靶比100∶1)。病理检测结果显示,治疗组荷瘤小鼠未见明显GVHD。结论:灭活的半相合CTL回输可以诱导机体抗肿瘤反应,降低小鼠移植肺癌转移,未出现明显的GVHD。     

HSV-tk基因转染过程对小鼠脾脏T细胞功能的影响

目的 :观察 HSV- tk基因转染过程对小鼠脾脏 T细胞增生、细胞毒效应和诱导移植物抗宿主病 (GVHD)能力的影响 ,为建立 HSV- tk/GCV系统治疗小鼠 GVHD模型作准备。方法 :将HSV- tk基因转染小鼠脾脏 T细胞 ,以未转染 T细胞作对照 ,比较两者增生能力 ;51 Cr释放试验测定TK+ T细胞和 T细胞的细胞毒效应 ;1× 1 0 6 雄性供鼠骨髓单个核细胞分别与 1× 1 0 7TK+ T细胞和 1× 1 0 7T细胞移植半相合雌性 Fl受鼠 ,观察两者诱导 GVHD的功能。结果 :TK+ T细胞和 T细胞增生能力和细胞毒效应无明显差异 ,诱导急性 GVHD能力相似。结论 :HSV- tk基因转染对小鼠脾脏 T细胞功能无明显影响     

黏附分子与移植物抗宿主病

GVHD是由移植物中淋巴细胞(主要是T细胞)识别宿主抗原而致敏、增生分化，直接或间接攻击受者靶组织而引起宿主的全身性疾病。GVHD主要发生于异基因造血干细胞移植，也见于其它组织器官移植，包括小肠移植、肝移……     

VPA-BSANPs对胶质瘤细胞系放射生物效应

目的 研究纳米VPA-BSANPs复合物对C6、U87胶质瘤细胞系的体外放射生物效应。方法 C6、U87细胞分别经不同浓度VPA、VPA-BSANPs作用12、24 h后MTT法检测群体细胞存活率,经不同浓度VPA、VPA-BSANPs联合X线0、2、4、6、8 Gy照射后克隆形成实验检测PE值,经不同浓度VPA、VPA-BSANPs作用12 h后X线0、4、8 Gy照射应用Annexin V-FITC/PI双染法流式细胞术检测细胞凋亡情况。应用蛋白免疫印迹方法检测VPA、VPA-BSANPs对射线诱导细胞凋亡蛋白表达影响。多组均数检验方差齐性后用单因素方差分析,两样本均数间进行t检验。结果 VPA、VPA-BSANPs对C6、U87胶质瘤细胞并无明显增殖抑制作用(P=0.328、0.920);VPA-BSANP联合照射的PE值明显低于VPA联合照射(P=0.000)。C6、U87细胞VPA-BSANPs联合照射的p53、Bax表达增加(C6细胞P=0.000、0.000和U87细胞P=0.010、0.002),Bcl-2表达降低(C6细胞P=0.008、U87细胞P=0.000)。Caspase-3激活片段仅见于VPA-BSANPs+照射、VPA+照射,且前者低于后者(P=0.004)。PPAR激活片段仅见于VPA-BSANPs+照射。结论 VPA-BSANPs可增加C6、U87胶质瘤细胞系放射生物效应,其机制与定向促进X线诱导的肿瘤细胞凋亡有关。     

活化半相合混合骨髓移植GVHD反应的动物实验研究

目的:观察活化半相合混合骨髓移植的GVHD反应.方法:以急性放射病615小鼠模型为受鼠,615×C57BL/6杂交F1代小鼠为半相合供鼠.半相合鼠骨髓和脾细胞中混合一定比例的同基因脾细胞,经白细胞介素-2体外活化后,进行活化半相合混合骨髓移植.观察移植后小鼠死亡率、白细胞系造血重建、脾结节、体内混合淋巴细胞培养、嵌合体及病理学改变,比较不同移植方式的GVHD反应.结果:活化半相合骨髓移植组与未活化半相合骨髓移植组具有明显的GVHD反应.供受鼠3:1活化半相合混合骨髓移植不能明显降低GVHD反应,供受鼠脾细胞比例1:1和2:1移植组可以明显减轻GVHD反应.表现为外周血白细胞及骨髓造血恢复快,体内混合淋巴细胞反应降低及嵌合体百分率升高.结论:活化半相合混合骨髓梃移植方式可以降低GVHD反应,并与同基因和异基因脾细胞比例有关.     

非清髓性异基因骨髓移植治疗白血病的动物实验研究

[目的]探讨非清髓性异基因骨髓移植及加供者淋巴细胞输注治疗小鼠白血病的疗效.[方法]荷L7212白血病的615(H-2K)小鼠,于接种白血病细胞后第2天接受60Co-γ射线全身照射(TBI 8.5Gy或5Gy)分为若干组,照射当天移植供鼠BALB/C(H-2d)小鼠的骨髓细胞(5×106)和脾细胞(1.5×107),移植后第2天腹腔注射环磷酰胺(200mg/kg);供者淋巴细胞输注组分别于移植后第7天、14天、21天再次输注供鼠脾细胞5×106、1×107、2×107,观察受鼠的移植物植入、移植物抗宿主病(GVHD)、受鼠生存时间及移植相关并发症等.[结果]非清髓性预处理能保证移植物的稳定植入,非清髓性异基因骨髓移植组小鼠生存时间为22.3±4.8天,与非清髓空白组14.7±3.4天和传统移植组18.3±3.2天比较均有显著性差异(P＜0.05),供者淋巴细胞输注(DLI)组小鼠平均生存时间明显延长,为34.3±2.5天,与非清髓移植组比较均有显著性差异(P＜0.05),且无明显GVHD表现和病理学改变,移植相关并发症减少.[结论]非清髓性异基因骨髓移植能在减轻GVHD的同时保留一定的移植物抗白血病(GVL)效应,移植后行DLI可在减轻移植相关并发症的基础上进一步增强GVL效应.     

PHA对CIK细胞体外扩增影响的研究

目的:观察植物血凝素PHA对细胞因子诱导的杀伤细胞(cytokineinducedkillercells,CIK)体外扩增的影响。方法:在外周血单个核细胞定向诱导CIK细胞时加或不加PHA,观察细胞增殖的变化,流式细胞仪检测细胞的免疫表型,MTT法测定细胞的杀伤活性。结果:PHA能明显提高CIK细胞的扩增倍数及体外杀伤活性,P=0.041。结论:为CIK细胞的过继性免疫治疗提供一种新方法。     

Depletion of alloreactive T-cells in vitro using the proteasome inhibitor bortezomib preserves the immune response against pathogens

Current graft-versus-host disease (GVHD) inhibition approaches lead to abrogation of pathogen-specific T-cell responses. We propose an approach to inhibit GVHD without hampering immunity against pathogens: in vitro depletion of alloreactive T cells with the preoteasome inhibitor bortezomib. We show that PBMCs stimulated with allogeneic cells and treated with bortezomib greatly reduce their ability to produce IFN-γ when re-stimulated with the same allogeneic cells, but mainly preserve their ability to respond to citomegalovirus stimulation. Unlike in vivo administration of immunosuppressive drugs or other strategies of allodepletion, in vitro allodepletion with bortezomib maintains pathogen-specific T cells, representing a promising alternative for GVHD prophylaxis.     

脐血与成人血成分、T淋巴细胞亚群及正常人骨髓集落形成的比较研究

目的：研究脐血,成人外周血及骨髓的特性,方法：采用全自动血细胞分析仪及流式细胞仪分别测定济血,成人外周血的血成分及T巴细胞亚群,采用集落形成试验观察脐血,外周因及骨髓的CTU－GM和BFU－E的产率变化,结果：脐血的各种血成分均显著高于外周血,CD,CD3明显低于外周血,而CD4,CD8的表达与外周血相似,经细胞因子诱导后,脐血中的CUF0－GM及BFU－E显著升高,结论：脐血富含造血干细胞,是重建造血与免疫系统的血细胞新来源。     

Proliferation of leukemic B cells in response to SAC and anti-mu. Evidence for different modes of action and comparison to proliferation and differentiation induced by conditioned medium

We investigated the proliferative responses and immunoglobulin production of highly purified E-rosette negative largely leukemic B cells from patients with CLL to Staphylococcus aureus Cowan I (SAC) or to SAC in combination with anti-mu or conditioned medium (CM). The latter was derived by stimulating human peripheral blood mononuclear leukocytes with PHA. We observed: (1) that purified E-rosette negative largely leukemic B cells from 25% (five out of 20) of the patients exhibited proliferative responses to SAC; (2) inhibition of SAC-induced proliferation by anti-mu in certain patients, whereas synergism between SAC and anti-mu in inducing proliferative responses in others; (3) the lack of synergism between SAC and CM in inducing proliferative responses, which is in contrast to the strong synergism that was observed between anti-mu and CM in inducing proliferation; and (4) induction or enhancement by SAC alone of Ig production by largely leukemic B-cell populations from few patients with CLL and purified tonsillar B lymphocytes, but not peripheral blood B cells from normal donors. These results suggest that SAC and anti-mu induce proliferation of B cells by different mechanisms and that B-cell proliferation and differentiation is dependent not only on the mitogen but also on the activation state of the cells.     

UVB irradiation regulates Cox-2 mRNA stability through AMPK and HuR in human keratinocytes

Considerable evidence has demonstrated that UVB irradiation is a strong carcinogen for nonmelanoma skin cancer. Up-regulation of cyclooxygenase-2 (Cox-2) has been shown to be a crucial event in human keratinocytes in their responses to UVB irradiation. To further understand the molecular mechanisms governing Cox-2 regulation, we found that UVB irradiation significantly increased Cox-2 mRNA stability by inducing cytoplasmic localization and protein abundance of human antigen R (HuR). We also found that AMP-activated kinase (AMPK) mediates these events and that UVB reduces AMPK activity by down-regulating LKB1 kinase. Finally, we propose a novel model in which UVB regulates Cox-2 mRNA stability through the LKB1/AMPK pathway.     

Persistent human papillomavirus infection is associated with a generalized decrease in immune responsiveness in older women

The development of cervical cancer and its precursors are linked to persistent infection with oncogenic types of human papillomavirus (HPV). Host immune responses seem to be determinants of risk for this disease. However, little is known about the immunologic determinants of HPV persistence. Here, we examined the association between lymphoproliferative responses to antigens/mitogens and persistent HPV infection in women older than 45 years. Women included in this study were participants in a 10,000-woman population-based cohort study of cervical neoplasia in Costa Rica. Women older than 45 years and HPV DNA positive at a screening visit were selected as cases (n = 283). We selected a comparably sized control group of HPV DNA-negative women, matched to cases on age and time since enrollment (n = 261). At an additional clinical visit, women were cytologically and virologically rescreened, and cervical and blood specimens were collected. Proliferative responses to phytohemagglutinin (PHA), influenza virus (Flu), and HPV16 virus-like particle (VLP) were lower among women with persistent HPV infection [median counts per minute (cpm): 72,849 for PHA, 1,241 for Flu, and 727 for VLP] than for the control group (median cpm: 107,049 for PHA, 2,111 for Flu, and 2,068 for VLP). The decreases were most profound in women with long-term persistence and were only observed for the oldest age group (>/=65 years). Our results indicate that an impairment in host immunologic responses is associated to persistent HPV infection. The fact that effects were evident for all studied stimuli is suggestive of a generalized effect.     

Postoperative Radiation Therapy and Adjuvant Chemoimmunotherapy in Breast Cancer Aspects of Timing and Immune Competence

The effects of radiation therapy and adjuvant chemoimmuno-therapy on the immune competence of patients with breast cancer were investigated. The tests performed included intradermal tuberculin tests, T- and B-lymphocyte counts, and lymphocyte blast transformation tests; phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) were used as mitogens. Enhancement in lymphocyte proliferative response to mitogenic stimulation by PHA and PWM was seen in patients after 3 courses of chemotherapy + levamisole, whereas irradiation given after chemotherapy caused long-lasting depression in response to PHA (p=0.039) and PWM (not significant). T-lymphocyte counts were also lower after irradiation than after chemoimmunotherapy (p=0.007). Clinically, the 16 patients treated with radiation therapy after chemotherapy exhibited a higher recurrence rate than the 24 patients treated first by irradiation. Enhanced reactivity to tuberculin tests occurred generally in patients receiving a planned treatment including irradiation, chemotherapy (5-fluorouracil, doxorubicin, cyclophosphamide) and levamisole. Enhancement of reactivity was seen more often in patients who had not relapsed.     

Therapeutic effects of Lycium barbarum polysaccharide (LBP) on irradiation or chemotherapy-induced myelosuppressive mice

AIM: The aim of this study was to investigate the effects of Lycium barbarum polysaccharide (LBP) on irradiation- or chemotherapy-induced myelosuppressive mice and cultured peripheral blood mononuclear cells (PBMCs). METHODS: In an in vivo experiment, mice were irradiated with a sublethal dose of 550 cGy X-ray or intraperitoneally (i.p.) injected with carboplatin (CB) 125 mg/kg to produce severe myelosuppression. Four to 6 hours after the irradiation or injection, mice were subcutaneously (s.c.) injected with LBP (50, 100, and 200 mg/kg) daily from day 0 to day 6. Blood samples were collected from the tail veins of mice at different time points, and peripheral white blood cells (WBC), red blood cells (RBC), and platelet (PLT) counts were monitored. In an in vitro experiment, human PBMCs were incubated with LBP at different concentrations in combination with phytohemagglutinin (PHA), and the production of granulocyte colony-stimulating factor (G-CSF) was tested. RESULTS: Compared to the control, 50 mg/kg LBP (LBP-L) significantly ameliorated the decrease of peripheral WBC of irradiated myelosuppressive mice on day 13, and 100 mg/kg LBP (LBP-M) did the same on days 17 and 21. All dosages of LBP significantly ameliorated the decrease of peripheral RBC of irradiated myelosuppressive mice on days 17 and 25. Two-hundred mg/kg LBP (LBP-H) and LBP-M significantly enhanced peripheral PLT counts of irradiated myelosuppressive mice on days 10, 13, 17, and 21, as did LBP-L on days 13 and 17. All dosages of LBP increased peripheral WBC counts of chemotherapy-induced myelosuppressive mice to some extent, but there was no statistic difference when compared to the control. LBP-H significantly ameliorated the decrease of peripheral RBC of chemotherapy-induced myelosuppressive mice on days 13, 15, 17, and 20, and LBP-M and LBP-L did the same on days 15 and 17. All dosages of LBP significantly enhanced peripheral PLT counts of chemotherapy-induced myelosuppressive mice on days 7 and 10, as did LBP-H on days 13, 15, and 17, and LBP-M on days 13 and 15. Also, LBP could obviously stimulate human PBMCs to produce G-CSF. CONCLUSIONS: LBP promoted the peripheral blood recovery of irradiation or chemotherapy-induced myelosuppressive mice, and the effects may be the result of the stimulation of PBMCs to produce G-CSF.     

外科手术对肿瘤患者血细胞凋亡和增殖活性的影响

目的：探讨外科手术对恶性肿瘤者外周血细胞动力学的影响。方法：用流式细胞术对221例手术前和287例手术后恶性肿瘤患者外周血细胞凋亡水平和增殖活性进行了对比分析。结果：手术后恶性肿瘤患者血细胞的细胞凋亡水平明显低于手术前患者（P＜0．01）,手术后患者血细胞S期细胞比率显著高于手术前患者（P＜0．01）,结论：外科手术能引起恶性肿瘤患者外周血细胞动力学发生明显改变。这些结果对判断肿瘤发展情况可提供一定的客观参考依据。     

脐血输注对急性白血病患者骨髓CFU－GM体外培养及血浆CSA、BPA的影响

以自身对比方法观察急性白血病化疗后骨髓衰竭患者输注脐血和外周血后，骨髓粒、单细胞系集落形成单位（ＣＦＵ－ＧＭ）、血浆集落刺激活性（ＣＳＡ）、爆式集落刺激活性（ＢＰＡ）及外周血白细胞计数（ＷＢＣ）的变化。结果脐血输注后可使患者ＣＦＵ－ＧＭ体外培养显著提高，ＣＳＡ、ＢＰＡ水平显著下降并趋于正常，可在输血量减少情况下，使ＷＢＣ显著增高。     

Cytomegalovirus interstitial pneumonitis following allogeneic peripheral blood stem cell transplantation

Objective： To explore the risk factors and prophylaxis and treatment of cytomegalovirus interstitial pneumonitis （CMV-IP） after allogeneic peripheral blood stem cell transplantation （allo-PBSCT）. Methods： 43 patients who received allo-PBSCT were allocated to either a Gancyclovir（GCV）-prophylaxis group （n=19） or a non-GCV prophylaxis group （n=24）. A comparison was made of the incidence of CMV-IP in patients given or not given prophylactic gancyclovir. Results： 9 patients in non-GCV prophylaxis group developed late CMV-IP （P〈0.05）. Graft-versus-host-disease （GVHD） may be associated with a high risk of CMV-IP. 5 cases of CMV-IP were successfully treated with GCV, but 3 cases died of CMV-IP. The most common adverse event of GCV was neutropenia, but was reversible. Conclusion： CMV infection was a major cause of interstitial pneumonitis after allo-PBSCT, which correlated strongly with the severity of GVHD. Gancyclovir was shown to be effective in both prophylaxis and treatment of CMV-IP.