Natural Killer Cell Function in Atopic Dermatitis

ABSTRACT. The natural killer cell activity was studied in 41 children with mild, moderate and severe atopic dermatitis (AD) and in 37 controls. The natural killer cell function of lymphocytes was reduced in atopic children (mean ± SD 21.92±6.18% vs. 43.87±5.80%; p <0.0001). This decrease was not related to the IgE serum level. A negative correlation was found between natural killer cell activity and the clinical severity of AD ( r =0.73; p <0.001). Natural killer cell function was re-evaluated after 9 months in 27 children during a quiescent phase of AD; it was still low, but to a lesser degree (27.66±5.42%, in the quiescent phase, vs. 43.87±5.80, controls; p <0,0001). The reduced natural killer cell activity seems related to the disease activity.     

丹参诱导人脐血间充质干细胞分化为神经样细胞

目的探讨丹参注射液对人脐血单个核细胞来源的间充质干细胞(MSCs)向神经细胞分化的诱导作用。方法利用FACScan流式细胞仪检测MSCs表面抗原CD29、CD44、CD59、CD33,用丹参注射液诱导人脐血原代细胞和脐血来源的间充质干细胞向神经细胞方向分化,并与神经生长因子(NGF)和神经常苷脂(GM1)的诱导作用比较,用免疫细胞化学方法对分化和未分化细胞进行鉴定。结果人脐血原代细胞中MSCs的表耐标记CD29、CD44、CD59阳性率分别为10.7%、37.27%和66.67%,而造血细胞的表面标记CD33阳性率仅为0.33%。人脐血传代细胞(第5代)MSCs的表面标记CD29、CD44、CD59的阳性率分别为40.2%、70.5%和95.4%。原代培养的贴壁细胞形态呈大小不等圆形和条形,用丹参诱导可表达神经细胞的标记。传代培养的间充质干细胞,可维持在未分化状态稳定增殖。用丹参可诱导这种细胞向神经样细胞分化,表达神经干细胞标记nestin,神经元的标记β-Ⅲ类神经微管(β-TubulinⅢ)、神经微丝(NF)和神经胶质细胞的标记胶质纤维酸性蛋白(GFAP)。与NGF和GMI的诱导作用比较,细胞形态类似,表达相同的神经细胞标记,丹参的诱导速度较快,诱导后细胞表达神经元标记的比例较高。结论人脐血是MSCs的来源之一,丹参可诱导人脐血干细胞分化为神经样细胞,丹参可作为神经诱导剂,人脐血干细胞可作为神经干细胞的来源。     

Transplantation of Umbilical Cord Blood in a Refractory Lymphoma

A successful cord blood transplantation combined with hematopoietic growth factor was performed in a boy presenting with refractory mediastinal T-cell lymphoma. Cord blood cells were collected from an HLA-identical sibling at the time of delivery. A transient and corticosensitive acute grade II graft versus host disease was observed. One year after transplantation, the child is still in remission with complete engraftment. This is the first report of cord blood transplantation in a patient with refractory lymphoma.     

Natural Killer Activity in Two Cases of Ataxia Telangiectasia

ABSTRACT. We report two cases of ataxia telangiectasia in which a small number of T cells and a slightly diminished response to phytohaemagglutinin or concanavalin A were observed. In one case, serum IgA was undetectable. In spite of T and/or B cell involvement, natural killer activity and the percentage of natural killer cells determined by monoclonal antibodies Leu 7 were normal in both cases.     

人脐带血间充质干细胞在缺氧缺血性脑病新生鼠脑内的定植

目的探讨脐带血间充质干细胞(MSCs)培养及其在缺氧缺血性脑病(HIE)新生鼠脑内的定植。方法用出生7 d的新生SD大鼠制作HIE模型,同时在当天分别用2种方法(定向注射和尾静脉注射方式)接受经Hoechst 33258标记24 h的MSCs移植,15~30 d观察MSCs存活情况。结果用改良Rice法可制备7日龄新生鼠单侧脑损伤为主的HIE模型;经定向注射和尾静脉注射移植的MSCs能在HIE脑内定植,分布在患侧大脑皮层、海马等部位,并主要以额叶为多,和宿主脑组织融合在一起。结论在体外培养条件下,人脐血MSCs可以生长,当MSCs移植到HIE模型鼠后,细胞能和脑实质融合在一起,并更多地向脑损伤部位迁移聚集。     

诱导脐血内皮祖细胞分化成内皮细胞的研究

目的探讨诱导脐血来源内皮祖细胞分化为内皮细胞,分析其用作组织工程心血管修复物种子细胞来源的可行性。方法采用6%羟乙基淀粉沉降法和密度梯度离心法联合应用分离脐血单个核细胞,在含有血管内皮生长因子(VEGF)等多种生长因子的培养液中培养扩增,通过形态学(光镜、电镜)、免疫荧光和流式细胞仪等技术对脐血来源内皮祖细胞分化的内皮细胞进行鉴定。结果从新鲜脐血中得到的单个核细胞数(3.4±2.1)×107/mL。贴壁细胞培养分化过程中形态发生改变,从小圆形变成梭形,最后分化成典型成熟内皮细胞的鹅卵石样形态,细胞数量可扩增达107。7 d后免疫荧光检测发现细胞表达内皮细胞特异性标志vWF和VEGFR-2。流式细胞仪分析单个核细胞经诱导分化后细胞表面表达CD133下降,从3.11%±1.05%下降至0.09%±0.02%。透射电镜观察到培养14 d的细胞胞浆中已出现典型的Weibel-Palade小体。结论脐血单个核细胞存在内皮祖细胞,并在体外可诱导分化为内皮细胞,初步具有用作构建组织工程心血管修复物种子细胞的可行性。     

三种不同方法分离脐血造血干细胞的效果比较

目的探讨一种较好的脐血造血干细胞(HSCs)的分离方法,以最大限度地分离出脐血中的HSCs,使其以高纯度、最佳生长状态用于治疗和研究。方法取本院足月妊娠健康产妇的脐血20份,每份均采用羟乙基淀粉沉淀(HES)法、淋巴细胞分离液(Ficoll)密度梯度离心法、免疫磁珠(MACS)法处理。比较3种方法分离后的单个核细胞(MNCs)回收率、锥虫蓝拒染率及CD34细胞阳性率;将以上3种方法所得细胞用完全培养基培养,不同时间点计数细胞,绘制生长曲线,并观察原代细胞的生长情况及其形态特征;并采用半固体琼脂培养法,计数其粒-巨噬细胞集落形成单位数。结果 HES法得到的MNCs回收率高于Ficoll密度梯度离心法和MACS法(Pa<0.05);锥虫蓝拒染率三者比较差异无统计学意义(P>0.05);MACS法所得细胞CD34阳性率明显高于另2种方法(Pa<0.05),并且其所得细胞生长状态好,集落数最多;HES法和Ficoll法所得细胞,部分呈贴壁生长,与贴壁细胞共培养的细胞生长状态好;Ficoll密度梯度离心法得到细胞生长状态最差,集落数最少。结论 HES法可作为一种首选常规分离脐血造血干细胞的方法;MACS法分离得到造血干细胞纯度高,适于实验及临床研究。     

早产儿脐血造血干/祖细胞特点

目的对12例早产儿和18例足月儿脐血采集量、有核细胞数量、CD34+细胞比例、体外形成造血细胞克隆的能力进行比较研究。方法采用密闭式脐血采集袋,经脐静脉穿刺收集脐带血,按体积比5:1与60 g.L-1羟乙基淀粉(HES)混合、浓缩有核细胞,流式细胞仪检测CD34+细胞比例,甲基纤维素半固体培养基检测其脐血形成造血细胞克隆能力。结果早产儿脐血采集量及分离前后有核细胞数量均低于足月儿脐血[脐血采集量分别为(76.52±22.48)mLvs(94.21±20.32)mL,P<0.05;分离前有核细胞数量分别为(4.78±2.30)×108vs(8.36±2.51)×108,P<0.01;分离后有核细胞数量分别为(3.72±1.71)×108vs(6.54±1.94)×108,P<0.01)]。早产儿CD34+细胞比例高于足月儿脐血[(0.49±0.16)%vs(0.32±0.13)%,P<0.01],早产儿脐血体外生成红系爆式集落形成单位和粒-单系集落形成单位的能力也高于足月儿脐血[分别为(29.58±10.54)/2×105MNCvs(19.27±7.26)/2×105MNC,P<0.01和(45.28±16.2...     

Natural Killer Cell Activity of Peripheral Blood and Bone Marrow Mononuclear Cells from Patients with Childhood Acute Lymphoblastic Leukemia

ABSTRACT. Patients with untreated acute lymphoblastic leukemia had highly significantly reduced natural killer cell activity in peripheral blood ( p <0.01) and bone marrow ( p <0.001) mononuclear cells compared with that in peripheral blood mononuclear cells in normal healthy controls. Patients in remission had normal killer cell activity ( p >0.5) both in peripheral blood and bone marrow mononuclear cells. Thus, the natural killer cell activity correlated well with the disease activity in the patients.     

人脐带血间充质干细胞体外诱导分化为神经元样细胞的可行性

目的探讨人脐带血来源间充质干细胞(MSCs)在体外诱导分化为神经元样细胞的可行性及条件。方法采用Fi- coll-Paque(1.077 g/mL)分离液密度梯度分离人脐带血中MSCs,体外扩增纯化后,二甲基亚砜、巯基乙醇及丁羟茴醚等试剂诱导其向神经细胞分化,显微镜下观察,细胞免疫化学法鉴定。结果诱导后脐血MSCs具有典型神经元形态细胞,免疫细胞化学显示神经丝蛋白(NF-M)染色呈强阳性,占(81．86±4．06)％;Nestin染色呈阳性细胞占(39．99±4．60)％;GFAP染色阴性。结论人脐带血MSCs在体外培养及诱导条件下可定向分化为神经元样细胞。     

树突状细胞与细胞因子诱导的杀伤细胞共培养细胞过继免疫治疗儿童急性白血病

目的观察树突状细胞(DC)与细胞因子诱导的杀伤细胞(CIK)共培养细胞(DC-CIK)过继免疫治疗儿童急性白血病的作用及安全性。方法回顾分析2005年5月-2011年5月本院确诊经联合化疗后达完全缓解的47例急性白血病患儿临床资料,观察DC-CIK过继免疫治疗过程中细胞采集、细胞培养、细胞回输的安全性,随访过程中通过定期检测其血常规、肝肾功能、心电图等来评估其远期安全性;DC-CIK过继免疫治疗后通过定期进行患儿骨髓细胞学、微小残留病(MRD)检测,初步评判其疗效。结果47例患儿接受DC-CIK治疗后44例患儿持续完全缓解(CCR),CCR率达93.62%;治疗前MRD阳性的9例患儿,8例治疗后转阴。47例患儿共接受DC-CIK过继免疫治疗61例次,不良反应轻微,单个核细胞采集术后局部有轻微疼痛,且均在术后2 h左右自行缓解。细胞培养过程中进行3次病原体检测,仅1例次培养出毛霉菌。细胞回输过程中23例次出现发热,10例次出现皮疹。中位随访41(5～72)个月未见心、肝、肾等重要器官功能损害,未见感染性疾病、第二肿瘤等的发生。结论 DC-CIK过继免疫治疗儿童急性白血病操作流程简便易行、安全性好,获得了满意疗效。     

Cryopreservation of Pluripotent and Committed Hemopoietic Progenitor Cells from Human Bone Marrow and Cord Blood

As one of the studies on autologous bone marrow transplantation for childhood cancer, the viabilities of several kinds of hemopoietic progenitor cells after cryopreservation was assayed using bone marrow and cord blood samples. The mean recovery rates of granulocyte-macrophage colony forming units (CFU-GM), erythroid burst forming units (BFU-E) and erythroid colony forming units (CFU-E) from bone marrow cells were 47.9%, 31.3% and 10.5%, respectively. The mean recovery rates of CFU-GM, BFU-E and CFU-E from cord blood cells were 46.7%, 39.1% and 22.4%, respectively. The ratio of primitive to mature BFU-E did not change after cryopreservation. The mean recovery rate of mixed colony forming units (CFU-mix) from marrow cells was 30.2%, and that from cord blood cells was 58.9%, demonstrating their sufficient proliferative activity after cryopreservation. These results demonstrate that CFU-mix, primitive BFU-E as well as CFU-GM, mature BFU-E can be well cryopreserved, supporting the usefulness of autologous marrow transplantation for the rescue of the ruined hemopoiesis after intensive cancer therapy.     

胎盘绒毛组织及脐血中造血细胞生长因子对人胎盘造血功能的影响

目的检测胎盘绒毛组织多种造血细胞生长因子(HGF)含量,探讨其对胎盘造血的意义。方法分别取早孕(6- 8周)、中孕(16-22周)、足月妊娠(37-42周)胎盘绒毛组织2 g各30份、脐血清30份,测定上清液中各种HGF含量。结果早期妊娠各种HGF均已产生;FL-13、IL-3在胎盘绒毛组织中含量随着胎龄增加,各胎龄组间比较,均有显著差异(P均< 0.01)。7种HGF在胎盘和脐血中含量,均有显著差异(P均<0.01)。结论F-T3配体(FL)、干细胞因子(SCF)、G-CSF等7 种HGF均参与胚胎期造血,尤以FL、IL-3、SCF最为重要。     

Highlights of the Third International Conference on Immunotherapy in Pediatric Oncology

The third International Conference on Immunotherapy in Pediatric Oncology was held in Frankfurt/Main, Germany, October 1–2, 2012. Major topics of the conference included (i) cellular therapies using antigen-specific and gene-modified T cells for targeting leukemia and pediatric solid tumors; (ii) overcoming hurdles and barriers with regard to immunogenicity, immune escape, and the role of tumor microenvironment; (iii) vaccine strategies and antigen presentation; (iv) haploidentical transplantation and innate immunity; (v) the role of immune cells in allogeneic transplantation; and (vi) current antibody/immunoconjugate approaches for the treatment of pediatric malignancies. During the past decade, major advances have been made in improving the efficacy of these modalities and regulatory hurdles have been taken. Nevertheless, there is still a long way to go to fully exploit the potential of immunotherapeutic strategies to improve the cure of children and adolescents with malignancies. This and future meetings will support new collaborations and insights for further translational and clinical immunotherapy studies.     

人脐带间充质干细胞向胰岛素分泌细胞分化过程免疫特性的研究

目的研究人脐带间充质干细胞(huMSCs)向胰岛素分泌细胞(IPCs)分化过程中的免疫学特性,为huMSCs作为胰岛细胞移植治疗1型糖尿病的细胞来源提供依据。方法以流式细胞术检测huMSCs及huMSCs源性IPCs的免疫表型;RT-PCR法检测huMSCs及huMSCs源性IPCs的HLA-A、HLA-DR基因的表达;CCK8法测定细胞增殖率,观察huMSCs及huMSCs源性IPCs对外周血单个核细胞增殖的影响。结果 huMSCs在体外培养条件诱导下,具有向胰岛素分泌细胞分化的潜能;huMSCs及huMSCs源性IPCs均不表达CD80、CD86、CD40、CD40L、HLA-DR,均表达HLA-A;huMSCs能够抑制PHA刺激的外周血单个核细胞的增殖,但huMSCs源性IPCs未见该作用。结论 huMSCs及huMSCs源性IPCs均具有低免疫原性,可作为胰岛细胞移植的细胞来源,huMSCs对免疫反应具有负调节作用,但huMSCs源性IPCs不具有该作用。     

缺氧缺血性脑病新生儿自然杀伤细胞与T细胞亚群的变化及其相关因素

目的了解缺氧缺血性脑病(HIE)新生儿自然杀伤(NK)细胞及T细胞亚群的变化,观察其与各种因素的相关性。方法应用流式细胞仪测定50例HIE和20例正常新生儿血NK细胞、CD3 、CD4 、CD8 细胞及CD4 /CD8 比值。结果HIE患儿NK细胞和T细胞亚群的表达均明显低于正常新生儿(P均<0.05);中、重度HIE患儿NK细胞和CD3 、CD4 细胞及CD4 /CD8 比值明显均低于轻度HIE(P均<0.05),而不同分度HIE的CD8 细胞则无统计学差异(P均>0.05);NK细胞、CD3 、CD4 及CD4 /CD8 与HIE病情分度均呈负相关(P均<0.05)。NK细胞表达与胎龄呈正相关;CD3 细胞与羊水污染呈负相关(P<0.05);CD4 细胞与羊水污染呈负相关,与出生体质量和Apgar评分呈正相关(P<0.05);CD4 /CD8 比值与分娩方式存在线性相关(P<0.05)。结论缺氧缺血可导致HIE患儿NK细胞活性抑制及T淋巴细胞亚群紊乱,某些因素对细胞免疫功能有一定影响。     

人胰岛素样生长因子1基因质粒载体的构建及其在人脐血源性神经干细胞中的表达

目的 构建人胰岛素样生长因子1(IGF-1)质粒表达载体,并观察重组体pcDNA3.1-IGF-1转染后的脐血源性神经干细胞(NSCs)中IGF-1基因的表达情况.方法 通过反转录-PCR(RT-PCR)方法从胎肝中提取IGF-1基因,胶回收方法分别纯化PCR产物(IGF-1基因)和质粒pcDNA3.1,二者分别由DNA限制性内切酶BamH Ⅰ与 Hind Ⅲ双酶切后,经T4 DNA Ligase连接的方法将IGF-1基因克隆到质粒表达载体pcDNA3.1中,采用测序方法及DNA限制性内切酶BamH Ⅰ与 Hind Ⅲ双酶切方法鉴定重组质粒,脂质体转染法将重组体pcDNA3.1-IGF-1及空质粒pcDNA3.1分别转染至脐血源性NSCs内,经G418抗性筛选后,利用免疫细胞化学法和RT-PCR法检测IGF-1基因在基因转染脐血源性NSCs内的表达情况.结果 IGF-1基因从胎肝中成功提取.重组体pcDNA3.1-IGF-1经基因测序及DNA限制性内切酶BamH Ⅰ与 Hind Ⅲ双酶切证实质粒表达载体pcDNA3.1-IGF-1构建正确.重组体经脂质体法转染脐血源性NSCs 24 h后,经G418筛选2周得到细胞抗性克隆,免疫细胞化学法检测到IGF-1基因在重组质粒表达载体pcDNA3.1-IGF-1转染的脐血源性NSCs中成功表达.RT-PCR方法检测到重组质粒pcDNA3.1-IGF-1转染的脐血源性NSCs中IGF-1 mRNA表达阳性,而空质粒pcDNA3.1转染的脐血源性NSCs中IGF-1 mRNA表达阴性.结论 IGF-1基因可在重组质粒表达载体pcDNA3.1-IGF-1转染的脐血源性NSCs内成功表达.     

Evaluation of cytotoxic function and apoptosis in interleukin (IL)-12/IL-15-treated umbilical cord or adult peripheral blood natural killer cells by a propidium-iodide based flow cytometry

Both deficient natural killer (NK) cell effector function and increased propensity to apoptosis of neonatal NK cells contribute to the increased susceptibility to infection in the neonates. Interleukin (IL)-12 and IL-15 are two immunoregulatory cytokines known to enhance cytolytic function of neonatal NK cells. The present study aims to simultaneously investigate the effect of IL-12/IL-15 on K562 cytotoxicity as well as NK cells apoptosis of enriched umbilical cord blood (CB) and adult peripheral blood (APB) NK cells, using flow cytometric cytotoxicity assays. The results indicated that (i) prior to cytotoxicity assays, CB NK cells underwent greater degree of spontaneous apoptosis than did APB NK cells; (ii) both IL-12 and IL-15 inhibited the spontaneous apoptosis of CB NK cells, while IL-15 promoted the apoptosis in APB NK cells; (iii) the deficient K562 cytotoxicity of CB NK cells could be enhanced to levels comparable with that of APB NK cells by IL-15; (iv) IL-15 increased the percentages of apoptosis in NK–K562 conjugates in a dose-dependant manner in both CB and APB with a greater effect seen with APB NK cells; (v) target-induced apoptosis was observed with APB NK cells which were further enhanced with IL-15. However, CB NK cells, unstimulated or IL-15-activated, were resistant to K562-induced apoptosis. Thus, the multi-parameter flow cytometry analysis not only demonstrates better for the deficient CB NK function but also provides greater details for cytotoxic mechanisms of NK cells.