The Cross-talk between ROS and p38MAPKα in the Ex Vivo Expanded Human Umbilical Cord Blood CD133~＋ Cells

This study investigated the correlation between and compared the effects of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase a (p38MAPKα) in the ex vivo expanded umbilical cord bl...     

Selective depletion of the allo-antigen specific T cells by Fas/FasL pathway by cytokine IFN-γ and IL-2

Summary To investigate the value of apoptosis of the allo-antigen specific T cells induced by Fas/FasL pathway in preventing graft-versus-host disease (GVHD), the CD34⁺ cells transfected with FasL or not, used as stimulus cells, were mixed with allo-antigen specific T lymphocytes in presence or absence of IFN-γ and IL-2. After 5 days, apoptosis of T cells was detected by TdT nick end mediated dUTP labeling (TUNEL) and flow cytometry (FCM). The affects of these two cytokines on CD34⁺ cells in the graft were also compared. The ratio of apoptosis of T cells was 12.1±1.5% when CD34⁺ cells transfected with FasL was used as stimulus cells, much higher than that of CD34⁺ cells non-transfected (3.2±1.1%,P<0.01). And in presence of IFN-γ or IL-2, the ratio reached 20.1±2.3%, 17.6±1.3% respectively (P<0.01). However, IFN-γ up-regulated Fas expression of CD34⁺ cells and increased the sensibility of CD34⁺ cells to soluble FasL(sFasL); IL-2 showed no such affect. It is possible to induce apoptosis of the allo-antigen specific T cells of grafts activated by allo-antigen by exogenous Fas ligand expressed on recipient cells and this might provide a new approach for preventing GVHD and IL-2 may be more suitable for clinical application. XIAO Juan, female, born in 1974, MD This project was supported by the grant from National Natural Science Foundation of China (No. 39770767).     

Expression and fuactional role of HERG1, K<Superscript>+</Superscript> channels in leukemic cells and leukemic stem cells

Summary In order to investigate the expression and functional role of HERG1 K⁺ channels in leukemic cells and leukemic stem cells (LSCs), RT-PCR was used to detect the HERG1 K⁻ channels expression in leukemic cells and LSCs. The functional role of HERG1 K⁺ channels in leukemic cell proliferation was measured by MTT assay, and cell cycle and apoptosis were analyzed by flow cytometry. The results showed that herg mRNA was expressed in CD34⁺/CD38⁻, CD123⁻ LSCs but not in circulating CD34⁺ cells. Herg mRNA was also up-regulated in leukemia cell lines K562 and HL60 as well as almost all the primary leukemic cells while not in normal peripheral blood mononuclear cells (PBMNCs) and the expression of herg mRNA was not associated with the clinical and cytogenetic features of leukemia. In addition, leukemic cell proliferation was dramatically inhibited by HERG K⁺ channel special inhibitor E-4031. Moreover, E-4031 suppressed the cell growth by inducing a specific block at the G1/S transition phase of the cell cycle but had no effect on apoptosis in leukemic cells. The results suggested that HERG1 K⁺ channels could regulate leukemic cells proliferation and were necessary for leukemic cells to proceed with the cell cycle. HERG1 K⁺ channels may also have oncogenic potential and may be a biomarker for diagnosis of leukemia and a novel potential pharmacological target for leukemia therapy. LI Huiyu, female, born in 1960, Associate Professor This project was supported by a grant from National Science Foundation for Distinguished Young Scholars of China (No. 30225038).     

Apoptosis of Leukemia Cells Induced by CD34^＋ Cells Transferred Exogenous Fas Ligand

Summary To assess the value of CD34⁺ cells transferred exogenous Fas ligand (FasL) in inducing apoptosis of human leukemic cells, the CD34⁺ cells transfected with FasL or without, pretreated with mitomycin C, was mixed with leukemic cell line U937 cells in presence or absence of daunorubicin (DNR) or cytosine arabinoside (Ara-C). After 18 h, apoptosis of cells was detected by FCM and TUNEL. Induced for 18 h by CD34⁺ cells transfected with FasL or without, the ratio of apoptosis of U937 cells was (5.0±1.3)%, (10.8±0.6)% (P<0.01), respectively. Induced by FasL⁺CD34⁺+DNR, FasL⁺CD34⁺+Ara-C, the ratio was (13.4±1.0) % (P<0.05), (17.9±1.3)% (P<0.01), respectively. The result demonstrated that CD34⁺ cells transfected with exogenous FasL could induce apoptosis of human leukemic cells and showed a cytotoxic synergistic effect when used in combination with chemotherapeutic drugs, suggesting that it was possible to develop a new method in treatment of leukemia. XIAO Juan, Female, born in 1974, Doctor in Charge This project was supported by the grant of National Nature Science Foundation of China (Serial No. 39770767).     

Liposome-mediated functional expression of multiple drug resistance gene in human bone marrow CD34<Superscript>+</Superscript> cells

Summary The expression and functional activity of multiple drug resistance (MDR1) gene in human normal bone marrow CD34⁺ cells was observed. Human normal bone marrow CD34⁺ cells were enriched with magnetic cell sorting (MACS) system, and then liposome-mediated MDR1 gene was transferred into bone marrow CD34⁺ cells. Fluorescence-activated cell sorter was used to evaluate the expression and functional activity of P-glycoprotein (P-gp) encoded by MDR1 gene. It was found that the purity of bone marrow CD34⁺ cells was approximately (91±4.56) % and recovery rate was (72.3±2.36) % by MACS. The expression of P-gp in the transfected CD34⁺ cells was obviously higher than that in non-transfected CD34⁺ cells. The amount of P-gp in non-transfected CD34⁺ cells was (11.2±2.2) %, but increased to (23.6±2.34) % 48 h after gene transfection (P<0.01). The amount of P-gp was gradually decreased to the basic level one week later. The accumulation and extrusion assays showed that the overexpression of P-gp could efflux Rh-123 out of cells and there was low fluorescence within the transfected cells. The functional activity of P-gp could be inhibited by 10 μg/ml verapamil. It was suggested that the transient and highly effective expression and functional activity of P-gp could be obtained by liposome-mediated MRD1 transferring into human normal bone marrow CD34⁺ cells. CAO Wenjing, female, born in 1968, Doctor in Charge     

Protective effects of trimetazidine on bone marrow mesenchymal stem cells viability in an ex vivo model of hypoxia and in vivo model of locally myocardial ischemia

Bone marrow mesenchymal stem cells (MSCs) have shown potential for cardiac repair following myocardial injury,but this approach is limited by their poor viability after transplantation.The present study was to investigate whether trimetazidine (TMZ) could improve survival of MSCs in an ex vitro model of hypoxia,as well as survival,differentiation,and subsequent activities of transplanted MSCs in rat hearts with acute myocardial infarction (AMI).MSCs at passage 3 were examined for their viability and apoptosis under a transmission electron microscope,and by using flow cytometry following culture in serumfree medium and exposure to hypoxia (5% CO2,95% N2) for 12 h with or without TMZ.Thirty Wistar rats were divided into 3 groups (n=10 each group),including groupⅠ(AMI control),groupⅡ (MSCs transplantation alone),and group Ⅲ (TMZ+MSCs).Rat MSCs (4×107) were injected into peri-infarct myocardium (MSCs group and TMZ+MSCs group) 30 min after coronary artery ligation.The rats in TMZ+MSCs group were additionally fed on TMZ (2.08 mg?kg-1?day-1) from day 3 before AMI to day 28 after AMI.Cardiac structure and function were assessed by echocardiography at 28th day after transplantation.Blood samples were collected before the start of TMZ therapy (baseline),and 24 and 48 h after AMI,and inflammatory cytokines (CRP,TNF-α) were measured.Then the sur-vival and differentiation of transplanted cells in vivo were detected by immunofluorescent staining.The cellular apoptosis in the peri-infarct region was detected by using TUNEL assay.Furthermore,apoptosis-related proteins (Bcl-2,Bax) within the post-infarcted myocardium were detected by using Western blotting.In hypoxic culture,the TMZ-treated MSCs displayed a two-fold decrease in apoptosis under serumfree medium and hypoxia environment.In vivo,cardiac infarct size was significantly reduced,and cardiac function significantly improved in MSCs and TMZ+MSCs groups as compared with those in the AMI control group.Combined treatment of TMZ with MSCs implantation demonstrated further decreased MSCs apoptosis,further increased MSCs viability,further decreased infarct size,and further improved cardiac function as compared with MSCs alone.The baseline levels of inflammatory cyto-kines (CRP,TNF-α) had no significant difference among the groups.In contrast,all parameters at 24 h were lower in TMZ+MSCs group than those in MSCs group.Furthermore,Western blotting indicated that the expression of antiapoptotic protein Bcl-2 was upregulated,while the proapoptotic protein Bax was down-regulated in the TMZ+MSCs group,compared with that in the MSCs group.It is suggested that implantation of MSCs combined with TMZ treatment is superior to MSCs monotherapy for MSCs viability and cardiac function recovery.     

Experimental study on the suppression of sodium nitroprussiate-induced chondrocyte apoptosis by Tougu Xiaotong Capsule (透骨消痛胶囊)-containing serum

Objective  

To study the mechanism of action of Tougu Xiaotong Capsule (透骨消痛胶囊, TGXTC) ex vivo in suppressing chondrocyte (CD) apoptosis induced by sodium nitroprussiate (SNP).     

Tumor Antigen-pulsed CD8α＋ Dendritic Cells Induce T Cell-mediated Graft-versus-tumor Effect In Vitro

The graft-versus-tumor (GVT) effect of T cells induced by tumor antigen-pulsed CD8α+dendritic cells (DCs) in vitro was investigated in this study.Immature CD8α+ DCs were prepared from C57BL/6 (H-2b) bo...     

甲状腺相关性眼病患者外周血CD4+、CD8+T细胞百分比及细胞表面程序性死亡蛋白1表达的改变及其意义

目的 观察甲状腺相关性眼病(TAO)患者外周血单个核细胞(PBMC)中CD4+、CD8+T细胞百分比及细胞表面程序性死亡蛋白1(PD-1)的表达水平,探索这两者变化在TAO发病机制及疾病诊断中的意义.方法 收集21例TAO患者、21例Graves病(GD)无眼病患者及20例健康对照者资料,采用流式细胞术检测所有受试者PBMC中CD4+、CD8+T细胞百分比及其表面PD-1的表达率,比较组间差异,分析其与患者病程、甲状腺功能、甲状腺相关抗体及TAO疾病活动性和严重程度之间的关系.结果 (1) TAO患者PBMC中CD4+、CD8+T细胞百分比分别为(35.9±14.5)％、(22.2±8.4)％,GD患者分别为(33.1±13.0)％、(18.6±9.2)％,均高于健康对照者[(17.4±7.4)％、(7.7±4.8)％,P＜0.01].(2) TAO及GD患者PBMC中CD4+T细胞表面PD-1表达率分别为(8.3±6.4)％、(28.0±26.6)％,均低于健康对照者[(52.2±28.6)％,P＜0.01.P＜0.05],且TAO患者低于GD患者(P＜0.05);TAO患者PBMC中CD8+T细胞表面PD-1表达率低于健康对照者r(12.7±13.4)％vs(38.2±24.2)％,P＜0.01].(3) TAO患者PBMC中CD8+T细胞百分比与病程呈正相关(r=0.478,P＜0.05).(4) TAO患者活动期组与非活动期组、轻度组与中重度组间CD4+、CD8+T细胞百分比及其表面PD-1表达率差异均无统计学意义(P＞0.05).结论 TAO患者PBMC中CD4+、CD8+T细胞百分比及细胞表面PD-1表达存在异常,可能参与TAO的自身免疫过程.     

大鼠骨髓内皮祖细胞的分离培养及其生物学特性

目的：探讨大鼠骨髓内皮祖细胞的分离培养方法及其生物学特性。方法：采用二次贴壁法体外扩增培养大鼠骨髓内皮祖细胞,观测细胞增殖能力;免疫化学检测细胞CD133、CD34、Flk1、CD31的表达;三维培养细胞并观察其体外成血管能力。结果：细胞培养第4天集落样生长,呈圆形或梭形;第10天细胞长满瓶底,呈鹅卵石样外观。原代血管内皮祖细胞CD133、CD34、Flk1均表达阳性;第2代细胞CD34、CD31、Flk1均表达阳性,CD133表达阴性。三维培养第2代血管内皮祖细胞在胶原内有＂出芽式＂生长现象及管腔样结构形成。结论：体外扩增法可以从骨髓中分离培养出内皮祖细胞,三维培养发现其具有体外成血管能力。     

小鼠骨髓内皮祖细胞的培养与鉴定

目的建立分离小鼠骨髓内皮祖细胞(endothelial progenitor cell,EPC)的方法。方法通过密度梯度分离法从小鼠骨髓中分离单个核细胞,行流式细胞检测,培养7d后,行DIL-acLDL和FITC-UEA-1双荧光染色、细胞移行实验和血管内皮网络掺入实验。结果流式细胞技术检测结果显示,骨髓来源的CD34/CD133/VEGFR2三阳细胞为0.029%±0.008%;细胞培养7d后,可见梭形细胞呈优势生长;DIL-acLDL与FITC-UEA-1荧光标记的双阳性细胞占细胞总数的比例为86.085%±5.622%;细胞移行实验结果可见平均每视野移行细胞数为(19.458±2.251)个;血管内皮网络掺入实验结果为,每视野掺入细胞数平均为(67.750±8.823)个。结论用流式细胞技术检测分离小鼠骨髓单个核细胞不同表面标志物的表达,用选择性内皮生长体系培养EPC,再通过细胞免疫及细胞功能检测所分离的细胞,是一种较为理想的分离与鉴定EPC的方法。     

A novel and feasible way to cultivate and purify endothelial progenitor cells from bone marrow of children with congenital heart diseases

Background  Endothelial progenitor cells (EPCs) are used in vascular tissue engineering and clinic therapy. Some investigators get EPCs from the peripheral blood for clinic treatment, but the number of EPCs is seldom enough. We have developed the cultivation and purification of EPCs from the bone marrow of children with congenital heart disease, to provide enough seed cells for a small calibre vascular tissue engineering study.

Methods  The 0.5-ml of bone marrow was separated from the sternum bone, and 5-ml of peripheral blood was collected from children with congenital heart diseases who had undergone open thoracic surgery. CD34+ and CD34+/VEGFR+ cells in the bone marrow and peripheral blood were quantified by flow cytometry. CD34+/VEGFR+ cells were defined as EPCs. Mononuclear cells in the bone marrow were isolated by Ficoll^® density gradient centrifugation and cultured by the EndoCult Liquid Medium Kit™. Colony forming endothelial cells was detected. Immunohistochemistry staining for Dil-ac-LDL and FITC-UEA-1 confirmed the endothelial lineage of these cells.

Results  CD34+ and CD34+/VEGFR+ cells in peripheral blood were (0.07±0.05)% and (0.05±0.02)%, respectively. The number of CD34+ and CD34+/VEGFR+ cells in bone marrow were significantly higher than in blood, (4.41±1.47)% and (0.98±0.65)%, respectively (P <0.0001). Many colony forming units formed in the culture. These cells also expressed high levels of Dil-ac-LDL and FITC-UEA-1.

Conclusion  This is a novel and feasible approach that can cultivate and purify EPCs from the bone marrow of children with congenital heart disease, and provide seed cells for small calibre vascular tissue engineering.

     

Effect of Cytokines Secreted by Human Adipose Stromal Cells on Endothelial Cells

Recently, several observations have pointed to the presence of a population named adipose-derived adult stem cells (ADAS) or adipose stromal cells (ASCs) in human adipose tissue. Subsequent studies revealed that ASCs were multipotent, differentiating along the cardiac myocyte, endothelial, neuronal, and other cells lineages[1, 2], and could secrete cytokines such as HGF and VEGF[3]. Since adipose tissue is an abundant, accessible, and re- plenishable source of adult stem cells that can b…     

Up-regulation of tim-3 expression contributes to development of burn-induced T cell immune suppression in mice

T cell immunoglobulin and mucin domain 3 (Tim-3) is well known to negatively regulate T cells responses,but its role in burn-induced T cells immune suppression remains unclear.In the present study,in o...     

丹酚酸B干预EPCs对BMSCs移植的AMI大鼠心肌VEGF、bFGF蛋白表达的影响

[目的]探讨中药丹酚酸B预处理的内皮祖细胞(EPCs)对骨髓间充质干细胞(BMSCs)移植后,急性心肌梗死(AMI)大鼠心肌血管新生的影响。[方法]密度梯度离心法和贴壁筛选法培养、纯化EPCs与BMSCs;免疫细胞化学法(CD34/CD133/CD44)分别鉴定两种细胞。结扎大鼠左冠状动脉,制作大鼠急性心肌梗死模型;丹酚酸B最佳药物浓度干预的EPCs,与BMSCs混合,在大鼠心肌梗死区周边分5点注射。免疫组织化学法检测心肌蛋白的表达。[结果]细胞移植4周后,EPCs与BMSCs共移植组血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)的积分光密度(IOD)分别13.179±3.053、23.634±4.705,与对照组差异具有统计学意义。[结论]丹酚酸干预EPCs提高BMSCs移植后大鼠心肌VEGF、bFGF蛋白表达,有效改善了BMSCs向心肌分化的血管微环境。     

Determination of CD30 expression on peripheral blood T lymphocyte subsets in patients with hemorrhagic fever with renal syndrome by FCM

Summary To determine the CD₃₀ expression on peripheral blood T lymphocyte subsets in patients with hemorrhagic fever with renal syndrome (HFRS) and its clinical implications, double immunofluorescence technique and flow cytometry were used. There was no significant difference among the severe group, mild-moderate group and normal control group in the CD+CD₃₀ T lymphocyte subset. While the CD₄ ⁺CD₃₀ ⁻ T cells of HFRS patients were increased and the difference between severe group and mild-moderate group or normal control group were very significant (P<0.01) and the difference between the mild-moderate group and normal control group was also significant (P<0.05). The CD₈ ⁺CD₃₀ ⁻ T cells were increased while the CD₈ ⁺CD₃₀ ^- T cells decreased obviously in HFRS patients, and the differences among three groups in both subsets were very significant (P<0.01). The results showed that the humoral immunity and cellular immunity are overactive in HFRS patients during acute phase. The loss of balance between T lymphocyte subsets may play an important role in the pathophysiology of HFRS and is closely correlated with the severity of the HFRS. This project is supported by the grant of the Ministry of Health (Serial No. 96-2-116).     

eNOS/NO途径在单侧输尿管梗阻小鼠肾间质微血管病变中的作用与机制

目的 探讨内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)和一氧化氮(nitric oxide, NO)在单侧输尿管梗阻(unilateral ureteral obstruction, UUO)肾间质纤维化小鼠微血管病变中的作用及机制。方法 64只KM小鼠随机分为两组:假手术组n=32只;单侧输尿管梗阻UUO组n=32只。观察4周,每周检测各组小鼠血BUN、Scr及一氧化氮,流式细胞计数外周血CD133⁺/VEGFR⁺内皮祖细胞(endothelial progenitor cells,EPCs)、Masson染色观察肾组织形态学变化,免疫组化法检测肾间质CD34⁺表达计数微血管密度,实时定量PCR检测肾皮质eNOS、VEGF mRNA表达。结果 UUO组血一氧化氮、内皮祖细胞计数、肾间质微血管密度、eNOS、VEGF mRNA表达水平持续下降,在第2、3、4周与对照组差异有统计学意义。一氧化氮水平与肾间质微血管密度呈正相关(r=0.715,P<0.05);eNOS mRNA表达水平与肾间质微血管密度(r=0.624,P<0.05)、内皮祖细胞计数(r=0.375,P<0.05)、VEGF mRNA(r=0.351,P<0.05)呈正相关。结论 eNOS/NO途径参与了UUO小鼠肾间质微血管的调节,其调节涉及对血管舒张功能影响、介导促血管肾脏因子VEGF mRNA表达及动员内皮祖细胞等机制。     

Establishment of nasal tolerance to heat shock protein-60 alleviates atherosclerosis by inducing TGF-��-dependent regulatory T cells

Mounting evidence supports that a newly identified regulatory T cell (Treg),CD4+LAP+ Treg,is associated with oral tolerance induction and following inhibition of atherosclerosis,but little is described about whether nasal tolerance to antigen likewise induces the novel Tregs production and the relevant antiatherosclerotic benefit.We investigated the effect of nasal administration of heat shock protein-60 (HSP60) on atherogenesis.HSP60 or phosphate buffer solution (PBS) was nasally adminis-tered to six-week-old male ApoE-/-mice.At the 10th week after the nasal administration,there was a significant decrease in atherosclerotic plaque areas of aortic roots in the HSP60-treated mice as com-pared with those in the PBS-treated mice.Atherosclerosis suppression was accompanied with a signifi-cant increase in CD4+LAP+ and CD4+CD25+Foxp3+ Tregs and a concurrently increased production of TGF-β in the HSP60-treated mice.The protective effect of HSP60 was offset by injection of anti-TGF-βantibody.It is concluded that nasal administration of HSP60 can inhibit atherosclerotic formation through immune tolerance which is established by Tregs depending on the induction of anti-inflammatory cytokine TGF-β.Immune tolerance induced by nasal administration of HSP60 may provide an alternative therapeutic method for atherosclerosis.     

Inhibitory Effect of Dexamethasone on TGF-β1 Expression of Rabbit Ciliary Pigment Epithelia Cultured in Vitro

As is well-known,transforming growth fac-tor-beta1(TGF-β1)is a multi-functioning factorwith sti mulating or inhibitory effects to majority ofcells in the body.In ocular,many cell types ex-pressed TGF-β1andits receptors,such as cornea,lens,trabecular meshwork and retinal cells.Par-ticularly,TGF-β1has been believed to play a cru-cial roleinthe developing of eye embryo[1].Due tothe double-direction regulating effect to differentcell types,the exact mechanism and regulatorypattern are uncle…     

Effect of Compound Zhebei Granule(复方浙贝颗粒)Combined with Chemotherapy on Surface Markers of Leukemia Stem Cell in Patients with Acute Myeloid Leukemia

Objective: To observe the effects of Compound Zhebei Granule(复方浙贝颗粒, CZBG) combined with chemotherapy on surface markers of leukemia stem cell(LSC) in the bone marrow of patients with acute myeloid leukemia(AML). Methods: Seventy-eight patients with AML received bone marrow aspiration and the percentages of CD34~+CD123~+ and CD33~+CD123~+ cells were tested using flow cytometry method. A total of 24 refractory or relapsed AML patients were enrolled and treated with one cycle of standard chemotherapy combined with CZBG. Bone marrow samples were obtained before and after treatment, and the percentages of CD34~+CD123~+ and CD33~+CD123~+ cells were examined by flow cytometry. Results: Compared with refractory or relapsed AML patients, patients achieved remission had a significant lower percentage of CD34~+CD123~+ cells(P0.01) and CD33~+CD123~+ cells(P0.01), indicating that controlling the LSC percentage may be important for patients with AML to achieve sustainable remission. Compared with those before treatment, the expression levels of CD34~+CD123~+ were significantly decreased after CZBG combined with chemotherapy treatment(P0.01). The percentages of CD34~+CD123~+ cells and CD33~+CD123~+ in patients achieving complete remission after CZBG combined with chemotherapy treatment were both significantly lower than those in patients with nonremission(P0.01). Conclusion: CZBG combining chemotherapy could reduce the percentages of CD34~+CD123~+ and CD33~+CD123~+ LSC, which might improve the clinical efficacy of refractory or relapsed AML.