羊水来源胎儿间充质干细胞定向诱导为脂肪细胞的研究

目的:研究体外诱导孕中期羊水来源胎儿间质干细胞(M SCs)分化为脂肪细胞。方法:机械手段挑取孕中期羊水细胞培养体系中的胎儿M SCs集落,培养扩增后,通过流式细胞仪检测表面抗原表达进行鉴定。取第3代胎儿M SCs,IBM X、胰岛素、氢化考的松等诱导其向脂肪细胞分化。倒置相差显微镜观察诱导后细胞形态的变化。诱导20 d后,油红O染色检测胞内中性脂肪并计算阳性细胞率,W estern Blot蛋白印记法检测PPA Rγ的表达。结果:机械手段分离出的胎儿M SCs为CD 44阳性,CD 34、CD 45、H LA-D R阴性,符合M SCs特点。诱导72 h后,细胞内有脂滴出现,随着时间延长,脂滴逐渐增加并融合为脂泡,细胞由梭形转变为圆形或多角性。20 d后85%以上胎儿M SCs变为油红O染色阳性,W estern Blot显示诱导20 d后,诱导组细胞PPA Rγ的表达量明显高于未诱导组。结论:经上述方法诱导20 d后,羊水来源胎儿M SCs可以分化为脂肪细胞。     

脐血间充质干细胞抑制外周血淋巴细胞增殖的作用

目的研究脐血间充质干细胞(UCBMSC)对成人外周血淋巴细胞增殖的影响。方法分离、扩增UCBMSC并鉴定其表面标志及向成骨细胞及脂肪细胞的分化能力。UCBMSC与淋巴细胞直接共培养并用植物血凝素(PHA)刺激,或在TranswellTM非接触体系中,用PHA刺激共培养。两个培养体系均分为阴性对照组(淋巴细胞单独培养组)、阳性对照组(淋巴细胞加PHA刺激组)、实验组(淋巴细胞加PHA和UCBMSC组)。四氮唑衍生物MTS还原法测定细胞增殖并计算抑制率。结果在共培养与TranswellTM非接触体系中不同数量UCBMSC对淋巴细胞增殖均具有抑制作用,且UCBMSC浓度越高,抑制作用越强(P0.05),具有剂量依赖性。但两体系中UCBMSC与淋巴细胞比值相同时,共培养体系抑制作用较非接触体系强,且差异具有统计学意义(P0.05)。结论 UCBMSC能抑制淋巴细胞增殖,且共培养体系抑制作用较非接触体系强。     

人羊膜间充质干细胞抑制异体淋巴细胞增殖并减少γ干扰素的分泌

目的观察人羊膜间充质干细胞(hAMSC)对体外培养的淋巴细胞功能的影响。方法通过酶消化法分离培养hAMSC,采用荧光团标记的小鼠抗人单克隆抗体结合流式细胞术鉴定细胞表面抗原;免疫荧光染色检测培养细胞波形蛋白(vimentin)和阶段特异表达抗原4(SSEA-4)的表达;分离培养hAMSC和外周血单个核细胞(PBMC),将刀豆蛋白(ConA)刺激的淋巴细胞与1×104、5×104、1×105个hAMSC进行共培养。CCK-8法测定淋巴细胞增殖,ELISA测定细胞上清液中IFN-γ的水平。结果5μg/mLConA能够引起淋巴细胞增殖;共培养条件下,hAMSC能够抑制ConA引起的淋巴细胞增殖,且随着hAMSC的数量增加,抑制效果更明显。培养72h后,CCK-8法结果表明,单纯ConA刺激后淋巴细胞数显著高于共培养细胞。选择抑制效果最佳的组别1×106个淋巴细胞与1×105个hAMSC共培养,ELISA测定1×105个hAMSC对淋巴细胞抑制72h后,上清液中IFN-γ分泌,共培养细胞上清液中IFN-γ水平显著低于单纯ConA刺激细胞。结论hAMSC能在体外抑制ConA引起的淋巴细胞增殖并减少IFN-γ分泌。     

大鼠骨髓间充质干细胞对淋巴细胞增殖影响的体外实验

目的：探讨大鼠骨髓间充质干细胞对淋巴细胞增殖的影响。方法：常规分离培养大鼠的骨髓间充质干细胞（MSCs）及淋巴细胞,灭活后的骨髓间充质干细胞和淋巴细胞混合培养48h后,MTT法检测淋巴细胞的相对细胞数,Hoechst 33258染色观察细胞核变化。结果：MTT结果显示,加入MSCs组OD值明显低于未加MSCs的对照组（p〈0．05）。Hoechst 33258染色显示MSCs组淋巴细胞有核回缩及凋亡小体出现。结论：MSCs对淋巴细胞的增殖有抑制作用,其作用机制可能与细胞凋亡有关。     

慢性再生障碍性贫血患者间充质干细胞对T细胞的抑制作用

目的比较来源于正常志愿者和慢性再生障碍性贫血(CAA)患者骨髓间充质干细胞(MSCs)免疫抑制作用的区别。方法培养5例正常志愿者和10例CAA患者的骨髓MSCs,比较两组MSCs的形态、细胞表型、细胞因子的表达,通过PHA刺激的T细胞增殖试验、混合淋巴细胞培养和T细胞周期检测比较两组MSCs对T细胞的抑制作用。结果两组MSCs形态、表型基本相同。CAA来源的MSCs对PHA和同种异体抗原诱导的T细胞的抑制作用均低于正常志愿者来源的MSCs,加入正常志愿者的MSCs后有更多的T细胞阻滞在G0/G1期,但CAA来源的MSCs作用较弱。两组MSCs表达的肝细胞生长因子(HGF)和转化生长因子(TGF-β2)无明显区别,但CAA患者的MSCs表达的TGF-β1、3较正常志愿者MSCs表达的明显减少。结论虽然CAA的MSCs在形态、增殖和细胞表型上基本正常,但其对T细胞的抑制作用减弱,在经过免疫抑制剂治疗后仍然存在,其异常是否与CAA的发病机制有关需要进一步研究。     

骨髓间充质干细胞培养上清液抑制异体淋巴细胞增殖

骨髓间充质干细胞（mesenchymal stem cells,MSCs）作为具有多向分化潜能的干细胞,具有广泛的临床应用前景。近年来的研究已经证实,MSCs具有免疫负调节作用,但其具体机制尚不清楚。我们试图通过研究MSCs培养上清液对异体淋巴细胞增殖的影响,观察MSCs的免疫调节作用,进一步探讨MSCs的免疫调控机制。     

人羊水来源间充质干细胞向成肌细胞诱导的体外实验研究

目的 探讨成肌细胞条件培养液体外诱导人羊水来源间充质干细胞向成肌细胞分化的可行性.方法 B超引导下穿刺抽得孕中期羊水,体外培养、分离得到羊水来源间充质干细胞.鼠成肌细胞体外培养后收集上清液,检测上清液中半乳糖凝集素-1（Galectin-1）含量,并制备成肌细胞条件培养液.实验组于成肌细胞条件培养液中培养,对照组于成肌细胞诱导培养液中培养.观察2组细胞形态学变化,免疫荧光染色、RT-PCR检测成肌细胞特异性标志物Pax7、MyoD、肌结蛋白（Desmin）、肌钙蛋白Ⅰ（Tn Ⅰ）及mRNA表达情况.结果 倒置相差显微镜下可见实验组细胞诱导第18天出现折光性强、体积较小的细胞,呈多角形,并带有突起,且逐渐成长条形;可见少量多核细胞.对照组细胞呈扁平多角形,胞体较大.诱导24 d免疫荧光染色及RT-PCR提示实验组细胞不同程度表达Pax7、MyoD、Desmin、TnⅠ及mRNA;对照组呈阴性.成肌细胞培养上清液中半乳糖凝集素-1含量较低.结论 成肌细胞条件培养液能诱导人羊水来源间充质干细胞向成肌细胞样细胞分化.     

脐血来源间充质干细胞可抑制异体T细胞的反应

目的 研究脐血间充质干细胞(HUCB-MSCs)对异体T细胞的抑制作用.方法 体外培养HUCB-MSCs,流式细胞术测表面标记;取正常人外周血,免疫磁珠分离CＤ３+T细胞,将分离的CD3+T与HUCB-MSCs 1:1混合培养5d,PHA刺激或不刺激,采用3H-TdR掺入法观察T细胞增殖,ELISA方法检测细胞因子,流...     

脐血来源间充质干细胞可抑制异体T细胞的反应

目的研究脐血间充质干细胞(HUCB-MSCs)对异体T细胞的抑制作用。方法体外培养HUCB-MSCs,流式细胞术测表面标记;取正常人外周血,免疫磁珠分离CD3+T细胞,将分离的CD3+T与HUCB-MSCs 1∶1混合培养5 d,PHA刺激或不刺激,采用3H-TdR掺入法观察T细胞增殖,ELISA方法检测细胞因子,流式细胞术观察细胞凋亡。结果 HUCB-MSCs呈纺锤样的细胞形态,不表达CD14、CD34、CD45、HLA-DR,而表达CD29、CD44、HLA-ABC。HUCB-MSCs抑制PHA引起的T细胞增殖(5 230±550 vs 10 500±800 counts/min,P<0.001);HUCB-MSCs还能抑制异体T细胞分泌IFN-γ(510±60 vs 1 580±100 pg/mL,P<0.001)和TNF-α(590±20 vs 1 180±30 pg/mL,P<0.001),上调IL-4(16.3±8.2 vs 4.1±1.8 pg/mL,P<0.001)和IL-10(105±5 vs 17±2 pg/mL,P<0.001)分泌;HUCB-MSCs不诱导T细胞的凋亡。结论 HUCB-...     

间充质干细胞的细胞表型及免疫学研究进展

间充质干细胞具有多分化潜能,参与构成骨髓的微环境,分泌大量细胞因子支持造血。最近的研究显示间充质干细胞免疫表型呈现动态的表达;同种异体细胞移植免疫排斥由于移植抗原差异而引起,但同种异体间充质干细胞却具有免疫抑制作用。综述了其可能的机制及作为组织工程种子细胞的间充质干细胞具有独特的生物学特性。     

人羊水来源干细胞生物学性状的研究

目的对人羊水标本进行体外分离培养,建立人羊水来源干细胞的体外培养体系,对其生物学形状进行研究。方法贴壁法体外分离获得人羊水来源干细胞,多次传代扩增后,采用流式细胞仪和RT-PCR技术检测细胞表面抗原的表达。结果羊水干细胞原代生长较慢,传代后生长迅速,体外倍增时间约36h,流式细胞仪检测证实细胞表达CD29、CD44、CD105等间充质干细胞标志,不表达造血干细胞标志CD45和CD133。RT-PCR检测显示羊水干细胞表达Oct-4、Nanog基因。结论实验成功分离获得羊水中具有干细胞性质的细胞群,采用贴壁法分离获得的干细胞体外增殖能力强,表达间充质干细胞表面标志,符合间充质干细胞的特点。     

外周血基质干细胞培养鉴定及其向施万细胞的诱导分化

目的研究体外分离培养大鼠外周血基质干细胞(PBMSCs)并诱导分化为施万细胞的潜能。方法从SD大鼠取血分离培养PBMSCs,采用流式细胞术和免疫细胞化学法对其细胞表面抗原进行检测与鉴定,并用免疫细胞化学法检测BrdU作用4h后BrdU的阳性率。PBMSCs经β-巯基乙醇、全反式维甲酸及复合条件培养基等三个步骤向施万细胞定向诱导后,用免疫细胞化学法检测S-100和P75的表达。结果流式细胞术显示培养获得的PBMSCs中CD11b、CD29、CD45、CD49d、CD90及CD106的阳性率分别为19.97%、99.96%、46.62%、5.46%、71.22%和10.76%。免疫细胞化学法显示PBMSCs呈CD34阴性,而BrdU阳性率为(34.1±4.3)%。PBMSCs经定向诱导后S-100和P75的阳性率分别是(75.2±4.1)%和(78.9±4.6)%。结论从外周血分离获得的干细胞符合基质干细胞的特性,这些细胞在特定的条件下可诱导分化成为施万细胞。     

HCV感染外周血单个核细胞及其对T淋巴细胞亚群的影响

目的：研究丙型肝炎病毒（ＨＣＶ）感染外周血单个核细胞（ＰＢＭＣ）的情况及其对Ｔ淋巴细胞亚群的影响。方法：运用非同位素原位杂交（ＮＩＳＨ）法和链酶亲和素－生物素（ＳＡＢＣ）法分别检测２０例慢性丙型肝炎患者ＰＢＭＣ中的ＨＣＶ－ＲＮＡ和非结构（Ｎｏｎｓｔｒｕｃｔｕｒａｌ,ＮＳ）蛋白ＮＳ５抗原,同时用ＳＡＢＣ法检测其Ｔ淋巴细胞亚群。结果：８例（４０．０％）患者的ＰＢＭＣ中ＨＣＶ－ＲＮＡ呈构（Ｎｏｎｓｔｒｕ     

Osteoblastic differentiation potential of human amniotic fluid-derived mesenchymal stem cells in different culture conditions

Osteoporosis is a bone degenerative disease characterized by a decrease in bone strength and an alteration in the osseous micro-architecture causing an increase in the risk of fractures. These diseases usually happen in post-menopausal women and elderly men. The most common treatment involves anti-resorptive agent drugs. However, the inhibition of bone resorption alone is not adequate for recovery in patients at the severe stage of osteoporosis who already have a fracture. Therefore, the combination of utilizing osteoblast micro mimetic scaffold in cultivation with the stimulation of osteoblastic differentiations to regain bone formation is a treatment strategy of considerable interest. The aims of this current study are to investigate the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid and to compare the monolayer culture and scaffold culture conditions. The results showed the morphology of cells in human amniotic fluid as f-type, which is a typical cell shape of mesenchymal stem cells. In addition, the proliferation rate of cells in human amniotic fluid reached the highest peak after 14 days of culturing. After which time, the growth rate slowly decreased. Moreover, the positive expression of specific mesenchymal cell surface markers including CD44, CD73, CD90, and also HLA-ABC (MHC class I) were recorded. On the other hand, the negative expressions of the endothelial stem cells markers (CD31), the hematopoietic stem cells markers (CD34, 45), the amniotic stem cells markers (CD117), and also the HLA-DR (MHC class II) were also recorded. The expressions of osteoblastogenic related genes including OCN, COL1A1, and ALP were higher in the osteogenic-induced group when compared to the control group. Interestingly, the osteoblastogenic related gene expressions that occurred under scaffold culture conditions were superior to the monolayer culture conditions. Additionally, higher ALP activity and greater calcium deposition were recorded in the extracellular matrix in the osteogenic-induced group than in the culture in the scaffold group. In summary, the mesenchymal stem cells derived from human amniotic fluid can be induced to be differentiated into osteoblastic-like cells and can promote osteoblastic differentiation using the applied scaffold.     

卡介苗多糖核酸对过敏性支气管哮喘外周血单个核细胞TH1/TH2反应的作用

目的观察卡介苗多糖核酸(BCG-PSN)对支气管哮喘患者外周血淋巴细胞TH1/TH2反应的作用,并与结核菌素纯蛋白衍生物(TB-PPD)进行比较. 方法缓解期过敏性支气管哮喘患者16例,对照组健康成人13例,分离外周血单个核细胞(PBMC),分别加入不同浓度的BCG-PSN(1、10、100、1000 μg/ml)、TB-PPD(10 μg/ml)、尘螨抗原(DerP, 10 μg/ml)体外培养4 d,不加刺激剂者为阴性对照.收集培养上清,ELISA检测IFN-γ、IL-5浓度的变化. 结果 PSN(1～100 μg/ml)刺激正常人PBMC分泌IFN-γ水平均高于哮喘患者(P＜0.05).BCG-PSN(10 μg/ml)可以刺激哮喘患者PBMC分泌IFN-γ(358.7 pg/ml,范围0～2433.0 pg/ml),但显著低于同等浓度的TB-PPD刺激作用(13 036 pg/ml,范围600.5～35 100.0 pg/ml,P＜0.01).PSN刺激PBMC分泌IFN-γ呈浓度依赖性,当浓度达到100 μg/ml时,与低浓度相比刺激作用显著增强(P＜0.01),与TB-PPD的刺激作用类似.DerP刺激哮喘患者PBMC分泌IL-5水平显著高于正常人(P＜0.05).BCG-PSN刺激PBMC分泌IL-5的作用较弱,显著低于TB-PPD和DerP的刺激作用. 结论 BCG-PSN具有一定的TH1刺激作用,但低于TB-PPD的刺激作用,有待对BCG-PSN组分进一步优化以增强疗效.     

系统性红斑狼疮患者外周血单个核细胞中白细胞介素-10mRNA表达的研究

目的 探讨白细胞介素(IL-10)在系统性红斑狼疮(SLE)中的作用。方法 采用逆转录多聚酶链反应(RT-PCR)及酶联免疫吸附法(ELISA)测定40例SLE患者和20例正常对照组外周血单核细胞(PBMC)IL-10mRNA表达及IL-10自发分泌水平。结果 SLE患者PBMC自发分泌IL-10水平及其IL-10mRNA表达水平均显著高于正常对照组(P<0.01),其中SLE活动期明显高于非活动期(P<0.01),而非活动期又明显高于正常对照组(P<0.01)。结论 IL-10在SLE发病中起重要作用,PBMC分泌IL-10水平对SLE诊断和病情活动性监测有重要临床意义,拮抗SLE患者体内IL-10水平,将为SLE治疗开辟一条新途径。     

乳腺癌外周血单个核细胞与间质免疫微环境的关系研究

目的探讨乳腺癌免疫微环境与外周血的关系。方法应用BRB-Array Tools软件对公共基因芯片数据库GEO中的乳腺癌间质及乳腺癌患者外周血单个核细胞基因芯片表达数据进行统计学分析,找出在乳腺癌间质及外周血单个核细胞均发生变化的基因,DAVID工具进一步分析其功能及参与的生物学通路。PINA蛋白质互作平台分析这些基因的蛋白质相互作用情况。结果比较后得到共同差异表达的103条基因,失调方向一致的基因70条,功能涉及炎症反应、髓系细胞分化、白细胞激活、抗原加工提呈等多种免疫相关的生物学过程。结论乳腺癌患者外周血单个核细胞基因表达改变,与肿瘤间质微环境具有一定相似度,有望建立基于外周血的免疫微环境分子预测,为乳腺癌的治疗靶点及预后判断的研究开辟新思路。     

Simvastatin induces interleukin-18 production in human peripheral blood mononuclear cells

The effects of statins on immune response depend on the inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase and leukocyte function-associated antigen (LFA)-1, which is a ligand of intercellular adhesion molecule (ICAM)-1. Simvastatin, an HMG-CoA reductase inhibitor with mild inhibition of LFA-1, induced the production of interleukin (IL)-18, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in human peripheral blood mononuclear cells (PBMC). The IL-18 production is located upstream of the cytokine cascade activated by simvastatin. Moreover, simvastatin concentration-dependently inhibited the expression of ICAM-1 and induced the expression of CD40 on monocytes. In the presence of IL-18, simvastatin suppressed the expression of ICAM-1 and CD40 as well as the production of IL-12, TNF-alpha and IFN-gamma in PBMC, contributing to the anti-inflammatory effect of simvastatin. The effects of simvastatin were abolished by the addition of the product of the HMG-CoA reductase, mevalonate, indicating the involvement of HMG-CoA reductase in the action of simvastatin.     

Optimisation of herpes simplex virus-based vectors for delivery to human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) represent a significant target for gene delivery both for therapeutic and experimental purposes. Thus far however, it has proved difficult to develop vectors capable of high efficient gene delivery to unstimulated PBMCs. We have tested a range of different vectors derived from herpes simplex virus (HSV) which differ in their degree of disablement in terms of their gene delivery efficiency to unstimulated human PBMCs and ability to deliver a reporter gene. None of the viruses had any significant toxic effect in PBMCs. However, optimal gene delivery to unstimulated PBMCs was obtained with a semidisabled virus lacking functional genes encoding ICP34.5 and Vmw65 which was more efficient than either nondisabled or more extremely disabled viruses. Expression of green fluorescent protein (GFP) with this virus was observed in up to 50% of PBMCs 1 day after infection, and reporter gene expression was detectable by Western blotting and immunofluorescence at undiminished levels at the longest time points tested, up to 5 days after infection. This optimised HSV vector may thus represent an effective tool for gene delivery to unstimulated PBMCs in culture.     

Increased gene expression of scavenger receptors and proinflammatory markers in peripheral blood mononuclear cells of hyperlipidemic males

Interactions between peripheral blood mononuclear cells (PBMCs) and those within plaques are suggested to be pathophysiologically relevant to lipid-induced arteriosclerosis. In this study, gene expressions of scavenger receptors (CD36, CD68), LPS receptor (CD14), proinflammatory (tumor necrosis factor alpha [TNFalpha], CD40, interleukin-1 beta [IL-1beta]) and oxidative stress-related (manganese superoxide dismutase [MnSOD]) markers were analyzed in PBMCs of clinically asymptomatic males with classical proatherogenic risk factors such as smoking and/or hyperlipidemia. PBMCs were isolated from venous blood of normolipidemic non-smokers (n = 10) and smokers (n = 8), and hyperlipidemic non-smokers (n = 9) and smokers (n = 8). RNA from PBMCs was used for PCR analyses. Plasma concentrations of oxidized low-density lipoproteins (oxLDL) were measured by ELISA. The gene expressions of CD36, CD68, CD40, TNFalpha, and MnSOD were significantly higher in PBMCs of hyperlipidemics than in normolipidemics, irrespective of whether they were smoking or not. The individual expression of these genes showed significant positive correlations with each other but also with serum cholesterol or plasma oxLDL concentrations. The higher expressions of scavenger receptors, proinflammatory and oxidative stress-related genes of PBMCs are suggested to result mainly from hyperlipidemia and the accompanied increase of oxLDL concentrations.