胞质转导肽-HBcAg18-27-Tapasin体外诱导HBV转基因小鼠髓源性树突状细胞成熟及在T淋巴细胞增殖中的作用

目的:观察融合蛋白胞质转导肽(cytoplasmic transduction peptide,CTP)-HBcAg18-27-Tapasin体外诱导HBV转基因小鼠髓源性树突状细胞(dendritic cell,DC)成熟和对T淋巴细胞增殖的作用.方法:体外分离、培养HBV转基因小鼠及近交系C57BL/6小鼠髓源性DC,加入重组粒细胞-巨噬细胞集落刺激因子和白介素(interleukin,IL)-4培养5d,再加入实验组10μg/mL CTP-HBcAg18-27-Tapasin、对照组10μg/mL CTP-HBcAg18-27、10μg/mL HBcAg18-27-Tapasin及空白组RPMI1640完全培养液.流式细胞术测定DC表面分子CD80、CD83、MHC-1的表达,ELISA法测定DC培养上清液中的IL-12p70的水平,细胞计数试剂盒(CCK-8)检测T淋巴细胞增殖反应,流式细胞仪检测增殖的T淋巴细胞内的细胞因子.结果:体外成功诱导小鼠髓源性DC;CTP-HBcAg18-27-Tapasin能明显上调DC表面分子CD80、CD83、MHC-1的表达;并且CTP-HBcAg18-27-Tapasin组诱导DC分泌的IL-12p70水平及诱导DC增殖T淋巴细胞增殖能力明显高于对照组及空白组[IL-12p70转基因小鼠(F=205.85,P=0.000);C57BL/6小鼠(F=406.20,P=0.000)];流式细胞仪检测实验组融合蛋白诱导的CTL水平也高于对照组[转基因小鼠(F=155.45,P=0.000);C57BL/6小鼠(F=392.90,P=0.000)],同时HBV转基因小鼠DC表面分子及在T淋巴细胞增殖中的作用要比C57BL/6小鼠低.结论:分子伴侣Tapasin修饰胞内化抗原肽能促进HBV转基因小鼠髓源性DC的分化、成熟,并能增强DC刺激T淋巴细胞增殖能力及诱导CTL的产生.     

人骨髓间充质干细胞治疗再生障碍性贫血的机制研究

目的探讨在体外骨髓间充质干细胞(MSC)治疗再生障碍性贫血(AA)的机制。方法2003-02~2005-04安徽医科大学第一附属医院血液内科从人骨髓分离培养MSC,用尼龙棉柱分离T淋巴细胞。分别以不同数量MSC加入到植物血凝素(PHA)刺激的AA患者T淋巴细胞增殖体系中,用MTT还原法测定MSC对AA患者T细胞增殖的影响,通过流式细胞术计算CD3+T细胞CD25(IL-2R)的表达率。结果当MSC为2×104/孔(A组)和1×104/孔(B组)时,与单独培养的AA患者T淋巴细胞(对照组)相比,MSC对AA患者T淋巴细胞增殖及CD25的表达呈明显抑制作用,差异有统计学意义(P<0·01)。当MSC为1×103/孔(C组)时,无统计学意义。结论MSC抑制AA患者T细胞的活化与增殖,且这种抑制作用具有数量依赖性。     

小鼠髓源性半成熟树突状细胞致免疫耐受的实验研究

三组体外培养的小鼠骨髓细胞,分别采用低剂量粒细胞巨细胞集落刺激因子（GM-CSF）诱导、高剂量GM—CSF诱导及脂多糖（LPS）刺激、低剂量GM—CSF诱导及LPS刺激,使其分化为树突状细胞（DC）。流式细胞术检测DC表面CD11c、CD25、CD40、CD80和CD86、MHC-Ⅱ,ELISA法检测细胞培养上清液中IL-10、IL-12,初次和再次混合淋巴细胞实验检测DC刺激T细胞增殖能力,术前回输DC行同种异位心脏移植并观察术后移植心存活时间。结果显示,低剂量GM—CSF能诱导骨髓细胞分化为CD11c^＋、CD25^-、CD40^low、CD80^low和CD36^low、MHC-Ⅱ^low未成熟表型DC,经LPS刺激后分化为CD11c^＋、CD25^-、CD40^mid、CD80^low和CD86^low、MHC-Ⅱ^mid。半成熟表型DC;半成熟表型DC培养上清液中IL-10／IL-12明显升高,该DC可明显抑制同种异型T细胞增殖反应,较未成熟DC更能延长移植心存活时间。认为小鼠骨髓细胞经低剂量GM—CSF体外诱导可分化为未成熟DC,经LPS刺激可转化为半成熟DC,拥有更强的致耐受性。     

IL-2联合热疗对SSMC7721细胞周期进程及凋亡的影响

目的 观察白细胞介素-2(IL-2)联合热疗诱导人肝癌细胞株(SSMC7721细胞)的增殖抑制作用.方法 应用MTT比色法检测不同条件下对SSMC7721细胞的增殖抑制率,流式细胞仪检测细胞周期进程的变化,荧光显微镜检测细胞凋亡率.结果 单纯热疗组,单纯IL-2组及IL-2联合热疗组对SSMC7721细胞的抑制率分别为18.4%、23.7%和46.7%,与对照组相比差别具有显著性意义.流式细胞仪结果显示G0/G1期细胞增多,S期细胞减少,G2/M期细胞相对增多,细胞凋亡率升高,组间比较差异显著.结论 IL-2联合热疗能显著提高SSMC7721细胞增殖抑制率,诱导SSMC7721细胞G1期向S期细胞转化进程,诱导SSMC7721细胞凋亡.     

人骨髓间充质干细胞体外培养及其生物学特性分析

目的探讨人骨髓间充质干细胞（MSC）的体外分离培养方法,并分析其生物学特性。方法无菌条件下抽取健康志愿者骨髓,采用密度梯度离心法分离骨髓单个核细胞,培养在含10%胎牛血清的DMEM培养基中,用贴壁筛选法纯化获得MSC。相差显微镜下观察细胞形态学变化,细胞计数法绘制生长曲线,流式细胞仪检测细胞表面抗原及细胞周期。结果原代和传代培养的细胞呈梭形,具有较强的生长增殖能力。细胞表面CD90、CD105表达阳性,CD34、CD11b阴性。第1、3、5代MSC生长曲线呈S形,均经历潜伏期、对数生长期和平台期。第3代MSC中,G0/G1期细胞约占77.42%,S＋G2/M期细胞约占22.58%。结论采用密度梯度离心结合贴壁筛选培养法可获得纯度较高的MSC,且该细胞增殖活性较强。密度梯度离心结合贴壁筛选培养法是一种简单、有效的分离纯化MSC的方法。     

细胞因子等对人原代培养肝细胞细胞间黏附分子-1表达的调控

目的　研究IFN γ、TNF α、IL 1和地塞米松对人原代培养肝细胞细胞间黏附分子 1(ICAM 1)表达的影响。方法　用IFN γ、TNF α和IL 1在体外诱导人原代培养肝细胞表达ICAM 1,并用细胞ELISA检测其ICAM 1表达水平 ;在用细胞因子刺激前先加入地塞米松 ,观察地塞米松对人肝细胞ICAM 1表达的影响。结果　未经刺激的人原代培养肝细胞ICAM 1表达水平很低 ;TNF α、IL 1、IFN γ刺激后 ,人原代培养肝细胞ICAM 1表达均明显增强 ,其表达水平与细胞因子刺激之间存在一定的剂量和时间依赖关系 ;地塞米松能部分抑制TNF α、IL 1、IFN γ所诱导的人原代培养肝细胞ICAM 1增强表达 ,其抑制率分别为 (4 0 .3± 5 .9) %、(3 8.1± 4.8) %和 (3 7.6± 6.7) %。结论　IFN γ、TNF α和IL 1能诱导人原代培养肝细胞ICAM 1增强表达 ,而地塞米松能抑制其表达。     

PTD-HBcAg体外诱导小鼠髓源性树突状细胞成熟及在T淋巴细胞增殖中的作用

目的 观察融合蛋白PTD-HBcAg诱导体外培养的小鼠髓源性树突状细胞(DC)成熟及对T淋巴细胞增殖的作用.方法 体外分离培养近交系BALB/C小鼠髓源性DC加入重组粒细胞-巨噬细胞集落刺激因子(rgM-CSF)、重组IL-4培养5 d,再加人TNF-a、HBcAg和PTD-HBcAg诱导DC成熟.激光共聚焦显微镜观察免疫荧光在细胞中的分布及定位,流式细胞计数仪测定DC表面分子表达,ELISA法测定DC培养上清液中IL-12 p70的水平,CCK-8试剂盒检测T淋巴细胞增殖反应.组间数据比较采用t检验.结果 成功体外诱导培养小鼠髓源性DC,HBcAg主要定位于DC膜表面,而PTD-HBcAg能够穿透DC膜进入细胞质.PTD-HBcAg能明显上调DC表面分子CD80、CD86和主要组织相容性复合体(MHC)II类分子表达;50 mg/L和100 mg/L PTD-HBcAg诱导DC分泌IL-12 p70水半分别为(142.50±18.31)ng/L和(124.30±15.12)ng/L,明显高于HBcAg诱导组的(42.31±4.21)ng/L(t=9.234和9.045,均P＜0.05);PTD-HBcAg诱导DC刺激T淋巴细胞增殖能力明显高于HBcAg组及阳性对照TNF-a组.结论 PTD-HBcAg具有穿透DC膜能力,并能促进DC分化、成熟,明显上调表面共刺激分子表达,增强DC刺激T淋巴细胞增殖能力及分泌IL-12 p70的水平.     

乙型肝炎病毒对干扰素-γ受体表达及其信号通路的影响

目的　探索乙型肝炎病毒 (HBV)持续存在和复制对干扰素 γ受体 (IFN γR)、IFN γ/STAT 1信号及MHC Ⅰ诱导表达的影响。方法　采用流式细胞术、Western blot及半定量逆转录 聚合酶链反应 (RT PCR)等方法 ,检测HepG2 .2 .15与HepG2肝母细胞瘤细胞株及人正常细胞株LO2的IFN γR表达 ;同时检测细胞IFN γR1对IFN γ的结合能力。观测不同时间段的IFN γ/p STAT1信号活化及IFN γ/MHC Ⅰ诱导效应。结果　HepG2 .2 .15细胞膜结合型及全细胞内IFN γR1表达高于其母源性细胞HepG2及正常肝细胞株LO2细胞 ;HepG2 .2 .15 细胞IFN γR1mRNA量显著高于HepG2及LO2细胞 (t=9.35 ,P <0 .0 1) ;HepG2 .2 .15 细胞全细胞IFN γR2表达低于HepG2及LO2细胞 ;两株肿瘤细胞存在内源性 p STAT1条带 ;HepG2 .2 .15细胞IFN γ/p STAT1、IFN γ/MHC Ⅰ诱导效应较母源细胞低下。拉米夫定抑制HBVDNA后 ,可上调HepG2 .2 .15细胞的IFN γ/MHC Ⅰ表达。结论　HBV持续存在和复制可降低IFN γ/STAT1及IFN γ/MHC Ⅰ诱导表达的敏感性 ;并能上调IFN γR1表达、下调IFN γR2表达。     

人脐带血间充质干细胞改善大鼠脑梗死免疫炎性反应的研究

目的研究人脐带血间充质干细胞(MSC)移植对大鼠局灶性脑缺血再灌注损伤炎性因子表达和调节性T细胞(Treg)数量及神经功能缺损的影响。方法选择健康雄性SD大鼠40只,随机分为假手术组、缺血再灌注组、MSC组、生理盐水组,每组10只。于缺血前、缺血后30min、缺血再灌注2、3、6h测定血清白细胞介素(IL)-6、干扰素γ(IFNγ)、IL-10、转化生长因子β(TGF-β)水平变化,于1、3、7、14d对大鼠神经功能进行评分,并检测外周血中Treg表达。结果 MSC组缺血后IL-6、IFNγ水平升高,分别在缺血再灌注2h、缺血后30min达高峰,显著小于缺血再灌注组和生理盐水组;缺血后缺血再灌注组和生理盐水组IL-10、TGF-β水平降低,MSC组升高。MSC组缺血再灌注3、7、14d神经功能缺损评分明显低于缺血再灌注组和生理盐水组(P0.05)。结论人脐带血MSC移植可促进缺血再灌注大鼠神经功能恢复,诱导CD4CD25Treg细胞增殖,促炎性因子IL-6、IFNγ水平下降,抗炎性因子IL-10、TGF-β水平升高,从而减轻缺血性脑损伤。     

α-干扰素对慢性粒细胞白血病树突状细胞分化及功能的影响

目的 :观察α 干扰素 (IFN α)对慢性粒细胞白血病 (CML)来源的树突状细胞 (DCs)分化及功能的影响。方法 :10例CML慢性期患者 ,Ph染色体阳性率均为 10 0 %。应用小牛血清体系培养CML来源的DCs,以重组干细胞因子 (rhSCF)、重组FIt 3配体 (rhFlt 3L)作为扩增体系的细胞因子 ,以rhGM CSF、rhTNF α、rhIL 4诱导DCs的生成 ,加或不加IFN α ,即分为A组 (0U/ml)、B组 (30 0U/ml) ,培养 2周后流式细胞仪检测培养细胞的免疫表型 ,G显带法显示Ph染色体 ,MTT法检测CML DCs刺激健康人外周血T淋巴细胞增殖情况。结果 :B组较A组培养细胞的总数减少 ,但DCs比例增加。Ph染色体两组比例分别为 10 0 %、81.75 %。B组较A组CD86、CD83、CD4 0、MHC Ⅰ类分子的表达明显升高 (P <0 .0 1)。同种混合淋巴细胞反应中 ,B组较A组A值亦明显升高 (P <0 .0 1)。结论 :IFN α可以加速CML DCs的成熟 ,增强CML DCs的功能。     

急性肝损伤肝脏炎症环境对骨髓间充质干细胞移植疗效的影响

目的:探讨猪急性肝损伤肝脏内不同程度炎症反应对移植骨髓间充质干细胞(mesenchymal stem cells,MSCs)存活率和疗效的影响.8):对照组和移植组.每组均按照低浓度D氨基半乳糖胺(D-gal,0.25 g/kg)、高浓度D氨基半乳糖胺(D-gal,0.35 g/kg)给药,分别建立2种急性肝损伤模型,诱导24 h后,对照组动物门静脉注射生理盐水40 mL,移植组动物门静脉移植40 mL PBS(约8×107同种异体GFP-MSCs).2 wk内观察猪肝功能变化、病理变化、血清炎症指标和移植MSCs定植存活情况.结果:低浓度D-gal组血清IL-1、TNF-α水平明显低于高浓度D-gal组,差异有显著性.低浓度D-gal移植组与低浓度D-gal对照组比较,ALT、TB和NH3在早期有显著差异(D2:232.6±57.6 vs 334.4±42.3,12.2±3.3 vs 16.0±1.2,79.7±9.3 vs 127.8±28.2,P<0.05);高浓度D-gal移植组与高浓度D-gal对照组比较,肝功能指标有改善,但无显著意义.低浓度D-gal移植组MSCs定植存活率和肝细胞增殖率显著高于高浓度D-gal移植组.结论:骨髓间充质干细胞移植治疗肝损伤的疗效在很大程度上取决于肝脏炎症反应的程度,炎症反应越严重,移植细胞越不易存活;相反,较低的体内炎症环境有益于MSCs定植、存活和肝细胞再生.     

Immunomodulatory Effects of Mice Mesenchymal Stem Cells on Maturation and Activation of Dendritic Cells

Background: Mesenchymal stem cells (MSCs) possess a wide range of immunomodulatory functions mostly in immune cells including dendritic cells (DCs). DCs are the key cells in the immune response and play an important role in initiating cell-mediated immunity. Objective: To evaluate the immunomodulatory effects of MSCs supernatant on maturation and function of DCs. Methods: Bone marrow derived mice MSCs were isolated and cultured. Twenty-four, forty-eight and seventy-two hours after passage 6, supernatants were collected and MSCs were assessed by cytometric analysis for the expression of CD34, CD44, CD45 and SCA-1. Splenic DCs were isolated using MACS and then co-cultured with MSCs supernatant. Expression of CD86, CD40 and MHC-II on DCs were also evaluated by cytometry. H 3-thymidine incorporation by proliferating T cells was determined in two separate MLR assay settings. In one setting, DCs were co-cultured with T cells in the presence of MSCs supernatant, and in the other setting DCs were treated with MSCs supernatant and then were co-cultured with T cells. Production of IL-12, IL-6 and IL-10 cytokines was measured in the supernatant of DCs treated with MSCs supernatant. We also measured IFN- γ and IL-4 levels in MLR supernatant. Results: The results showed that 72h MSCs supernatant could decrease the expression of MHC-II and CD86. The T cell proliferation was inhibited in the presence of MSCs supernatant and MSCs supernatant treated DCs as demonstrated by MLR assay. A significant increase in IL-4 level and a non significant decrease in IFN- γ level in MLR supernatant were observed. However, IL-6, IL-10 and IL-12 production did not change significantly. Conclusion: MSCs supernatant has a time dependent effect on the maturation of DCs. Also, it could alter cytokine production from responding T cells toward Th2. Generally, the findings of this study supported the immunomodulatory effect of MSCs supernatant on DCs maturation and function.     

Selected Stro‐1‐enriched bone marrow stromal cells display a major suppressive effect on lymphocyte proliferation

Mesenchymal stem cells (MSCs) have an immunosuppressive effect and can inhibit the proliferation of alloreactive T cells in vitro and in vivo. Cotransplantation of MSCs and hematopoietic stem cells (HSCs) from HLA‐identical siblings has been shown to reduce the incidence of acute graft‐vs.‐host disease. MSCs are heterogeneous and data on the inhibitory effects of different MSC subsets are lacking. The antigen Stro1 is a marker for a pure primitive MSC subset. We investigated whether Stro‐1‐enriched induce a more significant suppressive effect on lymphocytes in a mixed lymphocyte reaction (MLR), and whether this action is related to a specific gene expression profile in Stro‐1‐enriched compared to other MSCs. We demonstrated that the Stro‐1‐enriched population elicits a significantly more profound dose‐dependent inhibition of lymphocyte proliferation in a MLR than MSCs. One thousand expanded Stro‐1‐enriched induced an inhibitory effect comparable to that of 10 times as many MSCs. Inhibition by Stro‐1‐enriched was more significant in contact‐dependent cultures than in noncontact‐dependant cultures at higher ratio. The Stro‐1‐enriched inhibitory effect in both culture types was linked to increased gene expression for soluble inhibitory factors such as interleukin‐8 (IL‐8), leukemia inhibitory factor (LIF), indoleamine oxidase (IDO), human leukocyte antigen‐G (HLA‐G), and vascular cell adhesion molecule (VCAM1). However, tumor growth factor‐β1 (TGF‐β) and IL‐10 were only up‐regulated in contact‐dependant cultures. These results may support using a purified Stro‐1‐enriched population to augment the suppressive effect in allogeneic transplantation.     

骨髓间充质干细胞移植对急性胰腺炎后肠黏膜屏障的影响

目的 研究大鼠骨髓间充质干细胞(MSC)在急性胰腺炎后肠黏膜损伤修复中的作用.方法 从雄性SD大鼠股骨骨髓腔获取并培养MSC.20只雌性SD大鼠分成3组:急性重症胰腺炎(SAP)组(n=6)和MSC治疗组(n=8),均分2次以腹腔内注射L-精氨酸(2 g/kg)造模,成模12 h后,MSC治疗组予尾静脉注射MSC 5×...     

脂多糖对人外周血树突状细胞成熟的影响

目的 通过研究内毒素的不同作用方式,模拟慢性乙型肝炎肠源性内毒素血症,探讨脂多糖(LPS)对人外周血树突状细胞(DC)成熟的影响.方法 用重组人粒细胞集落刺激因子、重组人白细胞介素-4、酪氨酸激酶受体3配体和肿瘤坏死因子α体外诱导、培养人外周血单个核细胞.分为持续刺激组:于1、4、7、9 d加入LPS 1 μg/ml;短期刺激组:于7、8 d加入LPS 1 μg/ml;对照组:不加LPS.观察细胞形态,用流式细胞仪检测细胞表型,混合淋巴细胞反应检测DC刺激T淋巴细胞的能力,用酶联免疫吸附法检测DC分泌细胞因子的水平.组间比较采用SPSS10.0统计学软件进行单因素方差分析,进一步两两比较用SNK法.结果 DC表达人白细胞-DR抗原、CD86、CD80、CD83分子水平,持续刺激组分别为65.81%±10.96%、48.81%±18.13%、13.56%±5.48%、11.52%±5.09%,对照组分别为78.43%±20.34%、51.29%±15.75%、15.22%±5.53%、15.64%±5.26%,短期刺激组分别为89.83%±16.99%、69.90%±24.05%、25.97%±10.81%、25.96%±10.59%,持续刺激组DC表面分子表达水平低于短期刺激组和对照组,各组比较,F值分别为3.376、3.823、4.535、5.320,P值均＜0.05,差异均有统计学意义.3组DC诱导同种异体混合T淋巴细胞的增殖指数分别为1.593±0.303、1.949±0.240、1.548±0.365,各组比较,F=3.572,P=0.049,差异有统计学意义.3组DC表达干扰素γ水平短期刺激组为(40.52±11.38)pg/ml,高于对照组和持续刺激组[分别为(21.57±7.68)pg/ml、(15.6±5.83)pg/ml],各组比较,F=3.403,P=0.019,差异有统计学意义.3组DC表达白细胞介素12水平短期刺激组为(84.45±31.28)pg/ml,高于对照组和持续刺激组[分别为(54.42±20.34)pg/ml、(51.77±11.02)pg/ml],各组比较,F=2.212,P=0.088,差异有统计学意义.结论 LPS持续刺激可抑制DC的发育成熟,可能是肠源性内毒素血症患者细胞免疫功能低下的原因所在.     

乙型肝炎病毒转基因小鼠B7-H1表达水平与免疫耐受的相关性

目的 探讨HBV转基因(Tg)小鼠脾脏树突状细胞(DC)及肝脏B7-H1表达水平与HBV免疫耐受的相关性. 方法 制备小鼠脾脏DC,混合淋巴细胞反应检测其同种抗原刺激能力,流式细胞仪检测DC表面主要组织相容性复合物-Ⅱ(MHC-Ⅱ)及CD80、CD86、B7-H1等共刺激分子表达水平,酶联免疫吸附法检测白细胞介素-2(IL-2)、干扰素γ、IL-10水平,逆转录聚合酶链反应法和Western blot法检测肝组织B7-H1表达水平.计量数据组间比较采用f检验.结果 DC和T淋巴细胞比例分别为1:1、1:10、1:100时,HBV Tg小鼠脾脏DC刺激同种小鼠T淋巴细胞增殖数量(每分钟放射细胞数)分别为(865.4±39.3)个、(680.2±34.8)个和(320.0±12.7)个,正常小鼠刺激同种小鼠T淋巴细胞增殖数量分别为(22 385.6±99.7)个,(17 850.6±79.4)个和(760.0±32.1)个,HBV Tg小鼠脾脏DC刺激同种小鼠T淋巴细胞增殖能力明显均弱于正常小鼠DC,t值分别为16.674、19.674和21.712,P值均<0.01,差异有统计学意义.同时,HBV Tg小鼠MHC-Ⅱ,CD80表达下调,而CD86、B7-H1表达差异无统计学意义.HBV Tg小鼠分泌IL-2、干扰素γ、IL-10水平均降低,而HBV Tg小鼠和正常小鼠肝组织表达B7-H1的差异无统计学意义. 结论 HBV免疫耐受与MHC-Ⅱ、CD80下调表达导致的HBV Tg小鼠脾脏DC功能缺陷有关,而与负性共刺激分子B7-H1表达无关.     

调节性T淋巴细胞对成人乙型肝炎疫苗接种免疫应答的影响

目的 通过对乙型肝炎疫苗接种后不同免疫应答人群调节性T淋巴细胞(Treg细胞)Foxp3 mRNA的表达和细胞因子分泌的检测,探讨乙型肝炎疫苗接种后免疫应答与免疫调节细胞和细胞因子之间的内在联系.方法 采集不同反应人群全血,实时荧光定量PCR检测人外周血单个核细胞Foxp3 mRNA的表达;流式细胞术对外周血单核细胞的表面标志物CD4、CD25进行检测;酶联免疫法检测外周血单个核细胞植物血凝素(PHA)和HBsAg刺激后白细胞介素(IL)-4、IL-12、IL-18、干扰素(IFN)γ的产生水平.根据不同资料采用方差分析、q检验或相关分析进行统计学处理.结果 (1)PHA和HBsAg刺激前后,无应答组Foxp3的表达均高于应答组和对照组,差异有统计学意义(P＜0.05).(2)应答组外周血CD4+CD25+Treg细胞占CD4+T淋巴细胞的百分比(0.59%±0.46%)明显低于对照组(1.30%±1.44%),差异有统计学意义(F=2.990,P＜0.01).(3)各组外周血单个核细胞经PHA和HBsAg刺激后,无应答组的IFN γ浓度[(11.00±9.03)U/ml]明显低于应答组[(38.88±28.16)U/ml],差异有统计学意义(P＜0.01).(4)各组外周血单个核细胞经PHA和HBsAg刺激后,IL-18、IL-4、IL-12的浓度差异均无统计学意义.结论 CD4+CD25+Foxp3+Treg细胞在一定程度上参与了乙型肝炎疫苗接种应答的负性调控;乙型肝炎疫苗接种后无应答与IFN γ分泌不足有关;抗-HBs滴度水平与IFN γ和CD4+CD25+Foxp3表达无相关性.     

Decreased inducible expression of CD80 and CD86 in human monocytes after ultraviolet-B irradiation: its involvement in inactivation of allogenecity

Ultraviolet-B (UV-B) irradiation of antigen presenting cells (APCs) modifies their allogenecity, resulting in inhibition of the proliferative response of T cells in mixed lymphocyte reaction (MLR). Costimulation by the CD28 ligand CD80 (B7/B7-1) and CD86 (B70/B7-2) plays an important role during T-cell proliferation by augmenting synthesis of interleukin-2 (IL-2) and other cytokines. In this study, we demonstrated induced expression of both CD80 and CD86 during allogeneic MLR, though human freshly isolated monocytes express CD86 constitutively with a much lower level of CD80. A monoclonal antibody (MoAb) against CD86, but not CD80, efficiently inhibited allogeneic T- cell proliferative responses stimulated with highly purified monocytes. UV-B exposure (0 to 1,000 J/m2) of monocytes inhibited the proliferation of T lymphocytes in MLR in a dose-dependent manner. Flow cytometric analysis showed that UV-B exposure of monocytes impaired the constitutive expression of CD54 (intercellular adhesion molecule-1) by 24 hours after irradiation, but the effect on CD86 was relatively less. The surface expression of CD80, CD86, CD54, and HLA-DR on monocytes was further augmented by interferon (IFN)-gamma; this cytokine-induced expression was dose-dependently reduced by UV-B irradiation. Similarly, the upregulation of these molecules following allogeneic MLR was downregulated by UV-B irradiation. UV-B irradiation of monocytes inhibited the expression of IL-2 mRNA in monocyte-stimulated allogeneic MLR. In contrast, the addition of anti-CD28 MoAb at the onset of MLR prevented, at least partially, the reduction of IL-2 mRNA. These results strongly suggest that the impairment of inducible expression of CD86 and CD80 may contribute to the reduced MLR response following exposure of monocytes of UV-B.     

重组人生长激素和骨髓间质干细胞移植联合治疗阿霉素性心肌病心力衰竭的实验

目的 对比重组人生长激素(rhGH)与骨髓间质干细胞(MSCs)单用或联用对阿霉素性心肌病心力衰竭(心衰)大鼠的疗效.方法 将Wistar大鼠分为正常组6只、心衰组9只、rhGH组6只、MSCs组7只和rhGH与MSCs联合治疗(G+M)组7只.MSCs移植经心肌内点状注射,rhGH2 mg/kg,隔日1次,皮下注射.1个月后行左室功能、全心质量指数(HW/BW)及左室质量指数(LW/BW)测定.免疫组化观察MSCs在心肌内的分布,聚丙烯酰胺凝胶电泳分离心肌肌球蛋白重链(MHC)同工酶V1和V3型.治疗前后测定血清生长激素(GH)和B型利钠肽(BNP)浓度.结果 与心衰组比较,MSCs组心功能各指标均无有统计学意义的改变,rhGH组左室收缩压和左室内压力最大上升速率升高,G+M组左室收缩压和左室内压力最大上升及下降速率均升高,均为P<0.05.三个治疗组HW/BW和LW/BW均较心衰组有降低,但P>0.05.G+M组心肌内MSCs数量多于MSCs组.与心衰组比较,rhGH组V1型MHC(V1)、v3型MHC(V3)、V1/V3比值差异无统计学意义,MSCs组V3增加,G+M组V1和V3增加,均为P<0.05;与rhGH组比较,MSCs组V3增加,G+M组V1和V3均增加,均为P<0.05;与MSCs组比较,G+M组V1增加,P<0.05.GH水平在治疗前均较正常组降低,P<0.05;治疗后均较心衰组增高,P<0.05,但仅接近正常水平,各组间比较差异无统计学意义.BNP水平在治疗前均较正常组升高,P<0.05;治疗前后比较,rhGH组和G+M组差异有统计学意义,P<0.05和P=0.001.结论 rhGH治疗可改善阿霉素性心肌病心衰大鼠的心脏收缩功能,MSCs移植未见明显疗效;二者联合治疗可提高MSCs存活率,增加MHC含量,改善心脏收缩和舒张功能.     

HBV特异性抗原对HBV携带者T淋巴细胞免疫功能的影响

目的 通过分析不同类型HBV携带者外周血单个核细胞(PBMCs)的细胞免疫功能,分析HBV抗原对其的影响,探索HBV慢性感染的机制并寻求可能的免疫治疗方法.方法 用不同的抗原和(或)细胞因子刺激培养的无症状HBV携带者PBMCs,酶联免疫吸附法检测细胞培养上清液中不同细胞因子的水平;流式细胞术检测PBMCs的细胞表型.对数据进行t检验分析和相关性分析.结果 HBsAg刺激无症状HBV携带者PBMCs后产生的干扰素(IFN)γ为(48.3±19.8)Pg/ml,较健康对照人群低[(196.2±104.3)Pg/ml(t=3.023,P＜0.05)].HBsAg和HBcAg刺激HBeAg阳性患者PBMCs分泌的IFN y水平分别为(50.4±51.6)Pg/ml和(63.2±36.9)pg/ml,明显低于HBeAg阴性组[(86.2±42.3)Pg/ml和(101.4±32.5)pg/ml],t值分别为2.468和3.184,P值均＜0.05;HBeAg阳性患者组分泌白细胞介素(IL)-12 P70明显低于HBeAg阴性组(P＜0.05);补偿外源性IL-12可明显促进HBV携带者PBMCs分泌IFN γ(P＜0.01),IL-12协同HBV抗原可激活CD8+CD45RA+CCR7及CD8+CD45RA CD62L+细胞.结论 HBeAg阳性患者PBMCs分泌IL-12减少,这可能是HBV携带者持续感染的重要原因;外源性IL-12可促进HBV携带者PBMCs中的中枢记忆性T淋巴细胞的免疫功能.

Abstract：

Objective To investigate the effect of HBV antigens and pathological mechanism of chronic HBV infection by analyzing the cellular immune function of peripheral blood mononuclear cells (PBMCs) from HBsAg carriers. Methods PBMCs were prepared from individuals with chronic asymptomatic HBV infection and cultured in the presence of different antigens and/or cytokines. The levels of cytokines in culture supernatants were detected by ELISA method. The phenotype of the cells was detected by FACS.Results The levels of IFN γ secreted by PBMCs from HBsAg carriers were (48.3 ± 19.8) pg/ml, significantly lower than that from healthy controls (t = 3.023, P ＜ 0.05＝; The IFN γ produced by PBMCs from HBeAg positive patients due to HBsAg and HBcAg stimulation were (50.4±51.6) pg/ml and (63.2 ± 36.9)pg/ml, significantly lower than that of HBeAg negative patients (t = 2.468 and 3.184, P ＜ 0.05, respectively＝.The IL-12p70 secreted by PBMCs from HBeAg positive patients was also significantly lower than that of HBeAg negative patients (P ＜ 0.05＝; Exogenous IL-12 promoted significantly PBMCs to secrete IFN γ (P ＜0.01＝ and IL-12 combined with HBV antigens activated CD8+CD45RA+CCR7+ and CD8+CD45RA-CD62L+cells. Conclusion IL-12 secreted by PBMCs decreased in HBeAg positive patients, which may be the crucial reason of viral persistence in chronic HBV carriers. Exogenous IL-12 combined with specific HBV antigen could promote the central memory CD8+ T cells to produce IFN γ.