不同诱导条件对人骨髓基质干细胞向内皮分化的影响

目的探讨不同诱导条件对人骨髓基质干细胞向内皮分化的影响。方法采用密度梯度离心法分离培养人骨髓基质干细胞,用荧光激活细胞分选法分析骨髓基质干细胞CD34、CD105和CD166的表达率;用免疫荧光细胞化学法观察骨髓基质干细胞在细胞因子(50μgL血管内皮细胞生长因子、5μgL碱性成纤维细胞生长因子、100mgL内皮细胞生长补充因子)、内皮条件培养基或与成熟兔主动脉内皮共培养5天时vWF或和Flk1蛋白的表达。结果分离培养的骨髓基质干细胞CD34表达率为4.16%±0.16%,与阴性对照(4.06%±0.23%)相比无统计学差异,CD105表达率为90.20%±2.35%,CD166表达率为82.30%±3.22%,均明显高于阴性对照(P<0.05)。诱导前骨髓基质干细胞不表达vWF和Flk1蛋白,经血管内皮细胞生长因子、碱性成纤维细胞生长因子和内皮细胞生长补充因子等细胞因子联合诱导5天时,33.42%骨髓基质干细胞开始表达vWF蛋白;与成熟内皮共培养5天时,vWF染色仍为阴性,但25.71%骨髓基质干细胞开始表达Flk1;用内皮条件培养基培养5天时,骨髓基质干细胞vWF和Flk1染色均为阴性。结论细胞因子和成熟内皮细胞均能诱导骨髓基质干细胞向内皮分化,而内皮条件培养基不能诱导骨髓基质干细胞向内皮分化。     

紫杉醇联合骨髓基质干细胞种植体外修复内皮及对血管平滑肌增生的影响

目的探讨紫杉醇联合骨髓基质干细胞种植体外修复内皮的可行性及对血管平滑肌细胞增生的影响。方法培养兔主动脉内皮、平滑肌和人骨髓基质干细胞,通过细胞共培养将内皮/骨髓基质干细胞接种于下室、平滑肌细胞接种于上室模拟血管内皮修复过程,分别用3H TdR掺入和Westernblot检测紫杉醇(1,10,100nmol/L)干预20min后第10天平滑肌DNA合成和PCNA蛋白表达,用免疫荧光细胞化学法观察与紫杉醇干预内皮共培养的骨髓基质干细胞vWF和Flk1蛋白表达。结果骨髓基质干细胞种植组平滑肌细胞3H TdR掺入和PCNA蛋白光密度相对值均高于融合内皮组(n=6,P<0.05),低于对数内皮组(n=6,P<0.05)。共培养前骨髓基质干细胞不表达vWF和Flk1蛋白,与紫杉醇干预内皮共培养10天时vWF染色阴性,但部分骨髓基质干细胞开始表达Flk1蛋白。结论骨髓基质干细胞种植能部分抑制紫杉醇引起的平滑肌细胞延迟增生,与紫杉醇干预内皮共培养的骨髓基质干细胞有向内皮分化的能力。     

骨髓基质干细胞种植体外修复内皮及对血管平滑肌细胞增生的影响

目的探讨骨髓基质干细胞(MSCs)种植体外修复内皮的可行性及对血管平滑肌细胞增生的影响。方法培养兔血管内皮、平滑肌和人MSCs,通过细胞共培养模拟血管内皮修复过程,用流式细胞仪分析MSCs分子表型特征,免疫荧光细胞化学法观察与内皮共培养的MSCsFlk1和vWF蛋白表达,根据下室内皮生长状态及是否接种MSCs将其分为对照组、单纯MSCs组、融合内皮组、对数内皮组和MSCs种植组。氚胸腺嘧啶脱氧核苷(3HTdR)掺入检测平滑肌细胞DNA合成,Westernblot检测平滑肌细胞中增殖细胞核抗原蛋白表达。结果分离的MSCs表达基质细胞标志CD105和CD166,不表达造血干祖细胞和内皮细胞标志CD34、Flk1、vWF;与内皮共培养5天时,vWF染色仍为阴性,但约25.71%MSCs开始表达Flk1;MSCs种植组平滑肌细胞3HTdR掺入虽高于融合内皮组,但与对数内皮组比较显著降低;MSCs种植组平滑肌细胞PCNA蛋白吸光度相对值虽高于融合内皮组,但与对数内皮组比较明显减少。结论MSCs种植能抑制平滑肌细胞增生,种植在成熟内皮中的MSCs具有微环境依赖向内皮分化的能力。     

血管内皮生长因子及其受体随增龄在大鼠肾组织内表达的研究

目的　研究血管内皮细胞生长因子 (VEGF)及其受体Flt 1和Flk 1在大鼠肾组织内的表达及随增龄变化 ,探讨它们在肾脏衰老过程中的作用。　方法　应用 3、12、2 4月龄 (各 7只 )大鼠肾组织石蜡切片进行常规病理及免疫组织化学染色 ,定量分析肾组织内微血管变化及VEGF、Flt 1和Flk 1表达变化。应用逆转录聚合酶链反应技术 (RT PCR)检测肾组织内VEGF AmRNA的表达。　结果　 2 4月龄组与 3月龄组相比肾小球面积增大〔(15 6 35± 10 2 2 ) μm2 vs(72 0 5± 496 ) μm2 ,P <0 0 1〕 ,肾小球内毛细血管袢腔面积与肾小球面积百分比减少 (46 76 %± 4 91%vs 6 3 75 %±6 0 2 % ,P <0 0 1) ,肾小管周围毛细血管数量减少 (9 8± 2 6vs 14 7± 3 1,P <0 0 1) ;肾小球内VEGF阳性细胞数增多 (9 3± 2 4vs 6 4± 1 6 ,P <0 0 5 ) ;集合管中VEGF的表达则明显减少(9 35 %± 2 10 %vs 15 2 3%± 3 2 2 % ,P <0 0 5 ) ;Flk 1在肾小球血管袢上表达增加 (9 17%±2 0 2 %vs 1 0 3%± 0 35 % ,P <0 0 1) ,而Flt 1和Flk 1在肾小管上表达则明显减少 (7 6 4%±3 0 2 %vs 15 36 %± 2 5 4% ,2 48%± 0 86 %vs 9 0 1%± 2 6 3% ,P <0 0 1)。 2 4月龄组VEGF AmRNA较其他两组减少 (P <0 0 5 )。　结论　VEGF、Flk     

骨髓增生异常综合征T细胞早期激活及可溶性肿瘤坏死因子受体的研究

目的　研究骨髓增生异常综合征 (MDS)外周血CD+ 4 、CD+ 8T细胞早期激活标志CD69的表达及血清、骨髓可溶性肿瘤坏死因子受体 1、2 (sTNF R1、2 )的水平及其意义。方法　在植物血凝素 (PHA) 2 0mg/L条件下进行全血细胞培养 ,于 0h和 4h分别用流式细胞仪对CD+ 4 、CD+ 8T细胞CD69的表达进行分析。用ELISA法检测血清和骨髓sTNF R1、2的水平。结果　PHA刺激前难治性贫血 (RA)与难治性贫血伴环形铁粒幼细胞增多 (RAS)CD+ 4 、CD+ 8细胞CD69的表达率分别为 8 32 %、9 88% ,难治性贫血伴原始细胞增多 (RAEB)与转变中的RAEB(RAEB T)CD+ 8细胞CD69的表达率为7 92 %。PHA刺激后MDS患者CD+ 4 、CD+ 8细胞表达CD69明显增强 ,RA +RAS为 5 3 4 6 %、5 1 6 3% ;RAEB +RAEB T为 4 2 93%、4 1 96 % ,CD+ 4 与CD+ 8细胞CD69的表达率相似。MDS两种sTNF R1水平均明显升高 ,RA +RAS组sTNF R1血清为 (1 5 8± 0 6 8) μg/L ,骨髓为 (2 10± 0 2 6 ) μg/L ;sTNF R2血清为 (1 4 1± 0 5 0 ) μg/L ,骨髓为 (1 95± 0 6 4 ) μg/L ;RAEB +RAEB T组sTNF R1血清为 (2 6 2± 2 5 5 ) μg/L ,骨髓为 (3 12± 0 6 7) μg/L ;sTNF R2血清为 (1 96± 0 5 6 ) μg/L ,骨髓为(3 0 9± 0 6 2 ) μg/L。血清sTNF R2水平与PHA刺激     

射频消融对血管内皮及血小板功能的影响

目的　研究射频导管消融 (RFCA)术对血管内皮和血小板功能的影响。方法　应用放射性免疫法、酶联免疫法、单克隆抗体标记及流式细胞技术 ,观察 31例心动过速患者RFCA手术前、后血浆内皮素 (ET)、血管性假血友病因子 (vWF)水平及血小板α 颗粒膜蛋白 (CD62 P)、血小板溶酶体膜蛋白 (CD63 )表达的变化。结果　RFCA术前、后血浆ET、vWF水平无明显改变 ,但术后即刻血小板膜CD62 P、CD63 表达分别由术前的 (4 .75± 2 .32 ) %和 (9.6 2± 4 .0 8) %增高至 (7.6 4± 5 .2 5 ) % (t =3.0 5 ,P <0 .0 1)和 (12 .2 3± 5 .70 ) % (t=2 .10 ,P <0 .0 5 ) ,术后 4 7～ 115h(平均 6 5h)均降至基础水平。多元相关分析结果显示CD62 P表达变化与累积放电能量呈显著性正相关 (r =0 .30 ,P <0 .0 5 )。结论　RFCA术不引起明显的内皮损伤 ,但可导致血小板膜CD62 P、CD63 表达增加 ,促进血小板活化 ,其中手术累积放电能量是重要的影响因素。     

血管内皮生长因子的表达与胃癌浸润和转移的关系

目的　研究血管内皮生长因子165(VEGF)mRNA在胃癌中的表达 ,探讨VEGF与胃癌浸润和转移的关系。方法　采用RT PCR方法 ,对 31例胃癌及非癌组织手术标本中VEGF165mRNA的表达进行相对定量研究。结果　胃癌组织中VEGF165mRNA表达的平均相对量 (1.12 5± 0 .35 6 )明显高于非癌组织的表达量 (0 .76 0± 0 .2 78,P <0 .0 5 ) ,其中淋巴结转移组 (1.2 19± 0 .377)和Ⅲ、Ⅳ期组 (1.2 6 2±0 .386 )分别高于无淋巴结转移组 (0 .92 7± 0 .2 0 5 )和Ⅰ、Ⅱ期组 (0 .934± 0 .194 ,P均 <0 .0 5 )。VEGF高表达者中淋巴结转移率为 83.3% ,Ⅲ和Ⅳ期占 77.8% ,均明显高于VEGF低表达者的 4 6 .2 %和 33.8%(P <0 .0 5 )。结论　胃癌组织中有VEGF的高表达 ,VEGF的表达在胃癌浸润和转移过程中发挥重要作用。     

骨髓基质细胞的血管新生作用及对心功能的影响

目的　观察骨髓基质细胞在缺血心肌内诱导血管新生的作用及对心功能的影响。方法　复制兔心肌梗死动物模型 ,3d后将体外分离扩增的自体骨髓基质细胞用荧光标记物DAPI标记后移植到梗死周围缺血区心肌 (细胞移植组 ,n =8) ,对照组注射等量培养基 (n =6 )。 4W后 ,先进行心脏超声检查分别测定对照组和细胞移植组左室射血分数 (EF)和短轴缩短率 (FS) ,然后采集移植区心肌标本 ,用荧光示踪方法观察移植细胞在缺血坏死区心肌存活的情况 ,用CD31单克隆抗体免疫组化染色法测定毛细血管密度。结果　细胞移植组心肌组织和小血管内壁均可见蓝色荧光的DAPI标记的移植细胞 ;细胞移植组心肌毛细血管密度显著高于对照组 [(16 3 0 0± 2 5 85 )vs (96 0 0± 16 6 1) ,P <0 0 5 ];细胞移植组EF较对照组增高 [(0 5 0± 0 0 3)vs(0 4 6± 0 0 3) ,P <0 0 5 ],短轴缩短率FS也高于对照组[(2 7 0 2± 1 2 7)vs (2 3 85± 1 6 9) ,P <0 0 5 ]。结论　体外扩增的骨髓基质细胞可在缺心肌内存活 ,并能促进缺血组织血管新生、改善心功能。     

卡托普利对兔动脉粥样硬化斑块内碱性成纤维细胞生长因子和血管内皮生长因子的影响

目的　观察碱性成纤维细胞生长因子 (b FGF)和血管内皮生长因子 (VEGF)与动脉粥样硬化 (AS)的关系 ,以及卡托普利 (captopril)对 AS和 b FGF、VEGF表达的影响。方法　 36只兔随机分为 3组 :空白对照组 ( 组 )动物喂饲普通颗粒饲料 ;实验对照组 ( 组 )动物喂饲含 1g/d胆固醇和 3%猪油的饲料 ;实验组 ( 组 )喂饲含 1g/d胆固醇和 3%猪油的饲料 ,同时给予卡托普利 10 mg.kg- 1 .d- 1 。于 12周后处死动物 ,取出主动脉做 b FGF和 VEGF免疫组化定性和定量观察。结果　光镜下 组和 组有明显 AS形成。定量研究 :与 I组相比 , 组和 组 b FGF在主动脉内中膜的表达面积 (μm2 ) (2 6 999.6 8± 9931.82 ,2 4 0 75 .6 2± 2 4 787.6 8对 1386 8.14± 3180 .13)、密度 (5 .4 3±1.6 5 ,3.33± 1.15对 2 .0 7± 0 .78)和密度指数 (15 7886 .4 6± 113340 .0 5 ,73348.6 0± 4 6 0 4 8.81对 2 92 90 .78±15 0 16 .5 8)均有非常显著性增加 (P<0 .0 1) ,但 组与 组相比 ,其 b FGF表达的面积、密度和密度指数均有非常显著性降低 (P<0 .0 1)。与 组相比 , 组和 组 VEGF在主动脉内中膜的表达面积 (30 4 2 5 .4 3± 11114 .14 ,2 55 2 9.31± 10 30 5 .88对 1386 8.14± 3180 .13)、密度 (6 .10± 2 .0 9,6 .10± 2 .     

血管内皮细胞生长因子对小鼠CFU-GM、CFU-E、CFU-S生成的影响

目的 :研究血管内皮细胞生长因子 (VEGF)对小鼠骨髓单个核细胞 (MNC)粒 巨噬细胞系集落形成单位 (CFU GM)、红系集落形成单位 (CFU E)、脾集落形成单位 (CFU S)生成数量的影响。方法 :一部分小鼠骨髓MNC样本在加入或不加入外源性hVEGF的体系中预先培养 2 4h ,分别进行体外造血细胞集落培养和小鼠体内CFU S试验 ,计数CFU GM、CFU E、CFU S集落生成数目 ;另外对剩余骨髓样本不经处理直接在含rhVEGF的体系中进行体外CFU GM、CFU E集落培养 ,观察集落培养体系中添加VEGF对集落生成的影响。结果 :CFU GM、CFU E、CFU S数目分别为 33.2 1± 2 .84、72 .0 0± 4 .5 5和 12 .2 0± 1.32 ,显著高于未处理组 (分别为19.6 0± 2 .10、38.86± 2 .77和 6 .10± 1.5 2 ) (P <0 .0 5 ) ,但均低于正常对照组 (分别为 5 1.97± 2 .4 5、16 4 .2 0± 5 .70和 19.5 0± 2 .4 6 )。外源性hVEGF的存在使VEGF组CFU GM、CFU E产率较正常对照组显著提高 (73.12±3.80∶5 1.39± 2 .6 0 ,198.98± 4 .92∶16 4 .4 0± 5 .32 ) (P <0 .0 5 )。结论 :VEGF不仅对造血细胞集落生成能力具有保护作用 ,还具有促进造血细胞集落生成的作用。     

Mesenchymal stem cells participating in ex vivo endothelium repair and its effect on vascular smooth muscle cells growth

BACKGROUND: Previous studies have shown that mesenchymal stem cells (MSCs) transplantation can promote neovascularization and regenerate damaged myocardium. However, it remains unknown whether MSCs seeding can be used to repair injured cellular components in vascular diseases. In this study we explored the feasibility of applying MSCs to endothelium repair in endothelial damage and vasoproliferative disorders. METHODS: Ex vivo model of endothelium repair was developed in which rabbit vascular smooth muscle cells (SMCs) were inoculated into the upper chamber and rabbit endothelial cells (ECs)/human MSCs into the lower chamber of a co-culture system. 3H-TdR incorporation and PCNA protein expression were assayed and migrated number of SMCs was calculated to evaluate the effect of MSCs seeding on SMCs growth. Flk-1 and vWF protein expressions were observed to analyze the plasticity of the seeded MSCs along endothelial lineage. RESULTS: In this co-culture system, no vWF protein but Flk-1 protein was observed in the 25.71% of MSCs after having been co-cultured with mature rabbit ECs for 5 days. Compared with the control group, the proliferation and migration of SMCs was significantly increased by proliferative ECs but decreased by confluent ECs (n=6, P<0.01). MSCs seeding decreased the proliferation and migration of SMCs compatible with the effect of proliferative ECs (n=6, P<0.001). However, no inhibition on SMCs growth was observed with MSCs seeding in comparison to the effect of confluent ECs. CONCLUSIONS: MSCs seeding can inhibit the proliferation and migration of SMCs. MSCs co-cultured with mature ECs have the ability to undergo milieu-dependent differentiation toward ECs.     

小鼠内皮祖细胞的分离、培养与定向诱导分化

目的 建立一种稳定、高效,从小鼠骨髓中分离培养与定向诱导分化内皮祖细胞(EPCs)的方法。方法 从小鼠骨髓中密度梯度离心法分离单个核细胞,经差速贴壁结合特殊培养基扩增并向内皮细胞定向诱导分化EPCs。应用免疫荧光和流式细胞技术鉴定内皮细胞系列标志:CD34、CD31、Flk-1和祖细胞标志CD133。并通过检测其对FITC标记的UEA-1的吸附和内吞DiI-ac-LDL来进行细胞功能学的鉴定。对分化细胞行vWF、CD31 免疫组化染色鉴定,并与血管内皮细胞合成前列腺素能力进行比较。结果 经过梯度密度离心和贴壁法选择的细胞表达内皮细胞特异性抗原CD34、CD31、Flk-1,部分表达CD133。分离所得细胞经EBM-2专用培养基培养后,第4天可见集落形成,培养第9天流式细胞仪检测其CD34、CD133、CD31、Flk-1阳性率分别为(44±4)%、(18±3)%、(49±4)%和(79±6)%,细胞能特异性吸附FITC标记的荆豆凝集素并内吞DiI-ac-LDL,约3周左右可融合近80%,形成铺路石样内皮细胞特有形态。传代后vWF、CD31免疫组化染色阳性率分别为(66±5)%和(56±5)%。诱导后的内皮祖细胞的合成前列腺素能力与血管内皮细胞之间无显著差异。结论 从小鼠骨髓中分离培养与定向诱导分化EPCs的方法,效率高,稳定性和重复性好。     

心肌微环境对骨髓间充质干细胞的诱导分化作用

目的通过与心肌细胞(CMs)共同培养和采用含有CMs裂解液的培养基两种方法体外模拟心肌微环境,探讨心肌微环境对骨髓间充质干细胞(MSCs)分化的诱导作用。方法自新生乳鼠的心脏分离CMs,自成年大鼠的骨髓分离MSCs,将MSCs与CMs按1∶4的比例共同培养1周,观察细胞形态的改变,并通过免疫荧光方法检测共培养后MSCs表达心脏特异性肌钙蛋白T(cTnT)及CD31的情况;将分离的CMs反复冻融制成CMs裂解液,将MSCs在含有4倍CMs裂解液的培养基中培养1周,观察细胞形态的改变,并通过免疫化学方法检测MSCs表达心脏特异性肌钙蛋白T(cTnT)及CD31的情况;以仅用普通培养基所培养的MSCs作为对照。结果与CMs共培养的MSCs和CMs裂解液培养的MSCs逐渐伸展变长,形成肌细胞形态,培养1周后经抗cTnT和抗CD31免疫染色均呈阳性;对照组MSCs没有明显形态变化,抗cTnT和抗CD31免疫染色成阴性。结论与CMs共培养和采用含有CMs裂解液的培养基两种方法均可在体外模拟心肌微环境,诱导MSCs向心肌样细胞和内皮样细胞方向分化。     

Increases in circulating T lymphocytes expressing HLA-DR and CD40 ligand in patients with dilated cardiomyophthy

Inflammatory and immunological mechanisms are implicated in the development of idiopathic dilated cardiomyopathy (DCM). Since activated T lymphocytes express surface HLA-DR antigens, an increased level of these cells in the circulation could indicated an ongoing immune response. While the role of activated T lymphocytes in experimental myocarditis has been elucidated, the contribution of T lymphocyte activation in clinical DCM remains unclear. We therefore examined the role of T-cell activation in peripheral blood samples obtained from 10 patients with DCM (mean age, 49 ± 12 years) and from 10 age-matched healthy controls. Citrated whole blood was mixed with fluorescein isothiocyanate- or phycoerythrin-conjugated specific monoclonal antibodies and analyzed using a fluorescence-activated cell sorter (FACS). The ratio (%) of histocompatibility leukocyte antigen (HLA)-DR positive cells in the FACS gated lymphocyte population was significantly higher in DCM patients than in controls (7.9% ± 5.3% vs 2.0% ± 0.9%; P < 0.01). The expression of CD40L on T cells determined as mean fluorescence intensity (MFI) was also significantly higher in DCM patients than in controls (3.6 ± 2.1 vs 1.8 ± 0.4 MFI; P < 0.05). Furthermore, the ratios of T cells expressing HLA-DR and serum brain natriuretic peptide (BNP) levels closely correlated (P = 0.0008). We showed that HLA-DR on peripheral T cells significantly correlated with serum BNP levels and that high CD40L expression on T cells was concomitant with increased BNP levels (P < 0.05). Therefore the magnitude of T-cell expression, such as increased expression of HLA-DR and CD40L, contributes to myocardial dysfunction in DCM.     

Induction of Umbilical Cord Blood Mesenchymal Stem Cells into Neuron-Like Cells In Vitro

Mesenchymal stem cells (MSCs) in human umbilical cord blood are multipotent stem cells that differ from hematopoietic stem cells. They can differentiate in vitro into mesenchymal cells such as osteoblasts and adipocytes. However, differentiation into nonmesenchymal cells has not been demonstrated. Here, we report the isolation, purification, expansion, and differentiation of human umbilical cord blood MSCs into neurocytes in vitro. Cord blood samples were allowed to drain from the end of the cord into glass bottles with 20 U/mL preservative-free heparin. MSCs were isolated from human umbilical cord blood, purified, and expanded in Mesencult medium. Surface antigens of MSCs were analyzed by fluorescence-activated cell sorting (FACS). MSC passages 2,5, and 8 were induced to differentiate into neuron-like cells. Neurofilament (NF) and neuron-specific enolase (NSE) were detected by immunohistochemistry staining. Special Nissl bodies were observed by histochemical analysis. The results showed that 6.6 x 10(5) primary MSCs were expanded for 10 passages to obtain 9.9 x 10(8) MSCs, an increase of approximately 1.5 x 10(3)-fold. FACS results showed that the MSCs did not express antigens CD34, CD11a, and CD11b and expressed CD29 and CD71, an expression pattern identical to that of human bone marrow-derived MSCs. Induction results indicated that approximately 70% of the cells exhibited a typical neuron-like phenotype. Immunohistochemistry staining suggested that induced MSCs of different passages expressed NF and NSE. Special Nissl bodies were obvious in the neuron-like cells. These results suggest that MSCs in human umbilical cord blood are capable of differentiating into neuron-like cells in vitro.     

Cotransplantation of HLA-identical mesenchymal stem cells and hematopoietic stem cells in Chinese patients with hematologic diseases

Mesenchymal stem cells (MSCs) may be employed to support hematopoietic reconstitution and mitigate graft-vs.-host disease (GVHD) in transplantation of hematopoietic stem cells (HSCs). The aim of this study was to explore the feasibility and safety of cotransplantation culture-expanded MSCs and HSCs from the same human leukocyte antigen (HLA)-identical sibling donor in Chinese patients with hematologic diseases. Bone marrow mononuclear cells from healthy donors were cultured and expanded ex vivo. Immunophenotype, adipogenic and osteogenic differentiation potential, and karyotype of the harvested MSCs were detected on those who had been cotransplanted with HSCs and MSCs from the same donor. Hematopoietic reconstitutions, complications, and clinical outcomes were observed after cotransplantation in these patients. (1.77 ± 0.40) × 10⁶/kg (donor’s weight) MSCs were successfully expanded from 23.6 ± 5.96 ml of bone marrow samples. They had normal karyotypes with bi-lineages differentiation potential, and were CD73, CD90, and CD105 positive. Twelve patients underwent cotransplantation with no observable adverse response during and after the infusion of MSCs. Hematopoietic reconstitutions were rapid. Two patients developed grade II–IV acute GVHD, and two extensive chronic GVHD. Four patients suffered from cytomegalovirus infection but were cured eventually. Up to now, seven patients have been followed as long as 29–57 months and five patients died. It is concluded that MSCs can be expanded effectively by culture and it is safe and feasible to cotransplant patients with allogenic culture-expanded MSCs and HSCs.     

骨髓间质干细胞对系统性红斑狼疮患者调节性T细胞的免疫调节作用

目的探讨自体与异体骨髓间质干细胞（MSCs）对系统性红斑狼疮（SLE）患者调节性T细胞的免疫调节作用。方法用Percoll密度梯度离心法从14例SLE患者和15例健康人骨髓中分离MSCs,同时用免疫磁珠（MACS）分离SLE患者外周血CD4^＋CD25^＋T细胞。将SLE患者外周血分离的淋巴细胞或CD4^＋CD25^＋T细胞与自体、异体MSCs共培养,观察MSCs对淋巴细胞及CD4^＋CD未T细胞增殖的影响,同时测CD4^＋CD25^＋T细胞分泌IL-10、转化生长因子（TGFβ）水平及细胞毒T淋巴细胞相关抗原4（CTLA-4）表达。结果自体与异体MSCs均抑制淋巴细胞增殖,其抑制率分别为56．32％、65．46％。MSCs以数量依赖性方式促进纯化CD4^＋CD25^＋T细胞增殖,分泌IL-10、TGFβ水平升高。结论MSCs可纠正SLE活动时CD4＋CD25^＋T细胞免疫缺陷,并抑制淋巴细胞过度活化,可能在自身免疫病的外周血造血干细胞和间质干细胞共移植或MSCs单独移植中发挥重要的免疫调节作用。     

慢性乙型肝炎患者在HBV不同抗原负载下树突状细胞功能的变化

目的观察在不同HBV抗原负载下树突状细胞(DC)功能的变化及在核苷(酸)类药物抗病毒治疗后DC的功能变化。方法分离17例慢性乙型肝炎患者外周血单个核细胞(PBMC),并在不同抗原负载下(HBsAg、HBcAg、HBsAg联合HBcAg)诱导DC分化成熟,用流式细胞技术测定DC表面共刺激分子CD80、CD83、CD1a及HLA-Ⅱ类分子HLA-DR表达水平,同时用淋巴细胞增殖试验评估DC功能。结果与负载HBsAg诱导相比,HBcAg、HBsAg联合HBcAg诱导下DC共刺激分子表达明显增强(HLA-DR:85.12±1.55比98.37±1.27比99.21±1.33;CD1a:15.69±2.46比16.25±2.33比42.20±1.10;CD80:71.88±6.38比80.74±3.23比94.70±2.77;CD83:32.64±2.77比42.55±2.88比44.16±1.89)。淋巴细胞增殖试验提示DC功能改善更显著,尤其HBsAg联合HBcAg诱导DC功能改善更加明显(HBV DNA阳性:7.29±0.17比7.99±0.43比8.56±0.31;HBV DNA阴性:7.48±0.30比8.22±0.41比8.78±0.31)。将17例慢性乙型肝炎患者在核苷(酸)类药物抗病毒治疗前后DC功能进行比较,经抗病毒治疗在充分抑制HBV DNA复制的情况下,DC功能的恢复更趋于完善(HLA-DR:99.21±1.33比99.82±2.67;CD1a:42.20±1.10比71.33±5.89;CD80:94.70±2.77比96.42±3.56;CD83:44.16±1.89比68.34±2.11)。结论 HBsAg联合HBcAg诱导DC细胞功能明显得到恢复和增强,HBV DNA阴性时诱导自体DC功能恢复更趋完善。