提高间充质干细胞表面CXCR4表达的实验研究

目的：探索提高间充质干细胞（mesenchymal stem cells，MSCs）表面趋化因子受体4（CXCR4）表达的方法。方法：分别对兔骨髓间充质干细胞（Rabbit-MSCs）和人脐带间充质干细胞（HUC-MSCs）进行培养，采用3%低氧环境、不同浓度基质细胞衍生因子-1（SDF-1）（1 ng/mL，10 ng/mL，100 ng/mL）进行预处理，24 h后采用CCK-8试剂盒检测细胞活性，流式细胞仪及免疫荧光染色检测MSCs表面CXCR4表达情况。结果：两种预处理方式对MSCs活性无影响，经低氧和SDF-1预处理后MSCs表面CXCR4表达提高，低氧处理较SDF-1效果更好。结论：低氧环境对提高间充质干细胞表面CXCR4表达具有更好的效果。     

膜微粒抑制缺氧无血清诱导的骨髓间充质干细胞的凋亡

目的：明确心肌梗死后血液来源的膜微粒（MPs）抑制缺氧无血清诱导的骨髓间充质干细胞（BMSCs）凋亡的作用并探讨其相关机制。方法：利用结扎SD大鼠左冠脉前降支的方法来制作急性心肌梗死模型,然后提取血液中的MPs。提取培养SD大鼠的BMSCs,并建立缺氧无血清的干细胞凋亡模型。分别按照不同浓度（即0.5 μg/mL、1 μg/mL和2 μg/mL）的MPs对干细胞进行预处理,分别利用Hoechst染色、Tunel检测、AnnexinV/PI流式细胞学、Western blot检测caspase-3来揭示MPs抑制BMSCs的凋亡作用,并用Akt信号通路的通路抑制剂AZD5363来初步探讨其抗凋亡的相关机制。结果：大鼠心肌梗死后血液来源的MPs能够有效抑制缺氧无血清诱导的BMSCs的凋亡（P＜0.01）;并且其抗凋亡作用呈现出浓度依赖性,0.5 μg/mL 浓度的MPs亦能达到显著抗凋亡作用（P＜0.05）。其机制主要是通过激活Akt信号通路来实现。结论：心肌梗死后血液来源的MPs能有效抑制缺氧无血清诱导的BMSCs的凋亡。     

Inhibition of hypoxia and serum deprivation-induced apoptosis by salvianolic acid in rat mesenchymal stem cells

OBJECTIVE:To investigate the influence and mechanism of salvianolic acid B(SalB) on apoptosis inhibition in rat bone marrow-derived mesenchymal stem cells(BMSCs) induced by hypoxia and serum deprivation(hypoxia/SD).METHODS:SalB concentration of 0.1,1,10 or 100 mg/L(drug groups) were investigated for their ability to inhibit apoptosis in rat BMSCs.BMSCs in both the apoptosis model and drug groups were cultured under hypoxic conditions for 6 h,after which cell apoptosis and change in mitochondrial membrane potential(MMP) were detected using flow cytometry.Activation of caspase-3 was detected using western blot analysis.RESULTS:Hypoxia/SD induced apoptosis in rat BMSCs.The early apoptosis rate was lower in the drug groups compared to the apoptosis model group(P<0.05).SalB was found to inhibit the reduction in MMP and decrease the activation of caspase-3.CONCLUSION:0.1,1 and 10 mg/L of SalB inhibits activation of caspase-3 and early apoptosis of rat BMSCs induced by hypoxia/SD and could therefore enhance the survival rate of grafted stem cells.     

低氧预处理对大鼠骨髓间充质干细胞(BMSCs)增殖、凋亡和坏死的影响

 目的 探讨低氧预处理对大鼠骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMSCs)增殖、凋亡和坏死的影响及其可能机制。方法 采用全骨髓细胞贴壁法分离培养BMSCs,通过细胞成脂、成骨分化诱导及流式细胞仪检测其表面标志物(CD29、CD90、CD45)鉴定BMSCs;运用siRNA干扰技术沉默低氧诱导因子 1α(hypoxia inducible factor,HIF 1α)基因。将细胞分成6组:正常培养组、正常培养+转染阴性对照组、正常培养+HIF 1α RNA干扰组、低氧预处理组、低氧培养+转染阴性对照组及低氧培养+HIF 1α RNA干扰组,分别采用四甲基偶氮唑盐(MTT)法、流式细胞仪和乳酸脱氢酶(lactic acid dehydrogenase,LDH)活力测定等方法,检测各组BMSCs的增殖、凋亡和坏死情况;Western blot和real time PCR 检测各组BMSCs中HIF 1α、葡萄糖转运子 1(glucose transporter,GLUT 1)及血管内皮生长因子(vascular endothelial growth factor,VEGF)的mRNA和蛋白的表达。 结果 培养的BMSCs具有成脂、成骨等多向分化潜能;流式检测呈现CD29和CD90高表达,CD45低表达;低氧预处理可促进BMSCs增殖,减轻坏死,对细胞凋亡无明显影响;低氧预处理组HIF 1α、GLUT 1及VEGF的蛋白及mRNA表达明显高于常氧培养组(P<0.05);给予低氧预处理组siRNA HIF 1α后其HIF 1α、GLUT 1及VEGF的蛋白及mRNA表达均明显低于未干扰前(P<0.05),且细胞增殖受抑制,细胞凋亡和坏死加重(与未干扰前相比,P<0.05)。 结论 低氧预处理可促进BMSCs的体外增殖,减轻坏死,其机制可能与低氧预处理后HIF 1α表达增加及上调其下游基因GLUT 1和VEGF表达有关。     

HIF-1α对间充质干细胞SDF-1/CXCR4的调控作用研究

[目的]探讨缺氧诱导因子-1α（hypoxia inducible factor-1α,HIF-1α）对间充质干细胞基质细胞衍生因子-1（stromal cell-derived factor-1,SDF-1）/CXC趋化因子受体4（chemokine receptor 4,CXCR4）的调控作用。[方法]贴壁法培养ICR小鼠骨髓间充质干细胞（mesenchymal stem cells,MSCs）;采用RNA干扰技术用SuperFectinTMⅡ转染HIF-1αsiRNA于MSCs;实验分为五组：常氧组、缺氧组、转染Control siRNA组、转染HIF-1αsiRNA常氧培养组、转染HIF-1αsiRNA缺氧培养组;RT-PCR检测HIF-1α、SDF-1α、CXCR4 mRNA表达水平;Western blotting检测HIF-1α、SDF-1α、CXCR4蛋白表达水平。[结果]同常氧组比较,缺氧组HIF-1α、SDF-1α、CXCR4 mRNA及蛋白表达水平提高（P〈0.05）;同转染Control siRNA组比较,转染HIF-1αsiRNA常氧培养组HIF-1α、SDF-1α、CXCR4 mRNA及蛋白表达水平降低（P〈0.05）;同缺氧组比较,转染HIF-1αsiRNA缺氧培养组HIF-1α、SDF-1α、CXCR4 mRNA及蛋白表达水平降低（P〈0.05）。[结论]缺氧可使MSCs的HIF-1α、SDF-1α、CXCR4的表达提高,常氧与缺氧条件下抑制HIF-1α的表达可使SDF-1α、CXCR4的表达降低,HIF-1α在间充质干细胞中对SDF-1/CXCR4有调控作用。     

PEP-1-SOD1融合蛋白预处理对大鼠骨髓间充质干细胞在缺氧无血清条件下的保护作用

目的:研究PEP-1-SOD1融合蛋白转导入大鼠骨髓间充质干细胞(MSCs)的能力及其在缺氧无血清条件下对MSCs的保护作用。方法:将纯化后的SOD1和PEP-1-SOD1蛋白分别加入到培养及鉴定后的第3代MSCs中,免疫荧光法和Western blot分析PEP-1-SOD1穿透入MSCs的变化,黄嘌呤氧化酶法分析其SOD酶活性。将2.5μmol/L PEP-1-SOD1预处理MSCs 45 min后去除PEP-1-SOD1,缺氧无血清条件下再处理24 h,Annexin V/PI双染标记,流式细胞术分析MSCs凋亡情况。结果:PEP-1-SOD1融合蛋白可以转导入MSCs,呈时间和剂量依赖性分布于胞浆和胞核,缺氧无血清条件下MSCs凋亡率明显降低(P<0.05)。结论:PEP-1-SOD1融合蛋白可以高效、稳定转导入MSCs,并对缺氧无血清所诱导的MSCs凋亡具有显著保护作用。     

甲状腺素在骨髓间充质干细胞成软骨诱导过程中的作用

目的探讨甲状腺素在诱导骨髓间充质干细胞(BMSCs)软骨形成的作用。方法在两种条件培养基中培养和诱导猪BMSCs,对照组用成软骨培养基(含10-7 mol/L地塞米松和10 ng/mL TGFβ1),甲状腺素(T3)用成软骨培养基加100 nmol/L T3。MTT法检测BMSCs单层增殖情况。4周后取两组BMSCs Pellets,进行组织学染色和半定量基因表达分析。结果T3组BMSCs增殖明显高于对照组(P˂0.05)。T3组BMSCs Pellets形成典型软骨陷窝样结构,甲苯胺蓝染色阳性,并且优于对照组(P˂0.05)。与对照组相比,T3组第4周软骨生成标志基因Ⅱ型胶原(Col-Ⅱ)、聚集蛋白聚糖(aggrecan)和性别决定基因9(SOX9)的表达显著升高(P˂0.05),肥大标志基因Ⅹ型胶原(Col-Ⅹ),基质金属蛋白酶13(MMP13)基因的表达显著升高(P˂0.05),成骨基因Ⅰ型胶原(Col-Ⅰ),Runt相关转录因子2(RUNX2)表达在两组差异无统计学意义(P>0.05)。结论甲状腺素可促进间充质干细胞的增殖和软骨形成,同时诱导间充质干细胞分化的软骨细胞肥大,这些结果为研究软骨组织工程的诱导方案提供了有益的线索。     

骨髓基质细胞衍生因子1及其受体在骨髓增生异常综合征患者中的表达

目的 探讨基质细胞衍生因子1(SDF-1)与其受体趋化因子受体CXCR4在骨髓增生异常综合征(MDS)发病中的可能作用,为MDS的治疗寻找有意义的靶点.方法 收集2006年10月至2010年6月59例MDS病例,根据国际预后积分系统(IPSS)分为低危和高危两组,分别为33例和26例,以10份正常的骨髓标本作为对照.采集骨髓标本,检测骨髓血浆中SDF-1的含量、CD34+细胞CXCR4的表达率、CD34+细胞的凋亡率及血管内皮生长因子(VEGF)在骨髓血浆中的表达.结果 SDF-1在低危组和高危组患者骨髓血浆中的含量[(2301±413)、(1173 ±501)ng/L]显著高于对照组[(689±190)ng/L,P＜0.05],低危组显著高于高危组(P＜0.05).CD34+细胞CXCR4的表达在高危组(68.1％±18.8％)显著高于低危组(21.0％±9.7％)和对照组(19.4％±5.3％)(P＜0.05),在后两组的表达率差异无统计学意义(P＞0.05).CD34+细胞的凋亡率在低危组、高危组和对照组分别为54.8％±10.2％,24.3％±7.9％,l8.5％±8.7％,前组显著高于后两组(P＜0.05).骨髓血浆中VEGF的含量在低危组、高危组、对照组分别为(286±97)、(407±168)、(157±46)ng/L,差异有统计学意义(P＜0.05).相关分析显示:低危组CD34+细胞的凋亡率与骨髓血浆SDF-1的含量呈正相关(r =0.805,P＜0.05);高危组血浆VEGF的含量与CD34+细胞CXCR4的表达呈正相关(r=0.683,P＜0.05).结论 SDF-1/CXCR4在MDS中存在异常表达,且与骨髓细胞的凋亡和血管新成具有相关性,针对该生物轴的干预可为该病的治疗提供新的靶点.     

维甲酸和甲状腺激素在骨髓间充质干细胞软骨分化中的作用

目的 探讨甲状腺素(thyroxine, T3)和维甲酸(retinoic acid, RA)对骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)软骨形成的影响。方法 在3种条件培养基中培养和诱导猪BMSCs成软骨分化：对照组使用软骨生成培养基(含10^-7 mol/L地塞米松和10 ng/mL转化生长因子β1),RA组采用软骨生成培养基加1μmol/L RA,T3组采用软骨生成培养基加100 nmol/L T3。采用MTT法单层检测BMSCs的增殖情况。收获第3代BMSCs,离心形成细胞颗粒,分为3组分别进行软骨细胞诱导。4周后采集3组的细胞颗粒,通过组织学染色和半定量基因表达分析进行软骨形成评估。结果 T3组BMSCs增殖高于对照组(P<0.05);RA组BMSCs增殖低于对照组(P<0.05);对照组BMSCs颗粒中观察到软骨特征的类腔隙结构和甲苯胺蓝阳性染色,RA组未见;RA组软骨发生标志基因ColⅡ、蛋白聚糖(aggrecan)和Sox9的表达与对照组相比显著降低,而肥大标志基因ColX的表达...     

GDNF基因修饰的BMSCs对大鼠缺氧复氧神经细胞凋亡的实验研究

目的 研究腺病毒介导的外源性胶质细胞源性神经营养因子(GDNF)基因在大鼠骨髓间充质干细胞(BMSCs)中的表达及其对缺氧复氧神经细胞凋亡的影响.方法 将GDNF基因重组腺病毒感染大鼠BMSCs,采用酶联免疫吸附试验(ELISA)技术检测外源性GDNF基因在BMSCs中的表达水平;将GDNF基因修饰的BMSCs与缺氧复氧神经元共培养,观察神经细胞的凋亡情况.结果 GDNF基因感染组在感染后3～12 d能稳定表达GDNF蛋白,空质粒载体感染组和未感染组基本上无GDNF蛋白的表达;与BMSCs共培养组相比,BMSCs/GDNF分泌的GDNF能显著降低缺氧复氧神经细胞的凋亡率(复氧12 h至5 d,P<0.05).结论 腺病毒介导的基因感染技术能够有效地将GDNF基因转至BMSCs中,表达和分泌有生物学活性的GDNF蛋白,这为进一步研究GDNF基因修饰的BMSCs应用于中枢神经系统疾病的治疗研究奠定了基础.     

骨髓间充质干细胞对INS-1细胞凋亡的影响

目的探讨骨髓间充质干细胞(BMSCs)对过氧化氢(H2O2)诱导胰岛β细胞凋亡的影响。方法以H2O2作用于大鼠胰岛瘤细胞INS-1细胞构建凋亡模型,通过Transwell装置实现INS-1细胞与BMSCs共培养。实验分为4组:单独INS-1细胞培养组(A组);INS-1+H2O2处理组(B组);BMSC+INS-1Transwell共培养组(C组);BMSC+INS-1+H2O2Transwell共培养组(D组)。MTT法检测细胞存活率,Annexin-V/PI染色流式细胞技术检测细胞凋亡水平。结果 H2O2显著降低INS-1细胞存活率,引起INS-1细胞凋亡,且呈剂量依赖性,通过Tran-swell与BMSCs共培养后,由H2O2诱导的INS-1凋亡细胞比例下降,与单独H2O2刺激组相比,细胞凋亡率下降了约26.3%,差异有统计学意义(P<0.05)。结论 BMSCs通过Transwell共培养显著抑制H2O2诱导的INS-1细胞凋亡,其作用机制可能与BMSCs旁分泌作用有关,为进一步利用BMSCs防治糖尿病提供了理论依据。     

周围神经Wallerian变性不同时间点对干细胞归巢的趋化作用

目的 观察周围神经损伤后不同时间点Wallerian 变性对骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs） 归巢的影响。 方法 成年雄性SD 大鼠72 只,体质量220 ～ 250 g,按体质量编号随机分为6 组（n=12）,每组内再按注射细胞和药物的不同随机分为A、B 两组,A 组（n=6） 经尾静脉注射红色荧光间充质干细胞（red fluorescence proteinbone marrow mesenchymal stem cells,RFP-BMSCs） 和0.9% 氯化钠注射液,B 组（n=6） 经尾静脉注射RFP-BMSCs 和膜蛋白CXCR4 特异性拮抗剂（AMD3100） ;所有实验动物均在无菌条件下暴露坐骨神经,并于梨状肌下缘切断坐骨神经,近端结扎,远端旷置,然后分别在坐骨神经切断后的1 d、3 d、7 d、14 d、1 个月、2 个月行尾静脉RFP-BMSCs＋0.9% 氯化钠注射液（A 组） 和RFP-BMSCs＋AMD3100（B 组） 注射,并在注射后的第3 天行活体成像系统观察。 结果 随着时间的延长,RFPBMSCs在发生Wallerian 变性的坐骨神经中的聚集呈现先增高后降低的现象;坐骨神经Wallerian 变性的早期（1 ～ 3 d）,注射RFP-BMSCs＋AMD3100（B 组） 的RFP-BMSCs 聚集现象明显弱于RFP-BMSCs＋0.9% 氯化钠注射液（A 组）,其差异有统计学意义。 结论 干细胞可以向发生Wallerian 变性的神经组织聚集,在不同的时间点,其归巢现象存在先增高后降低的现象;在Wallerian 变性的早期,其归巢现象可能部分受到SDF-1-CXCR4 轴的调控,可以被CXCR4 特异性拮抗剂AMD3100 所抑制。     

Direct contact with bone marrow stromal cells promotes the invasions of SHI-1 leukemia cells

Background Interactions of tumor cells with the microenvironment were deemed to promote the tumor invasion and metastasis.CXC chemokine receptor 4 (CXCR4) and extracellular matrix metalloproteinase ind...     

CSE过表达保护低氧/无血清诱导骨髓间充质干细胞凋亡的机制

目的 探讨胱硫醚-γ-裂解酶(CSE)过表达保护低氧/无血清(H/SD)诱导骨髓间充质干细胞(MSCs)凋亡的机制.方法 体外分离培养大鼠MSCs,利用H/SD诱导MSCs凋亡,构建CSE基因的慢病毒表达载体pLV-ZsGreen-CSE lentivirus及空载体pLV-ZsGreen lentivus并感染MCSs(分别记为CSE MSCs、GFP MSCs).设立正常培养的MSCs(Norm MSCs组)、CSE MSCs组、GFP MSCs组,PI染色流式细胞术检测细胞凋亡率,Western blot法检测CSE、Bcl-2、Bax及细胞色素c(Cyt c)蛋白的表达.结果 与Norm MSCs组及GFP MSCs组相比,CSE MSCs组CSE蛋白表达显著增高(P<0.01),在H/SD 12 h状态下,CSEMSCs组凋亡率显著降低(P<0.01),CSEMSCs组Bcl-2蛋白表达水平显著高于GFP MSCs组及Norm MSCs组(P<0.01),CSEMSCs组Bax蛋白表达水平显著低于GFP MSCs组及NormMSCs组(P<0.01);CSEMSCs组线粒体Cyt c蛋白表达水平显著高于GFPMSCs组及NormMSCs组(P<0.01),CSEMSCs组胞质Cyt c蛋白表达水平显著低于GFP MSCs组及Norm MSCs组(P<0.01).NormMSCs与GFPMSCs组上述指标比较,差异均无统计学意义.结论 CSE过表达保护H/SD诱导MSCs凋亡,其机制与抑制线粒体凋亡途径有关.     

预缺氧对大鼠低压缺氧性脑损伤保护作用的实验研究

目的 探讨预缺氧对严重低压缺氧引起大鼠海马神经细胞损伤及学习记忆能力降低的保护作用.方法 ①Wistar大鼠分为对照组、单纯缺氧组、预缺氧复合缺氧组.光镜和电镜观察大鼠海马CA1区神经元形态变化;TUNEL法检测海马CA1区神经元凋亡.②单纯缺氧组和预缺氧复合缺氧组大鼠进行穿梭箱主动回避训练,随后进行低压缺氧,观察大鼠学习记忆能力的改变.结果 预缺氧复合缺氧组大鼠CA1区神经元病理改变较轻,神经元凋亡显著低于单纯缺氧组(P＜0.05);预缺氧复合缺氧组大鼠在严重缺氧后受电击次数和主动逃避时间的增加显著低于单纯缺氧组(P＜0.05).结论 缺氧引起大鼠海马CA1区神经元凋亡等病理改变,并导致学习记忆能力降低;预缺氧对海马CA1区神经元的缺氧性损伤具有保护作用,并能有效减轻严重缺氧所导致的学习记忆能力下降.     

IGF-1对2,5-己二酮诱导的骨髓间充质干细胞凋亡的拮抗作用研究

目的 研究胰岛素样生长因子-1（IGF-1）对2,5-己二酮（HD）暴露所致的骨髓间充质干细胞（BMSCs）凋亡的拮抗作用及其机制。方法 将BMSCs随机分成对照组（Control组）、IGF组、染毒组（HD组）和3个干预组（IGF+HD组）,即用生理盐水、50 ng/L IGF-1、40 mmol/L HD及不同浓度IGF-1（50,75,100 ng/L）共同处理24 h。然后,通过TUNEL法检测BMSCs的凋亡,应用试剂盒测定caspase-3的活性,应用western blot技术检测Akt和Bad的蛋白表达及磷酸化水平。结果 HD组BMSCs凋亡率及caspase-3活性显著高于Control组（P<0.05）。而经IGF-1干预后,上述指标显著低于HD组（P<0.05）。同时,HD组BMSCs的Akt和Bad蛋白磷酸化水平显著低于Control组（P<0.05）。而经IGF-1干预后,Akt和Bad的蛋白磷酸化水平显著高于HD组（P<0.05）。此外,各组间的Akt和Bad总蛋白的表达水平无统计学差异（P>0.05）。结论 IGF-1可通过调控Akt/Bad信号通路的活性,拮抗HD诱导的BMSCs凋亡。     

Proliferation and apoptosis property of mesenchymal stem cells derived from peripheral blood under the culture conditions of hypoxia and serum deprivation

Background  The proliferation and apoptosis property of mesenchymal stem cells derived from peripheral blood (PB-MSCs) were investigated under hypoxia and serum deprivation conditions in vitro so as to evaluate the feasibility for autologous PB-MSCs applications in cartilage repair.

Methods  MSCs were mobilized into peripheral blood by granulocyte colony stimulating factor (G-CSF) and AMD3100. The blood samples were collected from central ear artery of rabbits. Adhered cells were obtained by erythrocyte lysis buffer and identified as MSCs by adherence to plastic, spindle shaped morphology, specific surface markers, differentiation abilities into osteoblasts, adipocytes and chondroblasts in vitro under appropriate conditions. MSCs were cultured in four groups at different oxygen tension (20% O₂ and 2% O₂), with or without 10% fetal bovine serum (FBS) conditions: 20% O₂ and 10% FBS complete medium (normal medium, N), 20% O₂ and serum deprivation medium (D), 2% O₂ and 10% FBS complete medium (hypoxia, H), 2% O₂ and serum deprivation (HD). Cell proliferation was determined by CCK-8 assay. Apoptosis was detected by Annexin V/PI and terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) staining.

Results  Spindle-shaped adherent cells were effectively mobilized from peripheral blood by a combined administration of G-CSF plus AMD3100. These cells showed typical fibroblast-like phenotype similar to MSCs from bone marrow (BM-MSCs), and expressed a high level of typical MSCs markers CD29 and CD44, but lacked in the expression of hematopoietic markers CD45 and major histocompatibility complex Class II (MHC II). They could also differentiate into osteoblasts, adipocytes and chondroblasts in vitro under appropriate conditions. No significant morphological differences were found among the four groups. It was found that hypoxia could enhance proliferation of PB-MSCs regardless of serum concentration, but serum deprivation inhibited proliferation at the later stage of culture. Apart from that, hypoxia or serum deprivation could promote the apoptosis of PB-MSCs after 48 hours; the effect was stronger when these two conditions combined together. Furthermore, the effect of serum deprivation on apoptosis was stronger compared with that of hypoxia.

Conclusions  PB-MSCs possess similar phenotypes as BM-MSCs. Their differentiation and proliferation abilities make them a new source of seed cells for ischemia-related cell therapy and tissue engineering in the field of the articular cartilage repair.

     

COA:The proliferation and apoptosis property of MSCs derived from peripheral blood under the culture conditions of hypoxia and serum deprivation

Background We investigated the proliferation and apoptosis property of mesenchymal stem cells derived from peripheral blood (PB-MSCs) under hypoxia and serum deprivation conditions in vitro so as to evaluate the feasibility for autologous PB-MSCs applications in cartilage repair. Methods MSCs were mobilized into peripheral blood by Granulocyte Colony Stimulating Factor (GCSF) and AMD3100. The blood samples were collected from central ear artery of rabbits. Adhered cells were obtained by erythrocyte lysis buffer and identified as MSCs by adherence to plastic, spindle shaped morphology, specific surface markers, differentiation abilities into osteoblasts, adipocytes and chondroblasts in vitro under appropriate conditions. MSCs were cultured in four groups at different oxygen tension (20% O2 and 2% O2), with or without serum (10% FBS and 0% FBS) conditions: 20% O2 and 10% FBS complete medium (normal mediun, N), 20% O2 and serum deprivation medium (D), 2% O2 and 10% FBS complete medium (hypoxia, H), 2% O2 and serum deprivation (HD). Cell proliferation was determined by CCK-8 assay. Apoptosis was detected by Annexin V/PI and TUNEL staining. Results Spindle-shaped adherent cells were effectively mobilized from peripheral blood by a combined administration of GCSF plus AMD3100. These cells showed typical fibroblast-like phenotype similar to MSCs from bone marrow (BM-MSCs), and expressed a high level of typical MSC markers CD29 and CD44, but lacked in the expression of hematopoietic markers CD45 and MHC II. They could also differentiate into osteoblasts, adipocytes and chondroblasts in vitro under appropriate conditions. No significant morphological differences were found among the four groups. It was found that hypoxia could enhance proliferation of PB-MSCs regardless of serum concentration, but serum deprivation inhibited proliferation at the later stage of culture. Apart from that, hypoxia or serum deprivation could promote the apoptosis of PB-MSCs after 48 hours, the effect was stronger when these two conditions combined together. Furthermore, the effect of serum deprivation on apoptosis was stronger compared with that of hypoxia. Conclusions PB-MSCs possess similar phenotypes as BM-MSCs. Their differentiation and proliferation abilities make them a new source of seed cells for ischemia-related cell therapy and tissue engineering in the field of the articular cartilage repair.     

甘露醇对骨髓间充质干细胞治疗血管性痴呆大鼠行为学的影响

目的：观察甘露醇预处理对骨髓间充质干细胞（BMSCs）静脉移植治疗血管性痴呆模型大鼠行为学的影响。方法：以双侧颈总动脉永久性结扎法（2-VO）制作大鼠血管性痴呆模型。造模成功后,随机分成培养基组8只（注射1 mL无血清培养基）、BMSCs移植治疗组11只（注射1×10^6个BMSCs 1 mL）及甘露醇预处理BMSCs移植治疗组9只（注射1.5 g/kg甘露醇后,在10-30 min之内注射1×10^6个BMSCs 1 mL）。通过Morris水迷宫实验检测血管性痴呆大鼠模型移植治疗前与移植4周后空间学习记忆能力,比较各组之间差异。结果：与培养基组比较,BMSCs移植治疗组和甘露醇预处理BMSCs移植治疗组大鼠空间学习记忆能力都有改善,模型大鼠逃避潜伏期缩短（P〈0.05）,平台象限区滞留时间延长（P〈0.05）;与BMSCs移植治疗组比较,甘露醇预处理BMSCs移植治疗组大鼠空间学习记忆能力改善更加明显,逃避潜伏期缩短（P〈0.05）,平台象限区滞留时间延长（P〈0.05）。结论：甘露醇预处理后BMSCs静脉移植组治疗血管性痴呆与单独BMSCs静脉移植组大鼠比较,学习记忆能力显著改善。其机制可能与甘露醇能开放血脑屏障（BBB）,增加进入脑内的BMSCs数量相关。