人骨髓间充质干细胞体外心肌样细胞诱导分化中细胞周期与凋亡的分析

目的探讨体外诱导人骨髓间充质干细胞(hMSCs)向心肌样细胞分化过程中分化与凋亡的情况,为hMSCs临床移植治疗提供数据。方法采用体外纯化、扩增后的第4代hMSCs,分3组在体外分别经0μmol/L、2.5　μmol/L、5.0μmol/L 5-杂氮胞苷(5-Aza)诱导24 h后继续培养8周,相差显微镜下观察细胞形态变化,流式细胞仪检测各组细胞周期及凋亡指数,应用RT-PCR技术分析肌球蛋白重链(β-MHC)、肌钙蛋白T(cTNT)、凋亡相关基因(Bcl-2、Bax)的mRNA在分化过程中的动态表达。结果hMSCs诱导后凋亡指数随诱导后培养时间增加,低浓度诱导组低于标准浓度诱导组,组间差异有统计学意义(P<0.05),G_0/G_1期细胞比例诱导后较对照组显著减少(P<0.05);β-MHC和CTNT在诱导后表达开始增强,在第6周时均达到高峰,第8周时表达开始衰减,Bcl-2、Bax基因表达呈时间依赖性变化。结论hMSCs在体外经纯化、扩增和诱导后可表现心肌样细胞的生物学特性,但诱导过程中hMSCs发生细胞凋亡,采用2.5μmol/L 5-Aza低浓度诱导可减少细胞凋亡发生。     

人脐血间充质干细胞诱导分化成心肌细胞的研究

目的探讨体外脐血间充质干细胞诱导分化成心肌细胞的可行性和最佳方法。方法收集获知情同意的健康产妇脐血细胞,分离单个核细胞,从中进一步分离间充质干细胞,传代培养至第3代,应用免疫荧光流式细胞仪标记间充质干细胞特异性抗原CD34、CD44和CD90。5-氮胞苷诱导分化4周后,免疫组织化学染色和RT-PCR法分别检测心肌细胞标记物肌钙蛋白Ⅰ、转录因子GATA 4和β-肌球蛋白重链。结果在第3代细胞中,可检测到CD44、CD90的表达,未检测到CD34的表达。脐血间充质干细胞经5-氮胞苷诱导分化后,呈现成纤维细胞样形态和克隆增殖特点。免疫组织化学染色和RT-PCR可检测到肌钙蛋白Ⅰ,GATA 4和β-肌球蛋白重链的表达。结论脐血间充质干细胞能够被诱导分化成心肌样细胞,可成为干细胞移植的细胞来源。     

静脉注射人脐血间充质干细胞对大鼠脑损伤细胞凋亡的影响

目的观察脑损伤后静脉注射人脐血间充质干细胞(HUCBMSC)对大鼠脑损伤细胞凋亡的影响,探讨其脑保护的作用机制。方法清洁级健康雄性SD大鼠90只,随机分为假伤组、损伤组和注射HUCBMSC治疗组(治疗组)。每组30只。每组按时间再分为注射后3、7、14、21和28d,每个时间点6只。采用改进的Feeney自由落体法制作大鼠创伤性脑损伤模型。治疗组尾静脉注入3×106个HUCBMSC,损伤组及假伤组注入等体积的PBS液。在不同时间点,采用HE染色、免疫组织化学法、原位杂交法对大鼠脑组织形态学变化、5-溴脱氧尿核苷标记、细胞凋亡情况进行检测。结果假伤组细胞凋亡呈弱阳性表达。与假伤组比较,损伤组在相同时间点脑组织凋亡细胞明显增多,差异有统计学意义(P<0.05,P<0.01);与损伤组比较,治疗组在相同时间点脑组织凋亡细胞明显减少,差异有统计学意义(P<0.05,P<0.01)。结论在创伤性脑损伤中,静脉注射HUCBMSC可以减少损伤局部细胞凋亡,改善局部微环境,促进神经元的修复。     

微环境中人骨髓间充质干细胞向心肌细胞表型分化的实验研究

目的 探讨人骨髓间充质干细胞(hBMSC)在非接触共培养的微环境中向心肌细胞分化的能力.方法 密度梯度离心法分离培养hBMSC,用流式细胞仪鉴别其表面标记特征.采用transwell按1:10的比例共同培养hBMSC和SD乳鼠心室肌细胞.用聚合酶链反应检测心肌转录因子GATA4的基因表达水平.免疫细胞化学、免疫荧光检测横纹肌α-辅肌动蛋白、结蛋白、肌钙蛋白(cTn)T和cTnI等心肌细胞标志蛋白的表达.结果 hBMSC的标志物主要为CD29(98.64%±0.80%)和CD44(96.70%±1.50%),而极少表达CD105(2.21%±0.60%)、CD11b(0.80%±0.05%)、CD45(1.39%±0.13%)和CD34(1.73%±0.08%).与心肌细胞共培养后第7天可在hBMSC细胞中观察到少量搏动的细胞,随着诱导时间的增加,细胞的搏动更规则、有力.与心肌发育有关的转录因子GATA4基因在诱导后1、2、3周均有表达.共培养后2周,检测的心肌标志蛋白均有阳性表达,分别是结蛋白,α-辅肌动蛋白,cTnI和cTnT.荧光标记也检测到cTnT和cTnI的阳性表达.结论 hBMSC具有向心肌细胞分化的潜能,在共培养的微环境中,可以分化成心肌细胞表型,其机制是通过心肌细胞的旁分泌作用,而不需要hBMSC与心肌细胞的直接接触.     

间充质干细胞治疗心肌梗死研究进展

<正>2001年德国移植免疫和细胞治疗研究所Strauer等首次对心肌梗死(MI)患者尝试自体骨髓干细胞移植,10周后发现患者左心室周长缩小,心脏指数、LVEF、心输出量等均有明显改善,坏死心肌的平均面积由24.6%减至15.7%。目前,虽未能广泛应用于临床治疗,但已能预见其广阔前景。1 MI治疗现状     

系统性红斑狼疮患者骨髓间充质干细胞凋亡相关研究

目的 探讨系统性红斑狼疮(SLE)患者骨髓间充质干细胞(BMSCs)的凋亡变化及凋亡相关因子的表达.方法 密度梯度离心和贴壁筛选法分离培养SLE患者及健康人骨髓MSCs,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测细胞凋亡;流式细胞术检测细胞表面Fas、Bcl-2表达水平及Caspase 8活性;实时荧光定量聚合酶链反应(PCR)技术榆测Fas、Bcl-2、Bax、Bcl-w、Caspase 8及凋亡细胞蛋白酶激活因子(Apaf)-1 mRNA表达水平;免疫组织化学染色检测胞质细胞色素C表达水平;统计学采用Mann-Whitnev秩和检验.结果 SLE患者BMSCs凋亡明显高于健康对照组[(64±10)%和(14±9)%,U=0,P<0.05];SLE患者BMSCs抗凋亡因子Bcl-2蛋白表达显著低于健康对照组[(11±9)%和(56±18)%,U=0,P<0.05],其mRNA表达水平同样显著低于健康对照(0.2±0.2和2.4±0.7,U=24.P<0.05);SLE患者BMSCs胞质中细胞色素C表达显著增多[(56±21)%和(16±16)%,U=1,P<0.05].SLE患者BMSCs细胞内Caspase 8活性增强[(49±14)%和(16±12)%,U=0,P<0.05],但基因表达水平与健康对照组比较差异无统计学意义(U=28,P>0.05);SLE患者与健康对照组BMSCs均高表达Fas,差异无统计学意义(U=19,P>0.05).结论 SLE患者BMSCs凋亡增多,可能与Bcl-2表达减少、胞质中细胞色素C表达增多和Caspase 8活性增强,从而激活内源性及外源性凋亡途径相关.

Abstract：

Objective To investigate the apoptosis of bone marrow mesenchymal stem cells(BMSCs)from systemic lupus erythematosus(SLE)patients and the expression of apoptotic molecules.Methods BMSCs were isolated from bone marrow of SLE patients and normal controls by density eentrifugation and adhesive culture in vitro.The apoptosis of BMSCs were evaluated by TUNEL assay.The expressions of Fas.Bcl-2 and the activity of Caspase 8 were detected by flow cytometry.Real-time PCR technique was used to determine the gene expressions of Fas,Bcl-2,Bax,Bcl-w,Caspase 8 and Apaf-1.Meanwhile,cytochrome C was detected by immunocytochemistry.Statistical analysis was conducted with t-test and Mann-Whitney rank test.Results The percentage of apoptotic BMSCs increased in SLE patients compared with healthy donors[(64±10)%vs [14±9)%,U=0,P<0.05 ].The expression of Bcl-2 in BMSCs of SLE patients was lower than the normal controls at protein leve[(11±9)%vs(56±18)%,U=0,P<0.05 ],and mRNA level(0.2±0.2vs 2.4±0.7,U=24,P<0.05).More cytochrome C positive pellets in the cytosolic fraction could be detected in BMSCs from SLE patients compared with healthy controls [(56±21)%vs (16±16)%,U=1,P<0.05].The activity of Caspase 8 was enhanced[(49±14)%vs(16±12)%,U=0.P<0.05 ],although with no significant difierence at mRNA Ievel.Both groups expressed Fas but with no significant difference (U=19,P>0.05).Conclusion BMSCs from SLE patients undergo more apoptosis,the mechanisms may be associated with the down regulation of Bcl-2,up-regulation of Cytoehrome C in cytoplasm and the activation of Caspase 8,which directs the intrinsic and extrinsic apoptosis pathways.     

脐血间充质干细胞对T细胞分泌因子调节作用的实验研究

从脐血中分离和培养间充质干细胞(UCB-MSCs),流式细胞术检测其表面标志以鉴定纯度;用Brdu法测定细胞增殖率,观察UCB-MSCs对异体T淋巴细胞增殖的影响;且用ELISA法检测共培养上清细胞因子IL-4、INF-γ的表达水平.发现从脐血获得的MSCs免疫表型分析显示,其不表达HLA-Ⅱ抗原.UCB-MSCs对PHA刺激的脐带血T淋巴细胞的增殖反应具有抑制作用,呈细胞数量剂量依赖性;UCB-MSCs抑制了共培养体系中INF-γ分泌,然而促进了IL-4的分泌,与对照组比较具有统计学差异(P＜0.01).认为UCB-MSCs对免疫反应具负调节作用,可能为移植物抗宿主病(GVHD)的防治提供一条新途径.     

洛伐他汀抑制大鼠骨髓间充质干细胞凋亡的实验研究

目的体外以缺氧无血清条件模拟心肌梗死后的心脏缺血微环境,研究洛伐他汀是否能够抑制缺氧无血清引起的骨髓间充质干细胞（MSCs）凋亡。方法以Hoechst33342染色法及AnnexinV／PI流式细胞术检测洛伐他汀的抗凋亡作用,检测线粒体膜电位变化,进一步采用Western blot方法检测洛伐他汀对细胞色素C释放及caspase-3活化的影响。结果洛伐他汀0．01～1μmol／L能够有效地抑制缺氧无血清引起的MSCs凋亡。洛伐他汀抑制线粒体凋亡途径,增强线粒体膜电位水平、抑制细胞色素C释放,降低caspase-3活化水平。结论洛伐他汀能够抑制线粒体途径介导的凋亡,阻止caspase-3的活化,发挥抗缺氧无血清引起的MSCs凋亡,为提高移植干细胞的存活率提供了一种可能有效的干预措施。     

体外不同诱导条件对人脐血间充质干细胞分化为神经细胞的研究

目的观察体外不同诱导条件对人脐血间充质干细胞(MSCs)向神经细胞分化的作用,探讨最佳诱导方法。方法取第3代脐血MSCs进行诱导,分为化学诱导剂组、生长因子诱导组、丹参素联合生长因子诱导组。采用免疫细胞化学方法和免疫荧光方法检测诱导前后神经元特异性标志小鼠抗神经元核抗原(NeuN)、兔抗微管蛋白(β-TubulinⅢ)和星形胶质细胞特异性标志神经胶质纤维酸性蛋白(GFAP)表达的变化。结果 MSCs经3种方法诱导后出现类似神经元样细胞的形态改变,伸出长突起。免疫组织化学和免疫荧光方法鉴定显示,诱导后的细胞能特异性表达NeuN和β-TubulinⅢ,而GFAP阳性细胞较少。丹参素联合生长因子诱导组β-TubulinⅢ及NeuN的阳性细胞率均明显高于生长因子诱导组和化学诱导剂组,而GFAP阳性细胞率明显低于生长因子诱导组,差异均有统计学意义。结论丹参素联合表皮生长因子和碱性成纤维细胞生长因子在体外可定向诱导人脐血MSCs分化为神经元,诱导效果最佳。     

骨髓间充质干细胞移植对脑缺血大鼠脑组织凋亡相关蛋白的影响

目的 探讨经静脉移植的骨髓间充质干细胞(MSCs)在脑缺血大鼠脑组织的分布情况及对凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶3(Caspase 3)及Bcl-2蛋白表达的影响.方法 体外培养及扩增MSCs后,用绿色荧光染料羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)标记,通过静脉途径移植给大脑中动脉缺血2 h再灌注的SD大鼠,按不同时间点取材,荧光显微镜下观察MSCs在脑内的分布,免疫组化染色检测大鼠脑内Caspase 3、Bcl-2蛋白表达情况.结果 移植组6、12、24、72 h及7 d时,Caspase 3免疫组化阳性目标面密度分别为(2.81±0.35)%、(3.98±0.67)%、(5.58±0.92)%、(3.51±0.63)%、(1.64±0.29)%,明显低于对照组[(3.92±0.44)%、(5.23±0.30)%、(6.89±0.57)%、(4.39±0.57)%、(2.29±0.21)%],两组比较差异有统计学意义(t值分别为4.37、3.34、2.60、2.32、3.90,P＜0.05或＜0.01);移植组6、12 h及7 d,Bcl-2阳性目标面密度分别为(4.70±0.16)%、(5.61±0.26)%、(3.00±0.28)%,明显高于对照组[(3.28±0.27)%、(4.54±0.59)%、(2.15±0.62)%],两组比较差异有统计学意义(t值分别为8.32、3.25、2.54,P＜0.05或＜0.01).结论 MSCs可能通过下调Caspase 3蛋白表达、上调Bcl-2蛋白表达的方式对脑缺血再灌注损伤起保护作用.     

Safety and effect of umbilical cord blood –derived mesenchymal stem cells on apoptosis of human cardiomyocytes

Objective To study the safety and effect of the umbilical cord blood （UCB）-derived mesenchymal stem cells （MSCs） on apoptosis of human cardiomyocytes （HCM）. Methods UCB was collected at the time of delivery with informed consent obtained from 10 donors. The UCB-derived MSCs were treated with 5-azaserube （5-AZA） and were further induced to differentiate into cardiomyocytes. Telomerase activity, G-banding patterns of chromosomal karyotypes, tumor formation in nude mice, RT-PCR, and the effect of inhibiting apoptosis of HCM were investigated. Results MSCs derived from UCB were differentiated into cardiomyocytes in vitro, which possessed telomerase activity after 5-AZA induction, and no abnormal chromosomal karyotypes were observed. Expression of p53, cyclin A, cdk2, ~3 -actin, C-fos, h-TERT and c-myc were similar in MSCs before and after 5-AZA treatment. There was no tumor formation in nude mice after injection of UCB-derived MSCs. UCB-derived MSCs significantly inhibited apoptosis of HCM. Conclusion UCB-derived MSCs are a valuable, safe and effective source of cell-transplantation treatment .     

Mid-trimester fetal blood-derived adherent cells share characteristics similar to mesenchymal stem cells but full-term umbilical cord blood does not

Stem cell transplantation is a promising treatment for many conditions. Although stem cells can be isolated from many tissues, blood is the ideal source of these cells due to the ease of collection. Mesenchymal stem cells (MSCs) have been paid increased attention because of their powerful proliferation and pluripotent differentiating ability. But whether MSCs reside in blood (newborn umbilical cord blood and fetal or adult peripheral blood) is also debatable. The present study showed that MSC-like cells could be isolated and expanded from 16-26 weeks fetal blood but were not acquired efficiently from full-term infants' umbilical cord blood (UCB). Adherent cells separated from postnatal UCB were heterogeneous in cell morphology. Their proliferation capacity was limited and they were mainly CD45+, which indicated their haematopoietic derivation. On the contrary, MSC-like cells shared a similar phenotype to bone marrow MSCs. They were CD34- CD45- CD44+ CD71+ CD90+ CD105+. They could be induced to differentiate into osteogenic, adipogenic and neural lineage cells. Single cell clones also showed similar phenotype and differentiation ability. Our results suggest that early fetal blood is rich in MSCs but term UCB is not.     

Preferential expansion of human umbilical cord blood-derived CD34-positive cells on major histocompatibility complex-matched amnion-derived mesenchymal stem cells

Background

We previously found in a murine hematopoietic system that hematopoietic stem cells show high differentiation and proliferation capacity on bone marrow-derived mesenchymal stem cells/stromal cells (microenvironment) with “self” major histocompatibility complex (MHC).

Design and Methods

We examined whether amnion-derived adherent cells have the characteristics of mesenchymal stem cells, and whether these adherent cells can support the proliferation of umbilical cord blood-derived lineage-negative and CD34-positive cells (Lin^–CD34⁺ cells) obtained from the same fetus to a greater extent than those derived from other fetuses.

Results

Culture-expanded amnion-derived adherent cells expressed mesenchymal stem cell markers and HLA-ABC molecules and could differentiate into osteoblasts, adipocytes and chondrocyte-like cells, indicating that the cells have the characteristics of mesenchymal stem cells. The Lin^–CD34⁺ cells purified from the frozen umbilical cord blood were strongly positive for HLA-ABC, and contained a large number of hematopoietic stem cells. When the Lin^–CD34⁺ cells were cultured on the autologous (MHC-matched) or MHC-mismatched amnion-derived adherent cells in short-term assays (hematopoietic stem cell-proliferation) and long-term culture-initiating cell assays, greater expansion of the Lin^–CD34⁺ cells was observed in the MHC-matched combination than in MHC-mismatched combinations. The concentration of granulocyte-macrophage colony-stimulating factor in the culture supernatants of the long-term culture-initiating cell assays was significantly higher in the MHC-matched combination than in MHC-mismatched combinations.

Conclusions

It is likely that a MHC restriction exists between hematopoietic stem cells and mesenchymal stem cells/stromal cells in the human hematopoietic system and that granulocute-macropage colony-stimulating factor contributes to some extent to the preferential hematopoiesis-supporting ability of the MHC-matched amnion-derived adherent cells.     

早产胎儿脐血和脐带间充质干细胞分离培养的比较

目的对比早产胎儿脐血和脐带间充质于细胞（MSCs）的培养成功率,探讨早产胎儿MSCs获取的最佳途径。方法无菌条件下采集早产胎儿（不足37周）脐血,密度梯度离心法分离单个核细胞,采用贴壁培养法获得脐血MSCs;将脐带剪成1mm^3大小组织块,采用组织块贴壁法获得脐带MSCs;分别观察脐血和脐带MSCs的生物学特性,免疫荧光法检测MSCs表面标记物的表达情况。结果采用Mesencult^TM培养基,早产胎儿脐血MSCs培养成功率为100％,脐带MSCs培养成功率为67％,明显低于脐血MSCs;早产胎儿脐血和脐带MSCs均表达CD29、CD44和CD90不表达造血干细胞表面标志CD34。结论早产胎儿脐血中较易获得MSCs,为临床移植治疗提供了理想的细胞来源。     

骨髓间充质干细胞及Klotho基因移植对慢性缺氧大鼠心脏重构的影响

目的观察骨髓间充质干细胞(BMSCs)及抗衰老基因(Klotho基因)移植对慢性缺氧大鼠心脏重构的影响。方法建立慢性缺氧28 d大鼠心脏重构模型,分为模型组、BMSCs组和Klotho组,并另设正常对照组不给予缺氧处理。分离、培养、鉴定大鼠BMSCs;重组慢病毒介导Klotho基因。模型建立结束后分别通过尾静脉移植BMSCs及Klotho基因至慢性缺氧大鼠体内,4周后用ELISA法检测血清和心肌中Ⅰ型前胶原羟基端肽(P I CP)和Ⅲ型前胶原羟基端肽(PⅢCP)的浓度,HE染色法观察心肌细胞的形态,MASSON染色检测心肌胶原含量,Tunel检测心肌细胞凋亡率。采用SPSS 17.0软件进行数据处理。计量资料以均数±标准差(±s)表示,组间比较采用t检验。结果移植BMSCs和Klotho基因4周后,BMSCs组和Klotho组血清和心肌中的PⅠCP及PⅢCP浓度、胶原容积分数(CVF)和心肌细胞凋亡率均较模型组显著降低(P0.01)。与Klotho组比较,BMSCs组心肌细胞凋亡率显著降低(P0.01)。结论 BMSCs和Klotho基因移植均能有效逆转心脏重构,BMSCs在抗心肌细胞凋亡方面优于Klotho基因。     

脐带间充质干细胞 nestin mRNA表达的研究

目的：从基因角度求证脐带间充质干细胞（MSCs）神经巢蛋白nestin mRNA表达。方法取新鲜脐带组织行MSCs分离、培养及鉴定。采用胰酶消化法计数MSCs,流式细胞仪检测细胞活性,提取原代、一代、二代细胞的mR-NA,采用实时荧光定量PCR法检测nestin mRNA表达。结果脐带中分离出的细胞贴壁生长状况较好,其中大部分细胞呈梭形生长,传代后细胞与原代细胞生长形态相似,生长加快;个别细胞呈神经样生长。 MSCs呈nestin mRNA阳性表达,其表达量在传代后有明显减少;与nestin蛋白在神经前体细胞向神经细胞分化时的变化一致。结论脐带MSCs存在nestin mRNA表达;传代后nestin mRNA表达量虽减小,但仍可作为备选种子细胞进行移植。     

铁过载对脐带血来源的造血干祖细胞及造血支持细胞的作用观察

目的 探讨铁过载对脐带血(UCB)来源的造血干祖细胞及造血支持细胞,尤其是间充质干细胞(MSCs)的损伤作用.方法 体外培养脐带血单个核细胞(UCB-MNCs)和脐带血间充质干细胞(UCB-MSCs),向培养液中添加200 μmol/L的枸橼酸铁胺(FAC) 24 h建立铁过载模型.分为MNCs-CTL组、MNCs-FAC组、MSCs-CTL组、MSCs-FAC组,每组设3个复孔,实验重复3次.检测细胞内活性氧物质(ROS)水平变化、细胞增殖、分化、凋亡以及造血支持作用.结果 对UCB-MNCs进行铁过载,MNCs-FAC组造血集落形成单位(CFU-E、CFU-GM、BFU-E、CFU-mix)计数显著低于MNCs-CTL组(P＜0.05),MNCs-FAC组造血干细胞(CD+34)、髓系造血细胞(CD+33)、红系造血细胞(GlyA+)比例及计数均显著低于MNCs-CTL组(P均＜0.05);MNCs-FAC组的凋亡率高于MNCs-CTL组(P＜0.05).MSCs-FAC组的群体倍增时间明显长于MSCs-CTL组,且其凋亡率亦高于MSCs-CTL组(P＜0.05).结论 铁过载可抑制造血干祖细胞的增殖、分化,诱导其凋亡,也可抑制MSCs的增殖能力,诱导其凋亡,降低其造血支持能力,且此过程中ROS升高.     

脐血间充质干细胞的体外扩增及向类肝细胞分化的实验研究

目的探讨人脐带血间充质干细胞（UBC-MSCs）向类肝细胞定向分化的可能性。方法无菌条件下采集新生儿脐带血75份,采用密度梯度离心法与直接贴壁法相结合分离UBC－MSCs,体外培养传代,获得第4代集落生长细胞作流式细胞仪表面抗原测定,并应用a－FGF、b－FGF、HGF、OSM等多种成分,分阶段向肝细胞定向诱导分化。倒置相差显微镜观察细胞形态变化,取诱导后4周和6周的细胞分别采用RT-PCR法检测ALB、AFP和CK－19基因mRNA水平,分析类肝细胞诱导表达情况。结果按10。／ml接种,用含15％FBS的DMEM／F12培养基培养可成功获取UBC—MSCs。细胞呈长梭形,细胞表面抗原测定显示强烈表达CD29、CD44,而不表达造血细胞的标志物CD34、CD45。分阶段诱导6周后,细胞形态呈多角形,并检测到肝细胞表面标志物白蛋白、AFP和CK-19。结论新生儿脐血中可分离出MSCs,并可在体外进行培养扩增,分阶段诱导可分化为类肝细胞。