人脂肪干细胞体外培养及细胞载体复合物诱导培养

目的探讨人脂肪干细胞体外培养方法以及对细胞载体复合物诱导成软骨分化的潜能。方法从人脂肪组织分离人脂肪间充质干细胞(hADSCs),噻唑盐(MTT)法检测细胞生长情况,流式细胞术hADSCs细胞表面分子标志,hADSCs成脂、成骨和成软骨的诱导及鉴定检测。hADSCs与不同浓度胶原纤维蛋白溶液混合制成细胞载体复合物凝胶,14 d后进行细胞鉴定。结果体外分离、培养出高活性hADSCs,增值曲线呈S形。细胞CD29、CD90、CD105呈阳性表达,CD34、CD45呈阴性表达。hADSCs成脂诱导后细胞呈类圆形,在细胞浆内有透亮的脂滴出现,油红O染色脂滴呈鲜红色。成骨诱导后细胞呈为不规则形,免疫荧光检测细胞表达骨唾液酸蛋白、骨桥蛋白、骨连接蛋白。hADSCs成软骨诱导后细胞形态变为铺路石样,甲苯胺蓝染色细胞基质呈蓝色,免疫组化检测Ⅱ型胶原蛋白染色呈阳性。阿利新蓝染色细胞载体复合物成蓝色,免疫组化检测Ⅱ型胶原蛋白结果显示,混合胶原蛋白组较单一胶原蛋白组更强阳性表达。结论本研究成功分离培养出高度同源性hADSCs,具有多分化潜能。复合胶原纤维蛋白载体诱导hADSCs成软骨细胞分化,可能作为修复关节软骨缺损的理想载体支架材料。     

骨髓间充质干细胞体外诱导和端粒酶活性的研究

目的:建立骨髓间充质干细胞(MSCs)体外分离、培养、纯化的方法,探讨其体外诱导条件和端粒酶活性。方法:取正常成人骨髓用含10%小牛血清的LG-DMEM培养液培养、扩增。流式细胞仪行细胞表面抗原检测,细胞化学染色鉴定其生物学特性,在特定培养条件下检测其向成骨和脂肪细胞分化的能力,采用TRAP(Telomerase repeat amplification protocol assay)-银染法测定其端粒酶的活性。结果:分离培养获得的贴壁细胞,碱性磷酸酶染色阳性。流式细胞仪检测CD34、CD45表达阴性,CD29、CD44、CD115、CD166表达阳性。经向成骨和脂肪细胞诱导3周后,可得到成骨和脂肪细胞,经茜素红染色、油红O染色得到证实。TRAP-银染法证实MSCs表达一定的端粒酶活性。结论:体外分离培养的MSCs可以向成骨、脂肪诱导分化,表达一定的端粒酶活性,具有干细胞的生物学特性。     

传代和冻存对胎肺间充质干细胞生长及分化的影响

目的 观察传代和冻存对胎肺间充质干细胞（MSCs）生长及分化的影响.方法 以胶原酶消化法自流产胎儿肺组织中分离MSCs,在体外进行传代扩增、冻存、复苏;然后,以相差显微镜观察细胞形态学变化,以流式细胞仪检测表型变化,以神经分化诱导方案研究其向神经元样细胞分化的潜能.结果 经传代和冻存后,人胎肺MSCs的增殖能力、形态和表面标志物的表达均无明显变化,高表达MSCs特异性标志物,不表达造血和内皮细胞标志物,在适宜的神经诱导方案作用下可高效分化为神经元样细胞.结论 传代和冻存对人胎肺MSCs的形态、表型和向神经样细胞的分化潜能无明显影响,胎肺MSCs可作为细胞移植治疗神经系统疾病的备选来源.     

肿瘤坏死因子对于骨髓基质干细胞CXCR2表达的影响

目的观察肿瘤坏死因子(TNF-α)对于大鼠骨髓基质干细胞(MSCs)表达CXCR2的影响。方法采用有利于MSCs生长及增殖的全骨髓贴壁法分离及培养干细胞;成骨、成脂诱导检测其分化特性。采用TNF-α干预MSCs细胞培养。细胞分为:对照组、低剂量组(5 ng/m L)和高剂量组(10 ng/m L)。选取P3代细胞,培养10天后提取蛋白,Western blot分析MSCs表达CXCR2变化。结果所培养的MSCs成骨、成脂诱导后染色均阳性,证实所培养的即为MSCs细胞。提高培养液中TNF-α的浓度,则MSCs表达CXCR2的量显著增加,差异具有统计学意义。结论 TNF-α能够刺激MSCs表达CXCR2增加,这可能是急性肺损伤时,MSCs细胞动员和定植的机制之一。     

17β-雌二醇在大鼠骨髓间充质干细胞向成骨细胞转换中的作用

目的探讨17β-雌二醇对大鼠骨髓间充质干细胞(BMSCs)的作用。方法体外分离培养SD大鼠MSCs,诱导大鼠MSCs向成骨细胞定向分化,应用放免法、酶联免疫吸附法观察应用17β-E2对成年大鼠来源的MSCs成骨分化的影响。结果分离得出BMSCs细胞为长梭形或纺锤形,诱导7 d时,大部分细胞逐渐变为多边形,可见碱性磷酸酶(ALP)染色呈阳性;诱导约14 d,细胞互相聚集周围有钙盐晶体形成;诱导培养21 d,细胞聚集形成典型的骨小结,茜素红染色将骨结节中沉积的钙盐染成红色。结论密度梯度离心与贴壁筛选法相结合,是体外分离、纯化MSCs的理想方法;雌激素能通过增强MSCs的成骨分化能力来发挥促骨形成作用。     

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。     

再生障碍性贫血患者骨髓间充质干细胞成脂和成骨分化能力的变化

目的 通过观察再生障碍性贫血(再障)患者间充质干细胞(MSCs)成脂肪和成骨能力的变化,研究骨髓MSCs成脂分化的异常在再障患者红髓脂肪化中的作用.方法 分离培养再障患者及正常人的骨髓,观察其一般生物学特性,并在体外诱导其向脂肪、成骨细胞分化,同时用RT-PCR方法检测成脂肪、成骨特异基因的表达时间,比较再障患者和正常对照MSCs向脂肪细胞、成骨分化能力的不同.结果 原代培养7 d,再障组贴壁细胞克隆形成率为(19.30±4.77)/(5×105 MNCs),较正常对照组明显降低(47.72±3.46)/(5×105 MSCs)(P＜0.05).在培养初期,再障组MSCs与正常对照MSCs增殖能力相似,但在连续传8代后,其增殖能力降低.再障组MSCs体外诱导成脂滴早,诱导分化的脂肪细胞leptin(瘦素)基因表达早.再障组MSCs体外诱导成骨形成钙化结节少,碱性磷酸酶活性低,诱导的成骨细胞osteocalcin(骨钙素)基因表达晚.结论 再障患者MSCs成脂分化能力增强而成骨分化能力降低,可能在再障的病程中起一定作用.     

大鼠骨髓间充质干细胞的分离、培养和鉴定

目的 探讨体外分离和培养大鼠骨髓间充质干细胞(MSCs)的生物学特性,对其表型和生长曲线进行初步鉴定.方法 采用全骨髓培养法提取培养MSCs,绘制原代及三代细胞生长曲线,利用免疫组化法检测表面标志物CD34、CD44;流式细胞分析法检测表面标志物CD90.结果 成功培养出MSCs,免疫组化CD44阳性表达,CD34阴性表达;流式细胞术CD90阳性表达.结论 该实验分离培养的细胞群与MSCs的生物学特性相吻合,说明MSCs易于体外分离、培养和扩增.     

罗曼鹤鸡骨髓间充质干细胞的分离培养和鉴定

目的建立鸡骨髓间充质干细胞（BMSCs）的分离培养和鉴定体系。方法从1～14天龄罗曼鹤鸡骨髓中分离骨髓细胞,差速贴壁法纯化、扩增BMSCs。倒置显微镜下观察细胞形态特征,MTY法绘制生长曲线,流式细胞仪检测细胞标志物,并分别进行成骨、成脂诱导分化能力检测。结果分离培养的细胞呈成纤维细胞样或长梭形,生长状态良好;CD29阳性表达率为92．10％,CD34阳性表达率仅为0．80％;经成骨诱导分化,出现明显的钙化结节,茜素红染色阳性;经成脂诱导分化,油红O染色阳性,细胞内出现明显的脂质小滴。结论建立了操作简单、高效的鸡BMSCs分离培养和鉴定体系,为鸡BMSCs的进一步研究和应用提供了良好的基础。     

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。     

胎肝来源间充质干细胞的分离、培养与多向分化

目的从胎肝中分离培养间充质下细胞，并研究其牛物学特性。方法用优化的方法从胎肝中分离获得间充质干细胞。利用流式细胞仪分析细胞表型和细胞周期分布，并体外诱导成骨、成脂肪和成肝组织细胞分化。并用染色方法鉴定成骨、成脂肪分化结合形态学方法和RT-PCR方法鉴定成肝组织分化结果。结果从胎肝中分离培养的细胞为成纤维样，贴壁生长．表型相对均一，表面标志为CD90，CD44，CD147，而CD34，CD45，HLA-DR，具有向肝组织分化的潜能，并可向成骨和成脂肪分化。结论从胎肝中可分离培养得到具有多向分化潜能的间充质干细胞。     

Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow

Human mesenchymal stem/progenitor cells (MSCs) have been identified in adult bone marrow, but little is known about their presence during fetal life. MSCs were isolated and characterized in first-trimester fetal blood, liver, and bone marrow. When 10(6) fetal blood nucleated cells (median gestational age, 10(+2) weeks [10 weeks, 2 days]) were cultured in 10% fetal bovine serum, the mean number (+/- SEM) of adherent fibroblastlike colonies was 8.2 +/- 0.6/10(6) nucleated cells (69.6 +/- 10/microL fetal blood). Frequency declined with advancing gestation. Fetal blood MSCs could be expanded for at least 20 passages with a mean cumulative population doubling of 50.3 +/- 4.5. In their undifferentiated state, fetal blood MSCs were CD29(+), CD44(+), SH2(+), SH3(+), and SH4(+); produced prolyl-4-hydroxylase, alpha-smooth muscle actin, fibronectin, laminin, and vimentin; and were CD45(-), CD34(-), CD14(-), CD68(-), vWF(-), and HLA-DR(-). Fetal blood MSCs cultured in adipogenic, osteogenic, or chondrogenic media differentiated, respectively, into adipocytes, osteocytes, and chondrocytes. Fetal blood MSCs supported the proliferation and differentiation of cord blood CD34(+) cells in long-term culture. MSCs were also detected in first-trimester fetal liver (11.3 +/- 2.0/10(6) nucleated cells) and bone marrow (12.6 +/- 3.6/10(6) nucleated cells). Their morphology, growth kinetics, and immunophenotype were comparable to those of fetal blood-derived MSCs and similarly differentiated along adipogenic, osteogenic, and chondrogenic lineages, even after sorting and expansion of a single mesenchymal cell. MSCs similar to those derived from adult bone marrow, fetal liver, and fetal bone marrow circulate in first-trimester human blood and may provide novel targets for in utero cellular and gene therapy.     

人羊水来源c-kit+与c-kit-间充质干细胞生物学特征及体外心肌诱导分化能力的比较

目的比较人羊水来源c-kit＋与c-kit-间充质干细胞的生物学特征及体外心肌分化能力。方法通过常规产前诊断或自愿引产,以羊膜腔穿刺术获取14份孕中期羊水（15～31周）,通过流式细胞仪分选c-kit＋间充质干细胞,比较c—kit＋干细胞与c-kit-干细胞的细胞形态及生长曲线。流式细胞仪及免疫细胞化学分析对两种细胞进行表型鉴定,比较其体外向成骨、成脂及心肌样细胞诱导分化能力。RT-PCR和Westonblot检测相关基因及蛋白的表达。结果14份羊水标本中,有5份标本可以分选到c—kit＋羊水干细胞,分布在孕16-22周,分选的细胞数量占贴壁细胞的（3．30±1．24）％。羊水干细胞以成纤维状为主,可以稳定传代,体外培养可增殖到50代以上。经过c—kit分选后,细胞呈均匀一致的长纤维状,排列规律。C-kit＋及c-kit-羊水干细胞具有相似的生长曲线。两种细胞均具有间充质干细胞特征,表达间充质干细胞标记（CD29,CD44,CD73,CD90,CDl05）,不表达造血细胞系标记（CD34,CD45）,表达I类人类组织相容性抗原（HLA—ABC）,不表达II类人类组织相容性抗原（HLA—DR）。Oct-4及SSEA-4胚胎干细胞标记在c-kit＋干细胞的表达比率明显高于c．kit-干细胞。两种细胞在体外均可以向成脂细胞、成骨细胞分化。成脂细胞诱导后,可以看到细胞内脂滴聚集,油红0染色阳性。成骨细胞诱导后,可以看到钙盐沉积,VonKossa染色阳性。两种细胞分别与新生乳鼠心肌共培养,体外向心肌细胞诱导,c—kit＋羊水干细胞可以检测到GATA-4,α-actin,Cx43和cTnT基因及蛋白的表达。而c-kit-干细胞在诱导后只表达GATA-4的基因及蛋白。结论羊水来源c—kit＋及c—kit-干细胞均表达间充质干细胞表型特征,体外可以向成脂、成骨细胞分化。然而,c—kit＋干细胞的心肌样细胞分化能力明显强于c—kit-干细胞,可能与c—kit＋干细胞具有某些胚胎干细胞特征有关。     

Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage

OBJECTIVE: To determine the presence of mesenchymal progenitor cells (MPCs) in human articular cartilage. METHODS: Primary cell cultures established from normal and osteoarthritic (OA) human knee articular cartilage were analyzed for the expression of CD105 and CD166, cell surface markers whose coexpression defines mesenchymal stem cells (MSCs) in bone marrow and perichondrium. The potential of cartilage cells to differentiate to adipogenic, osteogenic, and chondrogenic lineages was analyzed after immunomagnetic selection for CD105+/CD166+ cells and was compared with bone marrow-derived MSCs (BM-MSCs). RESULTS: Up to 95% of isolated cartilage cells were CD105+ and approximately 5% were CD166+. The mean +/- SEM percentage of CD105+/CD166+ cells in normal cartilage was 3.49 +/- 1.93%. Primary cell cultures from OA cartilage contained significantly increased numbers of CD105+/CD166+ cells. Confocal microscopy confirmed the coexpression of both markers in the majority of BM-MSCs and a subpopulation of cartilage cells. Differentiation to adipocytes occurred in cartilage-derived cell cultures, as indicated by characteristic cell morphology and oil red O staining of lipid vacuoles. Osteogenesis was observed in isolated CD105+/CD166+ cells as well as in primary chondrocytes cultured in the presence of osteogenic supplements. Purified cartilage-derived CD105+/CD166+ cells did not express markers of differentiated chondrocytes. However, the cells were capable of chondrocytic differentiation and formed cartilage tissue in micromass pellet cultures. CONCLUSION: These findings indicate that multipotential MPCs are present in adult human articular cartilage and that their frequency is increased in OA cartilage. This observation has implications for understanding the intrinsic repair capacity of articular cartilage and raises the possibility that these progenitor cells might be involved in the pathogenesis of arthritis.     

雷奈酸锶对大鼠骨髓间充质干细胞增生和成脂分化的影响

目的从细胞和基因水平研究不同浓度雷奈酸锶对大鼠骨髓间充质干细胞成脂分化的影响。方法分离培养并鉴定大鼠骨髓间充质干细胞。以不同浓度雷奈酸锶（0、0．05、0．1、0．5、1．0和5．0mmol／L）刺激骨髓问充质干细胞,测定其增生活性。在成脂诱导条件下,加入不同浓度雷奈酸锶（0、1．0、5．0mmol／L）,倒置显微镜下检测油红染色并进行成脂相关基因检测。结果在非诱导条件下,浓度为1．0和5．0mmol／L的雷奈酸锶对细胞增生具有明显促进作用。在成脂诱导6及9d后,1．0和5．0mmol／L雷奈酸锶均对细胞成脂分化具有抑制作用,表现为细胞内脂滴缩小,油红染色阳性细胞数量减低,成脂分化关键基因PPAR-γ／EBP—β、aP-2以及Glut-4的表达量降低。结论在体外雷奈酸锶具有促进骨髓间充质干细胞增生作用,并抑制其向脂肪细胞分化,这可能与其具有促进前体干细胞成骨分化并维持成骨与成脂分化平衡作用有关。     

Immunomodulatory effects of human foetal liver-derived mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) have been suggested to decrease lymphocyte proliferation in vitro. We hypothesised that foetal MSCs (fMSCs) would have an immunosuppressive effect on allograft responses in vitro. Human MSCs were isolated and cultured from first-trimester foetal livers and characterised by flow cytometry. fMSC stained positive for CD29, CD44, CD166, CD105, SH-3 and SH-4, and negative for CD14, CD34 and CD45. When plated on adipogenic, chondrogenic and osteogenic media, fMSC differentiated into the respective cell lineage. Compared to adult MSC (aMSC), the proliferative capacity of fMSC was higher. Mitogen stimulation of PBL was inhibited by fMSC. The greatest inhibition (78%) was seen when 30,000 fMSCs were added to 150,000 lymphocytes stimulated by phytohaemagglutinin. Adult and fMSCs were added to mixed lymphocyte cultures (MLC) containing peripheral blood lymphocytes or foetal liver cells. Unlike aMSC, fMSCs did not inhibit MLC. fMSC could be culture-expanded several million folds with no loss of phenotype characteristics, which makes them ideal for ex vivo expansion. fMSC inhibit lymphocyte proliferation induced by mitogens, but not alloreactivity as measured by MLC.     

PI3K／Akt信号通路在骨髓间充质干细胞增殖及成骨分化调控中的作用

目的：研究PI3K/Akt信号通路抑制剂LY294002对骨髓间充质干细胞（mesenchymal stem cells,MSCs）增殖及分化的影响。方法采用贴壁法体外分离人骨髓间充质干细胞（hMSCs）,加入PI3K抑制剂LY294002（1、10μmol/L）,应用MTT法测定细胞增殖,常规成骨诱导分化培养3或7d,采用碱性磷酸酶（ALP）染色观察成骨分化水平,化学比色法测定ALP活性,茜素红染色后观察矿化钙结节数量并定量分析,Westernblot检测磷酸化Akt蛋白表达,应用Realtime-PCR检测各组细胞BMP2、Runx2、OPN及Osterix等成骨分化标记物的基因表达水平。结果从24至72h,LY294002对hMSCs增殖均产生显著抑制,随时间推延,可见抑制增殖效果增强（P＜0.05）。ALP染色和定量测定提示10μmol/L的ALP活性最强,在不同时间显著高于对照组和1μmol/L组（P＜0.05）。成骨诱导培养3和7d,1、10μmol/L组矿化量都显著高于对照组（P＜0.05）。10μmol/L组矿化量在成骨诱导7d也显著高于1μmol/L组（P＜0.05）。Westernblot检测结果证实成骨诱导可激活Akt磷酸化蛋白表达,但LY294002可抑制该蛋白磷酸化。成骨诱导分化7d,1、10μmol/L均明显促进BMP2、Runx2、OPN、Osterix4种基因mRNA表达（均P＜0.05）。结论PI3K/Akt信号通路参与hMSCs增殖和分化过程。成骨分化伴随下游Akt蛋白表达。PI3K抑制剂可抑制hMSCs增殖,但同时促进其向成骨分化和矿化。     

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.