年龄对人骨髓间充质干细胞体外增殖能力的影响

背景：人骨髓间充质干细胞的增殖和分化可以修复组织器官损伤,随着年龄增长人体组织修复能力减弱,是否人骨髓间充质干细胞增殖能力随年龄增长而减弱尚不清楚。 目的：观察年龄对人骨髓间充质干细胞体外增殖能力的影响。 方法：15份骨髓标本来源于青岛市中心医院骨折或行髋关节置换患者,其中儿童、成年人、老年人各5例,年龄分别为7~12岁,20~55岁,60~90岁。抽取人骨髓组织,采用密度梯度离心法分离出单核细胞并接种于培养瓶内,在第5天计数集落形成单位;当贴壁细胞融合后进行传代,第1代细胞分两种方式进行培养：192个细胞接种于2个96孔板观察单细胞克隆形成能力,12 500个细胞以5×103/cm2接种于培养瓶内并以此密度连续传代观察细胞增殖率。 结果与结论：不同年龄人群骨髓间充质干细胞体外培养集落形成单位和细胞增殖率无明显区别(P > 0.05),但单细胞克隆形成能力随年龄增长而下降(P  < 0.01)。提示骨髓间充质干细胞的数量和增殖能力没有随年龄的增长而下降,但其中能形成单细胞克隆未定向干细胞的数量随年龄的增长而减少。     

Comprehensive analysis of chemotactic factors for bone marrow mesenchymal stem cells

To understand which growth factors/cytokines can affect migration of mesenchymal stem cells (MSCs) to injured tissues, we compared the effects of many (26) growth factors/cytokines on the migration activity of rabbit and human MSCs using a microchemotaxis chamber. Among them, platelet-derived growth factor (PDGF)-BB, PDGF-AB, epidermal growth factor (EGF), HB-EGF, transforming growth factor (TGF-alpha), insulin growth factor (IGF-I), hepatocyte growth factor (HGF), fibroblast growth factor (FGF-2), and thrombin consistently enhanced the migration of rabbit and human MSCs at appropriate concentrations. PDGF-BB showed the greatest effect on migration. Various combinations of these factors further enhanced the migration of MSCs, whereas combinations of factors that shared common cell-surface receptors did not induce the additive stimulation. On the other hand, some combinations, including that of FGF-2 or thrombin with PDGF-BB, suppressed the migration activity of MSCs. These findings suggest that combinations of growth factors are important to eliciting the maximal chemotactic effect. The factors that induced the migration of MSCs also enhanced their proliferation, suggesting that migration and proliferation can take place simultaneously. The above factors were also effective in stimulating the migration of fibroblasts, but thrombin alone selectively enhanced the migration of MSCs, suggesting that thrombin is useful to stimulate migration of MSCs without migration of fibroblasts.     

Basic fibroblast growth factor controls migration in human mesenchymal stem cells

Little is known about the migration of mesenchymal stem cells (MSCs). Some therapeutic approaches had demonstrated that MSCs were able to regenerate injured tissues when applied from different sites of application. This implies that MSCs are not only able to migrate but also that the direction of migration is controlled. Factors that are involved in the control of the migration of MSCs are widely unknown. The migratory ability of isolated MSCs was tested in different conditions. The migratory capability was examined using Boyden chamber assay in the presence or absence of basic fibroblast growth factor (bFGF), erythropoietin, interleukin-6, stromal cell-derived factor-beta, and vascular endothelial growth factor. bFGF in particular was able to increase the migratory activity of MSCs through activation of the Akt/protein kinase B (PKB) pathway. The results were supported by analyzing the orientation of the cytoskeleton. In the presence of a bFGF gradient, the actin filaments developed a parallelized pattern that was strongly related to the gradient. Surprisingly, the influence of bFGF was not only an attraction but also routing of MSCs. The bFGF gradient experiment showed that low concentrations of bFGF lead to an attraction of the cells, whereas higher concentrations resulted in repulsion. This ambivalent effect of bFGF provides the possibility to a purposeful routing of MSCs.     

脂多糖诱导钛种植体周围骨髓间质细胞炎症中的巨噬细胞游走抑制因子

背景：目前大量研究证实,巨噬细胞游走抑制因子在多种生物活动中发挥着重要的作用,如肿瘤的发生发展等,近年来发现其在机体发生的炎症过程中也扮演者重要的角色,也取得了很多成绩,然而在口腔内牙种植体周围这一特殊的环境中,巨噬细胞游走抑制因子是否发挥着作用以及发挥着怎样的作用,尚不清楚。 目的：观察巨噬细胞游走抑制因子在经过脂多糖诱导纯钛材料周围生长的骨髓间质细胞发生的炎症中发挥的作用。 方法：首先将骨髓间质细胞接种于钛细胞培养爬片上,模拟口内种植体周围环境,实验分4组：对照组为不加任何刺激的骨髓间质细胞;脂多糖组利用脂多糖诱导骨髓间质细胞发生炎症;非特异性siRNA+脂多糖组利用脂多糖刺激转染过非特异性小干扰RNA的骨髓间质细胞;巨噬细胞游走抑制因子siRNA+脂多糖组利用脂多糖刺激转染过巨噬细胞游走抑制因子靶向小干扰RNA的骨髓间质细胞。 结果与结论：实验通过流式细胞筛选技术,筛选出细胞表面蛋白CD29、CD90阳性率大于95%,而且CD45表达低于5%的骨髓间质细胞作为实验对象。CCK8实验结果提示,脂多糖与巨噬细胞游走抑制因子靶向小干扰RNA对骨髓间质细胞的增殖活性几乎无影响,然而与对照组相比,脂多糖可明显刺激骨髓间质细胞发生炎症反应,为对照组的15-20倍(P < 0.01)。巨噬细胞游走抑制因子siRNA+脂多糖组白细胞介素1β、白细胞介素6、肿瘤坏死因子α表达低于脂多糖组(P < 0.01)。结果证实,脂多糖可以促进种植体周围骨髓间质细胞的炎症反应,而且巨噬细胞游走抑制因子靶向小干扰RNA沉默可以在一定程度上抑制炎症反应的发生。  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Chemokines stimulate bidirectional migration of human mesenchymal stem cells across bone marrow endothelial cells

Mesenchymal stem cells (MSCs) can be mobilized from the bone marrow and enter the circulation. Conversely, MSCs can be recruited from the circulation and into the bone marrow. For these migratory pathways, MSCs have to traverse the bone marrow endothelium, in a basal-to-apical and apical-to-basal direction, respectively. Here we describe the migratory cues that drive MSC transendothelial migration in both directions with focus on chemokines. Live cell imaging and electron microscopy were used to examine the interaction of human MSCs with human bone marrow endothelial cells (HBMECs), and MSC transmigration analyzed. Chemokines CXCL12, CXCL13, CXCL16, CCL11, and CCL22 significantly enhanced transendothelial migration in an apical-to-basal and basal-to-apical direction, showing preferences in terms of their capacity to stimulate the direction of migration. For apical-to-basal migration CXCL16 was the most effective (6-fold stimulation), with the rank order being CXCL16>CCL11>CXCL13>CCL22>CXCL12. In the basal-to-apical direction CCL22 was the most effective (5-fold enhancement), with the remaining chemokines being roughly equal. When MSCs interacted with HBMECs they flattened, extended long microvilli (filopodia) and podosome-like protrusions that inserted into the endothelial cells. In conclusion, chemokines enhance the migration of MSCs bidirectionally across HBMECs, with directional preferences shown for different chemokines.     

Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) constitute a population of multipotent adherent cells able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes, or chondrocytes. So far, the most common source of MSCs has been the bone marrow (BM); however BM-MSC harvesting and processing exhibits major drawbacks and limitations. Thus, identification and characterization of alternative sources of MSCs are of great importance. In the present study, we isolated and expanded fetal MSCs from second-trimester amniotic fluid (AF). We documented that these cells are of embryonic origin, can differentiate under appropriate conditions into cell types derived from all three germ layers, and express the pluripotency marker Oct-4, the human Nanog protein, and the stage-specific embryonic antigen-4 (SSEA-4). Furthermore, we systematically tested the immunophenotype of cultured MSCs by flow cytometry analysis using a wide variety of markers. Direct comparison of this phenotype to the one derived from cultured BM-MSCs demonstrated that cultured MSCs from both sources exhibit similar expression patterns. Using the two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approach, we have generated for the first time the protein map of cultured AF-MSCs by identifying 261 proteins, and we compared it directly to that of cultured BM-MSCs. The functional pattern of the identified proteins from both sources was similar. However, cultured AF-MSCs displayed a number of unique proteins related to proliferation and primitive phenotype, which may confer to the distinct features of the two types. Considering the easy access to this new cell source and the yield of expanded MSCs for stem cell research, AF may provide an excellent source of MSCs both for basic research and for potential therapeutic applications.     

不同年龄人骨髓间充质干细胞体外生长特性分析

目的建立人骨髓间充质干细胞（marrow mesenchymal stem cells，MMSCs）体外分离培养和扩增方法，观察不同年龄人MMSCs在体外培养的生长特性，为MMSCs的临床应用提供技术方法和依据。方法采集10～60岁之间的人骨髓血，密度梯度法分离出单个核细胞，利用DMEM／F12培养基添加胎牛血清进行黏附培养和扩增MMSCs，分析第3代MMSCs生长情况和形态变化，细胞计数，用MTT法观察其增殖及生长特征，免疫组化法分析MMSCs的纯度。结果10～60岁人MMSCs均贴壁生长，呈典型MMSCs形态和生长特征，传代细胞的增殖速度比原代细胞快，但随着年龄的增长，原代及传代培养MMSCs的增殖能力下降，生长周期延长，其中CD105阳性细胞均大于95％，但老年人的CD105阳性细胞比率有所降低。结论密度梯度离心结合贴壁培养法可获得高纯度MMSCs，成人MMSCs在传代培养的增殖活性随年龄增加而降低。     

人骨髓间充质干细胞和汗腺细胞的体外分离与培养*

背景：体外研究人骨髓间充质干细胞和汗腺细胞的分离培养与鉴定,可为探讨骨髓间充质干细胞再生汗腺的可行性打下基础。 目的：探寻在体外分离培养骨髓间充质干细胞和汗腺细胞的有效方法。 方法：采用直接贴壁法从成人骨髓中体外分离培养骨髓间充质干细胞,并进行扩增和鉴定。采用胶原酶消化法从人全层无烧伤皮肤中分离汗腺细胞,并进行扩增和鉴定。 结果与结论：倒置显微镜下见分离培养的骨髓间充质干细胞呈梭形,折光性强,免疫细胞化学染色显示细胞表达CD29、CD105,高表达CD44,不表达造血干细胞表面标志CD34和CD45。汗腺细胞呈扁平多角形,表达汗腺细胞表面标志细胞角蛋白7,8,18,19和癌胚抗原。说明直接贴壁法分离培养骨髓间充质干细胞和胶原酶消化法分离培养汗腺细胞是可行的。     

聚羟基丁酸/戊酸酯共聚物膜与人骨髓间充质干细胞的体外生物相容性

背景：聚羟基丁酸/戊酸酯共聚物是近年来受到重视的聚羟基脂肪酸族组织工程支架材料,具有免疫排斥反应低、生物相容性好和降解产物无毒副作用的优点。 目的：观察聚羟基丁酸/戊酸酯共聚物膜材料与人骨髓间充质干细胞的体外生物相容性。 方法：将第3代人骨髓间充质干细胞种植于聚羟基丁酸/戊酸酯共聚物膜上作为实验组,培养板单纯培养细胞作为对照组,计算1,2,4 h两组贴壁细胞的数量,得出细胞贴壁率。MTT比色法观察2,4,6,8 d两组细胞的增殖情况。采用Hoechst33258荧光法,检测3,6,9,12 d两组细胞内的DNA含量。将人骨髓间充质干细胞接种聚羟基丁酸/戊酸酯共聚物膜材料上5 d后,电镜扫描观察细胞在材料上的生长情况。 结果与结论：共培养1 h时,实验组的细胞贴壁率低于对照组;其他时间段两组之间细胞贴壁率差异无显著意义。两组各时点间的细胞增殖差异无显著意义。两组各时间点细胞内DNA含量差异无显著意义。扫描电镜观察人骨髓间充质干细胞在聚羟基丁酸/戊酸酯共聚物膜上生长良好,形态呈梭形,细胞间连接紧密,分泌较多细胞基质。证明聚羟基丁酸/戊酸酯共聚物膜材料与人骨髓间充质干细胞有良好的生物相容性。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

碱性成纤维细胞生长因子诱导人间充质干细胞向心肌样细胞分化

目的研究碱性成纤维细胞生长因子(bFGF)在体外诱导人骨髓间充质干细胞(MSCs)向心肌样细胞分化中的作用,及其对MSCs增殖的影响。方法用含5-氮杂胞苷(5-aza)的培养基(A组)、含bFGF的培养基(B组)、含5-aza bFGF的培养基(C组)以及普通培养基(对照组)培养人骨髓MSCs。观察细胞形态的改变;免疫细胞化学法检测α-actin、cTnT和connexin-43的表达;RT-PCR法检测Nkx2·5、GATA-4和cTnT的mRNA水平;MTT法检测MSCs的增殖。结果A组和C组的部分细胞呈肌细胞样改变,表达蛋白α-actin,cTnT和connexin43;A组和C组的cTnT、Nkx2·5和GATA-4的mRNA水平明显高于对照组,B组的Nkx2·5和GATA-4的mRNA水平明显高于对照组;B组细胞增殖明显高于对照组,A组和C组细胞增殖明显低于对照组,但C组明显高于A组。结论bFGF能明显促进MSCs增殖,与5-aza合用能更好地诱导人MSCs向心肌样细胞分化。     

人骨髓间充质干细胞体外分化为结膜上皮样细胞

目的 探讨体外诱导人骨髓基质干细胞(hMSCs)向结膜上皮样细胞(hCjEs)定向分化的可能性.方法 体外分离、培养hMSCs和人hCjEs,分别从细胞形态学和免疫细胞化学的方法对细胞进行鉴定;Transwell非接触共培养法诱导hMSCs向结膜上皮样细胞定向分化;倒置相差显微镜观察分化细胞的形态学改变;免疫细胞化学和反转录聚合酶链反应分别对分化细胞角蛋白13(CK13)的蛋白和mRNA表达进行检测;并用单独培养的hMSCs和hCjEs做对照.结果 实验获取的hMSCs以长梭形为主,融合呈漩涡状;hCjEs呈不规则圆形或多边形,融合的hCjEs成铺路石样;hMSCs阳性表达CD44和CD29,hCjEs阳性表达CK13;非接触共培养使hMSCs的形状由长梭形逐渐变为不规则多边形;免疫细胞化学和RT-PCR分别在蛋白和基因水平证实分化细胞表达CK13,与单独培养的结膜上皮细胞表达相似.结论 在一定条件下,可以诱导体外培养的hMSCs形态发生变化、向具有结膜上皮细胞表型的上皮样细胞定向分化.     

调节骨髓间充质干细胞向皮肤创面迁移过程中的血小板衍生生长因子

背景：血小板衍生生长因子具有创伤修复作用,对其研究大部分集中于骨组织的修复,在皮肤创伤愈合中的修复作用研究较少。 目的：观察血小板衍生生长因子在皮肤创伤愈合过程中调控骨髓间充质干细胞向创面迁移促进创面愈合的作用。 方法：培养大鼠骨髓间充质干细胞,免疫荧光检测细胞表面标记物CD34、CD44。选取健康雄性SD大鼠30只,随机分为5组,各组大鼠尾静脉注射PKH26标记的骨髓间充质干细胞。注射1周后,于大鼠背部正中线划长为3 cm的切口,制备皮肤创伤模型。造模后即刻于皮肤创伤处多点注射不同质量浓度血小板衍生生长因子干预药物,对照组注射等体积的生理盐水。注射14 d后留取皮肤创面组织进行相关指标检测。 结果与结论：荧光显微镜下观察血小板衍生生长因子能够剂量依赖性诱导骨髓间充质干细胞向皮肤创伤组织处迁移和聚集,进而促进皮肤创伤修复。Masson染色结果显示,随着血小板衍生生长因子干预质量浓度的增加,创面炎性细胞浸润减轻、胶原纤维数量不断增多。Western blot检测结果显示,血小板衍生生长因子能够抑制皮肤创伤组织基质金属蛋白酶1的表达,促进基质金属蛋白酶组织抑制因子1的表达,抑制胶原降解,发挥间接促愈合作用。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

人血管内皮生长因子165基因转染兔骨髓间充质干细胞的蛋白表达

背景：骨髓间充质干细胞是最好的组织工程种子细胞来源,含有血管内皮生长因子165(vascular endothelial growth factor 165,VEGF165)不仅对血管再生和启动成骨修复有重要意义,其持续稳定的释放还能够提高新生骨的矿化程度,增强修复组织的力学性能。 目的：观察hVEGF165基因转染的兔骨髓间充质干细胞分泌血管内皮生长因子的蛋白功能。 方法：体外分离、培养兔骨髓间充质干细胞,纯化并鉴定兔骨髓间充质干细胞;免疫荧光法检测细胞表面标志;传代培养后的骨髓间充质干细胞以pcDNA3.1-VEGF165质粒和脂质体1∶3比例的混合液转染,并分为3组：转染组应用pcDNA3.1-VEGF165转染细胞,空载体转染组应用pcDNA3.1-空载体转染,未转染组不处理。通过ELISA和Western-blot检测转染后细胞中外源性血管内皮生长因子的表达。 结果与结论：转染组与其他两组比较,VEGF165蛋白含量显著增高,差异有显著性意义(P < 0.05),但空载体转染组与未转染组之间差异无显著性意义(P > 0.05),转染组不同时间点之间VEGF165蛋白含量差异均有显著性意义(P< 0.05),hVEGF165基因转染的骨髓间充质干细胞能成功分泌VEGF165蛋白。提示采用基因转染技术可将hVEGF165基因转染到骨髓间充质干细胞中并可有效表达具有生物活性的VEGF165。     

骨髓间充质干细胞在肺腺癌微环境中的遗传稳定性及增殖能力

目的探讨人骨髓间充质干细胞(HMSC-bm)在肺腺癌微环境中遗传稳定性的变化。方法通过6孔板结合Transwell小室建立HMSC-bm和肺腺癌A549细胞的共培养体系,倒置相差显微镜下观察细胞的形态;MTT法检测细胞的增殖能力;常规染色体及G显带核型分析细胞的染色体;Western blot检测组蛋白去乙酰化酶4(HDAC4)的表达。以单独培养的HMSC-bm,A549作为对照组。结果共培养组细胞胞核大而深染,可见病理性核分裂象,HMSC-bm组无明显变化;细胞增殖曲线呈S型,共培养组细胞于第4天开始增殖速率快于HMSC-bm组;共培养组染色体数目为亚三倍体、三倍体,有明显的染色体异常;共培养组细胞HDAC4表达明显高于HMSC-bm组(P<0.01)。结论肺腺癌微环境可改变HMSC-bm的增殖速率和其遗传稳定性。     

不同浓度肝细胞生长因子及表皮细胞生长因子体外联合诱导兔骨髓间充质干细胞向肝细胞分化

背景：研究证实在多种微环境下可以将骨髓间充质干细胞诱导分化为成熟肝细胞,但诱导条件及诱导分化率尚无定论,选择合适的诱导剂及诱导剂浓度尤为重要。 目的：观察肝细胞生长因子和表皮细胞生长因子体外联合诱导兔骨髓间充质干细胞向肝细胞分化的最适浓度。 方法：不同浓度肝细胞生长因子、表皮细胞生长因子联合诱导培养原代兔骨髓间充质干细胞,观察细胞形态学变化,并检测肝细胞表面标志物甲胎蛋白、白蛋白表达及肝细胞合成功能。 结果与结论：随诱导时间延长,可观察到多极性的肝细胞样细胞。7 d时甲胎蛋白表达阳性,14 d达最大值后即开始下降(P < 0.05),此后各浓度组间表达无差别(P  > 0.05)。14 d时白蛋白表达阳性,随时间延长,阳性细胞数递增(P  < 0.05),且细胞生长因子浓度越高,阳性细胞数越高(P < 0.05)。诱导早期(9~15 d) 白蛋白上清液中白蛋白水平与诱导剂浓度成正比(P < 0.05)。18 d或21 d达高峰后下降,此时各浓度组间含量无差别(P  > 0.05)。结果显示高浓度的肝细胞生长因子及表皮细胞生长因子可提高骨髓间充质干细胞向肝细胞的分化率,诱导剂最适浓度为肝细胞生长因子60 μg/L、表皮细胞生长因子4.5 mg/L。     

骨髓间充质干细胞与髓核细胞的体外共培养

背景：细胞移植治疗椎间盘退行性变目前尚处在实验室研究阶段。 目的：通过SD大鼠骨髓间充质干细胞与退行性改变的髓核细胞体外共培养,探索骨髓间充质干细胞能否通过直接接触促进髓核细胞外基质的表达以及髓核细胞能否诱导骨髓间充质干细胞向类髓核细胞分化。 方法：取已自然退变的第6代髓核细胞与生长良好的第4代骨髓间充质干细胞按不同比例(75∶25,50∶50和25∶75)体外共培养7 d,单独髓核细胞(100∶0)与单独骨髓间充质干细胞(0∶100)分别作为阳性对照和阴性对照。 结果与结论：RT-PCR检测显示共培养组(75∶25和50∶50)蛋白多糖和Ⅱ型胶原蛋白条带均明显亮于单独髓核细胞培养组(100∶0),吸光度值比较差异有显著性意义;免疫组织化学检测显示Ⅱ型胶原蛋白在共培养组(75∶25和50∶50)胞浆内的表达明显高于单独髓核细胞培养组(100∶0)。共培养组中可见骨髓间充质干细胞由长梭形向多角形、类圆形转变,胞浆内异染颗粒增多。提示通过体外直接接触共培养,骨髓间充质干细胞能逆转髓核细胞退变,而且髓核细胞能诱导骨髓间充质干细胞向类髓核细胞转换。     

力生长因子促进骨髓间充质干细胞向成骨细胞的分化

BACKGROUND:Mechano growth factor has the potential to activate muscle satellite cells and promote myogenic cell growth, and has dual roles in maintaining bone mass and repairing bone defects. OBJECTIVE:To explore the mechanism underlying osteogenic differentiation of rabbit bone marrow mesenchymal stem cells promoted by the mechano growth factor. METHODS:The best concentration and time of mechano growth factor to promote osteogenic differentiation of rabbit bone marrow mesenchymal stem cells were detected by MTT. The mRNA and protein expressions of alkaline phosphatase and osteocalcin were detected by qPCR and western blot, respectively. The phosphorylation level of AKT and mTOR were detected by western blot assay. RESULTS AND CONCLUSION:The best concentration and time of mechano growth factor was 45 μg/L and 5 days for promoting the osteogenic differentiation of rabbit bone marrow mesenchymal stem cells. The expressions of alkaline phosphatase and osteocalcin at mRNA and protein levels were highest after 4-hour intervention with 45 μg/L mechano growth factor, and meanwhile, the phosphorylation levels of mTOR and AKT were also highest. These findings indicate that the mechano growth factor can promote the differentiation of rabbit bone marrow mesenchymal stem cells into osteoblasts via PI3K/AKT pathway, and its best concentration and time are 45 μg/L and 4 hours, respectively.     

体外诱导大鼠骨髓间充质干细胞向神经元样细胞的分化

背景：研究表明,移植后的骨髓间充质干细胞在新的环境中能够被诱导分化为神经元样细胞,从而替代损伤细胞重建神经环路。 目的：建立大鼠骨髓间充质干细胞和神经细胞的体外共培养系统,观察共培养条件下神经细胞对骨髓间充质干细胞向神经元样细胞分化的影响。 方法：体外培养Wistar大鼠脑组织神经细胞和股骨骨髓间充质干细胞,采用Transwell小室共培养分化诱导,观察骨髓间充质干细胞的组织形态变化,在共培养第5天免疫荧光染色检测骨髓间充质干细胞中神经细胞的特异标志物;与对照组单纯骨髓间充质干细胞培养结果相比较。 结果与结论：神经细胞共培养系统中的骨髓间充质干细胞生长伸展,呈放射状,互相形成连接,特异性烯醇化酶显示阳性结果,具有神经元样细胞的特性,表达特异性烯醇化酶阳性的神经元比例可达(33.0±10.5)%。而对照组骨髓间充质干细胞未形成神经元样细胞的形态结构,免疫荧光显示特异性烯醇化酶阴性。提示神经细胞提供的微环境对骨髓间充质干细胞分化为神经元样细胞具有诱导分化作用。      

转化生长因子β1与骨髓间充质干细胞向心肌样细胞的分化

背景：转化生长因子β1是一种具有调控细胞增殖、分化、黏附和凋亡,在生物发育及组织修复过程中具有重要作用的细胞因子。 目的：观察转化生长因子β1对诱导大鼠骨髓间充质干细胞定向分化为心肌样细胞的影响。 方法：取SD大鼠四肢骨骨髓,分离培养骨髓间充质干细胞,应用转化生长因子β1对第2代骨髓间充质干细胞定向诱导,以未诱导的骨髓间充质干细胞做对照。 结果与结论：①加入转化生长因子β1 诱导后的骨髓间充质干细胞培养7 d时,相差显微镜观察多紧密平行排列生长,呈类长柱形,少数呈不规则形;14 d后开始出现细胞间连接,形成数个相互连接的长柱形,28 d时骨髓间充质干细胞体积则变小,排列紧密呈条梭状。②转化生长因子β1 诱导4周后,免疫细胞化学技术检测骨髓间充质干细胞均可见结蛋白、原肌球蛋白及间隙连接蛋白43的阳性细胞,诱导组各指标的积分吸光度值均显著高于对照组(P均< 0.01)。③诱导组心肌肌钙蛋白I呈阳性表达。根据心肌肌钙蛋白I阳性率计算出的心肌样细胞转化率在诱导组显著高于对照组(P=0.000)。④荧光免疫双标技术检测结果显示,经转化生长因子β1诱导4周的骨髓间充质干细胞结蛋白及心肌肌钙蛋白I蛋白均定位于细胞质且呈共表达。⑤透射电镜结果显示,分化细胞的细胞质内可见到平行排列的肌丝,并富含线粒体、糖原和核糖体。提示骨髓间充质干细胞在转化生长因子β1诱导下可定向分化为心肌样细胞。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：