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目的应用血小板衍生生长因子BB(platelet-derived growthfactor BB,PDGF-BB),体外诱导人骨髓间充质干细胞(human bone marrowmesenchymal stemcells,hBMSCs)向血管平滑肌细胞表型分化,探讨该方法的可行性及诱导细胞作为组织工程血管平滑肌种子细胞的可行性。方法抽取健康成人志愿者骨髓,经密度梯度离心分离得单个核细胞,PDGF-BB(20ng/ml)诱导hBMSCs向血管平滑肌样细胞(vascular smooth muscle cells,VSMCs)分化,观察细胞形态变化。免疫荧光检测细胞内血管平滑肌肌动蛋白α(vascular smooth muscleα-actin,SMα-actin),血管平滑肌钙结合蛋白(vascular smoothmuscle calponin,SMcalponin),血管平滑肌肌球蛋白重链(vascular smooth muscle myosin heavy chain,SMMHC)和细胞血管平滑肌钙结合相关蛋白(smooth muscle22α,SM22α)表达情况;反转录聚合酶链式反应(RT-PCR)检测诱导后血管平滑肌细胞SMα-actin,SMcalponin,SMMHC和SM22α的mRNA表达。Western印记检测SM22α的表达。流式细胞分析技术(fluorescence activated cell sorter,FACS)分析诱导后细胞内SMα-actin,SMcalponin,SMMHC的表达。结果PDGF-BB20ng/ml诱导后可见单层培养的细胞形态呈“成纤维细胞样”。免疫荧光检测示SMα-actin,SMcalponin,SMMHC,SM22α表达阳性;RT-PCR检测SMα-actin,SMcalponin,SMMHC,SM22α的mRNA阳性表达。Western印迹检测SM22α的表达为阳性。FACS分析表明诱导后,SMα-actin,SMcalponin,SMMHC表达均增高,与未诱导组比较差异有统计学意义(P〈0.05,n=3)。结论人骨髓间充质干细胞在PDGF-BB的诱导下可向血管平滑肌细胞表型分化,有望成为血管组织工程血管平滑肌种子细胞的来源。 相似文献
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体外诱导大鼠骨髓间充质干细胞向血管内皮细胞分化的研究 总被引:1,自引:1,他引:1
目的:探讨大鼠骨髓间充质干细胞(MSCs)的体外培养方法和向血管内皮细胞分化的能力.方法:利用淋巴细胞分离液分离出MSCs,体外扩增,流式细胞仪检测MSCs表面抗原表达,以含VEGF、bFGF的诱导分化培养液定向诱导传代使细胞向血管内皮细胞分化,流式细胞仪进行内皮细胞特异性标志物鉴定,测定内皮细胞NO的释放量,电镜观测W-P小体.结果:MSCs在体外传代扩增后流式细胞仪检测结果显示CD29表达阳性,CD34、CD45为阴性,分化后的细胞具有内皮细胞的形态学特征并具特异性W-P小体,可表达CD34,而不表达CD45,能释放NO.结论:骨髓间充质干细胞可在体外诱导向内皮细胞方向分化. 相似文献
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目的 观察不同条件下骨髓间充质干细胞(MSC)体外诱导分化为肾小管上皮样细胞的差异。 方法 抽取SD大鼠的骨髓,经密度梯度离心分离,联合贴壁筛选法获取纯化的MSC。以流式细胞仪鉴定间充质干细胞表面标志。取扩增3代的MSC分组培养:(1)对照组:用含胎牛血清培养基;(2)全反式维甲酸(ATRA)组:胎牛血清+缺血再灌注肾脏匀浆上清+ATRA;(3)联合诱导组:胎牛血清+缺血再灌注肾脏匀浆上清+ATRA+表皮生长因子(EGF)+骨形成蛋白(BMP-7)。诱导7 d后,倒置显微镜下观察细胞形态变化;化学染色检测细胞碱性磷酸酶表达;免疫细胞化学法检测细胞角蛋白18(cytokeratin-18)、E钙黏蛋白(E-cadherin)的表达。 结果 流式细胞仪显示,体外分离培养的第3代MSC,CD44阳性细胞表达率为97.8%±0.9%;CD90阳性细胞表达率为96.8%±1.4%;CD29阳性细胞表达率为97.6%±2.4%;而CD11b/c阳性细胞表达率为13.2%±0.6%; CD34阳性细胞表达率为1.2%±0.5%。诱导7 d后,与对照组长梭形细胞相比,ATRA组部分细胞为圆形、短梭形单层排列;联合诱导组的大部分细胞为圆形、短梭形,细胞密集处呈鹅卵石样排列。碱性磷酸酶染色显示,对照组细胞为阴性;ATRA组部分细胞阳性;联合诱导组阳性细胞数明显增多。免疫细胞化学显示,ATRA组和联合诱导组细胞cytokeratin-18阳性表达率分别为29.47%±1.08%和47.52%±2.13%,显著高于对照组(P < 0.05);E-cadherin阳性表达率分别为14.88%±2.46%和36.15%±1.13%,也显著高于对照组(P < 0.05)。 结论 在体外模拟的急性肾衰竭微环境中加入ATRA可诱导MSC部分分化为肾小管上皮样细胞。联合EGF、BMP-7共同诱导能进一步促进MSC向肾小管上皮样细胞分化。 相似文献
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成人骨髓间充质干细胞诱导分化为神经元样细胞后的维持培养 总被引:2,自引:0,他引:2
目的:探讨体外诱导成人骨髓间充质干细胞(bonemarrowstromalcells,BMSCs)分化为神经元样细胞后长期培养的可行性。方法:自成人红骨髓分离培养BMSCs,通过克隆培养、成骨分化、成脂肪分化鉴定其特性,取第3代细胞重新接种,经碱性成纤维生长因子预诱导及β-巯基乙醇诱导后,换维持培养液维持培养,观察其分化及生长情况。结果:成神经元诱导分化0.5h后,多数细胞即开始呈现出神经元样外观,5h后绝大多数细胞表现出典型的神经元样外观,维持培养1周后,大多数细胞继续维持神经元样外观,免疫组织化学染色示65%±3.3%的细胞微管相关蛋白-2染色阳性,43%±2.1%的细胞神经微丝-160染色阳性。结论:成人BMSCs经诱导后半数左右的细胞可分化为神经元样细胞,在优化的培养体系中,分化的神经元样细胞可存活1周以上。 相似文献
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骨髓间充质干细胞定向肝细胞样分化的研究 总被引:17,自引:3,他引:17
目的探讨骨髓间充质干细胞定向肝细胞样分化的诱导条件,为肝组织工程提供新的种子细胞来源。方法分离小鼠骨髓间充质干细胞,在特定肝细胞培养液中用肝细胞生长因子(HGF)和表皮细胞生长因子(EGF)定向诱导。在倒置显微镜下观察细胞形态。应用免疫荧光法鉴定细胞性质。结果骨髓间充质干细胞经HGF和EGF诱导7d后变为肝细胞样圆形;2周后免疫荧光染色可见分化后细胞表达肝细胞标志物CK18和白蛋白。结论骨髓间充质干细胞在特定条件诱导下可以向肝细胞方向分化。有可能作为肝组织工程的种子细胞来源。 相似文献
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诱导骨髓间充质干细胞向软骨细胞分化的体外研究 总被引:6,自引:3,他引:6
目的 探讨转化生长因子β1(transforming growth factor β1,TGF—β1)、胰岛素样生长因子1(insulinlike growth factor1,IGF-1)在诱导骨髓间充质干细胞(marrow mesenchymal stem ceils,MSCs)向软骨细胞分化过程中的相互作用,并研究细胞密度对MSCs向软骨细胞分化的影响。方法 取健康昆明种小白鼠骨髓,用全骨髓贴壁法筛选获得MSCs,体外培养传代。采用特定的诱导培养使MSCs向软骨细胞分化,按培养基内添加生长因子的不同分成3个实验组和对照组。实验组分别为:TGF—β1+IGF-1联合应用组(TGF—β1 10ng/ml、IGF-1 50ng/m1);TGF—β1单独应用组(TGF—β1 10ng/m1);IGF-1单独应用组(IGF-1 50ng/m1);对照组不添加任何生长因子。TGF—β1+IGF-1联合应用组于诱导14d和21d,分别进行甲苯胺蓝染色及免疫荧光双染法鉴定;于诱导7、14和21d各组分别提取诱导细胞总RNA,进行RT—PCR扩增,检测TGF—β1、IGF-1对诱导细胞Ⅱ型胶原表达量的影响;比较MSCs在平板培养及细胞团培养时,Ⅱ型胶原表达量的差异。结果TGF—β1+IGF-1联合应用组诱导培养14d,诱导软骨细胞甲苯胺蓝染色呈阳性,免疫荧光染色可见诱导软骨细胞的细胞外基质含有Ⅱ型胶原。各组基因扩增产物的凝胶电泳可见,TGF—β1+IGF-1联合应用组和TGF—β1单独应用组Ⅱ型胶原扩增片段呈阳性;IGF-1单独应用组和对照组,未见Ⅱ型胶原扩增条带;凝胶成像系统灰度扫描示Ⅱ型胶原表达量TGF—β1+IGF-1联合应用组各时间点均比TGF—β1单独应用组明显增加(P〈0.05)。细胞团培养模式下,诱导细胞表达Ⅱ型胶原比平板培养模式更加显著。结论 MSCs向软骨细胞诱导分化时,IGF-1对TGF—β1有明显的促进作用;细胞培养密度提高有利于MSCs成软骨细胞表型。 相似文献
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Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7. 相似文献
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Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7. 相似文献
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Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7. 相似文献
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体外诱导骨髓间充质干细胞向肝细胞分化的实验研究 总被引:2,自引:0,他引:2
目的:探讨HGF和EGF对骨髓间充质干细胞向肝细胞方向的诱导分化作用,探索出合适的诱导条件,为肝细胞移植、生物人工肝、组织工程构建等提供基础。方法:骨髓来自杂种犬的髂骨,采用全血贴壁法分离培养骨髓间充质干细胞,流式细胞仪检测细胞CD标记;用含HGF和EGF的α—MEM培养液进行诱导培养,并于诱导后7、14、21d留取细胞;用免疫组化检测AFP、CK18,免疫荧光检测ALB,PAS反应检测糖原;流式细胞仪检测细胞分化率。结果:分离的骨髓间充质干细胞表面CD13、CD34和CD45皆为阴性,CD29为阳性。诱导至7d出现AFP表达.14d表达增高,21d时表达下降。CK18、ALB和糖原14d时出现表达,并随时间延长表达逐渐增加:21d时ALB表达阳性率可达50%以上。结论:HGF和EGF有诱导骨髓间充质干细胞向肝细胞分化的作用。 相似文献
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目的建立以30 ng/ml FGF-4和30 ng/ml OSM为主的诱导培养体系,研究体外骨髓干细胞向肝细胞的转化过程中细胞形态、结构、功能的变化.方法建立以30ng/ml FGF,30ng/mlOSM为主的小鼠骨髓干细胞诱导培养体系.观察细胞形态和数量的变化;RT-PCR、Western blot、免疫荧光染色检测肝细胞特异性标记物的表达;通过PAS糖原染色、白蛋白ELISA、尿素合成试验,检测细胞的合成和代谢功能.结果诱导12 d,观察到多极性的肝细胞样细胞,且逐渐增多、集落不断增大.在诱导过程中,不同的时间点可以分别检测到HNF-3βmRNA、ALBmRNA、CK18mRNA、TTRmRNA、G6-PasemRNA和TATmRNA的表达.诱导21 d,细胞表达HNF-3β、ALB、CK18蛋白;免疫荧光定位示ALB表达于胞浆和胞膜,而CK18表达于胞浆.诱导21 d,细胞胞浆内可见红染的糖原颗粒;在诱导过程中,不同的时间点检测到细胞分泌ALB、合成尿素的量的变化.结论我们建立的以30ng/ml FGF、30 ng/ml OSM为主的诱导培养体系,可以有效地促进骨髓干细胞体外定向转化为肝细胞. 相似文献
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目的全面了解目前体外诱导BMSCs向软骨分化的方法,为软骨组织工程研究提供参考。方法广泛查阅近年来有关软骨组织工程中诱导BMSCs向软骨分化的文献,并进行综合分析。结果目前BMSCs诱导成软骨方法主要是添加外源性生长因子,其中TGF-β家族被公认为最重要的诱导和调节因子。其他重要的诱导方法包括添加多种化学因子、物理因素、转基因技术和微环境诱导等方法,但这些方法仍存在诱导效率低、诱导效果不稳定的问题。结论诱导方法的进展促进了BMSCs在软骨组织工程中的应用,建立更高效、简便、安全的诱导方法仍是软骨组织工程领域重要研究课题之一。 相似文献
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骨髓间充质干细胞在糖尿病模型创面中向表皮细胞分化的初步研究 总被引:5,自引:1,他引:4
目的:探讨糖尿病皮肤创面微环境中的骨髓间充质干细胞分化为表皮细胞的可能性。方法:从大鼠骨髓中分离纯化骨髓间充质干细胞。选取第3—4代细胞,应用5-溴脱氧尿嘧啶(5-BrdU)标记技术进行细胞标记;同时采用一次性腹腔内注射链脲佐菌素制作糖尿病大鼠模型。2周后,在大鼠背部人工造成圆形创面,将已标记的骨髓间充质干细胞以注射方式回植入糖尿病大鼠创面组织周围,分别于注射后2、3周取材,常规石蜡包埋、连续切片,行BrdU和角蛋白免疫组织化学染色。结果:BrdU阳性细胞出现在表皮、真皮及皮下组织各层,连续切片表皮层中部分阳性细胞同时也表达角蛋白。结论:在糖尿病渍疡愈合过程中,骨髓间充质干细胞具有向表皮细胞分化的潜能。 相似文献
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目的探讨生长分化因子7(growth differentiation factor7,GDF-7)在体外能否促进BMSCs向肌腱细胞分化,为改善BMSCs修复肌腱疗效提供依据。方法采用密度梯度离心法从绿荧光大鼠骨髓组织中分离培养BMSCs,通过成骨、成脂和成软骨分化鉴定其多向分化能力。取第3代BMSCs根据成肌腱培养液中加入不同浓度GDF-7(0、12.5、25.0、50.0ng/mL),分为A、B、C、D组。体外诱导培养2周后,实时荧光定量PCR检测各组scleraxis、tenomodulin、tenascinC和Ⅰ型胶原mRNA表达,免疫细胞化学染色观察tenomodulin、tenascinC和Ⅰ型胶原蛋白表达,Western blot法检测A、C组tenomodulin蛋白的表达。结果经鉴定,分离培养的BMSCs具有成骨、成脂和成软骨分化的多向分化能力。实时荧光定量PCR检测结果示,B、C、D组的tenomodulin mRNA表达分别较A组增加了2.85、3.41、3.07倍(P<0.05),scleraxis mRNA表达增加了2.13、1.50、2.56倍(P<0.05),tenascin C mRNA表达增加了2.45、2.86、1.88倍(P<0.05),但B、C、D组间比较差异均无统计学意义(P>0.05)。B、C组Ⅰ型胶原mRNA表达较A组分别增高了1.92和2.45倍(P<0.05);D组较A组有所增高,但差异无统计学意义(P>0.05)。免疫细胞化学染色示,B、C、D组均有tenomodulin、tenascin C和Ⅰ型胶原蛋白表达,而A组均未表达;除C组Ⅰ型胶原表达高于B、D组外,其他蛋白表达B、C、D组间无明显差异。Western blot检测示C组比A组有更高的tenomodulin蛋白表达。结论 GDF-7在体外能促进大鼠BMSCs向肌腱细胞分化。 相似文献
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目的 BMSCs在体外分化为胰岛素分泌细胞存在分化效率低、成熟度差的问题。研究BMSCs在糖尿病猪胰腺微环境下分化为胰岛素分泌细胞的可行性。方法取1只4周龄雄性贵州小香猪骨髓,采用贴壁法制备BMSCs。15只8~10周龄雌性贵州小香猪,体重8~10 kg,随机分为正常组(A组)、糖尿病组(B组)和BMSCs移植组(C组),每组5只。B、C组连续3 d从耳缘静脉推注链脲菌素加四氧嘧啶溶液,连续2 d血糖>17 mmol/L提示糖尿病造模成功。C组将标记增强型绿色荧光蛋白(enhanced green?uorescent portein,EGFP)的第3代BMSCs(细胞密度为5×107个/mL)1.1 mL多点注射移植至胰腺包膜下,A、B组注射等量生理盐水。监测血糖至30 d后,取胰腺组织行HE染色观察并检测胰岛数目及直径;免疫荧光组织化学染色检测新生胰岛的胰岛素表达;激光捕获显微切割技术获得EGFP+细胞,提取总RNA,采用RT-PCR检测胰岛素mRNA和胰腺十二指肠同源框蛋白1(pancreatic and duodenal homeobox factor 1,PDX1)mRNA的表达;荧光原位杂交(fluorescence in situ hybridization,FISH)检测胰岛素基因和SRY(sexdetermining region ofthe Y chromosome)基因的共表达。结果移植18 d后C组血糖开始较B组明显降低,且随时间延长逐渐下降(P<0.05)。组织学观察发现BMSCs移植30 d后C组胰岛数目(10.9±2.2)个较B组(4.6±1.4)个明显增加,差异有统计学意义(P<0.05),与A组(12.6±2.6)个比较差异无统计学意义(P>0.05);C组新生胰岛直径(47.2±19.6)μm,明显小于A组(119.6±27.7)μm,差异有统计学意义(P<0.05),B组未见新生胰岛。免疫荧光组织化学染色显示C组新生胰岛有胰岛素表达。RT-PCR检测示C组EGFP+细胞有胰岛素mRNA和PDX1 mRNA表达。FISH检测C组细胞中有SRY基因和胰岛素基因共表达。结论 BMSCs在糖尿病猪胰腺微环境条件下可分化为胰岛素分泌细胞。 相似文献
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目的探讨兔骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs)体外培养、定向诱导分化为成骨细胞和成脂肪细胞的途径,为进一步的实验研究打下基础。方法抽取兔股骨骨髓,以全骨髓贴壁培养法进行体外培养,贴壁细胞传代,倒置显微镜下观察细胞形态。取第3代细胞向成骨细胞和成脂肪细胞诱导,14d后成骨细胞诱导组检测碱性磷酸酶,成脂肪细胞诱导组进行油红O染色。结果全骨髓贴壁培养法可获得BM-MSCs,原代和传代培养的BM-MSCs具有活跃的增殖能力。成骨细胞诱导组碱性磷酸酶检测表达阳性,成脂肪细胞诱导组油红O染色见胞浆内出现大量红染脂滴。结论全骨髓贴壁培养法可有效地分离和扩增BM-MSCs,分离培养的BM-MSCs生长稳定,增殖力强,可向成骨细胞和成脂肪细胞诱导分化。 相似文献
18.
目的 探讨仿生电刺激体外诱导人骨髓间充质干细胞株(UE7T-13)向肝细胞分化的作用.方法 采用静电纺丝及氧化聚合法制备聚吡咯/PLGA导电膜,并在其表面培养UE7T-13细胞株.实验按是否给予诱导因子及施加电刺激分为四组:电刺激+诱导因子组、电刺激组、诱导因子组以及空白对照组.诱导因子包括HGF、EGF、FGF及OSM等.电刺激为每日给予频率5Hz、脉宽5 ms、电压100 mV(电流强度100 μA)的仿生电刺激2h.每天常规收集培养液,检测其中白蛋白分泌、尿素合成、细胞色素P450活性.MTT法检测细胞增殖能力,细胞免疫荧光方法检测各组干细胞中白蛋白和甲胎蛋白的表达.结果 电刺激+诱导因子组及诱导因子组UE7T-13细胞均成功诱导出类肝样细胞.与单纯诱导因子组比较,电刺激+诱导因子组UE7T-13更早向肝细胞转化,其肝细胞特异功能表达及表面特异蛋白分子表达更强.UE7T-13细胞在有电刺激存在时增殖能力明显提高.结论 仿生电刺激可促进体外UE7T-13细胞增殖及其肝向分化,使其更早出现肝细胞特异性分子表达,并提高分化效率. 相似文献
19.
体外诱导骨髓间质干细胞向软骨方向分化的研究 总被引:7,自引:1,他引:6
目的 探讨体外诱导骨髓间充质干细胞 (BMSCs)向软骨方向分化的条件和方法。方法 抽取兔股骨骨髓 ,用密度梯度离心和贴壁分离法分离BMSCs ,分别在高密度 (1× 10 6/ml)和低密度 (1× 10 4/ml)培养条件下用转化生长因子 β1(TGF β1)诱导BMSCs向软骨方向分化。倒置显微镜、透射及扫描电子显微镜观察细胞 ,用免疫组织化学、原位杂交、特殊染色方法检测Ⅱ型胶原及软骨基质成分的表达。结果 高密度诱导的BMSCsⅡ型胶原免疫组织化学阳性率为 89.2 %,Ⅱ型胶原原位杂交阳性率为 82 .6%,黏多糖特殊染色阳性 ,低密度诱导的BMSCsⅡ型胶原免疫组织化学阴性 ,Ⅱ型胶原原位杂交阳性率为 3 .8%,黏多糖特殊染色阴性。结论 TGF β1可以诱导BMSCs向软骨方向分化 ,细胞密度是BMSCs分化的重要影响因素。 相似文献
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目的 观察低频振动对小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)成骨分化的影响。方法 取3周龄C57BL/6小鼠,分离及培养其BMSCs。将细胞分为两组,对照组及低频振动组,两组均诱导BMSCs进行成骨分化,低频振动组在诱导过程中实施低频振动干预(15 Hz,垂直加速度0.3 g),对照组不进行干预,分别于第7天、第14天收获细胞,将第7天的细胞进行碱性磷酸酶(alkaline phosphatase, ALP)染色,将第14天的细胞进行茜素红染色;并利用Real-time PCR检测相关基因表达。结果 低频振动组细胞第7天的ALP活性明显高于对照组,且在14 d形成的钙结节多于对照组;所检测基因中,低频振动组细胞Ⅰ型胶原蛋白、骨桥蛋白、骨钙素、骨保护素基因的表达均高于对照组,而破骨细胞分化因子(receptor activator of NF-κB ligand, RANKL)较对照组低。结论 低频振动可以促进BMSCs的成骨分化,并调节成骨细胞分泌的细胞因子,影响破骨细胞的生成。 相似文献