中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
CHINESE JOURNAL OF PATHOPHYSIOLOGY
2010年
3期
498-503
,共6页
陆利%张卫国%杨桂姣%闫八一%白丽敏
陸利%張衛國%楊桂姣%閆八一%白麗敏
륙리%장위국%양계교%염팔일%백려민
人骨髓间充质干细胞%26S蛋白酶体%神经元样细胞%细胞分化
人骨髓間充質榦細胞%26S蛋白酶體%神經元樣細胞%細胞分化
인골수간충질간세포%26S단백매체%신경원양세포%세포분화
Human bone marrow stromal cells%26S proteasome%Neuron-like cells%Cell differentiation
目的:研究人骨髓间充质干细胞(hBMSCs)向神经元样细胞逐步分化过程中26S蛋白酶体的作用.方法:贴壁分离获得的hBMSCs先经β-巯基乙醇(β-ME)预诱导24 h,随后用视黄酸(RA)诱导3 d,最后再用生长因子(GF,10 μg/L bFGF、20 μg/L NGF)持续培养3 d.免疫荧光染色观察不同诱导阶段神经前体细胞标志物nestin、早期神经元标志物Tuj1、成熟神经元标志物NF的表达.免疫荧光染色和RT-PCR分析不同诱导阶段26S蛋白酶体的表达变化.应用含26S蛋白酶体抑制剂的培养液(5 μmol/L MG132、10 μg/L bFGF、20 μg/L NGF)作用于经β-ME/RA诱导后的细胞,NF免疫荧光染色观察蛋白酶体抑制剂对hBMSCs向神经元样细胞分化的影响.结果:未诱导的hBMSCs几乎不表达nestin、Tuj1和NF;经β-ME/RA诱导后神经前体细胞标志物nestin和早期神经元标志物Tuj1表达率增高(34.41%±1.27%,27.79%±1.27%);经β-ME/RA/GF诱导后成熟神经元标志物 NF表达率迅速升高(56.72%±2.40%).免疫荧光染色和RT-PCR结果证实,未诱导hBMSCs 26S蛋白酶体表达水平较低;经β-ME/RA诱导后个别细胞26S蛋白酶体染色增强,26S蛋白酶体mRNA表达水平增至1.33倍;经β-ME/RA/GF诱导后 26S蛋白酶体深染细胞增多,26S蛋白酶体mRNA表达水平增至1.77倍.应用26S蛋白酶体抑制剂MG132后,NF阳性细胞表达率显著降低(37.59%±1.52%).结论:hBMSCs在β-ME/RA/GF诱导下可逐步向神经元样细胞分化,26S蛋白酶体在诱导过程中表达水平逐步增高,26S蛋白酶体抑制剂能够抑制hBMSCs向成熟神经元分化,提示26S蛋白酶体可能参与诱导hBMSCs向神经元样细胞的成熟分化.
目的:研究人骨髓間充質榦細胞(hBMSCs)嚮神經元樣細胞逐步分化過程中26S蛋白酶體的作用.方法:貼壁分離穫得的hBMSCs先經β-巰基乙醇(β-ME)預誘導24 h,隨後用視黃痠(RA)誘導3 d,最後再用生長因子(GF,10 μg/L bFGF、20 μg/L NGF)持續培養3 d.免疫熒光染色觀察不同誘導階段神經前體細胞標誌物nestin、早期神經元標誌物Tuj1、成熟神經元標誌物NF的錶達.免疫熒光染色和RT-PCR分析不同誘導階段26S蛋白酶體的錶達變化.應用含26S蛋白酶體抑製劑的培養液(5 μmol/L MG132、10 μg/L bFGF、20 μg/L NGF)作用于經β-ME/RA誘導後的細胞,NF免疫熒光染色觀察蛋白酶體抑製劑對hBMSCs嚮神經元樣細胞分化的影響.結果:未誘導的hBMSCs幾乎不錶達nestin、Tuj1和NF;經β-ME/RA誘導後神經前體細胞標誌物nestin和早期神經元標誌物Tuj1錶達率增高(34.41%±1.27%,27.79%±1.27%);經β-ME/RA/GF誘導後成熟神經元標誌物 NF錶達率迅速升高(56.72%±2.40%).免疫熒光染色和RT-PCR結果證實,未誘導hBMSCs 26S蛋白酶體錶達水平較低;經β-ME/RA誘導後箇彆細胞26S蛋白酶體染色增彊,26S蛋白酶體mRNA錶達水平增至1.33倍;經β-ME/RA/GF誘導後 26S蛋白酶體深染細胞增多,26S蛋白酶體mRNA錶達水平增至1.77倍.應用26S蛋白酶體抑製劑MG132後,NF暘性細胞錶達率顯著降低(37.59%±1.52%).結論:hBMSCs在β-ME/RA/GF誘導下可逐步嚮神經元樣細胞分化,26S蛋白酶體在誘導過程中錶達水平逐步增高,26S蛋白酶體抑製劑能夠抑製hBMSCs嚮成熟神經元分化,提示26S蛋白酶體可能參與誘導hBMSCs嚮神經元樣細胞的成熟分化.
목적:연구인골수간충질간세포(hBMSCs)향신경원양세포축보분화과정중26S단백매체적작용.방법:첩벽분리획득적hBMSCs선경β-구기을순(β-ME)예유도24 h,수후용시황산(RA)유도3 d,최후재용생장인자(GF,10 μg/L bFGF、20 μg/L NGF)지속배양3 d.면역형광염색관찰불동유도계단신경전체세포표지물nestin、조기신경원표지물Tuj1、성숙신경원표지물NF적표체.면역형광염색화RT-PCR분석불동유도계단26S단백매체적표체변화.응용함26S단백매체억제제적배양액(5 μmol/L MG132、10 μg/L bFGF、20 μg/L NGF)작용우경β-ME/RA유도후적세포,NF면역형광염색관찰단백매체억제제대hBMSCs향신경원양세포분화적영향.결과:미유도적hBMSCs궤호불표체nestin、Tuj1화NF;경β-ME/RA유도후신경전체세포표지물nestin화조기신경원표지물Tuj1표체솔증고(34.41%±1.27%,27.79%±1.27%);경β-ME/RA/GF유도후성숙신경원표지물 NF표체솔신속승고(56.72%±2.40%).면역형광염색화RT-PCR결과증실,미유도hBMSCs 26S단백매체표체수평교저;경β-ME/RA유도후개별세포26S단백매체염색증강,26S단백매체mRNA표체수평증지1.33배;경β-ME/RA/GF유도후 26S단백매체심염세포증다,26S단백매체mRNA표체수평증지1.77배.응용26S단백매체억제제MG132후,NF양성세포표체솔현저강저(37.59%±1.52%).결론:hBMSCs재β-ME/RA/GF유도하가축보향신경원양세포분화,26S단백매체재유도과정중표체수평축보증고,26S단백매체억제제능구억제hBMSCs향성숙신경원분화,제시26S단백매체가능삼여유도hBMSCs향신경원양세포적성숙분화.
AIM: To investigate the process of human bone marrow stromal cells (hBMSCs) differentiation into neural-like cells and to determine the role of 26S proteasome in neuronal differentiation. METHODS: Purified hBMSCs were treated with β-mercaptoethanol (β-ME) for 1 day and retinoic acid (RA) for 3 days, followed by growth factor (10 μg/L bFGF or 20 μg/L NGF) for another 3 days. Immunofluorescence was performed to detect the expression of nestin (a neural precursor cells marker), Tuj1 (a premature neuronal marker), and neurofilament (NF, a mature neuronal marker) at all stages of induced differentiation. Immunostaining and RT-PCR were used to analyze the expression of 26S proteasome during neuronal differentiation of hBMSCs. To further confirm the role of 26S proteasome in hBMSCs differentiation, cells were treated with β-ME/RA and then followed by protesome inhibitor MG132 and growth factor. Immunostaining was performed to detect NF-positive cells. RESULTS: Quantification results showed that the untreated cells were almost never positive for nestin, Tuj1 and NF. After treated with β-ME/RA, the numbers of nestin-positive cells (34.41%±1.27%) and Tuj1-positive cells (27.79%±1.27%) were increased. Notably, the numbers of NF-positive cells were significantly increased to 56.72%±2.4% after induction with β-ME/RA/GF. Immunofluorescence analysis showed that undifferentiated hBMSCs cells were weakly stained by antibody against 26S proteasome, but the numbers of cells with high-intensity of 26S proteasome were increased after treated with β-ME/RA. The RT-PCR result of 26S proteasome further confirmed that the mRNA level of the cells differentiated by β-ME/RA (1.33), as well as by β-ME/RA/GF (1.77), was significantly increased compared to the undifferentiated cells. Moreover, hBMSCs incubated with protesome inhibitor MG132 significantly decreased the numbers of NF-positive cells (37.59%±1.52%). CONCLUSION: After induction with β-ME/RA/GF, hBMSCs can be differentiated into neural-like cells, which is concomitant with the increase in 26S proteasome expression. Inhibitor of 26S protesome prevents hBMSCs differentiation, suggesting that 26S proteasome may be involved in the differentiation of hBMSCs into neural-like cells.
