碱性成纤维生长因子联合全反式维甲酸诱导脐血单个核细胞分化为神经细胞的实验研究
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摘要
目的：1.探讨脐血单个核细胞(CB-MNCs)的体外分离、培养、扩增的规律和特点,初步探讨脐血单个核细胞(CB-MNCs)在体外向神经细胞分化的条件。2.采用全反式维甲酸(All-Trans-Retinoic Acid,RA)和碱性成纤维细胞生长因子(bFGF)作为诱导剂,对脐血单个核细胞(CB-MNCs)进行体外诱导实验。探讨RA和bFGF体外诱导脐血单个核细胞分化为神经细胞的机制,为脊髓损伤的细胞移植治疗提供实验基础。
方法：1.收集脐血：收集妊娠足月正常分娩的新生儿脐带血,按卫生部颁布的《献血体检标准》检验合格,于第3产程自脐静脉消毒后全封闭采集脐血,CPD21抗凝,平均采量80-130ml。2脐血分离：新鲜脐血在24小时内分离。新鲜血液与PBS按1：l比例混匀并制成细胞悬液,叠加到淋巴细胞分离液上,2000 r/min离心20 min,然后取出白膜层,1000 r/min离心10 min,弃上清,然后调整细胞浓度为1×106／ml。3.分组：将上述CB-MNCs以3×106个细胞接种于6孔培养板,每孔终体积3ml(含20%FBS的H.DMEM。双抗生素青、链霉素各100U／ml);按诱导体系中诱导因素的不同组合分4组,A组：对照组,不加任何因子。B组：加全反式维甲酸。C组：加碱性成纤维生长因子。D组：加碱性成纤维生长因子和全反式维甲酸。每日在倒置显微镜下观察细胞的形态变化,每3天更换培养基,弃未贴壁细胞；2周后观察结果。4.细胞免疫荧光鉴定：采用细胞免疫荧光方法,对以上四组细胞诱导2周后检测神经元标志物Tuj-1和星形胶质细胞标志物GFAP的表达情况,荧光显微镜下观察并拍照记录。荧光显微镜下在各组细胞随机取100个细胞,计数Tuj-1及GFAP反应阳性细胞个数之和,统计学分析：数据均以x±s表示,并用SPSS17.0软件进行单因素方差分析。
结果：1,倒置显微镜下形态观察结果：刚分离的CB-MNCs呈小、圆形,1d后,少量细胞贴壁生长,未见明显形态变化；6d时B、C、D组均可见一些细胞伸出胞突,有的呈梭状,有的伸出细丝状的突起；10d时,D组细胞可见一些细胞胞体变大、胞突伸长,有的细胞双极,有的多极；14d后,A组细胞未见明显变化,而B、C、D组可见一些胞体较大、有数个粗长突起的细胞生长,相邻细胞突起连成网状,类似神经元。2,细胞免疫荧光结果：CB-MNCs诱导为神经细胞,经统计学分析,(1)诱导后D组分化率最高,与其它组有显著差异(P<0.05)；(2)诱导后B、C、D组与A组分化率有显著差异(P<0.05)；(3)B和C组分化率无显著差异(P>0.05)。
结论：1.全反式维甲酸与碱性成纤维生长因子均能诱导脐血单个核细胞分化成神经细胞。2.碱性成纤维生长因子联合全反式维甲酸能协同诱导脐血单个核细胞分化成神经细胞,提高脐血单个核细胞的神经细胞转化率。
Objective:1.To discuss the law and the feature of Separation、cultivation and amplification of the cord blood mononuclear cells (CB-MNCs)in vitro,and discuss the conditions of CB-MNCs differentiating into neural cells basically in vitro.2. Carry out the induced experiments in vitro of cord blood mononuclear cells (CB-MNCs) with all-trans-retinoic acid (RA) and basic fibroblast growth factor (bFGF) as an inducer. To discuss the mechanism of induced CB-MNCs differentiating into neural cells in vitro, which provide the experimental basis to the cell transplantation treatment of spinal cord injury.
Methods:1.Collection of cord blood:Collecting cord blood of newborn who full-term pregnancy and normal delivery and passed the test according to the "blood medical standards" promulgated by the Ministry of Health, to collect umbilical cord blood completely closed after disinfection in the 3 stages of labor,use CPD21 anticoagulant and taken the average amount 80-130ml.2.Cord blood separation:Isolating fresh cord blood within 24 hours.Fresh blood mix with PBS by 1:1 ratio to produce a cell suspension, superimposed on Lymphocyte separation medium,2000 r/min centrifugation 20 min,then remove the white film, 1000 r/min centrifugation 10 min, discard supernatant, and then adjust the cell concentration to 1×106/ml.3 Group:Using Above-mentioned CB-MNCs with 3×106 cells inoculated in 6-hole plate, each hole final volume 3ml (containing 20% fetal bovine serum(FBS) in H-DMEM,Two antibiotic,penicillin and streptomycin each 100 U/ml);Divide into 4 groups according to the different combinations of induction factors in induction system. Group A:control group, without any factor. Group B: Add RA.Group C:add bFGF. Group D:add RA and bFGF. Observing the morphological changes of cells daily under the inverted microscope, and changed the medium every 3 days, discard the cells which not adherent. Observing the results after 2 weeks.4.Immunofluorescence identification:Detecting the expression of the neuronal markers Tuj-1 and the astrocyte marker GFAP, after induced the cells above-mentioned four groups 2 weeks later, using the method of immunofluorescence, observe by the Fluorescence microscope and take photographs.Checking 100 cells randomly in each group under the Fluorescence microscopy, and counting the amount of the cells Tuj-1-positive and GFAP-positive. Statistical analysis:Data are Expressed by x±s,and analysis single factor variance with SPSS 17.0 software.
Results:1,The result observed under the microscope morphological: newly isolated CB-MNCs were small and round.1 day later, Small number of cells adhered to the wall with no significant morphological changes at the early culture;in the sixth day,the B,C,D groups were seen some cells out of synapses, and some were spindle,and some out of thin filamentous processes;in the tenth day, group D cells of some cells become larger cell body, some cells has elongation, some cells double poles, and some multi-polar; Fourteen days later, cells of A group have no significant changes,while B,C,D group showed a greater number of cell body, some has several long and big processes, even has the adjacent reticular cell processes, similar to neural cells.2 immunofluorescence results:CB-MNCs induced to neural cells, by way of statistical analysis, 1)The differentiation rate of D group after induced is the highest, and significantly different with other groups (P<0.05);(2) B,C,D group differentiation rate were significantly different with A group (P<0.05);(3) B and C groups showed no significant difference (P> 0.05).
Conclusion:1.Both RA and bFGF could induce CB-MNCs to differentiate into neural cells.2.RA and bFGF can synergistically induced CB-MNCs to differentiate into neural cells,and the difference with the other groups is statistically significant.
方法：1.收集脐血：收集妊娠足月正常分娩的新生儿脐带血,按卫生部颁布的《献血体检标准》检验合格,于第3产程自脐静脉消毒后全封闭采集脐血,CPD21抗凝,平均采量80-130ml。2脐血分离：新鲜脐血在24小时内分离。新鲜血液与PBS按1：l比例混匀并制成细胞悬液,叠加到淋巴细胞分离液上,2000 r/min离心20 min,然后取出白膜层,1000 r/min离心10 min,弃上清,然后调整细胞浓度为1×106／ml。3.分组：将上述CB-MNCs以3×106个细胞接种于6孔培养板,每孔终体积3ml(含20%FBS的H.DMEM。双抗生素青、链霉素各100U／ml);按诱导体系中诱导因素的不同组合分4组,A组：对照组,不加任何因子。B组：加全反式维甲酸。C组：加碱性成纤维生长因子。D组：加碱性成纤维生长因子和全反式维甲酸。每日在倒置显微镜下观察细胞的形态变化,每3天更换培养基,弃未贴壁细胞；2周后观察结果。4.细胞免疫荧光鉴定：采用细胞免疫荧光方法,对以上四组细胞诱导2周后检测神经元标志物Tuj-1和星形胶质细胞标志物GFAP的表达情况,荧光显微镜下观察并拍照记录。荧光显微镜下在各组细胞随机取100个细胞,计数Tuj-1及GFAP反应阳性细胞个数之和,统计学分析：数据均以x±s表示,并用SPSS17.0软件进行单因素方差分析。
结果：1,倒置显微镜下形态观察结果：刚分离的CB-MNCs呈小、圆形,1d后,少量细胞贴壁生长,未见明显形态变化；6d时B、C、D组均可见一些细胞伸出胞突,有的呈梭状,有的伸出细丝状的突起；10d时,D组细胞可见一些细胞胞体变大、胞突伸长,有的细胞双极,有的多极；14d后,A组细胞未见明显变化,而B、C、D组可见一些胞体较大、有数个粗长突起的细胞生长,相邻细胞突起连成网状,类似神经元。2,细胞免疫荧光结果：CB-MNCs诱导为神经细胞,经统计学分析,(1)诱导后D组分化率最高,与其它组有显著差异(P<0.05)；(2)诱导后B、C、D组与A组分化率有显著差异(P<0.05)；(3)B和C组分化率无显著差异(P>0.05)。
结论：1.全反式维甲酸与碱性成纤维生长因子均能诱导脐血单个核细胞分化成神经细胞。2.碱性成纤维生长因子联合全反式维甲酸能协同诱导脐血单个核细胞分化成神经细胞,提高脐血单个核细胞的神经细胞转化率。
Objective:1.To discuss the law and the feature of Separation、cultivation and amplification of the cord blood mononuclear cells (CB-MNCs)in vitro,and discuss the conditions of CB-MNCs differentiating into neural cells basically in vitro.2. Carry out the induced experiments in vitro of cord blood mononuclear cells (CB-MNCs) with all-trans-retinoic acid (RA) and basic fibroblast growth factor (bFGF) as an inducer. To discuss the mechanism of induced CB-MNCs differentiating into neural cells in vitro, which provide the experimental basis to the cell transplantation treatment of spinal cord injury.
Methods:1.Collection of cord blood:Collecting cord blood of newborn who full-term pregnancy and normal delivery and passed the test according to the "blood medical standards" promulgated by the Ministry of Health, to collect umbilical cord blood completely closed after disinfection in the 3 stages of labor,use CPD21 anticoagulant and taken the average amount 80-130ml.2.Cord blood separation:Isolating fresh cord blood within 24 hours.Fresh blood mix with PBS by 1:1 ratio to produce a cell suspension, superimposed on Lymphocyte separation medium,2000 r/min centrifugation 20 min,then remove the white film, 1000 r/min centrifugation 10 min, discard supernatant, and then adjust the cell concentration to 1×106/ml.3 Group:Using Above-mentioned CB-MNCs with 3×106 cells inoculated in 6-hole plate, each hole final volume 3ml (containing 20% fetal bovine serum(FBS) in H-DMEM,Two antibiotic,penicillin and streptomycin each 100 U/ml);Divide into 4 groups according to the different combinations of induction factors in induction system. Group A:control group, without any factor. Group B: Add RA.Group C:add bFGF. Group D:add RA and bFGF. Observing the morphological changes of cells daily under the inverted microscope, and changed the medium every 3 days, discard the cells which not adherent. Observing the results after 2 weeks.4.Immunofluorescence identification:Detecting the expression of the neuronal markers Tuj-1 and the astrocyte marker GFAP, after induced the cells above-mentioned four groups 2 weeks later, using the method of immunofluorescence, observe by the Fluorescence microscope and take photographs.Checking 100 cells randomly in each group under the Fluorescence microscopy, and counting the amount of the cells Tuj-1-positive and GFAP-positive. Statistical analysis:Data are Expressed by x±s,and analysis single factor variance with SPSS 17.0 software.
Results:1,The result observed under the microscope morphological: newly isolated CB-MNCs were small and round.1 day later, Small number of cells adhered to the wall with no significant morphological changes at the early culture;in the sixth day,the B,C,D groups were seen some cells out of synapses, and some were spindle,and some out of thin filamentous processes;in the tenth day, group D cells of some cells become larger cell body, some cells has elongation, some cells double poles, and some multi-polar; Fourteen days later, cells of A group have no significant changes,while B,C,D group showed a greater number of cell body, some has several long and big processes, even has the adjacent reticular cell processes, similar to neural cells.2 immunofluorescence results:CB-MNCs induced to neural cells, by way of statistical analysis, 1)The differentiation rate of D group after induced is the highest, and significantly different with other groups (P<0.05);(2) B,C,D group differentiation rate were significantly different with A group (P<0.05);(3) B and C groups showed no significant difference (P> 0.05).
Conclusion:1.Both RA and bFGF could induce CB-MNCs to differentiate into neural cells.2.RA and bFGF can synergistically induced CB-MNCs to differentiate into neural cells,and the difference with the other groups is statistically significant.
引文
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