神经干细胞体外定向分化为多巴胺能神经元的实验研究
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摘要
目的:在探讨神经干细胞(neural stem cells,NSCs)分离培养和扩增的基础上,重点研究抗坏血酸(ascorbic acid,AA)和胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)对体外培养的NSCs定向分化为多巴胺(dopamine,DA)能神经元的影响,为DA能神经元的定向分化研究提供基础的实验依据。
方法:(1)从新生24h内的SD大鼠脑组织分离NSCs,采用无血清悬浮培养法进行NSCs体外扩增培养。倒置相差显微镜观察细胞形态,通过绘制细胞生长曲线观察NSCs的自我更新和增殖能力,采用免疫细胞化学法检测NSCs标志物神经上皮干细胞蛋白(neuroepithelial stem cell protein,Nestin)的表达及分化后细胞神经元特异性烯醇化酶(neuron specialenolase,NSE)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和2,3-环核甘酸磷酸二脂酶(cyclic nucleotide phosphohydrolase,CNP)的表达;(2)第二代NSCs诱导培养基中分别给予不同浓度的AA,10ng/mlGDNF及100μmol/1 AA+10ng/ml GDNF。10d后终止诱导,进行DA能神经元特异性标记物酪氨酸羟化酶(tyrosine hydroxylase,TH)和多巴胺转运蛋白(dipamine transporter,DAT)的免疫细胞化学检测和TH基因的RT-PCR检测。
结果:(1)从新生SD大鼠脑组织分离的细胞在无血清的培养基中形成悬浮的神经球。神经球具有自我更新和表达Nestin的能力,分化后的细胞能表达神经元、星型胶质细胞及少突胶质细胞的特异性抗原。(2)与对照组比较,不同浓度的AA组、10ng/ml GDNF组及100μmol/l AA+10ng/ml GDNF联合诱导组均能增加NSCs向TH阳性细胞分化的比率(p＜0.05)。与单独运用100μmol/l AA或10ng/ml GDNF组比较,100μmol/l AA+10ng/ml GDNF联合诱导组可明显提高NSCs向TH阳性细胞分化的比率(p＜0.05)。各诱导组均检测到TH mRNA的表达。
结论:(1)从新生大鼠的脑组织中成功分离出NSCs,其具有在体外自我更新和增殖、多向分化潜能及表达Nestin的能力。(2)在本实验中,不同浓度AA组均能促进NSCs向DA能神经元分化。(3)GDNF能促进NSCs向DA能神经元分化,与AA联合诱导后分化作用得到进一步加强。
Objective:To investigate the neural stem cells(NSCs)isolation,culture and expansion.To provide the experimental bases for the directional differentiation of dopaminergic(DA)neurons,we studied the effects of ascorbic acid(AA)and glial cell line-derived neurotrophic factor(GDNF)on the differentiation of NSCs into DA neurons in vitro.
Methods:(1)Isolated the primary NSCs from whole brain of neonatal Sprague Dawley rats(within 24h after birth),the cells were suspended and cultured in serum-free medium in vitro.The cell morphology was observed with inverted phase-contrast microscope.Ploting the growth curve to observe the capacity of self-renewing and proliferation of NSCs.Immunocytochemistry was performed to detect the expression of neuroepithelial stem cell protein(Nestin), neuron specific enolase(NSE),glial fibrillary acidic protein(GFAP),and cyclic nucleotide phosphohydrolase(CNP).(2)The P 2(passage 2)NSCs were seeded in the differentiation medium supplemented with different concentration of AA or 10ng/ml GDNF or 100μmol/l AA+10ng/ml GDNF respectively.After these cells were induced for 10 days,the specific antigens of DA neurons including tyrosine hydroxylase(TH)and dopamine transporter(DAT)were detected by Immunocytochemistry.TH mRNA was detected by RT-PCR.
Results:(1)The proliferating cells isolated from neonatal Sprague Dawley rats grew into floating neurospheres in the serum-free medium.The neurospheres had the capacities of self-renewing and expressing Nestin.The cells differentiated from these neurospheres could express the specific antigens of neurons,astrocytes,and oligodendrocytes.(2)Each group of different concentration of AA,10ng/ml GDNF and 100μmol/l AA+ 10ng/ml GDNF could increase the proportion of NSCs differentiated into TH-positive cells compared with the control group(p＜0.05).When treated with 100μmol/l AA +10ng/ml GDNF,the proportion of NSCs differentiated into TH-positive cells was significantly higher than 100μmol/l AA or 10ng/ml GDNF group alone(p＜0.05).Each group could detect the expression of TH mRNA.
Conclusions:(1)The NSCs were isolated from neonatal rats successfully. In vitro,they showed the capacities of self-renewing and proliferation, pluripotentiality of differentiation,and expressed the Nestin antigen.(2)In our studies,each group of AA could promote the differentiation of NSCs into DA neurons.(3)GDNF could promote the differentiation of NSCs into DA neurons, when treated with GDNF + AA,the effect was stronger than AA or GDNF group alone.
方法:(1)从新生24h内的SD大鼠脑组织分离NSCs,采用无血清悬浮培养法进行NSCs体外扩增培养。倒置相差显微镜观察细胞形态,通过绘制细胞生长曲线观察NSCs的自我更新和增殖能力,采用免疫细胞化学法检测NSCs标志物神经上皮干细胞蛋白(neuroepithelial stem cell protein,Nestin)的表达及分化后细胞神经元特异性烯醇化酶(neuron specialenolase,NSE)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和2,3-环核甘酸磷酸二脂酶(cyclic nucleotide phosphohydrolase,CNP)的表达;(2)第二代NSCs诱导培养基中分别给予不同浓度的AA,10ng/mlGDNF及100μmol/1 AA+10ng/ml GDNF。10d后终止诱导,进行DA能神经元特异性标记物酪氨酸羟化酶(tyrosine hydroxylase,TH)和多巴胺转运蛋白(dipamine transporter,DAT)的免疫细胞化学检测和TH基因的RT-PCR检测。
结果:(1)从新生SD大鼠脑组织分离的细胞在无血清的培养基中形成悬浮的神经球。神经球具有自我更新和表达Nestin的能力,分化后的细胞能表达神经元、星型胶质细胞及少突胶质细胞的特异性抗原。(2)与对照组比较,不同浓度的AA组、10ng/ml GDNF组及100μmol/l AA+10ng/ml GDNF联合诱导组均能增加NSCs向TH阳性细胞分化的比率(p＜0.05)。与单独运用100μmol/l AA或10ng/ml GDNF组比较,100μmol/l AA+10ng/ml GDNF联合诱导组可明显提高NSCs向TH阳性细胞分化的比率(p＜0.05)。各诱导组均检测到TH mRNA的表达。
结论:(1)从新生大鼠的脑组织中成功分离出NSCs,其具有在体外自我更新和增殖、多向分化潜能及表达Nestin的能力。(2)在本实验中,不同浓度AA组均能促进NSCs向DA能神经元分化。(3)GDNF能促进NSCs向DA能神经元分化,与AA联合诱导后分化作用得到进一步加强。
Objective:To investigate the neural stem cells(NSCs)isolation,culture and expansion.To provide the experimental bases for the directional differentiation of dopaminergic(DA)neurons,we studied the effects of ascorbic acid(AA)and glial cell line-derived neurotrophic factor(GDNF)on the differentiation of NSCs into DA neurons in vitro.
Methods:(1)Isolated the primary NSCs from whole brain of neonatal Sprague Dawley rats(within 24h after birth),the cells were suspended and cultured in serum-free medium in vitro.The cell morphology was observed with inverted phase-contrast microscope.Ploting the growth curve to observe the capacity of self-renewing and proliferation of NSCs.Immunocytochemistry was performed to detect the expression of neuroepithelial stem cell protein(Nestin), neuron specific enolase(NSE),glial fibrillary acidic protein(GFAP),and cyclic nucleotide phosphohydrolase(CNP).(2)The P 2(passage 2)NSCs were seeded in the differentiation medium supplemented with different concentration of AA or 10ng/ml GDNF or 100μmol/l AA+10ng/ml GDNF respectively.After these cells were induced for 10 days,the specific antigens of DA neurons including tyrosine hydroxylase(TH)and dopamine transporter(DAT)were detected by Immunocytochemistry.TH mRNA was detected by RT-PCR.
Results:(1)The proliferating cells isolated from neonatal Sprague Dawley rats grew into floating neurospheres in the serum-free medium.The neurospheres had the capacities of self-renewing and expressing Nestin.The cells differentiated from these neurospheres could express the specific antigens of neurons,astrocytes,and oligodendrocytes.(2)Each group of different concentration of AA,10ng/ml GDNF and 100μmol/l AA+ 10ng/ml GDNF could increase the proportion of NSCs differentiated into TH-positive cells compared with the control group(p＜0.05).When treated with 100μmol/l AA +10ng/ml GDNF,the proportion of NSCs differentiated into TH-positive cells was significantly higher than 100μmol/l AA or 10ng/ml GDNF group alone(p＜0.05).Each group could detect the expression of TH mRNA.
Conclusions:(1)The NSCs were isolated from neonatal rats successfully. In vitro,they showed the capacities of self-renewing and proliferation, pluripotentiality of differentiation,and expressed the Nestin antigen.(2)In our studies,each group of AA could promote the differentiation of NSCs into DA neurons.(3)GDNF could promote the differentiation of NSCs into DA neurons, when treated with GDNF + AA,the effect was stronger than AA or GDNF group alone.
引文
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3 Palmer TD,Takahashi J,Gage FH.The adult rat hippocampus contains primordial neural stem cells[J].Mol Cell Neurosci,1997,8:389-404.
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