BACKGROUND: Midbrain-derived neural stem cells (mNSCs) can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson's disease. OBJECTIVE: ...BACKGROUND: Midbrain-derived neural stem cells (mNSCs) can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson's disease. OBJECTIVE: To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING: An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital (China) from March to November 2007. MATERIALS: Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein (GFAP) antibody and cyclic nucleotide 3'-phosphohydrolase (CNPase) antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) were provided by R&D Systems. METHODS: The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/FI 2, 1% N: supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10ug/mL polylysine and induced to differentiate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES: The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for β Ⅲ tubulin, GFAP and CNPase. RESULTS: Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tubulin. CONCLUSION: Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells following induction by EGF, FGF2 and N: additive.展开更多
为深入揭示同型半胱氨酸(hom ocysteine,HCY)的致畸性及作用机理,应用大鼠胚胎中脑细胞微团培养法探讨了HCY(0~10m m ol/L)对胚胎中脑细胞增殖和分化的影响。结果显示:随着剂量的增加,HCY对胚胎中脑细胞分化具有促进和抑制双相作用,半...为深入揭示同型半胱氨酸(hom ocysteine,HCY)的致畸性及作用机理,应用大鼠胚胎中脑细胞微团培养法探讨了HCY(0~10m m ol/L)对胚胎中脑细胞增殖和分化的影响。结果显示:随着剂量的增加,HCY对胚胎中脑细胞分化具有促进和抑制双相作用,半数抑制分化浓度(IC50 )为4.3m m ol/L,当加入肝微粒体S9时,IC50减至2.3m m ol/L;在实验剂量范围内,HCY对中脑细胞增殖无明显抑制作用。上述结果说明HCY 对大鼠胚胎中脑细胞分化的影响明显高于对细胞增殖的影响。展开更多
基金the National Natural Science Foundation of China,No.30672151
文摘BACKGROUND: Midbrain-derived neural stem cells (mNSCs) can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson's disease. OBJECTIVE: To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING: An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital (China) from March to November 2007. MATERIALS: Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein (GFAP) antibody and cyclic nucleotide 3'-phosphohydrolase (CNPase) antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) were provided by R&D Systems. METHODS: The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/FI 2, 1% N: supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10ug/mL polylysine and induced to differentiate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES: The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for β Ⅲ tubulin, GFAP and CNPase. RESULTS: Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tubulin. CONCLUSION: Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells following induction by EGF, FGF2 and N: additive.
文摘为深入揭示同型半胱氨酸(hom ocysteine,HCY)的致畸性及作用机理,应用大鼠胚胎中脑细胞微团培养法探讨了HCY(0~10m m ol/L)对胚胎中脑细胞增殖和分化的影响。结果显示:随着剂量的增加,HCY对胚胎中脑细胞分化具有促进和抑制双相作用,半数抑制分化浓度(IC50 )为4.3m m ol/L,当加入肝微粒体S9时,IC50减至2.3m m ol/L;在实验剂量范围内,HCY对中脑细胞增殖无明显抑制作用。上述结果说明HCY 对大鼠胚胎中脑细胞分化的影响明显高于对细胞增殖的影响。
文摘目的观察针刺对海洛因复吸大鼠脑神经细胞凋亡的影响。方法采用剂量递增法复制海洛因成瘾大鼠模型,将40只Wistar大鼠平均分成正常组、模型组、针刺组、药物组,于实验第39天取4组大鼠海马、中脑腹侧被盖区(ventral tegmental area,VTA)脑组织,光镜下观察神经细胞坏死情况,采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase mediated nick end labeling,TUNEL)检测脑神经细胞凋亡情况。结果光镜下可见模型组大鼠脑海马、VTA神经细胞丢失、变性较严重,核溶解消失,神经细胞变性坏死及间质水肿明显,并可见筛状软化灶。针刺组未见明显筛状软化灶,神经细胞变性坏死及间质水肿程度较模型组、药物组减轻。针刺组神经细胞间质水肿较轻,未见明显筛状软化灶,神经细胞水肿坏死变性较少量。与正常组比较,模型组大鼠脑海马、VTA中TUNEL染色阳性细胞数显著增多(P<0.01);与模型组、药物组比较,针刺组大鼠脑海马、VTA中TUNEL染色阳性细胞数显著减少(P<0.01)。结论海洛因成瘾可致大鼠脑神经细胞凋亡,针刺"百会"、"大椎"穴可抑制神经细胞凋亡。