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背景:有证据表明羊膜上皮细胞能够表达神经系统细胞的几乎全部特异性抗原,且可以分泌多种神经营养因子及递质。若羊膜上皮细胞能够代替神经细胞,其神经营养作用必将为治疗神经推行性病变带来广阔前景。目的:检测神经组织细胞特异性抗原在大鼠羊膜上皮细胞中是否表达?设计:重复测量设计。单位:吉林大学第二临床医学院,吉林大学基础医学院组织与胚胎学教研室。材料:实验于2004-10/2005-10在吉林大学基础医学院组织与胚胎学教研室完成。选取清洁级孕12～14d的Wistar大鼠1只,将分离出的羊膜上皮细胞用于实验。小鼠抗大鼠神经元特异性抗原微管相关蛋白、星形胶质细胞特异性抗原胶质原纤维酸性蛋白、乙酰胆碱转移酶单克隆抗体、兔抗大鼠神经元特异性烯醇化酶和NT-3多克隆抗体(武汉博士德公司);大鼠抗大鼠Musashi抗体(日本庆应义塾大学岗野荣之教授惠赠)。方法:取孕12～14d的Wistar大鼠胎盘,剥离羊膜获得上皮细胞,在37℃、体积分数为0.05的CO2环境下,胰蛋白酶消化5min,加入DMEM/F12培养液,以5×109L-1的浓度接种于培养瓶中。培养3d后,细胞以1×108L-1的浓度接种于预先涂有多聚赖氨酸的直径35mm平皿中,用质量浓度为40g/L的多聚甲醛固定20min。采用免疫细胞化学染色方法对神经元特异性抗原微管相关蛋白、神经元特异性烯醇化酶、星形胶质细胞特异性抗原胶质原纤维酸性蛋白、乙酰胆碱转移酶在羊膜上皮细胞中的表达情况进行检测。主要观察指标:①大鼠羊膜上皮细胞不同培养时间的形态观察。②神经组织细胞特异性抗原在大鼠羊膜上皮细胞中的表达情况。结果:①大鼠羊膜上皮细胞不同培养时间的形态观察:羊膜上皮细胞培养24h后,细胞扁平呈成纤维细胞样。3～5d后,胞体饱满,核大而圆,核仁清晰,突起发达,并互相连成网状。②神经组织细胞特异性抗原在大鼠羊膜上皮细胞中的表达情况:培养4d后免疫细胞化学染色结果可见,羊膜上皮细胞表达Nestin、神经元特异性烯醇化酶、乙酰胆碱转移酶以及Musashi、神经元特异性抗原微管相关蛋白、星形胶质细胞特异性抗原胶质原纤维酸性蛋白。结论:羊膜上皮细胞与神经组织细胞具有一定的同源性,可望作为治疗神经系统疾病新的细胞来源。
BACKGROUND: There is evidence that amniotic epithelial cells are capable of expressing almost all of the specific antigens of nervous system cells and can secrete a variety of neurotrophic factors and neurotransmitters. If the amniotic membrane epithelial cells can replace nerve cells, its neurotrophic effect will certainly have broad prospects for the treatment of neuropathic lesions. Aims: To detect whether neural tissue-specific antigen is expressed in rat amniotic epithelial cells. Design: Repeat measurement design. Unit: Second Clinical College of Jilin University, College of Basic Medical Sciences, Jilin University Department of Histology and Embryology. MATERIALS: The experiment was performed at the Department of Tissue and Embryology, College of Basic Medical Sciences, Jilin University from October 2004 to October 2005. One Wistar rat of 12 ~ 14 days of pregnancy was selected and the isolated amniotic epithelial cells were used in the experiment. Mouse anti-rat neuron-specific antigen microtubule-associated protein, astrocyte-specific antigen glial fibrillary acidic protein, acetylcholine transferase monoclonal antibody, rabbit anti-rat neuron-specific enolase and NT -3 polyclonal antibody (Wuhan Boster Company); rat anti-rat Musashi antibody (a gift of Professor Konno Yoshinori of Japan’s Keio University). Methods: Placenta of Wistar rats were taken from 12 to 14 days of pregnancy, and the amniotic membrane was dissected to obtain epithelial cells. The cells were trypsinized at 37 ℃ and CO2 concentration of 0.05 for 5 minutes. DMEM / F12 medium was added to 5 × 109L-1 Inoculated into culture flasks. After culturing for 3 days, the cells were seeded at a density of 1 × 10 8 L -1 into 35 mm diameter poly-lysine pre-coated plates and fixed with paraformaldehyde at a concentration of 40 g / L for 20 min. The expression of neuron-specific antigen microtubule-associated protein, neuron-specific enolase, astrocyte-specific glial fibrillary acidic protein and acetylcholinesterase in amniotic epithelial cells were detected by immunocytochemical staining The situation is tested. MAIN OUTCOME MEASURES: ① Morphological observation of rat amniotic epithelial cells in different culture time. ② expression of neural tissue-specific antigen in rat amniotic epithelial cells. Results: (1) Morphological observation of amniotic epithelial cells in different culture time: After cultured for 24 hours, the cells were flat and fibroblast-like. After 3 ~ 5d, the cell body is full, the nucleus is big and round, the nucleolus are clear, the processus is developed, and are interconnected into the network. ② The expression of neural tissue-specific antigen in rat amniotic epithelial cells: Immunocytochemical staining results after 4 days showed that amniotic epithelial cells expressed Nestin, neuron-specific enolase, acetylcholine transferase and Musashi, neurons Specific antigen microtubule-associated protein, astrocyte-specific antigen glial fibrillary acidic protein. Conclusion: The amnion epithelial cells have certain homology with nerve tissue cells, which is expected to be a new source of cells for the treatment of nervous system diseases.