CAJ | 학술논문

背景：研究发现细胞质膜微囊蛋白1在哺乳动物脑内表达，参与脑的正常发育，能影响脑内神经干细胞的增殖。目的：从细胞质膜微囊蛋白1敲除小鼠脑内获取神经干细胞并观察其生物学特性。方法：分别取E14-E16正常C57BL/6胚胎小鼠和细胞质膜微囊蛋白1敲除C57BL/6胚胎小鼠全脑，采用酶消化法获得单细胞悬液，置神经干细胞条件培养基中培养，原代培养7 d后，加入含体积分数10%胎牛血清的DMEM/F12培养基诱导7 d。结果与结论：从两种胎鼠脑内获取的细胞悬液培养1d后较多细胞发生死亡，可见单个细胞漂浮于培基中，透光度较好，3 d后逐渐形成悬浮生长的多细胞团。传代后培养板底部可见少量细胞发生贴壁，7 d后可见大量细胞团出现，且细胞质膜微囊蛋白1敲除胎鼠源细胞增殖速度更明显。免疫细胞化学检测显示，两种胎鼠来源细胞团均为巢蛋白阳性，分化细胞可见神经丝蛋白200、胶质纤维酸性蛋白或O4阳性表达；正常C57BL/6胎鼠来源细胞团呈细胞质膜微囊蛋白1阳性表达，而细胞质膜微囊蛋白1敲除C57BL/6胎鼠来源细胞团呈细胞质膜微囊蛋白1表达阴性，且神经干细胞成球速度和成球数量优于正常胎鼠神经干细胞。说明实验成功从细胞质膜微囊蛋白1敲除胎鼠脑内培养出细胞质膜微囊蛋白1缺失神经干细胞，细胞质膜微囊蛋白1可促进神经干细胞的增殖，并抑制其分化。
배경：연구발현세포질막미낭단백1재포유동물뇌내표체，삼여뇌적정상발육，능영향뇌내신경간세포적증식。목적：종세포질막미낭단백1고제소서뇌내획취신경간세포병관찰기생물학특성。방법：분별취E14-E16정상C57BL/6배태소서화세포질막미낭단백1고제C57BL/6배태소서전뇌，채용매소화법획득단세포현액，치신경간세포조건배양기중배양，원대배양7 d후，가입함체적분수10%태우혈청적DMEM/F12배양기유도7 d。결과여결론：종량충태서뇌내획취적세포현액배양1d후교다세포발생사망，가견단개세포표부우배기중，투광도교호，3 d후축점형성현부생장적다세포단。전대후배양판저부가견소량세포발생첩벽，7 d후가견대량세포단출현，차세포질막미낭단백1고제태서원세포증식속도경명현。면역세포화학검측현시，량충태서래원세포단균위소단백양성，분화세포가견신경사단백200、효질섬유산성단백혹O4양성표체；정상C57BL/6태서래원세포단정세포질막미낭단백1양성표체，이세포질막미낭단백1고제C57BL/6태서래원세포단정세포질막미낭단백1표체음성，차신경간세포성구속도화성구수량우우정상태서신경간세포。설명실험성공종세포질막미낭단백1고제태서뇌내배양출세포질막미낭단백1결실신경간세포，세포질막미낭단백1가촉진신경간세포적증식，병억제기분화。
BACKGROUND:Caveolin-1 is expressed in mammalian brain and involved in the normal development of the brain, which can affect the proliferation of neural stem cells in the brain. OBJECTIVE:To acquire neural stem cells from caveolin-1 knockout embryonic mice in vitro and study their biological characteristics. METHODS:The whole brain was separated from C57BL/6 mice and caveolin-1 knockout C57BL/6 mice respectively at encyesis 14-16 days. Single cellsuspension was obtained by enzyme digestion, and cultured in the conditioned medium of neural stem cells. Fol owing 7 days of primary culture, the cells were induced in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10%fetal bovine serum for 7 days. RESULTS AND CONCLUSION:The major cells of the cellsuspensions from the fetal mouse brain were dead at 1 day after culture, and some single cells floated in the medium and their transmittance were better, and then they gradual y formed multicellular bal s after 3 days. A smal amount of cells were adhered at the bottom of culture plate after passage, and a great amount of cellbal s appeared after 7 days. The proliferation rate of neural stem cells from caveolin-1 knockout mice was higher than that from normal mice. The cellbal s were nestin-positive and their differentiated cells was positive for neurofilament 200, glial fibril ary acidic protein or O4, respectively. Al of the cells from normal mouse brain were positive for caveolin-1, but the cells from caveolin-1 knockout mice were negative for caveolin-1 by immunocytochemistry. Moreover, the speed of cellbal formation and the number of cellbal s in neural stem cells from caveolin-1 knockout mice were better than those from normal mice. Caveolin-1 negative neural stem cells were cultured successful y from caveolin-1 knockout mouse brain, and the results show that caveolin-1 can promote the proliferation of neural stem cells and inhibit their differentiation in vitro.