CAJ | 학술논문

目的探讨脉冲强磁场对体外新生大鼠神经干细胞(NSCs)向神经元方向分化的作用.方法体外分离培养新生3d内的SD大鼠脑室下区的NCSs,无血清培养14d后,随机分为实验组和对照组.实验组置于脉冲强磁场条件下用前期探索的促增殖参数(0.1 Hz、4T、8次)进行干预,每次脉冲放电20 ms.对照组置于相同条件下但不做干预处理.干预后第1天,采用10％胎牛血清诱导贴壁分化,显微镜下观察不同时间段细胞形态学变化;贴壁分化后第7天,通过免疫荧光染色、蛋白免疫印迹法(Western Blot)及实时定量聚合酶链反应技术(RT-PCR)检测NCSs分化后第7天神经元βⅢ微管蛋白(TUJ1)及星形胶质细胞相关特异性标志神经胶质纤维酸性蛋白(GFAP)的表达.结果干预后,免疫荧光染色示实验组TUJ1阳性细胞数为(33.4±5.1)％,较对照组[(26.5 ±7.0)％]明显增多(P＜0.05),实验组的GFAP阳性细胞率[(23.9±5.0)％]较对照组[(36.2±2.2)％]明显减少(P＜0.05).RT-PCR检测显示,实验组TUJ1的mRNA表达量是对照组的(1.682 ±0.086)倍,GFAP的mRNA相对表达量是对照组的(0.590±0.157)倍,且组间差异有统计学意义(P＜0.05).Western Blot检测结果与RT-PCR检测一致,实验组TUJ1蛋白表达较对照组增多,GFAP蛋白表达较对照组减少.结论脉冲强磁场具有促进NCSs向神经元分化,并抑制其向星形胶质细胞分化的作用.
목적 탐토맥충강자장대체외신생대서신경간세포(NSCs)향신경원방향분화적작용.방법 체외분리배양신생3d내적SD대서뇌실하구적NCSs,무혈청배양14d후,수궤분위실험조화대조조.실험조치우맥충강자장조건하용전기탐색적촉증식삼수(0.1 Hz、4T、8차)진행간예,매차맥충방전20 ms.대조조치우상동조건하단불주간예처리.간예후제1천,채용10％태우혈청유도첩벽분화,현미경하관찰불동시간단세포형태학변화;첩벽분화후제7천,통과면역형광염색、단백면역인적법(Western Blot)급실시정량취합매련반응기술(RT-PCR)검측NCSs분화후제7천신경원βⅢ미관단백(TUJ1)급성형효질세포상관특이성표지신경효질섬유산성단백(GFAP)적표체.결과 간예후,면역형광염색시실험조TUJ1양성세포수위(33.4±5.1)％,교대조조[(26.5 ±7.0)％]명현증다(P＜0.05),실험조적GFAP양성세포솔[(23.9±5.0)％]교대조조[(36.2±2.2)％]명현감소(P＜0.05).RT-PCR검측현시,실험조TUJ1적mRNA표체량시대조조적(1.682 ±0.086)배,GFAP적mRNA상대표체량시대조조적(0.590±0.157)배,차조간차이유통계학의의(P＜0.05).Western Blot검측결과여RT-PCR검측일치,실험조TUJ1단백표체교대조조증다,GFAP단백표체교대조조감소.결론 맥충강자장구유촉진NCSs향신경원분화,병억제기향성형효질세포분화적작용.
Objective To Investigate the influence of high-intensity pulsed electromagnetic field (HIPEMF) on neural differentiation of neonatal rats neural stem cells in vitro.Methods Neural stem cells (NSCs) were isolated from the subventricular zone of 3-day-old neonatal rats and cultured with serum-free condition medium for 14 days.All the NSCs were then randomly classified into an experimental group which received the stimulation of HIPEMF (0.1 Hz,4 T,8 pulses) and a control group which received no special intervention.Differentiation of the culture was induced by addition of 10％ fetal bovine serum on the first day after intervention,the morphological changes of cells were observed under the microscope at different time points.The NSCs adhered to the wall and differentiated for seven days,the immunofluorescence was employed to observed and calculate the ratio of differentiated cells with astrocyte marker GFAP or neuronal markers TUJ1.RT-PCR and western blotting were used to measure the expression levels of the differentiated cells based on gene and protein levels,respectively.Results Immunofluorescence staining showed the number of TUJI positive cells in the experimental group(33.4％ ± 5.1％)was significantly more than the control group (26.5％ ± 7.0％),while the number of GFAP positive cells was decreased(23.9％ ± 5.0％) as compared with the control group(36.2％ ± 2.2％).RT-PCR showed that the TUJ1 mRNA expression levels in the experimental group was (1.682 ± 0.086) times of the control group.Western blot showed that the expression of TUJ1 (0.729 ±0.061) in the experimental group was higher than in the control group (0.590 ± 0.157),while the expression of GFAP in the experimental group (0.566 ± 0.056) was less than in the control group(1.034 ± 0.051).Conclusions HIPEMF facilitates differentiation of neural stem cells to neurons,at the cost of reducing astrocytic differentiation.