摘要
目的:研究核因子-κB(NF-κB)在刺五加诱导骨髓基质细胞(MSCs)分化为神经元样细胞中的作用。方法:试验分对照组和刺五加诱导组。采用倒置显微镜观察细胞形态;免疫荧光检测MSCs表型、诱导后神经细胞标记蛋白表达和NF-κB亚基p65核移位情况;Western blot法检测诱导组、对照组IκBα的表达。结果:刺五加诱导1 h,可见部分细胞体积缩小,立体感增强,胞体收缩成锥形或球形;诱导5 h,较多细胞成三角形或球形,细胞微丝收缩,伪足形成细长突起,局部交织成网,部分细胞脱落、死亡。诱导7 d,多数细胞成锥形、三角形、球形,交织成网,形成较典型的神经元样细胞。对照组5 h时细胞无明显形态变化,7 d时偶见锥形细胞。免疫荧光检测到诱导组诱导1 h时nestin表达即为强阳性,到第7天仍为阳性,而β-ⅢTubulin在第1小时为弱阳性,3,5 h则为强阳性,并持续到7 d仍为阳性。神经细胞特异性烯醇化酶(NSE)在第3小时为弱阳性,第5,7天则为强阳性,未见胶质细胞标记蛋白GAFP的表达;对照组各标记蛋白表达均为阴性或可疑或弱阳性。对照组出现p65由胞浆向胞核移位,而诱导组p65信号主要仍在胞浆中表达。Western blot发现诱导前IκBα蛋白相对IA为1.0,诱导后刺五加组IκBα蛋白相对IA为0.455 6±0.100 8,较诱导前明显下降(P<0.01)。诱导后对照组IκBα蛋白相对IA为0.029 6±0.009 9,较诱导前及诱导组明显下降(P<0.01)。结论:刺五加可诱导MSCs分化为神经元样细胞。在此过程中,刺五加可以抑制NF-κB的活化。
Objective： To study whether the nuclear factor-kappa B （NF-κB） play a role in differentiation of marrow stroma cells （MSCs） into neuron-like cells induced by Ciwujia injection in vitro. Method： Rats were randomly divided into control group and Ciwujia induced group. Cell morphology was observed with invert microscope; MSCs phenotype, expression of neural marker protein and nucleus translocation of NF-κB subunit RelA （p65） after induction were observed by immunofluorescence; Expression of IκBα in Ciwujia induced group and control group was detected by Western blot. Result： The decreased volume of some MSCs, strengthened three-dimensional structure and shrinkage of cell body with formation of spire or round was observed after induction by Ciwujia for 1 hour. After 5-hour exposure to Ciwujia, changes in the morphology of MSCs were as follows ： appearance of more triangular or round shapes cells, retraction of microfilament in MSCs, formation of slender process from pseudopod with reticular structure, exfoliation and necrosis. After 7-day induction, most MSCs became triangular and round shapes, exhibiting a typical neuronal structure. Immunofluorescence method showed that nestin expression displayed intensively positive in Ciwujia group at 1 hour, and still remain positive at 7 day . However, β-ⅢTubulin expression was weakly positive at 1 hour and intensive positive at 3 and 5 hour. It was still positive at 7 day. NSE expression was weakly positive at 3 hour and intensive positive at 5 hour and 7 day. GFAP expression was negative. Howev-er, in the control group, every expression of marker protein was negative or weakly positive. Expression of 1365 was in cytoplasm. Western blot method showed IκBα protein relative IA was 1. 000 before induction. It was 0. 455 6 ± 0. 100 8 after induction in Ciwujia group and decreased significantly than before induction（P 〈 0.01 ）. It was 0. 029 6 ±0.009 9 after induction in control group and decreased significantly than before induction and in Ciwujia group （P 〈 0.01 ）. Conclusion： The data showed that MSCs can induce differentiation into neuron-like cells by Ciwujia, and Ciwujia could inhibit the translocation of NF-κB from the cytoplasm to the nucleus, which may be relate to the differentiation of MSCs into neuron-like cells.
出处
《中国中药杂志》
CAS
CSCD
北大核心
2008年第15期1859-1865,共7页
China Journal of Chinese Materia Medica
基金
重庆市卫生局科学基金(04-2-143)
重庆市自然科学基金(CSTC
2005BB5315)
