大鼠成骨细胞向神经细胞转化的体外诱导研究

发布时间：2018-04-27 06:35

本文选题：成骨细胞 + 神经细胞　；参考：《成都中医药大学》2006年硕士论文

【摘要】：目的考察大鼠骨膜中的成骨细胞经漏芦总黄酮体外诱导后向神经细胞转化的可行性，为临床中枢和外周神经损伤的修复治疗提供新的选择。方法从大鼠胫骨内侧获取骨膜，组织块培养法得到成骨细胞，经鉴定确认后体外纯化扩增培养，然后借助MTT(噻唑蓝)比色法了解漏芦总黄酮对体外培养成骨细胞生长的影响，选出合适的诱导浓度，分别加入0.0613％漏芦总黄酮和2％二甲基亚砜(DMSO)进行诱导，于诱导24h、48h、72h时相差显微镜下观察细胞形态变化，并于72h时通过逆转录聚合酶链反应技术(RT-PCR法)检测经诱导后的细胞中是否具有神经细胞特有的神经元烯醇化酶(NSE)和特异性胶质纤维酸性蛋白(GFAP)的mRNA表达，以此确定神经细胞和神经胶质细胞的存在。结果细胞毒实验中，在0.09％到1.92％的浓度范围内，与空白组比较，各浓度漏芦总黄酮组活细胞吸光度值(optical density，OD)明显减小(P＜0.01)。折线图显示随着漏芦总黄酮浓度加大，OD值呈现持续上升状态，对细胞的抑制率呈现下降趋势。筛选合适诱导浓度(0.0613％)诱导72h后，漏芦总黄酮组和二甲基亚砜组细胞形态均发生了改变，由原来的不规则三角形变为突触样生长；RT-PCR法显示经诱导后的细胞NSE、GFAP的mRNA表达阳性，，且在NSE的mRNA表达上，漏芦总黄酮组强于二甲基亚砜组，而在GFAP的mRNA表达上，二甲基亚砜组强于漏芦总黄酮组；空白组细胞在形态上无明显变化，NSE和GFAP的mRNA有极微弱表达。结论在0.09％到1.92％的浓度范围内，漏芦总黄酮对体外培养成骨细胞的生长有一定抑制作用，但呈现浓度越大抑制率越小的趋势，考虑不仅与漏芦总黄酮对细胞的毒性作用有关，也与其对细胞的诱导作用有关。成骨细胞经0.0613％漏芦总黄酮体外诱导后有向神经细胞和神经胶质细胞转化的可行性，且更倾向于向神经细胞转化，因此有望成为神经种子细胞的另外一个来源。
[Abstract]:Objective to investigate the feasibility of osteoblast in rat periosteum induced by total flavone in vitro and to provide a new choice for the repair and treatment of central and peripheral nerve injury. Methods Osteoblasts were obtained from the medial tibia of rats, and osteoblasts were obtained by tissue mass culture. After identification, the osteoblasts were purified and cultured in vitro. Then the effect of total flavonoids on the growth of osteoblasts cultured in vitro was investigated by MTT colorimetry. The suitable induction concentration was selected and 0.0613% total flavone and 2% DMSO were added respectively. The morphological changes of the cells were observed under a phase-contrast microscope at 24 h, 48 h or 72 h after induction. At 72 h, the mRNA expression of neuronal enolase (NSE) and specific glial fibrillary acidic protein (GFAP) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). This determines the existence of nerve cells and glial cells. Results in the cytotoxicity test, in the range of 0.09% to 1.92%, compared with the blank group, the absorbance of living cells in the total flavone group decreased significantly (P < 0.01). The curve diagram showed that the OD value increased with the increase of total flavonoids concentration, and the inhibition rate of the cells decreased. After 72 hours of induction, the morphology of the cells in the total flavone group and the dimethyl sulfoxide group changed from irregular triangle to synaptic growth. RT-PCR showed that the mRNA expression of NSEG FAP was positive in the induced cells. In the mRNA expression of NSE, the total flavone group was stronger than that in the dimethyl sulfoxide group, while the mRNA expression in the GFAP group was stronger than that in the GFAP group, and the mRNA expression in the blank group had no obvious change in morphology. Conclusion in the range of 0.09% to 1.92%, the total flavone of Leymus chinensis has a certain inhibitory effect on the growth of osteoblasts cultured in vitro, but the higher the concentration is, the smaller the inhibition rate is, which is related not only to the toxicity of the total flavonoids to the cells, but also to the growth of osteoblasts in vitro. It is also related to its induction to cells. Osteoblasts induced by 0.0613% total flavonoids have the feasibility of transforming into nerve cells and glial cells, and are more inclined to convert to nerve cells, so it is expected to be another source of neural seed cells.
【学位授予单位】：成都中医药大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329

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