嗅黏膜神经干细胞及嗅鞘细胞生物学特性的体外研究

发布时间：2018-07-20 20:50

【摘要】： 研究目的：建立体外分离培养嗅黏膜神经干细胞的方法,优化嗅黏膜神经干细胞传代培养条件,诱导神经干细胞分化为多巴胺能神经元,为嗅黏膜神经干细胞或多巴胺能神经元移植治疗帕金森病提供初步的基础研究资料。建立体外分离纯化嗅黏膜嗅鞘细胞的方法,从神经营养角度鉴定其生物学特性,论证嗅黏膜嗅鞘细胞作为辅助细胞进行移植治疗的可能性。研究方法：1.自成年大鼠鼻中隔活体剪取部分鼻粘膜制成细胞悬液,以含10%胎牛血清的DMEM/F12完全培养基培养3天后换为含生长因子的神经干细胞培养基继续培养,动态观察原代培养细胞的生长过程。以免疫荧光染色法检测细胞中神经干细胞特异性分子标记的表达。 2.应用2%琼脂糖悬浮培养传代嗅黏膜神经球,在免疫荧光染色法和免疫印迹的辅助下,研究传代培养神经球的生物学特性。 3.以神经生长因子、全反式维甲酸和音猬因子诱导嗅黏膜神经球分化,鉴定分化细胞中酪氨酸羟化酶的表达,确认其为多巴胺能神经元。 4.自成年大鼠鼻中隔获取嗅黏膜,制成细胞悬液,以完全培养基体外培养,三次应用差速贴壁法纯化混合细胞中的嗅鞘细胞,对嗅鞘细胞的分子标记进行鉴定。 5.以免疫荧光染色法和免疫印迹比较第1代和第10代嗅黏膜嗅鞘细胞中神经营养素、神经肽Y和血管内皮生长因子的表达情况。研究结果：1.嗅黏膜混合细胞接种于培养瓶后,首先贴壁生长的细胞大多为扁平多边形细胞,此后可见散在的球形细胞贴壁。培养3天更换成神经干细胞培养基,贴壁细胞中出现大量的圆球形细胞,该细胞有聚集生长的特点。大约培养7天后出现半贴壁的神经球,呈鸟巢状。免疫荧光染色发现贴壁细胞中存在较多的Nestin和CD133染色阳性的圆形细胞,Nestin主要分布于该细胞的细胞质,CD133主要分布于细胞膜,半贴壁的神经球也阳性表达Nestin和CD133。 2以2%琼脂糖悬浮培养的神经干细胞以神经球的方式旺盛增殖,传代后的神经球形态规则,大小较均匀,界限明显。免疫荧光染色和免疫印迹均证明悬浮培养的第4代神经球经6小时快速贴壁后,仍然阳性表达Nestin和CD133。 3使用含细胞因子的培养液作用贴壁的神经干细胞24小时后,部分细胞向四周发出细长的突起,突起之间相互交织成网。免疫荧光染色发现,这些多突起细胞的胞体和细长突起均含神经丝和酪氨酸羟化酶,免疫印迹也证明了这一点。 4.体外扩增经过三次差速贴壁纯化的嗅鞘细胞,可得到大量梭形双极细胞,细胞排列规则,形态一致,呈鱼群样。免疫荧光染色显示,第1代和第10代的嗅黏膜嗅鞘细胞均阳性表达GFAP、S-100和p75NTR，且两代细胞间无差异。 5.第1代嗅黏膜嗅鞘细胞与第10代嗅鞘细胞均能表达各种神经营养素、神经肽Y和血管内皮生长因子,且各因子第1代的表达水平均高于第10代。研究结论：1.通过大鼠嗅黏膜神经干细胞的体外分离与筛选实验,我们从嗅黏膜中获得了阳性表达神经干细胞分子标志的半贴壁神经球。这些神经球可以通过特殊的琼脂糖悬浮培养法在传代过程中得到不断扩增。 2.源于嗅黏膜的神经球能够被诱导分化为TH阳性的神经元,证明嗅黏膜来源的神经干细胞能够在体外诱导分化为多巴胺能神经元。 3.以完全培养基体外分离培养嗅黏膜能获得复杂的细胞群体,通过三次差速贴壁筛选法可以纯化其中的嗅鞘细胞,经鉴定嗅黏膜嗅鞘细胞能够表达多种神经营养素,且能够合成神经肽Y和血管内皮生长因子。 4.本实验成功地在体外培养获得了嗅黏膜神经干细胞和嗅鞘细胞,并将神经干细胞诱导分化为多巴胺能神经元,为临床帕金森病细胞移植治疗提供了新的种子细胞来源。
[Abstract]:Objective : To establish a method for the isolation and culture of olfactory mucosa neural stem cells in vitro , optimize the culture conditions of olfactory mucosa neural stem cells , induce the differentiation of neural stem cells into dopaminergic neurons , and provide preliminary basic research data for the transplantation of olfactory mucosa neural stem cells or dopaminergic neurons in the treatment of Parkinson ' s disease .

Methods : 1 . The cell suspension was made from nasal mucosa of rat nasal septum , and cultured in DMEM / F12 medium containing 10 % fetal bovine serum for 3 days . The growth process of primary cultured cells was observed dynamically . The expression of specific molecular markers of neural stem cells in cells was detected by immunofluorescence staining .

2 . Using 2 % agarose suspension to culture the olfactory mucosa neurosphere , the biological characteristics of the cultured neurospheres were studied with the aid of immunofluorescence staining and Western blot .

3 . The expression of tyrosine hydroxylase in differentiated cells was identified by the differentiation of nerve growth factor , all - trans retinoic acid ( ATRA ) and sound Hedgehog ' s factors in olfactory mucosa , and it was confirmed that it was dopaminergic neuron .

4 . The olfactory mucosa was obtained from the nasal septum of the rat . The cell suspension was prepared in vitro . The olfactory ensheathing cells in the mixed cells were purified three times by differential adherent method , and the molecular markers of olfactory ensheathing cells were identified .

5 . The expression of neurotrophin , neuropeptide Y and vascular endothelial growth factor in olfactory ensheathing cells of olfactory mucosa in the first and 10th passages were compared by immunofluorescence staining and Western blot .

The results were as follows : 1 . After inoculation of olfactory mucosa mixed cells in culture flask , most of the cells were flattened polygonal cells . After 3 days , the cells were replaced by neural stem cell culture medium . The cells were cultured for 3 days to replace them into neural stem cell culture medium .

2 % agarose suspension cultured neural stem cells proliferate in the form of neurospheres , the morphology of the neurospheres after passage is uniform and the limit is obvious . Both immunofluorescence staining and Western blot prove that the 4th generation nerve cells in suspension culture are able to express Nestin and CD133 after 6 hours .

3 . After 24 hours of using a cell containing a cytokine - containing culture solution to act on the wall , a portion of the cells sent an elongated protrusion around each other , and the protrusions were intermeshed with each other . Immunofluorescence staining showed that both the cell body and the elongated protrusion of these multiple - protruding cells contained both nerve fibers and tyrosine hydroxylase , and the Western blot demonstrated this .

4 . In vitro amplification of olfactory ensheathing cells purified by triple differential adherent cells can obtain a large number of spindle - shaped bipolar cells , the cell arrangement rules and the morphology are consistent , and are in the form of a fish group . Immunofluorescence staining shows that the olfactory mucosa olfactory ensheathing cells of the 1st and 10th passages are positive for GFAP , S - 100 and p75NTR , and there is no difference between the two generations .

5 . Both the olfactory ensheathing cells and the 10th generation olfactory ensheathing cells express various neurotrophic factors , neuropeptides Y and vascular endothelial growth factors , and the expression level of each factor in the 1st generation is higher than that of the 10th generation .

Conclusion : 1 . In vitro isolation and screening experiment of olfactory mucosa neural stem cells in rats , we obtained semi - adherent neurospheres expressing the molecular markers of positive expression neural stem cells from the olfactory mucosa . These neurospheres can be amplified by special agarose suspension culture in the passaging process .

2 . Neural stem cells derived from olfactory mucosa can be differentiated into TH - positive neurons , and the olfactory mucosa - derived neural stem cells can induce differentiation into dopaminergic neurons in vitro .

3 . In vitro isolation and culture of olfactory mucosa from culture medium to obtain complex cell population , olfactory ensheathing cells can be purified by three times differential wall screening method , and the olfactory mucosa olfactory ensheathing cells can express various neurotrophin , and neuropeptides Y and vascular endothelial growth factors can be synthesized .

4 . The olfactory mucosa neural stem cells and olfactory ensheathing cells were obtained successfully in vitro and the neural stem cells were induced into dopaminergic neurons .
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329.28

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