六味地黄丸联合bFGF诱导骨髓间充质干细胞向神经元样细胞分化的研究

发布时间：2019-06-21 15:00

【摘要】：目的：通过细胞生物学技术,探讨分离、培养、纯化和鉴定大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)的方法,观察滋补肝肾中药六味地黄丸联合碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)体外诱导大鼠BMSCs分化为神经元样细胞的作用,探索获得较高比例神经元样细胞的诱导方法。方法：采用全骨髓贴壁分离法分离、培养SD大鼠BMSCs,以纯化的BMSCs为研究对象,观察细胞形态变化、生长增殖状态,MTT法检测绘制细胞生长曲线,对第3代细胞进行表面标志物CD44、CD34免疫细胞化学染色法检测,鉴定BMSCs。鉴定后,第3代细胞经bFGF预诱导24小时后按空白组、bFGF组、六味地黄丸组、bFGF+六味地黄丸组分别加入相应诱导液进行诱导,观察细胞形态变化；诱导培养7天,采用免疫细胞化学染色法检测相关标志物巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)的表达。结果：1.采用全骨髓贴壁分离法获得原代BMSCs,随多次换液弃悬浮细胞传代弃其它贴壁细胞后得到纯化的BMSCs.经分离培养的第3代细胞增殖旺盛,具有较好的活力,利于诱导进行。免疫细胞化学染色结果显示CD44呈阳性表达,CD34呈阴性表达。 2.生长良好的第3代BMSCs经bFGF预诱导24小时后与空白组相比,细胞数量增多,立体感增强；各组BMSCs分别以不同诱导液进行诱导,2-3天细胞胞体收缩,形成圆形、不规整的锥形或三角形；4-5天,部分细胞逐渐伸出突起,多为2-3极,呈现较典型的神经元样细胞形态；6-7天,神经元样细胞数量增多,bFGF+六味地黄丸组更为明显。空白组细胞形态1-3天未出现明显改变,但生长速度明显减慢,其后出现老化、死亡脱落。 3. bFGF组、六味地黄丸组和bFGF+六味地黄丸组均有Nestin、NSE、GFAP阳性表达,bFGF+六味地黄丸组各指标阳性率高于bFGF组、六味地黄丸组,差异有统计学意义(P0.05)。结论：1.经全骨髓贴壁分离法分离、培养的骨髓间充质干细胞能逐渐纯化,细胞活性较高、生物学性状稳定,适于做进一步研究。 2.六味地黄丸能诱导骨髓间充质干细胞向神经元样细胞定向分化,六味地黄丸和碱性成纤维细胞生长因子联合诱导可获得较高比例的神经元样细胞。
[Abstract]:Objective: To study the methods of separation, culture, purification and identification of bone marrow mesenchymal stem cells (BMSCs) in rats by cell biology technique. BFGF was used to induce the differentiation of BMSCs into neuron-like cells in vitro, and to explore the induction method of high-scale neuron-like cells. Methods: BMSCs of SD rats were cultured by full-bone marrow adherent separation. BMSCs from SD rats were cultured. The morphological changes of cells, growth and proliferation of BMSCs were observed, and the growth curves of the cells were detected by MTT method. 4. CD34 immune cell chemical staining method for detection and identification of BMSC and s. after the identification, the third generation cell is pre-induced for 24 hours through the bFGF, and the corresponding inducing liquid is added into the blank group, the bFGF group, the six-flavor rehmannia pill group, the bFGF + Liuwei Dihuang pill group, and the change of the cell morphology is observed; and the induction culture 7 The expression of Nestin, neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) was detected by immunocytochemical staining. Dida. junction Fruit:1. The primary BMSCs were obtained by using the full-bone marrow adherent separation method, and the purified BM was obtained after the cells were discarded and the other adherent cells were discarded. SCs. The third generation of cells isolated and cultured has vigorous proliferation and has better activity and is beneficial to inducing The results showed that the positive expression of CD44 and the negative of CD34 were positive in the immunocytochemical staining. 2. In the third generation BMSCs with good growth, the number of cells increased and the three-dimensional effect was enhanced after 24 hours of pre-induction with bFGF. BMSCs were induced by different induction liquid, and the cell bodies of each group were contracted to form a circular, irregular cone or triangle. 4-5 days, some of the cells gradually protrude, most of which are 2-3 poles, presenting more typical neuron-like cell morphology;6-7 days, the number of neuron-like cells is increased, and bFGF + Liuwei Dihuang Wan The group was more obvious. The cell morphology of the blank group did not change significantly in 1-3 days, but the growth rate was significantly reduced, and then the aging was observed. 3. The positive expression of Nestin, NSE and GFAP in the bFGF group, the Liuwei Dihuang pill group and the bFGF + Liuwei Dihuang pill group were higher than that of the bFGF group and the Liuwei Dihuang pill group. (P0.0 5) Conclusion:1. The bone marrow-derived mesenchymal stem cells can be gradually purified by the whole bone marrow adherent separation method, and the cell activity is high and the biological character is stable. 2. Liuwei Dihuang Wan can induce the directional differentiation of bone marrow mesenchymal stem cells into neuron-like cells, and the combination of Liuwei Dihuang Wan and basic fibroblast growth factor can be obtained.
【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】：2012
【分类号】：R329

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