干扰Hes1基因对骨髓基质细胞向胆碱能神经元分化前期的影响

发布时间：2017-12-31 20:02

  本文关键词：干扰Hes1基因对骨髓基质细胞向胆碱能神经元分化前期的影响　出处：《山西医科大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： 骨髓基质细胞 基因表达 Hes1基因 Mash1基因 RNAi 骨髓基质细胞 胆碱能神经元 诱导分化 实时定量PCR Hes1

【摘要】：目的：本部分研究旨在通过对骨髓基质细胞的分离培养，观察其生物学特性，了解其增殖分化和体外培养获得大量扩增的条件，为进一步研究骨髓基质细胞的定向分化奠定基础。在体外环境下干扰骨髓基质细胞中Hes1基因的表达，检测Mash1基因的变化情况，探讨骨髓基质细胞体外培养条件下Hes1对Mash1的调控机制。方法：采用全骨髓培养法体外分离培养获得骨髓基质细胞，倒置显微镜下观察其形态变化，应用实时定量PCR技术分析在干扰Hes1基因表达的情况下Mash1基因表达的变化。 结果：经体外全骨髓分离培养的细胞贴壁能力强，细胞均一性好，呈旋涡状聚集，几乎都表达CD71。应用200nM，100nM，50nM，25nM四种不同浓度梯度的siRNA干扰抑制Hes1基因，与对照组相比Hes1表达均降低。200nM干扰后Hes1基因降低的程度是原来的0.036(P0.05)，50nM干扰后Hes1基因降低的程度是原来的0.076(P0.05)，优于其他浓度的干扰效果。Hes1降低可使Mash1基因表达增加，且Hes1降低越显著，Mash1表达增加越高。 结论：干扰Hes1基因对Mash1基因的表达有影响，且呈负相关调控关系。 目的：通过检测体外培养的骨髓基质细胞定向诱导分化为胆碱能神经元过程中干扰Hes1基因对Sept-9，Stmn1基因的表达变化的分析，探讨影响骨髓基质细胞定向分化为胆碱能神经元的机制。 方法：取体外培养的第三代的骨髓基质细胞，，接种于多聚赖氨酸包被过的6孔培养板中，通过在培养基中添加诱导因子RA、SHH和bFGF，诱导其向胆碱能神经元分化。应用实时定量PCR技术分析骨髓基质细胞向胆碱能神经元诱导分化的过程中干扰Hes1基因对Sept-9，Stmn1基因表达的影响。 结果：Hes1经单纯诱导后表达增高；在干扰后诱导组中Hes1表达显著增高。用50nM的siRNA干扰Hes1后Sept-9基因表达增高；单纯诱导分化P3的BMSCs后Sept-9基因表达情况与对照组相比无统计学意义；而干扰Hes1后诱导P3的BMSCs后Sept-9基因表达增高，且增高最显著。用50nM的siRNA干扰Hes1后Stmn1基因表达显著增高；单纯诱导分化P3的BMSCs后Stmn1基因表达稍有增高；而干扰Hes1后诱导P3的BMSCs后Stmn1基因表达显增高，且其增高幅度介于干扰组与单纯诱导组之间。 结论：Hes1表达的高低影响发育相关基因Sept-9，Stmn1的表达，并且发挥了重要的作用。
[Abstract]:Objective: to investigate the biological characteristics of bone marrow stromal cells (BMSCs) by isolation and culture, and to understand the conditions of proliferation, differentiation and proliferation in vitro. In order to further study the bone marrow stromal cells directional differentiation. In vitro interference of bone marrow stromal cells in the expression of Hes1 gene detection of Mash1 gene changes. Methods: bone marrow stromal cells were isolated and cultured in vitro to obtain bone marrow stromal cells (BMSCs) under the condition of bone marrow stromal cells (BMSCs) cultured in vitro. The morphologic changes were observed under inverted microscope, and the changes of Mash1 gene expression under interference of Hes1 gene expression were analyzed by real-time quantitative PCR technique. Results: the cells isolated from whole bone marrow in vitro had strong adherent ability, good homogeneity and swirl aggregation, almost all expressed CD71. The cells were treated with 200nM, 100nM, 50nM. Four kinds of siRNA interference with different concentration gradient of 25nm inhibited Hes1 gene. Compared with the control group, the expression of Hes1 decreased. 200nM interference, the degree of the decrease of Hes1 gene was the same as that of the original 0.036nM P0.05). The decrease of Hes1 gene after 50 nm interference was the same as that of 0.076 P0.05, which was better than that of other concentrations of interference. The decrease of Hes1 could increase the expression of Mash1 gene. The lower the Hes1, the higher the expression of Mash1. Conclusion: interfering Hes1 gene has an effect on the expression of Mash1 gene and has a negative regulatory relationship. Objective: to detect the expression of Sept-9 Stmn1 gene by interfering Hes1 gene during the differentiation of bone marrow stromal cells into cholinergic neurons in vitro. To explore the mechanism of affecting the directional differentiation of bone marrow stromal cells into cholinergic neurons. Methods: the third generation of bone marrow stromal cells were cultured in vitro and inoculated into the poly-lysine coated 6-well culture plate. The induction factors RAANGSHH and bFGF were added to the culture medium. It was induced to differentiate into cholinergic neurons. The interference of Hes1 gene to Sept-9 during the differentiation of bone marrow stromal cells into cholinergic neurons was analyzed by real-time quantitative PCR. The effect of Stmn1 gene expression. Results the expression of 1% Hes1 was increased after simple induction. The expression of Hes1 was significantly increased in the group induced by interference, and the expression of Sept-9 gene was increased after Hes1 was interfered with 50 nm siRNA. There was no significant difference in the expression of Sept-9 gene between P3-induced BMSCs and control group. After interfering with Hes1, the expression of Sept-9 gene increased after P3 BMSCs. The expression of Stmn1 gene increased significantly after Hes1 interference with 50nM siRNA. The expression of Stmn1 gene increased slightly after P3-induced BMSCs. The expression of Stmn1 gene in P3 induced BMSCs was significantly increased after Hes1 interference, and the increase was between the interference group and the simple induction group. Conclusion the expression of 1: Hes1 affects the expression of development related gene Sept-9 Stmn1 and plays an important role.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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