本文關鍵詞：干擾Hes1基因對骨髓基質細胞向膽堿能神經(jīng)元分化前期的影響　出處：《山西醫(yī)科大學》2011年碩士論文　論文類型：學位論文

  更多相關文章： 骨髓基質細胞 基因表達 Hes1基因 Mash1基因 RNAi 骨髓基質細胞 膽堿能神經(jīng)元 誘導分化 實時定量PCR Hes1

【摘要】：目的：本部分研究旨在通過對骨髓基質細胞的分離培養(yǎng)，觀察其生物學特性，了解其增殖分化和體外培養(yǎng)獲得大量擴增的條件，為進一步研究骨髓基質細胞的定向分化奠定基礎。在體外環(huán)境下干擾骨髓基質細胞中Hes1基因的表達，檢測Mash1基因的變化情況，探討骨髓基質細胞體外培養(yǎng)條件下Hes1對Mash1的調控機制。方法：采用全骨髓培養(yǎng)法體外分離培養(yǎng)獲得骨髓基質細胞，倒置顯微鏡下觀察其形態(tài)變化，應用實時定量PCR技術分析在干擾Hes1基因表達的情況下Mash1基因表達的變化。 結果：經(jīng)體外全骨髓分離培養(yǎng)的細胞貼壁能力強，細胞均一性好，呈旋渦狀聚集，幾乎都表達CD71。應用200nM，100nM，50nM，25nM四種不同濃度梯度的siRNA干擾抑制Hes1基因，與對照組相比Hes1表達均降低。200nM干擾后Hes1基因降低的程度是原來的0.036(P0.05)，50nM干擾后Hes1基因降低的程度是原來的0.076(P0.05)，優(yōu)于其他濃度的干擾效果。Hes1降低可使Mash1基因表達增加，且Hes1降低越顯著，Mash1表達增加越高。 結論：干擾Hes1基因對Mash1基因的表達有影響，且呈負相關調控關系。 目的：通過檢測體外培養(yǎng)的骨髓基質細胞定向誘導分化為膽堿能神經(jīng)元過程中干擾Hes1基因對Sept-9，Stmn1基因的表達變化的分析，探討影響骨髓基質細胞定向分化為膽堿能神經(jīng)元的機制。 方法：取體外培養(yǎng)的第三代的骨髓基質細胞，，接種于多聚賴氨酸包被過的6孔培養(yǎng)板中，通過在培養(yǎng)基中添加誘導因子RA、SHH和bFGF，誘導其向膽堿能神經(jīng)元分化。應用實時定量PCR技術分析骨髓基質細胞向膽堿能神經(jīng)元誘導分化的過程中干擾Hes1基因對Sept-9，Stmn1基因表達的影響。 結果：Hes1經(jīng)單純誘導后表達增高；在干擾后誘導組中Hes1表達顯著增高。用50nM的siRNA干擾Hes1后Sept-9基因表達增高；單純誘導分化P3的BMSCs后Sept-9基因表達情況與對照組相比無統(tǒng)計學意義；而干擾Hes1后誘導P3的BMSCs后Sept-9基因表達增高，且增高最顯著。用50nM的siRNA干擾Hes1后Stmn1基因表達顯著增高；單純誘導分化P3的BMSCs后Stmn1基因表達稍有增高；而干擾Hes1后誘導P3的BMSCs后Stmn1基因表達顯增高，且其增高幅度介于干擾組與單純誘導組之間。 結論：Hes1表達的高低影響發(fā)育相關基因Sept-9，Stmn1的表達，并且發(fā)揮了重要的作用。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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