共培养法诱导骨髓间充质干细胞向毛细胞样细胞分化的实验研究
发布时间：2018-07-21 09:42
【摘要】：目的:建立体外共培养法定向诱导骨髓间充质干细胞(BMSCs)向毛细胞样细胞分化的方法。 方法:采用全骨髓贴壁法分离培养小鼠骨髓中的间充质干细胞。并传代和纯化。第三代用荧光免疫组化法检测细胞表面标志蛋白CD44、CD34、CD106、Vimentin的表达。采新生小鼠脑胶质细胞(ASTs)体外培育到第三代,用荧光免疫组化法检测胶质细胞表达标志蛋白GFAP的表达。采新生小鼠耳基底膜细胞(BAMCs)体外扩增培育至第三代备用。取第三代的BMSCs与ASTs共培养,并联合RA (DMEM/F12/RA +10%FBS)诱导培养7天,使BMSCs向神经前体细胞分化。诱导后的细胞用荧光免疫组化法检测神经前体标志蛋白(Nestin)的表达情况。诱导后所得的细胞再与BAMCs共培养于DMEM/F12+10%FBS中14天,诱导其向毛细胞样细胞分化。并用荧光免疫组化法检测听毛细胞标记物(Math1和MyosinVIIa)蛋白的表达。采用RT-PCR法检测Math1和MyosinVIIa mRNA的表达。 结果:经ASTs共培养并联合RA诱导后的BMSCs表达神经前体细胞特异性蛋白Nestin。此种神经前体细胞与BAMCs共培养后,部份细胞表达毛细胞特异性标志蛋白Math1、MyosinVIIa mRNA和蛋白。 结论: BMSCs经与ASTs共培养并联合RA诱导后,所得细胞再与BAMCs共培养,能诱导分化出部份表达Math1和MyosinVIIa mRNA和蛋白的毛细胞样细胞。共培养法能诱导BMSCs向毛细胞样细胞分化。
[Abstract]:Aim: to establish a method of coculture to induce bone marrow mesenchymal stem cells (BMSCs) to differentiate into hair cell like cells. Methods: mesenchymal stem cells were isolated from mouse bone marrow by whole bone marrow adherent method. Passage and purification. In the third generation, the expression of CD44, CD34, CD106, Vimentin, a marker protein on the cell surface, was detected by fluorescence immunohistochemical method. Brain glial cells (ASTs) of newborn mice were cultured to the third generation in vitro. The expression of GFAP, a marker of glial expression, was detected by fluorescence immunohistochemistry. Newborn mouse ear basement membrane cells (BAMCs) were amplified and cultured to the third generation. The third generation BMSCs were co-cultured with ASTs and co-cultured with RA (DMEM / F _ (12) / RA _ (10) for 7 days to differentiate BMSCs into neural precursor cells. The expression of neural precursor marker protein (nestin) was detected by fluorescence immunohistochemistry. The cells were co-cultured with BAMCs in DMEM / F1210S for 14 days to differentiate into hair-cell-like cells. The expression of auditory hair cell markers (Math1 and Myosin VIIa) was detected by fluorescence immunohistochemistry. The expression of Math1 and Myosin VIIa mRNA was detected by RT-PCR. Results: BMSCs co-cultured with ASTs and induced by RA expressed Nestin. After co-cultured with BAMCs, some of the cells expressed Math1- Myosin VIIa mRNA and protein. Conclusion: BMSCs were co-cultured with ASTs and induced by RA, and then co-cultured with BAMCs to induce some hair cell-like cells expressing Math1 and Myosin VIIa mRNA and protein. Co-culture can induce BMSCs to differentiate into hair cell-like cells.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764.43
本文编号：2135115
[Abstract]:Aim: to establish a method of coculture to induce bone marrow mesenchymal stem cells (BMSCs) to differentiate into hair cell like cells. Methods: mesenchymal stem cells were isolated from mouse bone marrow by whole bone marrow adherent method. Passage and purification. In the third generation, the expression of CD44, CD34, CD106, Vimentin, a marker protein on the cell surface, was detected by fluorescence immunohistochemical method. Brain glial cells (ASTs) of newborn mice were cultured to the third generation in vitro. The expression of GFAP, a marker of glial expression, was detected by fluorescence immunohistochemistry. Newborn mouse ear basement membrane cells (BAMCs) were amplified and cultured to the third generation. The third generation BMSCs were co-cultured with ASTs and co-cultured with RA (DMEM / F _ (12) / RA _ (10) for 7 days to differentiate BMSCs into neural precursor cells. The expression of neural precursor marker protein (nestin) was detected by fluorescence immunohistochemistry. The cells were co-cultured with BAMCs in DMEM / F1210S for 14 days to differentiate into hair-cell-like cells. The expression of auditory hair cell markers (Math1 and Myosin VIIa) was detected by fluorescence immunohistochemistry. The expression of Math1 and Myosin VIIa mRNA was detected by RT-PCR. Results: BMSCs co-cultured with ASTs and induced by RA expressed Nestin. After co-cultured with BAMCs, some of the cells expressed Math1- Myosin VIIa mRNA and protein. Conclusion: BMSCs were co-cultured with ASTs and induced by RA, and then co-cultured with BAMCs to induce some hair cell-like cells expressing Math1 and Myosin VIIa mRNA and protein. Co-culture can induce BMSCs to differentiate into hair cell-like cells.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764.43
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