【摘要】：目的:建立體外共培養(yǎng)法定向誘導(dǎo)骨髓間充質(zhì)干細(xì)胞(BMSCs)向毛細(xì)胞樣細(xì)胞分化的方法。 方法:采用全骨髓貼壁法分離培養(yǎng)小鼠骨髓中的間充質(zhì)干細(xì)胞。并傳代和純化。第三代用熒光免疫組化法檢測(cè)細(xì)胞表面標(biāo)志蛋白CD44、CD34、CD106、Vimentin的表達(dá)。采新生小鼠腦膠質(zhì)細(xì)胞(ASTs)體外培育到第三代,用熒光免疫組化法檢測(cè)膠質(zhì)細(xì)胞表達(dá)標(biāo)志蛋白GFAP的表達(dá)。采新生小鼠耳基底膜細(xì)胞(BAMCs)體外擴(kuò)增培育至第三代備用。取第三代的BMSCs與ASTs共培養(yǎng),并聯(lián)合RA (DMEM/F12/RA +10%FBS)誘導(dǎo)培養(yǎng)7天,使BMSCs向神經(jīng)前體細(xì)胞分化。誘導(dǎo)后的細(xì)胞用熒光免疫組化法檢測(cè)神經(jīng)前體標(biāo)志蛋白(Nestin)的表達(dá)情況。誘導(dǎo)后所得的細(xì)胞再與BAMCs共培養(yǎng)于DMEM/F12+10%FBS中14天,誘導(dǎo)其向毛細(xì)胞樣細(xì)胞分化。并用熒光免疫組化法檢測(cè)聽毛細(xì)胞標(biāo)記物(Math1和MyosinVIIa)蛋白的表達(dá)。采用RT-PCR法檢測(cè)Math1和MyosinVIIa mRNA的表達(dá)。 結(jié)果:經(jīng)ASTs共培養(yǎng)并聯(lián)合RA誘導(dǎo)后的BMSCs表達(dá)神經(jīng)前體細(xì)胞特異性蛋白Nestin。此種神經(jīng)前體細(xì)胞與BAMCs共培養(yǎng)后,部份細(xì)胞表達(dá)毛細(xì)胞特異性標(biāo)志蛋白Math1、MyosinVIIa mRNA和蛋白。 結(jié)論: BMSCs經(jīng)與ASTs共培養(yǎng)并聯(lián)合RA誘導(dǎo)后,所得細(xì)胞再與BAMCs共培養(yǎng),能誘導(dǎo)分化出部份表達(dá)Math1和MyosinVIIa mRNA和蛋白的毛細(xì)胞樣細(xì)胞。共培養(yǎng)法能誘導(dǎo)BMSCs向毛細(xì)胞樣細(xì)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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R764.43

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