【摘要】：目的：建立豚鼠骨髓間充質(zhì)干細(xì)胞(BMSCs)的體外培養(yǎng)體系,體外脂質(zhì)體2000介導(dǎo)神經(jīng)營(yíng)養(yǎng)因子-3(NT-3)轉(zhuǎn)染BMSCs,為內(nèi)耳移植提供前期研究基礎(chǔ)。 方法：(1)采用全骨髓貼壁法培養(yǎng)豚鼠BMSCs,傳代培養(yǎng),取P3代BMSCs做生長(zhǎng)曲線(xiàn)圖,在流式細(xì)胞儀上檢測(cè)BMSCs的表面標(biāo)志物CD29,CD90以及造血細(xì)胞表面標(biāo)志物CD45。(2)按照不同質(zhì)粒-脂質(zhì)體配比介導(dǎo)pEGFP-N1-NT3轉(zhuǎn)染P3代BMSCs,轉(zhuǎn)染后48h后計(jì)算轉(zhuǎn)染效率,選擇最佳質(zhì)粒-脂質(zhì)體配比。(3)按照最佳質(zhì)粒-脂質(zhì)體配比將pEGFP-N1-NT3質(zhì)粒轉(zhuǎn)染P3代BMSCs,轉(zhuǎn)染后1d,3d,7d,14d,觀(guān)察其熒光表達(dá)情況。(4)轉(zhuǎn)染后48h的BMSCs提取蛋白,采用Western Blot來(lái)驗(yàn)證NT-3蛋白表達(dá)。 結(jié)果：(1)全骨髓貼壁法獲得細(xì)胞數(shù)量多,增殖快,通過(guò)傳代可以純化細(xì)胞。(2)生長(zhǎng)曲線(xiàn)呈S型,基本符合Logistic生長(zhǎng)曲線(xiàn)。(3)流式細(xì)胞儀檢測(cè)細(xì)胞CD29和CD90陽(yáng)性表達(dá),CD45陰性表達(dá),符合BMSCs特征。(4)不同質(zhì)粒-脂質(zhì)體配比中,質(zhì)粒：脂質(zhì)體為4μg：12μl時(shí)轉(zhuǎn)染率最高,是最佳質(zhì)粒-脂質(zhì)體配比。(5)轉(zhuǎn)染后48小時(shí)熒光表達(dá)較強(qiáng),7d可見(jiàn)熒光表達(dá),但較前明顯減弱,到14d基本未見(jiàn)明顯熒光表達(dá)。(6)Western Blot驗(yàn)證NT-3基因成功表達(dá)。 結(jié)論：(1)全骨髓貼壁法可以成功培養(yǎng)出增殖快,細(xì)胞純度高的BMSCs。(2)脂質(zhì)體法成功介導(dǎo)NT-3真核質(zhì)粒在BMSCs內(nèi)表達(dá),BMSCs可能作為內(nèi)耳基因治療的理想載體。
[Abstract]:Aim: to establish a culture system of guinea pig bone marrow mesenchymal stem cells (BMSCs) in vitro. Liposome 2000 mediated neurotrophin-3 (NT-3) transfection into BMSCs, provides a preliminary basis for inner ear transplantation. Methods: (1) Guinea pig BMSCs, was cultured by whole bone marrow adherent method. BMSCs of P3 generation was taken as growth curve. CD29, the surface marker of BMSCs, was detected by flow cytometry. The transfection efficiency of CD90 and hematopoietic cell surface marker CD45. (2) was calculated 48 hours after transfection of P3 generation BMSCs, with pEGFP-N1-NT3 mediated by different plasmide-liposome ratios. Select the best plasmide-liposome ratio. (3) according to the best plasmide-liposome ratio, the pEGFP-N1-NT3 plasmid was transfected into P3 generation BMSCs, for 1 day, 3 days, 7 days and 14 days after transfection, and the fluorescent expression was observed. (4) the BMSCs protein was extracted 48 hours after transfection. Western Blot was used to verify the expression of NT-3 protein. Results: (1) the cells obtained by whole bone marrow adherent method were numerous and proliferative, and the cells could be purified by passage. (2) the growth curve was S-shaped, which basically accorded with the growth curve of Logistic. (3) the positive expression of CD29 and CD90 was detected by flow cytometry. The negative expression of CD45 was consistent with the characteristics of BMSCs. (4) among the different plasmide-liposome ratios, the transfection efficiency was the highest when the liposome was 4 渭 g: 12 渭 l, which was the best ratio of plasmids to liposomes. (5) the fluorescence expression was stronger at 48 hours after transfection. Fluorescence expression was observed on day 7, but weakened significantly, and no obvious fluorescence expression was found at day 14. (6) NT-3 gene was successfully expressed by) Western Blot. Conclusion: (1) BMSCs. (2) liposome with high cell purity and rapid proliferation can be successfully cultured by whole bone marrow adherent method to mediate the expression of NT-3 eukaryotic plasmid in BMSCs. BMSCs may be an ideal vector for inner ear gene therapy.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R764.43

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