豚鼠骨髓间充质干细胞培养及体外转染神经营养因子-3的研究

Study on in vitro culture bone marrowderived mesenchymal stem cells of guinea pig and transfection with neurotrophic factor-3

摘要:

目的建立豚鼠骨髓间充质干细胞（bonemesenchymal stem cells，BMSCs）的体外培养体系，体外脂质体2000介导神经营养因子3（neurotrophic factor3,NT3）转染BMSCs，为内耳移植提供前期研究基础。方法①采用全骨髓贴壁法培养豚鼠BMSCs并传代，取P3代做生长曲线图，流式细胞仪上检测BMSCs的表面标志物CD29，CD90以及造血细胞表面标志物CD45；②按照不同质粒-脂质体配比介导pEGFPN1NT3转染P3代BMSCs，转染后48 h计算转染效率，选择最佳配比将pEGFPN1NT3质粒转染P3代BMSCs，转染后1、3、7、14 d，观察其荧光表达情况；③转染后3 d的BMSCs提取蛋白，Western Blot验证NT3蛋白表达。结果①全骨髓贴壁法获得细胞数量多，增殖快，传代可以纯化细胞。生长曲线符合Logistic生长曲线。流式细胞仪检测细胞CD29和CD90阳性表达，CD45阴性表达，符合BMSCs特征；②不同配比中质粒：脂质体为4 μg:12 μl时转染率最高，为最佳配比；③转染后48 h荧光表达较强，7 d荧光表达但较前明显减弱，14 d基本未见明显荧光表达。Western Blot验证NT3基因成功表达。结论①全骨髓贴壁法可以成功培养出增殖快，细胞纯度高的BMSCs；②脂质体法成功介导NT3真核质粒在BMSCs内表达，BMSCs可能作为内耳基因治疗的理想载体。

Abstract:

ObjectiveTo provide foundation for further study of inner ear transplantation via establishing in vitro culture system of guinea pig bone marrowderived mesenchymal stem cells (BMSCs) and transfecting neurotrophic factor3 (NT3) by Lipofectamin 2000 into BMSCs.Methods①Guinea pig BMSCs were extracted, cultured and passaged by whole bone marrow direct adherent method in flasks. The third generation cells were used for detection of the surface markers CD29, CD90 of BMSCs and the surface antigen CD45 of hematopoietic cell by flow cytometry. ②BMSCs of P3 were transfected with pEGFPN1NT3 by plasmidliposome mixtures of different ratio. In 48 h after transfection, the transfection efficiencies of different mixtures were calculated and that with the best efficiency was chosen to transfect BMSCs of P3 with pEGFPN1NT3. On day 1, 3, 7 and 14 after transfection, the fluorescent expressions were observed. ③Proteins of BMSCs were extracted on day 3 after transfection, and the expression of NT3 was validated by Western Blot.Results①By whole bone marrow direct adherent method in flasks, large amount of BMSCs were harvested. The BMSCs could proliferate quickly and be purified via passage. Their growth curves accorded with Logistic growth curve. The flow cytometric detection showed positive expressions of CD29 and CD90, negative expression of CD45, which matched the characteristics of BMSCs.②The transfection efficiency was the highest with the ratio of plasmidliposome (μg:μl) at 4:12, making it the best ratio of plasmidliposome. ③Fluorescent expression was the strongest in 48 hours after transfection, and was visible but significantly decreased in day 7, and could not been observed in day 14. Western Blot identified successful expression of NT3 gene in BMSCs.Conclusions①BMSCs of guinea pig can be cultured by whole bone marrow direct adherent method in flasks with high purity and quick proliferation. ②Eukaryotic plasmid of NT3 can be successfully mediated by Lipofectamin 2000 and expressed in BMSCs. BMSCs may be ideal carriers of gene therapy for inner ear.