摘要
目的建立C57/BL6小鼠骨髓间充质干细胞(marrowmesenchymalstemcells,MSCs)的体外分离和培养体系,探讨酒精及其代谢物对MSCs的毒性。方法从C57/BL6小鼠的股骨骨髓中分离MSCs,通过优化细胞的培养方法进行有效培养、传代,最终获得纯度较高的MSCs,并对第4代细胞进行CD90和CD34免疫组织化学染色鉴定。对第2代细胞以4×105个/ml密度接种于96孔板,每孔200μl,第2天以用乙醇及乙醛进行染毒,剂量设计为乙醇5.7、17.0、50.0、100.0、150.0mmol/L,乙醛4.5、0.9、0.18、0.036、0.0072、0.00144、0.000288mmol/L,对照组为MSCs以不加乙醇及乙醛的10%胎牛血清的α-MEM培养液培养。3d后MTT检测细胞增殖活性。结果MSCs在前6代表现出较好的稳定性和旺盛的自我增殖能力,免疫组织化学染色显示第4代MSCsCD90呈阳性、CD34呈阴性。MTT检测乙醇在17.0、100.0及150.0mmol/L时出现细胞增殖抑制,与对照组比较,差异有统计学意义(P<0.05);乙醛>0.18mmol/L时随着浓度增加细胞增殖力减弱,在4.5mmol/L时出现了细胞增殖抑制,与对照组比较,差异有统计学意义(P<0.05)。结论优化培养体系能有效分离和培养MSCs,乙醇和乙醛均能抑制其增殖,表现出毒性作用,乙醛的毒性作用较乙醇强,其造成的损伤更不容忽视。
Objective To optimize the in vitro culture system of C57/BL6 marrow mesenchymal stem cells （MSCs） and to investigate the effect of alcohol and acetaldehyde on MSCs. Methods The MSCs were isolated from the femur marrow of C57/BL6 mice and were cultured in the optimized system, so that highly-purified MSCs were harvested and identified by immunohistochemistry. Then, MSCs were cultured in the medium containing alcohol or its metabolic product acetaldehyde, with the following concentration groups： alcohol 5.7, 17.0, 50.0,100.0 and 150.0 mmol/L; acetaldehyde 4.5, 0. 9, 0. 18, 0. 036, 0. 007 2, 0. 001 44, 0. 000 288 mmol/L. MSCs were cultured with α-MEM as the control group. After 3 days, their proliferation activity was measured by the MTT method. Results MSCs within 6 passages had a good stability and a high proliferation activity. They were identified to express CD90 but no CD34. The MTT assay showed that alcohol at the concentration greater than 100.0 mmol/L and acetaldehyde at the concentration greater than 4.5 mmol/L could inhibit proliferation of MSCs（P〈0.05）. But the proliferation activity might rise with an increase in the acetaldehyde concentration smaller than 0. 18 mmol/L（P〈0.05）. Conclusion The optimized culture system can effectively isolate and culture MSCs. Both alcohol and acetaldehyde can inhibit proliferation of MSCs but toxicity of acetaldehyde is more serious.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2006年第4期471-474,共4页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(30000139)~~
关键词
骨髓间充质干细胞
乙醇
乙醛
细胞毒性
Marrow mesenchymal stem cells Alcohol Acetaldehyde Cell toxicity
