水平振搖在骨髓間充質干細胞提純中的作用

·基礎研究·

水平振搖在骨髓間充質干細胞提純中的作用

王偉恒，鄧國英，徐立璋，程自申，楊向群，葉曉健

作者單位：200003上海， 第二軍醫大學附屬長征醫院骨科

通信作者：葉曉健yexj2002@163.com

【摘要】目的研究水平振搖對全骨髓貼壁分離大鼠骨髓間充質干細胞在細胞數量、純度、凋亡及遠期增殖及分化能力的影響，探索更為簡單、高效的間充質干細胞提純方法。 方法在全骨髓貼壁法的基礎上，使用水平搖床，以180 r/min、4 h為干預條件，水平振搖后計算貼壁細胞總數量；使用流式細胞儀測定貼壁細胞純度及凋亡情況；使用MTT法測定振搖后貼壁細胞24 h的增殖情況；對分離純化后細胞使用定向誘導培養基進行誘導分化。結果經振搖處理后，獲得的細胞總量有所下降，細胞純度顯著提升，凋亡略有增加，增殖情況變化不大，具有向成骨細胞和脂肪細胞分化的能力。結論水平振搖法可顯著提高全骨髓貼壁法的提純效率，具有推廣價值。

【關鍵詞】大鼠; 骨髓; 間質干細胞; 分離和提純;

基金項目：國家高技術研究發展計劃(863計劃)(2013AA032203)

作者簡介：王偉恒(1989— )， 碩士在讀，醫師

【中圖分類號】R 329.28 【文獻標志碼】 A

DOI【】

收稿日期：(2014-10-12)

Exploration of horizontal shaking for bone marrow mesenchymal stem cells purificationWANGWei-hang,DENGGuo-ying,XULi-zhang,CHENGZi-shen,YANGXiang-qun,YEXiao-jian.DepartmentofOrthopaedics,ChangzhengHospital,SecondaryMilitaryMedicalUniversity，Shanghai200003，China

Abstract【】ObjectiveTo study the effects of horizontal shaking on cell purity, activity, apoptosis, and differentiation in the process of the in vitro isolation and purification of rat bone marrow mesenchymal stem cells (BMSCs). Even the shaking effect for the cells proliferation, differentiation ability in the future. To establish a simpler and more efficient method for BMSCs purification. Methods Based on the whole bone marrow adhesion method, BMSCs isolated and purified by using different attachment method and shaking treatment of 180 r/min for 4 h, and then analyzed the adherent cell state including cell counting by cells counter; cell purity and apoptosis by flow cytometry; cells proliferation after shaking 24 h by MTT method; cells differentiation by directional induction medium. ResultsAfter shaking, cell count was decrease, purity was increased, apoptosis was increased, proliferation had no change, and the harvested cells had the ability to differentiate into osteoblasts and adipocytes. ConclusionThe whole bone marrow adhesion method combined with appropriate shaking is more efficient and worth popularizing.

【Key words】Rats; Bone marrow; Mesenchymal stem cells; Isolation & purification;

J Spinal Surg, 2015,13(1):45-49

近年來，骨髓間充質干細胞(bone marrow mesenchymal stem cells, BMSCs)作為種子細胞在脊髓損傷[1-3]、椎間盤退變[4-6]和纖維環修復[7-8]等脊柱外科領域備受關注。但因其在骨髓中含量低，體外培養易分化和老化[9],獲得足夠數量的高純度、高活性、低分化的間充質干細胞是進行相關研究的重要基礎。

目前從骨髓中提純BMSCs方法主要包括：全骨髓貼壁法[10]、密度梯度離心法[11]、流式細胞儀分離法[12]、免疫磁珠分離法[13]?！?br>