应用免疫磁珠负选法分离与纯化小鼠骨髓间充质干细胞

目的：拟建立体外分离纯化小鼠骨髓间充质干细胞的新方法，从而得到高纯度的骨髓间充质干细胞。 方法：实验于2002—09／2005-03在解放军第二军医大学附属长海医院烧伤科实验室完成。①选取清洁级7d龄FVB／N小鼠15只，利用免疫磁珠法（CD11b负选）从小鼠的骨髓细胞中分离纯化骨髓间充质干细胞：颈椎脱臼处死后无菌条件下取出小鼠双侧股骨，消毒离断后去除上清及脂肪层，吹打成单细胞悬液后接种于培养基中。贴壁细胞达到90％左右融合后，用胰蛋白酶和乙二胺四乙酸进行消化，将悬浮的细胞与CD11b抗体避光反应2min。移入已预置入磁场的LD管，不加压通过，收集管中的细胞成分，用培养基重悬并吹打成单细胞悬液后以1×10^9L^-1-2×10^9L^-1的密度接种于培养基中培养。②培养传代后流式细胞仪检测细胞周期、表面标记物，并利用诱导剂观察其定向诱导为脂肪细胞和成骨细胞的能力。 结果：①细胞形态学观察：免疫磁珠法获得的细胞几乎无圆形细胞夹杂。贴壁率高。形态单一呈典型成纤维样、漩涡状生长。体外增殖迅速。②细胞生长曲线绘制情况：细胞贴壁后一两天为细胞生长缓滞期，之后进入对数增长期，六七天后进入平台期。③细胞周期分析结果：G0／G1期细胞约86％，S＋G2＋M期约14％。④细胞袭面标记物检测结果：第3代骨髓间充质干细胞3％表达CD45，68．7％表达CD29，3％表达CD34，89．6％表达CD105。⑤骨髓间充质干细胞向脂肪细胞与成骨细胞定向分化情况：在地塞米松、1-甲基-3-异丁基-黄嘌呤、吲哚美辛等诱导剂作用下，脂肪细胞＋成骨细胞诱导组细胞第5天开始油红O染色显示细胞核呈蓝色，细胞内脂滴呈橙色；在β-甘油磷酸钠、2-磷酸-抗坏血酸等诱导剂的作用下，第10天开始AKP染色显示细胞内有致密颗粒形成，Von Kossa染色显?

Isolation and purification of mesenchymal stem cells of mice with immunomagnetic beads negative selection technique

AIM：To establish a new method to isolate and purify mcscnehymal stem cells （MSCs） of mice in vitro so as to acquire highly purified MSCs.
METHODS：The experiment was conducted at the Laboratory of Department of Bum, Affiliated Changhai Hospital, Second Military Medical University of Chinese PLA from September 2002 to March 2005. （1）Totally 15 FVB/N mice aged 7 days of clean grade were selected. MSCs were separated and purified from bone marrow cells with immunomagnetic beads method （CD11b negative selection）. After killing by cervical vertebra luxation, bilateral femur was obtained aseptically from mice, disinfected, mutilated, removed supernatant and fat layer, blew and stroke into monoplast suspension, and then cultured in medium. Adherent cells of nearly 90% confluence were treated with trypsinase and ethylene diamine tetracetic acid. The suspended cells were mixed＇with CD11b antibody away from light for 2 minutes, moved into LD tube pre-set with magnetic field, crossed without adding pressure. Cells of the tube were collected, and then cultured in medium with the density of 1 ×10^9 L^-1-2×10^9 L^-1 after medium re-suspended and blew and struck into monoplast suspension. （2）Cell cycle and surface marker were detected with flow cytometry after culture passage. The muhipotential differentiation ability into adipose ceils and osteoblasts was observed with inductor.
RESULTS： （1） Cell morphological observation： Cells gained by immunomagnetic beads method were mostly without round cells, with high adherent rate, simple form, typical fibroblast shape, whirlpool growth, extraorgan proliferation rapidly. （2）Grewth curve drawing in ceils： One or two days after cell adherence was arrearage stage, followed by increased logarithmic phase, six or seven days later entered platform stage. （3） Analytic result of cell cycle： The ratio of G0/G1 phase cell was about 86%, the ratio of S＋G2＋M phase was about 14%, （4）Detection of cell surface markers showed that of all the cells