Huang Qian, Wu Tao, Liang Jie, Yang Keyue, Jin Dan
Lnstitute of Plastic and Aesthetic Surgery, Guangdong Medical College, Zhanjiang Guangdong 524023, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Nov;23(11):1360-4.
To make a comparative study on the effects of whole bone marrow culture method and density gradient centrifugation method in isolating hBMSCs.
hBMSCs were obtained from healthy adult volunteers and isolated by whole bone marrow culture method and density gradient centrifugation method. Primary cell morphology was observed using inverted phase contrast microscope and the cells in the 2nd passage were stained with HE after being cultured for 7 days. And then, the generation time of the primary, 2nd and 3rd passage hBMSCs was compared between two methods and the surface markers were detected by flow cytometer. In addition, the ALP expression in osteoinductive hBMSCs were evaluated by ALP activity kit at 3, 6 and 9 days and ALP staining was used for osteoinductive hBMSCs with Kaplow method at 9 days.
Primary cells isolated with whole bone marrow culture method showed aggregation growth while cells isolated with density gradient centrifugation method showed diffusion growth. HE staining showed no significant difference in the morphology of the 2nd passage cells between these two methods. The generation time of primary cells isolated by whole bone marrow culture method (15.36 +/- 1.67) days was significantly shorter than that of cells isolated by density gradient centrifugation method [(18.57 +/- 1.05) days] (P < 0.01), while the generation time of the 2nd and 3rd passage cells showed no statistically significant differences between these methods (P > 0.05). The consent of positive surface markers (CD29, CD44, CD71, CD105, CD166) and negative surface marker CD34 in the 2nd cells showed no significant difference between these two isolation methods (P > 0.05); however, negative markers CD14 and CD45 showed significant difference (P < 0.01). The ALP expression in osteoinductive cells showed no statistical significant (P > 0.05) at 3, 6 and 9 days; and the ALP staining positive cell ratio of whole bone marrow culture method was basically in accordance with that of density gradient centrifugation method at 9 days.
hBMSCs could be isolated by whole bone marrow culture method, and the cell isolation effects of whole bone marrow culture method are equivalent with density gradient centrifugation method.
比较全骨髓培养法和密度梯度离心法分离人骨髓间充质干细胞（hBMSCs）的效果。
从健康成年志愿者获取hBMSCs，采用全骨髓培养法和密度梯度离心法进行分离。利用倒置相差显微镜观察原代细胞形态，第2代细胞培养7天后进行HE染色。然后，比较两种方法分离的原代、第2代和第3代hBMSCs的倍增时间，并通过流式细胞仪检测表面标志物。此外，在第3、6和9天使用碱性磷酸酶（ALP）活性试剂盒评估成骨诱导hBMSCs中ALP的表达，在第9天采用Kaplow法对成骨诱导hBMSCs进行ALP染色。
全骨髓培养法分离的原代细胞呈聚集生长，而密度梯度离心法分离的细胞呈扩散生长。HE染色显示两种方法第2代细胞的形态无显著差异。全骨髓培养法分离的原代细胞倍增时间（15.36±1.67）天显著短于密度梯度离心法分离的细胞[（18.57±1.05）天]（P<0.01），而两种方法第2代和第3代细胞的倍增时间无统计学差异（P>0.05）。两种分离方法第2代细胞中阳性表面标志物（CD29、CD44、CD71、CD105、CD166）和阴性表面标志物CD34的一致性无显著差异（P>0.05）；然而，阴性标志物CD14和CD45存在显著差异（P<0.01）。成骨诱导细胞中ALP的表达在第3、6和9天无统计学差异（P>0.05）；第9天全骨髓培养法的ALP染色阳性细胞比例与密度梯度离心法基本一致。
全骨髓培养法可分离hBMSCs，且全骨髓培养法的细胞分离效果与密度梯度离心法相当。
