Hao Zichen, Wu Hao, Li Yang, Wang Shanzheng, Lu Jun
Medical School of Southeast University, Nanjing Jiangsu, 210009, P.R.China.
Department of Orthopaedics, Zhongda Hospital, Medical School of Southeast University, Nanjing Jiangsu, 210009, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2017 Apr 15;31(4):473-480. doi: 10.7507/1002-1892.201611021.
To compare the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) and anterior cruciate ligament derived mesenchymal stem cells (ACL-MSCs) from rats .
Ten male SPF-level BN rats, weighing 200-220 g, were selected to obtain anterior cruciate ligaments and bone marrows, and ACL-MSCs and BMSCs were isolated for passage culture respectively under sterile condition. The cell morphology was observed, and the cells at passage 3 were used to detect the surface markers of CD34, CD45, CD90, and CD29 by flow cytometry, the ability of cell proliferation by cell counting kit 8 (CCK-8), and colony formation ability by clone forming test. The mRNA levels of differentiation related genes [alkaline phosphatas (ALP), bone gamma-carboxyglutamate protein, runt related transcription factor 2, bone morphogenetic protein 2 (BMP-2), secreted phosphoprotein 1 (Spp1), collagen type II α1 (Col2α1), Aggrecan (Acan), Sox9, peroxisome proliferator activated receptor γ2 (PPARγ2), and CCAAT-enhancer-binding protein-α] were also determined by real-time fluorescent quantitative PCR.
BMSCs and ACL-MSCs had similar morphology, adherent cells displaying long fusiform. The immunoprofile of ACL-MSCs and BMSCs at passage 3 was positive for CD29 and CD90 and was negative for CD45 and CD34. The absorbance ( ) value of ACL-MSCs (1.11±0.08) was significantly higher than that of BMSCs (0.78±0.05) ( =3.599, =0.023); the number of colonies of ACL-MSCs [(53.00±5.51)/hole] was significantly more than that of BMSCs [(30.67±4.84)/hole] ( =3.045, =0.038). The results of toluidine blue staining, alizarin red staining, and oil red O staining were positive in BMSCs and ACL-MSCs at 21 days after osteogenic, chondrogenic, and adipogenic induction. The mRNA expressions of BMP-2, Spp1, Col2α1, Acan, Sox9, and PPARγ2 in ACL-MSCs were significantly higher than those in BMSCs ( <0.01).
The proliferation potential of ACL-MSCs is greater than that of BMSCs, and the former is apt to differentiate into chondrocytes. ACL-MSCs are promising cells to promote tendon-bone healing.
比较大鼠骨髓间充质干细胞（BMSCs）和前交叉韧带来源的间充质干细胞（ACL-MSCs）的生物学特性。
选取10只体重200 - 220 g的雄性SPF级BN大鼠，获取其前交叉韧带和骨髓，分别在无菌条件下分离ACL-MSCs和BMSCs并进行传代培养。观察细胞形态，取第3代细胞通过流式细胞术检测CD34、CD45、CD90和CD29的表面标志物，采用细胞计数试剂盒8（CCK-8）检测细胞增殖能力，通过克隆形成试验检测集落形成能力。还通过实时荧光定量PCR测定分化相关基因[碱性磷酸酶（ALP）、骨γ-羧基谷氨酸蛋白、 runt相关转录因子2、骨形态发生蛋白2（BMP-2）、分泌性磷蛋白1（Spp1）、Ⅱ型胶原α1（Col2α1）、聚集蛋白聚糖（Acan）、Sox9、过氧化物酶体增殖物激活受体γ2（PPARγ2）和CCAAT增强子结合蛋白-α]的mRNA水平。
BMSCs和ACL-MSCs形态相似，贴壁细胞呈长梭形。第3代ACL-MSCs和BMSCs的免疫表型CD29和CD90呈阳性，CD45和CD34呈阴性。ACL-MSCs的吸光度（ ）值（1.11±0.08）显著高于BMSCs（0.78±0.05）（ =3.599， =0.023）；ACL-MSCs的集落数[（53.00±5.51）/孔]显著多于BMSCs[（30.67±4.84）/孔]（ =3.045， =0.038）。在成骨、软骨和成脂诱导21天后，BMSCs和ACL-MSCs的甲苯胺蓝染色、茜素红染色和油红O染色结果均为阳性。ACL-MSCs中BMP-2、Spp1、Col2α1、Acan、Sox9和PPARγ2的mRNA表达显著高于BMSCs（ <0.01）。
ACL-MSCs的增殖潜能大于BMSCs，且前者易于分化为软骨细胞。ACL-MSCs是促进腱骨愈合的有前景的细胞。
