中国修复重建外科杂志

中国修复重建外科杂志

大鼠骨髓来源与前交叉韧带来源 MSCs 体外生物学特性比较研究

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目的 比较大鼠骨髓来源与前交叉韧带(anterior cruciate ligament,ACL)来源的 MSCs 体外增殖及成骨、成软骨、成脂分化潜能的差异。 方法 取 SPF 级 6 周龄雄性 BN 大鼠 10 只,体质量 200~220 g,无菌条件下分别取大鼠骨髓及 ACL,贴壁法培养获取 BMSCs 与 ACL 来源 MSCs 并进行传代,倒置相差显微镜下观察细胞形态变化。取生长良好的第 3 代细胞,流式细胞仪检测细胞免疫表型 CD34、CD45、CD90 和 CD29 表达;细胞计数试剂盒 8(cell counting kit 8,CCK-8)法检测细胞体外增殖能力,克隆形成实验检测细胞体外克隆形成能力;行成骨、成软骨及成脂体外诱导多向分化能力检测;实时荧光定量 PCR 检测成骨[ALP、骨钙蛋白、RUNX2、BMP-2、分泌性磷蛋白 1(secreted phosphoprotein 1,Spp1)]、成软骨[Ⅱ 型胶原 α1(collagen type Ⅱ α1,Col2α1)、软骨聚糖蛋白(Aggrecan,Acan)、Sox9]及成脂[过氧化物酶体增殖物激活受体 γ2(peroxisome proliferator activated receptor γ2,PPARγ2)、CCAAT/增强子结合蛋白 α]相关基因 mRNA 相对表达量。 结果 第 3 代 ACL 来源 MSCs 与 BMSCs 形态相似,均表现为贴壁生长的长梭形细胞。流式细胞仪检测示,两种细胞均表达 CD29、CD90,基本不表达 CD45 及 CD34。CCK-8 法检测示,ACL 来源 MSCs 的吸光度(A)值(1.11±0.08)显著高于 BMSCs(0.78±0.05),差异有统计学意义(t=3.599,P=0.023);ACL 来源 MSCs 的细胞集落数[(53.00±5.51)个/孔]亦明显多于 BMSCs[(30.67±4.84)个/孔](t=3.045,P=0.038)。经成骨、成软骨、成脂体外诱导培养 21 d 后,BMSCs 及 ACL 来源 MSCs 均表现为茜素红、甲苯胺蓝及油红 O 染色阳性。实时荧光定量 PCR 检测示,ACL 来源 MSCs 的 BMP-2、Spp1、Col2α1、Acan、Sox9 及 PPARγ2 mRNA 相对表达量显著高于 BMSCs,差异均有统计学意义(P<0.01)。 结论 ACL 来源 MSCs 比 BMSCs 具有更强的体外增殖能力,在相同体外条件下更易向软骨分化,是一种有潜力的促进腱骨愈合的种子细胞。

Objective To compare the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) and anterior cruciate ligament derived mesenchymal stem cells (ACL-MSCs) from ratsin vitro. Methods 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. Results 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 (A) value of ACL-MSCs (1.11±0.08) was significantly higher than that of BMSCs (0.78±0.05) (t=3.599,P=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] (t=3.045,P=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 (P<0.01). Conclusion 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.

关键词: BMSCs; 前交叉韧带来源 MSCs; 增殖分化; 大鼠

Key words: Bone marrow mesenchymal stem cells; anterior cruciate ligament derived mesenchymal stem cells; proliferation and differentiation; rat

引用本文: 郝滋辰, 吴浩, 李阳, 王善正, 陆军. 大鼠骨髓来源与前交叉韧带来源 MSCs 体外生物学特性比较研究. 中国修复重建外科杂志, 2017, 31(4): 473-480. doi: 10.7507/1002-1892.201611021 复制

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