骨髓间质干细胞和脂肪间质干细胞混合培养后的成骨能力

Osteogenic ability study on co-culture of adipose-derived mesenchymal stem cells and bone marrow mesenchymal stem cells

目的探讨骨髓间质干细胞（bone mesenchymal stem ceils, B MSC）和脂肪间质干细胞（adipose-derived mesenchymal stem cells，ADSC）混合培养后的成骨分化能力。方法以贴壁法和酶消化法获得BMSC和ADSC，用流式细胞术鉴定细胞表型；对两种细胞进行成脂肪、成软骨、成骨诱导，分别以油红“O”染色、甲苯胺蓝染色、茜素红染色进行鉴定；碱性磷酸酶（alkaline phosphatase，ALP）、茜素红染色鉴定成骨分化。取第3代BMSC和ADSC细胞加入成骨诱导液，根据不同处理方法分为四组：BMSC未诱导组、BMSC成骨诱导组、ADSC成骨诱导组、BMSC＋ADSC成骨诱导组。成骨诱导第1、3、5、7、9、11天检测ALP活性，第7、14、21、28天检测钙离子吸光度，第14天采用RT-PCR和Western Blot检测骨钙素（Osteocalcin，OCN）、Runx2蛋白的表达。将BMSC、ADSC或BMSC＋ADSC细胞植入羟基磷灰石，壳聚糖／聚乳酸（Hydroxylapatite／Chitosan／Poly L-latic acid，HA/CS/PLLA）支架材料，植入大鼠下肢骨缺损模型，术后8周取材检测复合材料的新骨形成面积。结果ADSC和BMSC两种细胞在成脂、成软骨、成骨诱导后染色均为阳性。诱导第1、3天，各组细胞ALP活性增加不明显，第5天以后活性开始明显增强，其中BMSC＋ADSC组高于其他三组。诱导第7天，各组细胞钙离子吸光度无明显变化，第14天开始逐渐增高，其中BMSC＋ADSC成骨诱导组高于其他三组。诱导第14天，BMSC＋ADSC成骨诱导组OCN和Runx2基因表达（分别为78．24±8．11，1180．13±121．16）和蛋白表达（分别为6．54±0．59，4．43±0．51）较其他三组升高，差异有统计学意义（P〈0．05）。材料植入体内8周后，BMSC＋ADSC组有大量板状骨形成，支架材料基本降解完全，新骨形成面积为（497．75±7．44）μm2，明显大于其他三组，差异有统计学意义（P〈0．05）。结论BMSC与ADSC两种细胞混合培养较单一细胞的体内和体外成骨能力增强。

Objective To assess the osteogenic ability after co-culture BMSC and ADSC in vivo and in vitro. Methods ADSC and BMSC were obtained by adherent screening method and enzymatic digestion method. Flow cytometry was used to con- firm the phenotypes of ADSC and BMSC. Oil red O was used to induce MSC to fat. Alkaline phosphatase （ALP） and alizarin red staining were used in osteogenic group. This sample was divided into four groups, no-induced stem ceils group; BMSC osteogenic induction group; ADSC osteogenic induction group; co-culture of BMSC and ADSC osteogenic induction group. ALP activities and Calcium absorbance were determined during different periods of osteogenic introduction. OCN and Runx2 expression level were tested via RT-PCR and western blot methods after osteogenic induction for 2 weeks. Furthermore, cells in each group were seeded on HA/CS/PLLA composite scaffolds, and the scaffolds with cells were planted into bone defects in rat models. The rats were sacri- riced by overdose anesthesia at 8 weeks after surgery and the scaffolds were removed for further analysis. Results Oil red O stain- ing demonstrated red after adipogenic induction. Alkaline phosphatase and Alizarin red staining showed flaky red under condition of osteogenic induction. There had no statistical change among each group after osteogenic induction for 3 days, and ALP activity significantly increased after osteogenic induction for 5 days. Meanwhile, the ALP activity in co-culture of BMSC and ADSC group was markedly higher than the other three groups. However, there had no significant change in A value of calcium absorbance among each group after osteogenic induction for 7 days, while it increased at 14th day and ALP activity in co-culture of BMSC and ADSC group was significantly higher than the other three groups. After osteogenic induction for 2 weeks, the mRNA expression of OCN and Runx2 in co-culture of BMSC and ADSC group was 78.24±8.11 and 1 180.13±121.16 respectively, and the protein ex- pression of OCN and Runx2 was 6.54±0.59 and 4.43±0.51. These mRNA and protein expression level in co-culture of BMSC and ADSC group enhanced significant compared with the other 3 groups. Histological assay demonstrated that the new bone tissues formed in co-culture of BMSC and ADSC group were 497.75±7.44 μm2, which was larger than that in the other 3 groups at 8 weeks after implantation. Conclusion Co-culture BMSC and ADSC may up-regulated the osteogenic ability in vivo and in vitro.