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本研究培养小鼠密质骨间充质干细胞(MSC)并分析其免疫功能和分化潜能。分离小鼠双侧股骨和胫骨,反复冲洗,取出骨髓细胞,用胶原酶消化骨碎片,分离有核细胞并接种于6孔板。对第3代MSC进行免疫表型测定、免疫抑制功能分析及进行成脂细胞、成骨细胞和成软骨细胞诱导分化实验。结果表明,培养3周内可自小鼠密质骨分离高纯度的MSC,后者具有向成脂细胞、成骨细胞和成软骨细胞分化的潜能,具有抑制混合淋巴细胞反应的功能。BALB/c T细胞经C57BL/6 T细胞刺激后对照组液体闪烁计数仪检测每分钟计数(CPM)值为(2.56±0.31)×104,而与细胞比例为100∶1和10∶1的C57BL/6第3代密质骨MSC共孵育组的CPM值分别为(0.47±0.12)×104和(0.28±0.09)×104,MSC处理组CPM值与对照组CPM值相比差别有统计学意义(P<0.001),MSC抑制功能呈剂量依赖性。结论:小鼠密质骨富含MSC,原代培养MSC造血细胞污染程度低,具有抑制混合淋巴细胞反应功能,具有更广泛的实验研究应用价值。
In this study, mouse mesenchymal stem cells (MSCs) were cultured and their immune function and differentiation potential were analyzed. Bilateral femur and tibia were separated from mice, rinsed repeatedly, and bone marrow cells were removed. Collagenase was used to digest bone fragments and nucleated cells were isolated and seeded on 6-well plates. The third generation of MSCs were immunophenotype assay, immunosuppressive function analysis and adipogenic, osteoblastic and chondrogenic differentiation induction experiments. The results showed that high purity of MSCs could be isolated from the mouse bovine cancellous bone within 3 weeks after culture, which has the potential to differentiate into adipogenic, osteogenic and chondrogenic cells and inhibit the mixed lymphocyte reaction. BALB / c T cells were stimulated with C57BL / 6 T cells in the control group with a liquid scintillation counter (CPM) of (2.56 ± 0.31) × 104, while C57BL cells with ratios of 100: 1 and 10: 1 / 6 third generation of dense bone marrow co-incubation group CPM values were (0.47 ± 0.12) × 104 and (0.28 ± 0.09) × 104, the CPM value of MSC treatment group compared with the control group CPM value was statistically significant (P <0.001). The inhibitory function of MSC was dose-dependent. CONCLUSION: MSCs with MSCs are enriched in MSCs, and the degree of contamination of MSCs in primary culture is low, which has the function of inhibiting mixed lymphocyte reaction and has more extensive experimental research value.