小鼠脂肪源间充质干细胞在体外对造血功能影响的研究

发布时间：2018-05-21 02:47

本文选题：脂肪源间充质干细胞 + 成骨诱导　；参考：《新疆医科大学》2013年硕士论文

【摘要】：目的：探讨小鼠脂肪源间充质干细胞对造血干/祖细胞体外扩增的影响并对其机制进行初步研究。方法：无菌条件下获取C57BL/6小鼠的脂肪源干细胞（ADSCs），培养至P3代：①细胞形态学分析；②流式细胞术检测细胞表面免疫表型；③脂肪源干细胞定向成骨、成脂细胞诱导。将小鼠成纤维细胞、骨髓间充质干细胞、成骨诱导前后的脂肪源干细胞经丝裂霉素C处理后作为滋养层，以骨髓单个核细胞（BMNCs）作为长期培养起始细胞（LTC-IC），对其进行35天长期体外共培养，将本实验分4组：A组：BMNCs加小鼠成纤维细胞，，B组：BMNCs加骨髓间充质干细胞（BMSCs），C组：BMNCs加ADSCs，D组：BMNCs加成骨诱导后的脂肪源干细胞（osteo-ADSCs）；分别于0d、14d、28d、35d行甲基纤维素14d短期处理分析造血干/祖细胞集落增殖状态；同时应用酶联免疫吸附法（ELISA）检测上清液中的基质细胞衍生因子-1（SDF-1）、N-钙黏素（N-cadherin）、β-连环蛋白（β-catenin）、Jagged1蛋白（JAG1）的表达情况。结果：1. ADSCs的生物学分析：ADSCs在体外呈梭形生长并可稳定传代；成骨、成脂诱导均为阳性；流式结果分析：CD29高表达（95.37±0.95)％；CD34低表达（8.7±0.4）％。2.细胞体外二维共培养后对造血干/祖细胞（HSPCs）扩增的影响：①不同时间点造血干/祖细胞（HSPCs）形成的集落数不同，且成骨诱导后的脂肪源干细胞更具有造血支持作用；②SDF-1、N-cadherin、β-catenin、JAG1的表达情况在不同时间点不同，SDF-1、β-catenin的表达结果显示：C、D组表达明显高于A、B两组（P 0.05）；且C组与D组比较有统计学意义（P0.05）；而N-cadherin、JAG1在各组间比较无统计学意义（P＞0.05）。结论： C57BL/6小鼠的脂肪源干细胞及成骨诱导后的脂肪源干细胞在体外可有效地刺激造血。
[Abstract]:Aim: to investigate the effect of adipose derived mesenchymal stem cells (MSCs) on hematopoietic stem / progenitor cell expansion in vitro and its mechanism. Methods: adipose derived stem cells from C57BL/6 mice were obtained under aseptic condition and cultured to P3: 1 cell morphology. Flow cytometry was used to detect the surface immunophenotype of adipose derived stem cells to orient osteogenesis and adipogenic cells to induce adipogenic cells. Mouse fibroblasts, bone marrow mesenchymal stem cells and adipose stem cells before and after osteogenesis were treated with mitomycin C as trophoblast layer. Bone marrow mononuclear cells (BMNCs) were cocultured in vitro for 35 days with bone marrow mononuclear cells (BMNCs) as the starting cells for 35 days. The experiment was divided into 4 groups: group A, w: BMNCs plus mouse fibroblast cells, group B: BMNCs + bone marrow mesenchymal stem cells, group C: BMNCs + ADSCsC, group C: BMNCs added to adipose derived stem cells induced by bone osteo-ADSCsC, bone derived stem cells osteo-ADSCs1 were added to group C, respectively, on day 14, 14 days, 28 days after 35 days of methylcellulose treatment, hematopoiesis was analyzed. Stem / progenitor cell colony proliferation; At the same time, the expression of stromal cell derived factor-1, SDF-1, N-cadherin, 尾-catenin (尾-catenin1 protein) in the supernatant was detected by enzyme linked immunosorbent assay (Elisa). The result is 1: 1. The biological analysis of ADSCs showed fusiform growth and stable passage in vitro, osteogenesis and adipogenic induction were positive, flow analysis showed that the high expression of CD29 was 95.37 卤0.95% and the low expression of CD34 was 8.7 卤0.40.0.2. Effect of two dimensional co-culture of cells on the expansion of hematopoietic stem / progenitor cells HSPCsThe colony number of hematopoietic stem / progenitor cells was different at different time points, and adipose derived stem cells induced by osteogenesis had more hematopoietic support. 2the expression of SDF-1N-cadherin, 尾 -cateninine JAG1 was different at different time points. The expression of 尾 -catenin in group B was significantly higher than that in group Agna B (P 0.05), and that in group C was significantly higher than that in group D (P 0.05), but the expression of N-cadherin in JAG1 was not significantly higher than that in group A (P > 0.05). Conclusion: adipose stem cells of C57BL/6 mice and adipose stem cells induced by osteogenesis can stimulate hematopoiesis effectively in vitro.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329.2

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