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目的:研究大鼠骨髓间充质干细胞(MSCs)对脾单个核细胞(MNC)的免疫调节作用,并初步探讨其作用机制。方法:从大鼠骨髓中分离培养间充质干细胞,通过瑞氏-姬姆萨染色进行形态学观察。应用流式细胞术(FCM)检测鉴定其细胞表面特征分子。以刀豆蛋白A(ConA)作为刺激原,用MTT法测定不同数量MSCs对脾MNC增殖能力的影响。ELISA法检测MSCs对脾MNC分泌IL-2和IL-10水平的影响。用FCM分析MSCs对脾MNC细胞周期分布以及p27、cyclin E表达水平的影响。用乳酸脱氢酶释放法检测MSCs对脾MNC杀伤活性的影响。结果:经不同数量的MSCs作用后,脾MNC增殖水平明显低于阳性对照组(P<0.01),而且MSCs比例越高,其抑制作用越强(P<0.01)。经MSCs作用后,脾MNC分泌IL-2的水平明显降低,而分泌IL-10的水平明显升高(P<0.01),且这种作用随MSCs比例的增加而增强。在ConA刺激下,MSCs可以使脾MNC阻滞于G0/G1期,抑制其进入S期(P<0.01)。MSCs可使脾MNC表达p27的水平明显上升,cyclin E表达的水平明显下降(P<0.05)。与MSCs共培养后,脾MNC对colon26和H22细胞的杀伤活性和单独培养组相比明显下降(P<0.05)。结论:MSCs能够抑制脾MNC体外增殖,该作用可能与MSCs改变MNC分泌细胞因子的水平及其对MNC细胞周期的调节有关。
Objective: To study the immunomodulatory effect of rat bone marrow mesenchymal stem cells (MSCs) on splenic mononuclear cells (MNCs) and to explore its mechanism. METHODS: Mesenchymal stem cells (MSCs) were isolated and cultured from rat bone marrow and observed by Wright-Giemsa staining. Flow cytometry (FCM) detection of cell surface markers. Using ConA as stimulus, the effects of different numbers of MSCs on the proliferation of splenic MNCs were determined by MTT assay. The effect of MSCs on the secretion of IL-2 and IL-10 by spleen MNC was detected by ELISA. The effects of MSCs on the cell cycle distribution and the expression of p27 and cyclin E in splenic MNC were analyzed by FCM. Effect of MSCs on cytotoxicity of splenic MNC by lactate dehydrogenase release assay. Results: The proliferation of splenic MNC was significantly lower than that of the positive control (P <0.01). The higher the proportion of MSCs, the stronger the inhibition was (P <0.01). After MSCs treatment, the level of IL-2 secreted by splenic MNC was significantly decreased, while the level of IL-10 secretion was significantly increased (P <0.01), and this effect was enhanced with the increase of MSCs proportion. Under the stimulation of ConA, MSCs could arrest the MNCs in G0 / G1 phase and inhibit their entry into S phase (P <0.01). MSCs could significantly increase the expression of p27 in the MNCs and the expression of cyclin E in the MNCs (P <0.05). After co-cultured with MSCs, the cytotoxic activity of splenic MNC on colon26 and H22 cells was significantly decreased compared with that of cultured cells alone (P <0.05). CONCLUSION: MSCs can inhibit the proliferation of splenic MNC in vitro, which may be related to the changes of MNC-secreting cytokines and the regulation of MNC cell cycle.