低剂量LPS预处理的间充质干细胞对胰岛保护作用的研究

Co-transplantation with LPS Preconditioned Mesenchymal Stem Cells Promote the Efficiency of Islets Grafts

目的:探讨低剂量脂多糖(LPS)预处理的间充质干细胞(MSCs)对于胰岛移植物的保护作用及其机制。方法:给予MSCs不同浓度的LPS预处理,利用流式细胞仪检测不同处理组的间充质干细胞在低氧条件下的凋亡情况,筛选出最佳刺激浓度。通过ELISA检测低氧条件下LPS预处理组与未处理组的MSCs促生长因子的分泌情况。利用Western blot的方法检测不同处理组的MSCs在低氧条件下bax,bcl-2的表达。体外低氧条件下共培养不同处理的MSCs和胰岛细胞,检测胰岛细胞内部CD31阳性细胞的表达。以F344大鼠为供者,以Balb/c裸鼠为受者,制作胰岛联合MSCs移植模型,连续观察21天检测胰岛功能的恢复情况。结果:当以500ng/mL的LPS刺激MSCs能够减少MSCs在低氧条件下的凋亡水平(P<0.05),此时为最适浓度。LPS预处理的MSCs相较于未处理组能够在低氧条件下分泌更多的HGF,IGF-1,VEGF。LPS预处理的MSCs能够上调bcl-2下调bax的表达(P<0.05)。LPS预处理的MSCs能够明显保护低氧条件下胰岛细胞内部的CD31阳性的内皮细胞的数量。胰岛细胞联合LPS预处理的MSCs能够明显提高胰岛功能的改善。结论:胰岛细胞联合LPS预处理的MSCs能够明显提高移植效率。

Objective： To investigate the effect of LPS preconditioned MSCs on the transplantation efficiency of islets grafts and the possible mechanism. Methods： Different concentration of LPS was used to precondition the MSCs. Apoptosis rates were measured by the FACS, and the optimum concentration of LPS was determined by the lowest apoptosis rate under hypoxia. ELISA was used to detect the secretion of growth factor by the MSCs. Western blot and PCR were used to detect the expression of the bax and bcl-2. In vitro, MSCs was co-cultured with islets under hypoxic condition. CD31 expression in the intraislets was detected by the immunofluorescence. xenogenic islets transplantation models were made using C57BL/6 as donor mice and Balla/c nude as recipient mice. Grafts fiaxxction was measured by the level of blood glucose. Results： 500ng/ml was the optimum concentration to precondition MSCs. Pretreated with LPS could reduce the apoptosis of MSCs under hypoxic condtion. Compared with that in the non-treatment group, the MSCs preconditioned with LPS secret more HGF, IGF-1 and VEGF. In vitro results showed that MSCs preconditioned with LPS could preserve the CD31 positive cells in the intraislets tmder hypixa. Co-transplantation with LPS preconditioned MSCs could hasten the recovery of the islets grafts. Conclusion： MSCs preconditioned with low dose of LPS could improve the efficiency of islets grafts.