缺氧和无血清培养对骨髓间充质干细胞内源性硫化氢生成的影响

Effect of hypoxia and serum deprivation on endogenous hydrogen sulfide production in rat bone marrow mesenchymal stem cells

背景:大鼠骨髓间充干细胞移植后的低存活率多与缺血微环境相关,而硫化氢可以对抗多种细胞与组织的凋亡和损伤模型。目的:检测大鼠骨髓间充质干细胞在不同缺氧和无血清培养时间后的细胞凋亡、细胞活力、硫化氢含量及其合成酶体系的变化情况。方法:取第3代大鼠骨髓间充质干细胞,设立5个(0,3,6,12和24 h)不同的缺氧和无血清培养时间点。用SubG1法检测细胞的凋亡率,用CCK-8法检测细胞的活力,并检测细胞培养基中硫化氢的含量以及细胞中硫化氢合成酶体系的表达变化情况。结果与结论:与正常培养组相比,缺氧和无血清培养不同时间后,细胞的凋亡率显著增高,细胞活力明显下降。缺氧和无血清时间越长,细胞凋亡越多,活力越低,并且细胞培养基中硫化氢含量和细胞中硫化氢合成酶体系的表达也越低,差异有显著性意义。表明缺氧和无血清培养可以抑制大鼠骨髓间充质干细胞中硫化氢及其合成酶体系的生成和表达。

BACKGROUND：Ischemia microenvironment contributes mostly to the low survival rate of rat bone marrow mesenchymal stem cells after transplantation. Hydrogen sulfide （H2S） can protect various cells and tissue models against apoptosis and injury. OBJECTIVE：To detect the cellapoptosis and viability, content of H2S in supernatant, and the expression of H2S synthetase after different time of hypoxia and serum deprivation cultivation of rat bone marrow mesenchymal stem cells. METHODS：The passage 3 rat bone marrow mesenchymal stem cells were divided into five different cultivation time groups：0-, 3-, 6-, 12-and 24-hour groups. After enough hypoxia and serum deprivation cultivated time, the cellapoptosis was detected by SubG1, the cellviability was determined by cellcounting kit-8, the content of H 2S in supernatant was measured by N,N-dimethyl-p-phenylenediamin and the expression of H2S synthetase by RT-PCR and western blot. RESULTS AND CONCLUSION：Compared to the normal cultivation group, after different hypoxia and serum＆amp;nbsp;deprivation cultivated time, the cellapoptosis increased and cellviability decreased significantly. The longer hypoxia and serum deprivation cultivated time caused the more cellapoptosis and the lower cellviability. The contents of H2S and its synthetase were also suppressed by hypoxia and serum deprivation cultivation. The difference was statistical y significant. These findings suggest that hypoxia and serum deprivation cultivation can inhibit the generation of H 2 expression of its synthetase.