小鼠神经细胞中胱硫醚β合成酶催化生成的H_(2)S对脑微血管内皮细胞增殖、迁移的影响及与VEGFR _(2)的关系

Effect of CBS-derived H_(2)S from mouse nerve cells on proliferation and migration of brain microvascular endothelial cells and its relationship with VEGFR_(2)

目的探讨神经细胞中的胱硫醚β合成酶(CBS)催化生成的H_(2)S对小鼠脑血管内皮细胞增殖、迁移的影响及其与VEGFR 2的关系。方法用CCK-8法检测细胞增殖,细胞划痕法和Transwell法分别检测细胞迁移,甲基蓝法检测H_(2)S含量和钙荧光成像法检测内皮细胞的细胞内游离Ca^(2+)浓度,HT22细胞和bEnd.3细胞进行Transwell细胞共培养。结果H_(2)S供体NaHS(1×10^(-8)—1×10^(-3.5) mol·L^(-1))可明显增加HUVEC细胞和bEnd.3细胞的增殖、迁移面积和迁移的细胞数,而VEGFR 2阻断剂SU5416(10μmol·L^(-1))可明显抑制NaHS促进增殖和迁移的作用;在共培养体系中,H_(2)S合酶底物L-cys(100μmol·L^(-1))可明显促进bEnd.3细胞增殖和迁移及细胞内Ca^(2+)荧光强度,并提高体系中的H_(2)S含量,但CBS抑制剂AOAA(1 mmol·L^(-1))和SU5416则明显减弱L-cys的增殖和迁移,促进作用及细胞内Ca^(2+)荧光强度的增高。结论神经细胞中CBS催化生成的H_(2)S可促进脑血管内皮细胞增殖和迁移,其作用可能与激活VEGFR 2,导致内皮细胞的胞内游离Ca^(2+)浓度升高有关。

Aim To study the effect of H_(2)S synthase CBS-derived H_(2)S from mouse nerve cells on the proliferation and migration of the brain vascular endothelial cells and its relationship with VEGFR 2.Methods CCK-8 method was used to detect cell proliferation,cell scratch method and Transwell method were used to detect cell migration,methyl blue method was used to detect H_(2)S content,and calcium fluorescence imaging method was used to detect intracellular free Ca^(2+)concentration.HT22 cells were co-cultured with bEnd.3 cells by using Transwell system.Results The H_(2)S donor NaHS(1×10^(-8)—1×10^(-3.5) mol·L^(-1))significantly increased the proliferation and migration of HUVEC cells and bEnd.3 cells,while the VEGFR 2 blocker SU5416(10μmol·L^(-1))markedly inhibited the NaHS-increased proliferation and migration;in the co-culture system,the substrate of H_(2)S synthase CBS,L-Cys(100μmol·L^(-1)),significantly promoted the proliferation and migration of bEnd.3 cells,and increased intracellular Ca^(2+)fluorescence intensity in bEnd.3 cells and the H_(2)S content in the co-culture.However,CBS inhibitor AOAA(1 mmol·L^(-1))and SU5416 significantly attenuated the effects of L-Cys on the proliferation and migration and intracellular Ca^(2+)fluorescence intensity.Conclusions The CBS-derived H_(2)S from neurons can promote the proliferation and migration of mouse cerebral vascular endothelial cells,which may be related to activation of VEGFR 2 and subsequently increase of intracellular free Ca^(2+)concentration.