目的:探讨持续缺氧阵发加剧的缺氧形式(PI hypoxia)导致人肺动脉内皮细胞(HPAECs)受损的具体机制,以及过氧化物酶体增殖物激活受体γ(PPARγ)激动剂罗格列酮可能的保护作用。方法:将HPAECs置于持续15%供氧并间歇低氧至5%的PI hypoxia环...目的:探讨持续缺氧阵发加剧的缺氧形式(PI hypoxia)导致人肺动脉内皮细胞(HPAECs)受损的具体机制,以及过氧化物酶体增殖物激活受体γ(PPARγ)激动剂罗格列酮可能的保护作用。方法:将HPAECs置于持续15%供氧并间歇低氧至5%的PI hypoxia环境中培养72 h。PPARγ激动剂治疗组的HPAECs培养基中添加了5μM的罗格列酮并同样接受72 h PI hypoxia处理。通过慢病毒感染HPAECs,得到过表达Smad3的HPAECs并接受同样的缺氧处理。在细胞培养结束后使用免疫印迹技术检测细胞裂解中PPARγ的相对含量;高通流式细胞仪测定HPAECs细胞凋亡;CCK-8法测定细胞活力;β-半乳糖苷酶染色衡量细胞老化程度;定量聚合酶链式反应检测端粒相对长度。结果:经历72 h PI hypoxia的HPAECs细胞裂解液中PPARγ表达显著下降。与正常供氧的HPAECs相比,接受72 h PI hypoxia的HPAECs高表达特异性细胞老化因子β-半乳糖苷酶,细胞凋亡显著增加,HPAECs细胞活性明显降低。与PI hypoxia的细胞相比,接受5μM罗格列酮治疗的HPAECs细胞凋亡减轻,细胞活力改善,但是细胞老化程度并没有变化。PI hypoxia环境中培养的HPAECs细胞较正常供氧的HPAECs细胞端粒显著延长,且与接受罗格列酮治疗的细胞端粒长度没有差异。过表达Smad3能显著降低β-半乳糖苷酶,减少细胞凋亡,改善HPAECs细胞活性,但是并没有改变缺氧细胞PPARγ的表达水平。结论:PPARγ激动剂治疗能减轻PI hypoxia诱导的HPAECs细胞凋亡和细胞活力下降,但是并不能改善细胞老化水平。展开更多
The MTEC1 cell line, established in our laboratory, is a normal epithelial cell line derived from thymus medulla of Balb/c mice and these cells constitutively produce multiple cytokines. The selection of thymic microe...The MTEC1 cell line, established in our laboratory, is a normal epithelial cell line derived from thymus medulla of Balb/c mice and these cells constitutively produce multiple cytokines. The selection of thymic microenvironment on developing T cells was investigated in an in vitro system. Un-separated fresh thymocytes from Balb/c mice were cocul-tured with MTECl cells or/and MTEC1-SN,then,the viability, proliferation and phenotypes of cultured thymocytes were assessed. Without any exogenous stimulus, both MTECl cells and MTECl -SN were able to maintain the viability of thymocytes, while only the MTECl cells, not the MTECl -SN, could directly activate thymocytes to exhibit moderate proliferation, indicating that the proliferative signal is delivered through cell surface interactions of MTECl cells and thymocytes. Phenotype analysis on FACS of viable thymocytes after coculture revealed that MTECl cells preferentially activate the subsets of CD4+ CDS', CD4+ CD8+ and CD4- CD8- thymocytes; whereas MTEC1- SN preferentially maintained the viability of CD4+ CD8- and CD4-CD8+ thymocyte subsets.For the Con A-activated thymocytes, both MTEC1 cells and MTEC1-SN provided accessory signal(s) to significantly increase the number of viable cells and to markedly enhance the proliferation of thymocytes with virtually equal potency, phenotyped as CD4+CD8-, CD4-CD8+, and CD4- CD8- subsets. In summary, MTEC1 cells displayedselective support to the different thymocyte subsets , and the selectivity is dependent on the status of thymocytes.展开更多
文摘目的:探讨持续缺氧阵发加剧的缺氧形式(PI hypoxia)导致人肺动脉内皮细胞(HPAECs)受损的具体机制,以及过氧化物酶体增殖物激活受体γ(PPARγ)激动剂罗格列酮可能的保护作用。方法:将HPAECs置于持续15%供氧并间歇低氧至5%的PI hypoxia环境中培养72 h。PPARγ激动剂治疗组的HPAECs培养基中添加了5μM的罗格列酮并同样接受72 h PI hypoxia处理。通过慢病毒感染HPAECs,得到过表达Smad3的HPAECs并接受同样的缺氧处理。在细胞培养结束后使用免疫印迹技术检测细胞裂解中PPARγ的相对含量;高通流式细胞仪测定HPAECs细胞凋亡;CCK-8法测定细胞活力;β-半乳糖苷酶染色衡量细胞老化程度;定量聚合酶链式反应检测端粒相对长度。结果:经历72 h PI hypoxia的HPAECs细胞裂解液中PPARγ表达显著下降。与正常供氧的HPAECs相比,接受72 h PI hypoxia的HPAECs高表达特异性细胞老化因子β-半乳糖苷酶,细胞凋亡显著增加,HPAECs细胞活性明显降低。与PI hypoxia的细胞相比,接受5μM罗格列酮治疗的HPAECs细胞凋亡减轻,细胞活力改善,但是细胞老化程度并没有变化。PI hypoxia环境中培养的HPAECs细胞较正常供氧的HPAECs细胞端粒显著延长,且与接受罗格列酮治疗的细胞端粒长度没有差异。过表达Smad3能显著降低β-半乳糖苷酶,减少细胞凋亡,改善HPAECs细胞活性,但是并没有改变缺氧细胞PPARγ的表达水平。结论:PPARγ激动剂治疗能减轻PI hypoxia诱导的HPAECs细胞凋亡和细胞活力下降,但是并不能改善细胞老化水平。
基金grants from the China medical board, USA,and from the national foundation of natural scirnces,China
文摘The MTEC1 cell line, established in our laboratory, is a normal epithelial cell line derived from thymus medulla of Balb/c mice and these cells constitutively produce multiple cytokines. The selection of thymic microenvironment on developing T cells was investigated in an in vitro system. Un-separated fresh thymocytes from Balb/c mice were cocul-tured with MTECl cells or/and MTEC1-SN,then,the viability, proliferation and phenotypes of cultured thymocytes were assessed. Without any exogenous stimulus, both MTECl cells and MTECl -SN were able to maintain the viability of thymocytes, while only the MTECl cells, not the MTECl -SN, could directly activate thymocytes to exhibit moderate proliferation, indicating that the proliferative signal is delivered through cell surface interactions of MTECl cells and thymocytes. Phenotype analysis on FACS of viable thymocytes after coculture revealed that MTECl cells preferentially activate the subsets of CD4+ CDS', CD4+ CD8+ and CD4- CD8- thymocytes; whereas MTEC1- SN preferentially maintained the viability of CD4+ CD8- and CD4-CD8+ thymocyte subsets.For the Con A-activated thymocytes, both MTEC1 cells and MTEC1-SN provided accessory signal(s) to significantly increase the number of viable cells and to markedly enhance the proliferation of thymocytes with virtually equal potency, phenotyped as CD4+CD8-, CD4-CD8+, and CD4- CD8- subsets. In summary, MTEC1 cells displayedselective support to the different thymocyte subsets , and the selectivity is dependent on the status of thymocytes.
