摘要
目的 探讨DNase 2α在缺氧诱导大鼠肺动脉平滑肌细胞(rat pulmonary artery smooth muscle cells, RPASMCs)增殖中的作用及机制。方法 培养原代RPASMCs,采用3%O;缺氧处理细胞构建缺氧诱导细胞增殖模型。采用小干扰RNA(small interfering RNA sequence, siRNA)敲低Dnase2a的表达,采用质粒过表达上调细胞中Dnase2a和Hif1a的表达。采用外源性加入线粒体DNA(mitochondrial DNA,mtDNA)诱导细胞增殖,使用脯氨酸羟化酶抑制剂ROXA作为HIF-1α激动剂上调细胞中HIF-1α的表达。采用MTS法检测各组细胞增殖能力,实时荧光定量PCR法检测Dnase2a的mRNA表达,Western blot法检测DNase 2α、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)和细胞周期蛋白(cell cycle protein D1,Cyclin D1)的表达。结果 缺氧可诱导RPASMCs增殖,并上调DNase 2α的表达(P<0.05)。siRNA可显著降低RPASMCs中Dnase2a的表达(P<0.05),并显著增强缺氧诱导的RPASMCs增殖(P<0.05),同时显著上调PCNA和Cyclin D1的蛋白表达(P<0.05)。过表达Dnase2a可显著上调RPASMCs中DNase 2α的表达(P<0.05),并显著抑制缺氧诱导RPASMCs增殖(P<0.05)。外源性加入mtDNA可诱导RPASMCs增殖,而过表达Dnase2a可显著抑制mtDNA诱导的RPASMCs增殖(P<0.05)。过表达Hif1a或使用脯氨酸羟化酶抑制剂ROXA可显著上调HIF-1α表达,并显著上调DNase 2α的蛋白表达(P<0.05)。结论 DNase 2α可抑制缺氧或线粒体DNA诱导的大鼠肺动脉平滑肌细胞增殖,缺氧诱导的DNase 2α表达上调与HIF-1α有关。
Objective To explore the role and mechanism of DNase 2α on hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells(RPASMCs). Methods Primary RPASMCs were cultured with 3%O;to establish a hypoxia-induced cell proliferation model. Small interference RNA(siRNA) was used to decrease the expression of Dnase2 a, and corresponding plasmids were employed to up-regulate the expression of Dnase2 a and Hif1a. Cell proliferation was induced by exogenous mitochondrial DNA(mtDNA). Proline hydroxylase inhibitor ROXA was adopted to enhance the expression of HIF-1α. The proliferation of cells in each group was detected by MTS assay, the mRNA level of Dnase2 a was detected by qPCR, and the protein levels of DNase 2α, proliferating cell nuclear antigen(PCNA) and cell cycle protein D1(Cyclin D1) were detected by Western blotting. Results Hypoxia induced the proliferation of RPASMCs and up-regulated the expression of DNase 2α. siRNA significantly decreased the expression of Dnase2 a(P<0.05) and promoted the hypoxia-induced proliferation of RPASMCs(P<0.05), and the protein expression of PCNA and Cyclin D1 was up-regulated(P<0.05). Overexpression of Dnase2 a up-regulated the expression of Dnase2 a(P<0.05) and inhibited the proliferation of RPASMCs induced by hypoxia(P<0.05). Exogenous mtDNA also promoted the proliferation of RPASMCs, while, overexpression of DNase 2α inhibited the above proliferation(P<0.05). Overexpression of Hif1a or proline hydroxylase inhibitor ROXA could significantly up-regulate the protein expression of HIF-1α and DNase 2α(P<0.05). Conclusion DNase 2α inhibits the proliferation of RPASMCs induced by hypoxia or mtDNA, whose expression induced by hypoxia is associated with HIF-1α.
作者
耿建慧
陈建
张二龙
阳一栋
陈德伟
吴刚
王羽
赵文琦
高钰琪
GENG Jianhui;CHEN Jian;ZHANG Erlong;YANG Yidong;CHEN Dewei;WU Gang;WANG Yu;ZHAO Wenqi;GAO Yuqi(Department of Medicine and Equipment for High Altitude Region,Faculty of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China;Key Laboratory of Extreme Environmental Medicine of Ministry of Education,Faculty of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China;Department of High Altitude Physiology,Faculty of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China;Department of Pathophysiology,Faculty of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2022年第8期731-739,共9页
Journal of Army Medical University
基金
国家自然科学基金重点项目(81830062)。