HAND2对脑微血管发育阶段周细胞增殖和迁移的影响
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摘要
目的探讨人脑原代微血管周细胞(HBVPs)和人肾脏原代微血管周细胞(HRVPs)差异表达基因及其对脑微血管周细胞迁移和增殖功能的影响。方法利用RNA-seq检测对HBVPs和HRVPs进行转录组测序,qPCR及Western blot检测HAND2 mRNA及蛋白水平,构建HAND2基因的sh-RNA下调脑微血管周细胞HAND2表达,分别利用CCK-8检测HBVPs增殖活性和Transwell小室迁移实验检测迁移能力。结果 RNA-seq检测结果显示胚胎期HBVPs相对HRVPs有1 906个高表达基因,进一步的聚类分析发现这些基因可显著地聚类为4类功能富集的亚群;与血管发育密切相关的第Ⅱ群中HAND2在HBVPs中显著高表达(lg2~(FoldChange)=11.93,P<0.05)。此外,qPCR和/或Western blot检测发现在小鼠胚胎第10.5天HAND2表达量较高,在第14.5天时最高,出生后几乎不表达(P<0.05),并且HAND2主要表达在HBVPs(P<0.05)。当sh-RNA下调HBVPs的HAND2表达后,发现其在24 h时迁移能力增强(P<0.05),48 h和72 h时增殖活性显著增加(P<0.05)。结论 HAND2对胚胎脑微血管发育和成熟期周细胞的增殖和迁移能力具有重要的调控作用。
Compared with renal microvascular pericytes, cerebral microvascular pericytes play a unique role in promoting the development and maturation of microvascular barrier structure, which play an important role in inhibiting the damage of peripheral blood immune inflammatory cells and inflammatory molecules on neurons and maintaining the stability of central nervous system. Thus, this study was designed to investigate the gene differentially expressed in human brain primary microvascular pericytes(HBVPs) and human renal microvascular pericytes(HRVPs) and its effect on the cerebral microvascular pericytes migration and proliferation. Genes differentially expressed in HBVPs and HRVPs were measured by RNA-seq; the expression of HAND2 mRNA and protein were measured by qPCR and Western blot. Down-regulation of HAND2 in cerebral microvascular pericytes was conducted by HAND2 sh-RNA. RNA-seq data indicated that there were 1 906 genes with high expression in embryonic HBVPs compared with HRVPs,and cluster analysis indicated that there were four gene functional enrichment subgroups. HAND2 in the second subgroup(closely related to vascular development)was significantly higher expressed in HBVPs compared with HRVPs(lg2~(FoldChange)= 11.93, P<0.05). In addition, HAND2 in mice started to increase at E10.5, peaked at E14.5, hardly expressed after birth(P<0.05), and mainly expressed in HBVPs(P<0.05). After down-regulation of HAND2 expression, the proliferation activity of HBVPs increased at 48 h and 72 h(P<0.05), and the migration ability increased at 24 h(P<0.05). Taken together, HAND2 regulates pericytes proliferation and migration in the stages of embryonic cerebral microvascular development and maturation.
Compared with renal microvascular pericytes, cerebral microvascular pericytes play a unique role in promoting the development and maturation of microvascular barrier structure, which play an important role in inhibiting the damage of peripheral blood immune inflammatory cells and inflammatory molecules on neurons and maintaining the stability of central nervous system. Thus, this study was designed to investigate the gene differentially expressed in human brain primary microvascular pericytes(HBVPs) and human renal microvascular pericytes(HRVPs) and its effect on the cerebral microvascular pericytes migration and proliferation. Genes differentially expressed in HBVPs and HRVPs were measured by RNA-seq; the expression of HAND2 mRNA and protein were measured by qPCR and Western blot. Down-regulation of HAND2 in cerebral microvascular pericytes was conducted by HAND2 sh-RNA. RNA-seq data indicated that there were 1 906 genes with high expression in embryonic HBVPs compared with HRVPs,and cluster analysis indicated that there were four gene functional enrichment subgroups. HAND2 in the second subgroup(closely related to vascular development)was significantly higher expressed in HBVPs compared with HRVPs(lg2~(FoldChange)= 11.93, P<0.05). In addition, HAND2 in mice started to increase at E10.5, peaked at E14.5, hardly expressed after birth(P<0.05), and mainly expressed in HBVPs(P<0.05). After down-regulation of HAND2 expression, the proliferation activity of HBVPs increased at 48 h and 72 h(P<0.05), and the migration ability increased at 24 h(P<0.05). Taken together, HAND2 regulates pericytes proliferation and migration in the stages of embryonic cerebral microvascular development and maturation.
引文
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[14]Zhao Z,Nelson AR,Betsholtz C,et al.Establishment and dysfunction of the blood-brain barrier[J].Cell,2015,163(5):1064-1078.
[15]Hall CN,Reynell C,Gesslein B,et al.Capillary pericytes regulate cerebral blood flow in health and disease[J].Nature,2014,508(7494):55-60.
[16]Yemisci M,Gursoy Ozdemir Y,Vural A,et al.Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery[J].Nat Med,2009,15(9):1031-1037.
[17]Anderson KM,Anderson DM,McAnally JR,et al.Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development[J].Nature,2016,539(7629):433-436.
[18]Shen H,Bocksteins E,Kondrychyn I,et al.Functional antagonism of voltage-gated K+channel alpha-subunits in the developing brain ventricular system[J].Development,2016,143(22):4249-4260.
[19]Niday Z,Tzingounis AV.Potassium channel gain of function in epilepsy:An unresolved paradox[J].Neuroscientist,2018,24(4):368-380.
[20]Segarra M,Aburto MR,Cop F,et al.Endothelial Dab1signaling orchestrates neuro-glia-vessel communication in the central nervous system[J].Science,2018,361(6404):eaaO2861.
[21]Osterwalder M,Speziale D,Shoukry M,et al.HAND2targets define a network of transcriptional regulators that compartmentalize the early limb bud mesenchyme[J].Dev Cell,2014,31(3):345-357.
[22]Alvarez JI,Dodelet Devillers A,Kebir H,et al.The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence[J].Science,2011,334(6063):1727-1731.
[23]Ben Zvi A,Lacoste B,Kur E,et al.Mfsd2a is critical for the formation and function of the blood-brain barrier[J].Nature,2014,509(7501):507-511.
[2]Sweeney MD,Ayyadurai S,Zlokovic BV.Pericytes of the neurovascular unit:key functions and signaling pathways[J].Nat Neurosci,2016,19(6):771-783.
[3]王高宁,夏志伦,时伟,等.马索罗酚对实验性自身免疫性脑脊髓炎小鼠血脑屏障的保护作用及机制研究[J].免疫学杂志,2017,33(2):118-123.
[4]Nation DA,Sweeney MD,Montagne A,et al.Blood-brain barrier breakdown is an early biomarker of human cognitive dysfunction[J].Nat Med,2019,25(2):270-276.
[5]Hill J,Rom S,Ramirez SH,et al.Emerging roles of pericytes in the regulation of the neurovascular unit in health and disease[J].J Neuroimmune Pharmacol,2014,9(5):591-605.
[6]Dalkara T,Alarcon Martinez L.Cerebral microvascular pericytes and neurogliovascular signaling in health and disease[J].Brain Res,2015,1623:3-17.
[7]Yamagishi H,Olson EN,Srivastava D.The basic helixloop-helix transcription factor,dHAND,is required for vascular development[J].J Clin Invest,2000,105(3):261-270.
[8]Daneman R,Zhou L,Kebede AA,et al.Pericytes are required for blood-brain barrier integrity during embryogenesis[J].Nature,2010,468(7323):562-566.
[9]Armulik A,GenovéG,M?e M,et al.Pericytes regulate the blood-brain barrier[J].Nature,2010,468(7323):557-561.
[10]Obermeier B,Daneman R,Ransohoff RM.Development,maintenance and disruption of the blood-brain barrier[J].Nat Med,2013,19(12):1584-1596.
[11]田春梅,张林,范万峰,等.儿童病毒性脑炎血清免疫学研究[J].免疫学杂志,2013,29(10):880-882.
[12]Holm A,Heumann T,Augustin HG.Microvascular mural cell organotypic heterogeneity and functional plasticity[J].Trends Cell Biol,2018,28(4):302-316.
[13]Yamazaki T,Mukouyama YS.Tissue specific origin,development,and pathological perspectives of pericytes[J].Front Cardiovasc Med,2018,5:78.
[14]Zhao Z,Nelson AR,Betsholtz C,et al.Establishment and dysfunction of the blood-brain barrier[J].Cell,2015,163(5):1064-1078.
[15]Hall CN,Reynell C,Gesslein B,et al.Capillary pericytes regulate cerebral blood flow in health and disease[J].Nature,2014,508(7494):55-60.
[16]Yemisci M,Gursoy Ozdemir Y,Vural A,et al.Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery[J].Nat Med,2009,15(9):1031-1037.
[17]Anderson KM,Anderson DM,McAnally JR,et al.Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development[J].Nature,2016,539(7629):433-436.
[18]Shen H,Bocksteins E,Kondrychyn I,et al.Functional antagonism of voltage-gated K+channel alpha-subunits in the developing brain ventricular system[J].Development,2016,143(22):4249-4260.
[19]Niday Z,Tzingounis AV.Potassium channel gain of function in epilepsy:An unresolved paradox[J].Neuroscientist,2018,24(4):368-380.
[20]Segarra M,Aburto MR,Cop F,et al.Endothelial Dab1signaling orchestrates neuro-glia-vessel communication in the central nervous system[J].Science,2018,361(6404):eaaO2861.
[21]Osterwalder M,Speziale D,Shoukry M,et al.HAND2targets define a network of transcriptional regulators that compartmentalize the early limb bud mesenchyme[J].Dev Cell,2014,31(3):345-357.
[22]Alvarez JI,Dodelet Devillers A,Kebir H,et al.The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence[J].Science,2011,334(6063):1727-1731.
[23]Ben Zvi A,Lacoste B,Kur E,et al.Mfsd2a is critical for the formation and function of the blood-brain barrier[J].Nature,2014,509(7501):507-511.