5153997033內(nèi)皮祖細(xì)胞的研究進(jìn)展

1、department of pathophysiology hongmei tan nov, 2008 definition they are circulating, bone marrow-derived cells that are functionally and phenotypically distinct from mature endothelial cells they can differentiate into endothelial cells in vitro, as assessed by expression profiles and functional cha

2、racteristics they can contribute to in vivo vasculogenesis and / or vascular homeostasis 內(nèi)皮祖細(xì)胞是能直接分化為血管內(nèi)皮細(xì)胞的前體細(xì)胞內(nèi)皮祖細(xì)胞是能直接分化為血管內(nèi)皮細(xì)胞的前體細(xì)胞 embryonic epc 胚外中胚層卵黃囊血島胚外中胚層卵黃囊血島 與造血干細(xì)胞存在共同的前體與造血干細(xì)胞存在共同的前體 adult epc bone marrow (3% bm-mnc) peripheral blood (0.2% mnc) origin and differentiation of endothelia

3、l progenitor cells hematopoietic stem cells cd133+ cd34+ cd45 cd14+ cd45+ cd45+cd14+ cd45+ cd14+ cd133+ kdr+ cd34+ cd14+ cd133+ endothelial markers+ endothelial markers+ epcs epcs myeloid precursormonocytesmacrophage epcs myeloid subtype mature ec ? ? ? ? cd14low cd34low cd133- endothelial markers+

4、mesenchymal stem cells c-kit-, cd34- tissue-resident stem cells c-kit+ epcs development molecular mechanisms transcription factor scl / tal 是參與原血干細(xì)胞分化的基本的轉(zhuǎn)錄因子。是參與原血干細(xì)胞分化的基本的轉(zhuǎn)錄因子。 a basic helix-loop-helix transcription factor 小鼠小鼠scl基因無效突變的純合子引起死亡基因無效突變的純合子引起死亡 表現(xiàn)為卵黃囊毛細(xì)血管內(nèi)皮形成障礙、表現(xiàn)為卵黃囊毛細(xì)血管內(nèi)皮形成障礙、 早期造血

5、不能發(fā)育早期造血不能發(fā)育 vegfr and vegf receptor vegfr-1 (flt-1) vegfr-2 (flk-1/kdr) vegfr-3 (flt-4) ligand vegf、 vegf-b, c, d, e epcs development vegfr and vegf 研究顯示研究顯示vegfr2缺陷鼠在缺陷鼠在 e8.5e9.5時因缺乏時因缺乏 內(nèi)皮和造血細(xì)胞而死亡內(nèi)皮和造血細(xì)胞而死亡。 flk1-/-的胚胎干細(xì)胞離體不能分化為的胚胎干細(xì)胞離體不能分化為epc。 胚胎干細(xì)胞向內(nèi)皮細(xì)胞分化對胚胎干細(xì)胞向內(nèi)皮細(xì)胞分化對vegf呈劑量依賴性，呈劑量依賴性， vegf濃

6、度增加可增加原血干細(xì)胞向濃度增加可增加原血干細(xì)胞向epc轉(zhuǎn)化，轉(zhuǎn)化， 而而 相應(yīng)減少造血干細(xì)胞的生成。相應(yīng)減少造血干細(xì)胞的生成。 vegfr1純合突變鼠也因內(nèi)皮細(xì)胞不能形成管樣結(jié)純合突變鼠也因內(nèi)皮細(xì)胞不能形成管樣結(jié) 構(gòu)而使胚胎在構(gòu)而使胚胎在e9.5e10死亡。死亡。 vegf是唯一已知的在胚胎雜合子狀態(tài)致死的常染色體基因。是唯一已知的在胚胎雜合子狀態(tài)致死的常染色體基因。 tie receptor and ligand：受體酪氨酸激酶家族：受體酪氨酸激酶家族 receptor tie-1 (tie) tie-2 (tek) tie-2 ligand：angiopoietin (ang，血管生成素

7、血管生成素 ) ang-1：血管內(nèi)皮化：血管內(nèi)皮化 ang-2：血管周圍細(xì)胞和內(nèi)皮細(xì)胞分離：血管周圍細(xì)胞和內(nèi)皮細(xì)胞分離 epcs development tie receptor and ligand：受體酪氨酸激酶家族：受體酪氨酸激酶家族 tie-2缺陷的胚胎不能建立血管結(jié)構(gòu)的完整性缺陷的胚胎不能建立血管結(jié)構(gòu)的完整性 ang-1缺陷鼠也表現(xiàn)為血管生成缺陷缺陷鼠也表現(xiàn)為血管生成缺陷 epcs development ephs family erythropoietin producing hepatocyte receptor (eph) ligand: ephrins (eph a、b) e

8、ph b可調(diào)節(jié)可調(diào)節(jié) ang-1 和和 tie-2的表達(dá)的表達(dá) epcs development 促紅細(xì)胞生成素肝細(xì)胞受體及其配體，促紅細(xì)胞生成素肝細(xì)胞受體及其配體， 是酪氨酸激酶家族中的最大成員。是酪氨酸激酶家族中的最大成員。 基因缺失及體外血管形成實驗表明：基因缺失及體外血管形成實驗表明： ephbephb和和ephrinbephrinb在胚胎血管分化及成人在胚胎血管分化及成人 病理性血管形成中發(fā)揮重要作用。病理性血管形成中發(fā)揮重要作用。 cd34+, flk-1+ cd34+, flk-1+, ac133+ cd34+ cd34+, flk-1+, ac133- stem cell ep

9、chsc epc, ec, hsc circulating epc, ec epc ec cd34-selected culture-dish non-adherent putative epc (cdnac) change to a more mature endothelial phenotype during differentiation while the progenitor phenotype remains stable and the monocytic phenotype decreases. cd34-selected putative epc from culture-

10、dish adherent cells (cdac) express lower levels of endothelial and progenitor markers than cdnac. vegfr-2/flk-1+ cd31+ / tie-2+ ve-cadeherin+ / tie-1+ ac133+ ac133- cd34+/ vegfr-2+ cells can behave as epcs. cd133+/ cd34+/ vegfr-2+ cells represent a more primitive epc. putative epcs take up acetylate

11、d low density lipoprotein (ac-ldl) and bind the endothelial specific lectin uea-1. mobilization of epcs determined by local microenvironment (stem cell niche) fibroblastsosteoblastsendothelial cells stromal cells mobilization of epcs stromal cells stem cells mobilizing cytokines vegf, sdf-1, g-csf,

12、epo statins, estrogen, exercise hamper transendothelial migration elastase, cathepsin, mmps blood circulating chemotaxis, migration and invasion sdf-1 vegf adhesion integrin selectin differentiation vegf sdf-1: stromal cell-derived factor-1 epcs were originally thought to be present only during embr

13、yonic development. evidence accumulating over several years suggests that they can persist in adult life. this has generated interest in the use of epcs for neovascularization of ischemic or injured tissue and for the clinical assessment of risk factors for various diseases. epc tumor angiogenesis i

14、schemic vasculogenesis/ wound healing vascular homeostasis embryo vascular network expand revascularization v angiogenesis v vasculogenesis 血管血管發(fā)生發(fā)生/ /生成生成 血管血管生成生成/ /新生新生/ /增生增生 不但是正常生理變化中不但是正常生理變化中( (如生長、傷口愈合如生長、傷口愈合) )所必所必 需有的過程，和腫瘤的發(fā)展也有密切的關(guān)系。需有的過程，和腫瘤的發(fā)展也有密切的關(guān)系。 v angiogenesis a regulated proces

15、s involving the proliferation,and migration of endothelial cells from adjacent pre-existing blood vessels. 血管生成血管生成 指由已經(jīng)存在的血管指由已經(jīng)存在的血管成熟內(nèi)皮細(xì)胞成熟內(nèi)皮細(xì)胞通過局部芽生或通過局部芽生或 套疊的形式生成新的毛細(xì)血管，是血管套疊的形式生成新的毛細(xì)血管，是血管從少到多從少到多的的 過程，是血管新生的過程，是血管新生的經(jīng)典理論經(jīng)典理論。 v vasculogenesis a regulated process following differentiation of

16、endothelial progenitor cells from mesodermal precursors / endothelial progenitor cells (epcs) 血管發(fā)生血管發(fā)生 這種方式最早發(fā)生在胚胎期的血管生成，直接由這種方式最早發(fā)生在胚胎期的血管生成，直接由 epcs或更早來源的血管母細(xì)胞或更早來源的血管母細(xì)胞（angioblast）長成，長成， 是血管是血管從無到有從無到有的過程。最近證據(jù)表明，的過程。最近證據(jù)表明，epcs參參 與的血管發(fā)生亦參與了出生后機體局部缺血組織與的血管發(fā)生亦參與了出生后機體局部缺血組織 的修復(fù)。的修復(fù)。 vasculogenesis

17、和和angiogenesis是血管發(fā)育過是血管發(fā)育過 程中的兩個階段，但在中文翻譯時出現(xiàn)了意思程中的兩個階段，但在中文翻譯時出現(xiàn)了意思 重疊和相近、不便于區(qū)別的問題。重疊和相近、不便于區(qū)別的問題。 傳傳 統(tǒng)統(tǒng) angiogenesis主要是主要是ec遷移增殖參與的，遷移增殖參與的，vasculogenesis 在胚胎期和生后均存在，主要依靠在胚胎期和生后均存在，主要依靠epc分化增殖參與。分化增殖參與。 現(xiàn)現(xiàn) 在在 1997年，年， angiogenesis概念被打破，概念被打破，asahara等證實除等證實除 有有ec遷移增殖參與，還有遷移增殖參與，還有epc的參與。的參與。 decreas

18、e of endothelial progenitor cells in the blood is a risk factor for vascular disease. depletion or senescence of endothelial progenitor cells may contribute to blood vessel disease. epcs and endothelial regeneration in the past, endothelial regeneration has been attributed to the migration and proli

19、feration of neighboring ec in 1997, asahara first demonstrated epc contributed to endothelial regeneration epc and neovascularization transplantation of genetically modified cell (rosa-26, gfp, lac z) basal incorporation rate: extremely low in ischemic tissue, the incorporation rate: up to 90% high

20、rate was predominantly detected in models of tumor angiogenesis therapeutic application of cells by infusion ( iv ) of purified bm mononuclear cells or expanded epcs led to a higher incorporation rate ( 7 20%) as compared with endogenously mobilized bm - engrafted cells (2%) mechanisms by which epcs

21、 improve neovascularization incorporation of epcs release of proangiogenesis factors in paracrine manner vegf hgf igf-1 vegf: vascular endothelial growth factor hgf: hepatocyte growth factor igf: insulin-like growth factor i bone marrow histopaque 1083 methods mononuclear cells (mncs) were isolated

22、from mouse b o n e m a r r o w w i t h histopaque 1083 by density gradient centrifugation. bone marrow histopaque 1083 resuspend in egm2 plate 5x106 cells/well on fibronectin-coated 6-well dish methods mncs were cultured in egm2 with the supplement of vegf, b-fgf, igf-1 on fibronectin-coated 6-well

23、plate for 2 days to remove mature endothelial cells and monocytes. bone marrow histopaque 1083 resuspend in egm2 plate 5x106 cells/well on fibronectin-coated 6-well dish 2 days collect no-adherent cells plate 1x106 cells/well on fibronectin-coated 24-well dish methods the non-adherent cells (which contain epcs) are then harvested and plated on fibronectin- coated 24-well plates for 3 days. bone marrow histopaque 1083 resuspend in egm2 plate 5x106 cells/well on fibronectin-coated 6-well dish 2 days collect no-adherent cells plate 1x106 ce