中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
CHINESE JOURNAL OF PATHOPHYSIOLOGY
2015年
5期
769-776
,共8页
朱光旭%周芳%阮光萍%宋明宝%杨建勇%黄岚%康华莉%潘兴华
硃光旭%週芳%阮光萍%宋明寶%楊建勇%黃嵐%康華莉%潘興華
주광욱%주방%원광평%송명보%양건용%황람%강화리%반흥화
内皮祖细胞%平滑肌细胞%细胞表型%细胞增殖%细胞迁移
內皮祖細胞%平滑肌細胞%細胞錶型%細胞增殖%細胞遷移
내피조세포%평활기세포%세포표형%세포증식%세포천이
Endothelial progenitor cells%Smooth muscle cells%Cell phenotype%Cell proliferation%Cell migra-tion
目的：探讨老龄和年轻个体来源融合生长状态内皮祖细胞（EPCs）对血管平滑肌细胞（SMCs）表型转换以及增殖和迁移的调节作用。方法：脱臼处死1～2月龄、19～26月龄SD大鼠，应用含15％ FBS的DMEM／F12培养基（含内皮细胞生长添加剂100 mg／L、肝素100 mg／L、青霉素、链霉素各1×105 U／L）培养EPCs，取1～2月龄大鼠腹主动脉，组织块法培养血管SMCs，应用DiI-Ac-LDL与FITC-UEA-1荧光双染以及α-SM-actin免疫荧光分别对EPCs和SMCs进行鉴定。建立细胞共培养体系，上室为融合生长状态的EPCs，下室为SMCs，实验分4组：（1）第3代SMCs（P3）组；（2）第4代SMCs（P4）组；（3）第4代SMCs与年轻大鼠来源EPCs共培养（P4YE）组；（4）第4代SMCs与老龄大鼠来源EPCs共培养（ P4AE）组。 Western blotting检测α-SM-actin和osteopontin蛋白的表达；[3 H ]-TdR掺入法检测SMCs增殖；细胞划痕实验检测SMCs的迁移能力。结果：与P3组相比， P4组的SMCsα-SM-actin表达显著下调，而osteopontin表达显著增强；P4YE组SMCs的α-SM-actin及osteopontin表达与P3组比较未见有显著差别；与P4组相比，年轻和老龄大鼠来源的EPCs均显著促进第4代SMCs的α-SM-actin和下调osteopontin的表达，抑制第4代SMCs的增殖和迁移；与老龄大鼠来源的EPCs相比，年轻大鼠来源的EPCs更能够显著延迟SMCs表型由收缩型向合成型转换，抑制SMCs增殖和迁移。结论：共培养融合生长状态的EPCs使血管SMCs表型转换延迟、抑制SMCs增殖和迁移，年轻大鼠来源的EPCs较老龄大鼠来源的EPC更显著延迟血管SMCs表型由收缩型向合成型转换，并具有更强的抑制血管SMCs增殖和迁移的能力。
目的：探討老齡和年輕箇體來源融閤生長狀態內皮祖細胞（EPCs）對血管平滑肌細胞（SMCs）錶型轉換以及增殖和遷移的調節作用。方法：脫臼處死1～2月齡、19～26月齡SD大鼠，應用含15％ FBS的DMEM／F12培養基（含內皮細胞生長添加劑100 mg／L、肝素100 mg／L、青黴素、鏈黴素各1×105 U／L）培養EPCs，取1～2月齡大鼠腹主動脈，組織塊法培養血管SMCs，應用DiI-Ac-LDL與FITC-UEA-1熒光雙染以及α-SM-actin免疫熒光分彆對EPCs和SMCs進行鑒定。建立細胞共培養體繫，上室為融閤生長狀態的EPCs，下室為SMCs，實驗分4組：（1）第3代SMCs（P3）組；（2）第4代SMCs（P4）組；（3）第4代SMCs與年輕大鼠來源EPCs共培養（P4YE）組；（4）第4代SMCs與老齡大鼠來源EPCs共培養（ P4AE）組。 Western blotting檢測α-SM-actin和osteopontin蛋白的錶達；[3 H ]-TdR摻入法檢測SMCs增殖；細胞劃痕實驗檢測SMCs的遷移能力。結果：與P3組相比， P4組的SMCsα-SM-actin錶達顯著下調，而osteopontin錶達顯著增彊；P4YE組SMCs的α-SM-actin及osteopontin錶達與P3組比較未見有顯著差彆；與P4組相比，年輕和老齡大鼠來源的EPCs均顯著促進第4代SMCs的α-SM-actin和下調osteopontin的錶達，抑製第4代SMCs的增殖和遷移；與老齡大鼠來源的EPCs相比，年輕大鼠來源的EPCs更能夠顯著延遲SMCs錶型由收縮型嚮閤成型轉換，抑製SMCs增殖和遷移。結論：共培養融閤生長狀態的EPCs使血管SMCs錶型轉換延遲、抑製SMCs增殖和遷移，年輕大鼠來源的EPCs較老齡大鼠來源的EPC更顯著延遲血管SMCs錶型由收縮型嚮閤成型轉換，併具有更彊的抑製血管SMCs增殖和遷移的能力。
목적：탐토노령화년경개체래원융합생장상태내피조세포（EPCs）대혈관평활기세포（SMCs）표형전환이급증식화천이적조절작용。방법：탈구처사1～2월령、19～26월령SD대서，응용함15％ FBS적DMEM／F12배양기（함내피세포생장첨가제100 mg／L、간소100 mg／L、청매소、련매소각1×105 U／L）배양EPCs，취1～2월령대서복주동맥，조직괴법배양혈관SMCs，응용DiI-Ac-LDL여FITC-UEA-1형광쌍염이급α-SM-actin면역형광분별대EPCs화SMCs진행감정。건립세포공배양체계，상실위융합생장상태적EPCs，하실위SMCs，실험분4조：（1）제3대SMCs（P3）조；（2）제4대SMCs（P4）조；（3）제4대SMCs여년경대서래원EPCs공배양（P4YE）조；（4）제4대SMCs여노령대서래원EPCs공배양（ P4AE）조。 Western blotting검측α-SM-actin화osteopontin단백적표체；[3 H ]-TdR참입법검측SMCs증식；세포화흔실험검측SMCs적천이능력。결과：여P3조상비， P4조적SMCsα-SM-actin표체현저하조，이osteopontin표체현저증강；P4YE조SMCs적α-SM-actin급osteopontin표체여P3조비교미견유현저차별；여P4조상비，년경화노령대서래원적EPCs균현저촉진제4대SMCs적α-SM-actin화하조osteopontin적표체，억제제4대SMCs적증식화천이；여노령대서래원적EPCs상비，년경대서래원적EPCs경능구현저연지SMCs표형유수축형향합성형전환，억제SMCs증식화천이。결론：공배양융합생장상태적EPCs사혈관SMCs표형전환연지、억제SMCs증식화천이，년경대서래원적EPCs교노령대서래원적EPC경현저연지혈관SMCs표형유수축형향합성형전환，병구유경강적억제혈관SMCs증식화천이적능력。
AIM:To explore the effects of confluent endothelial progenitor cells (EPCs) derived from young and aged rats on the phenotype conversion, proliferation and migration of vascular smooth muscle cells ( SMCs) .METH-ODS:Mononuclear cells were obtained from the bone marrow of young (1~2 month old) and aged (19 to 26 month old) Sprague-Dawley rats and cultured with medium DMEM/F12 ( containing 15% fetal bovine serum, endothelial cell growth supplements (ECGs) 100 g/L, 1 ×105 units/L of penicillin and streptomycin, respectively).EPCs were characterized as double positive for DiI-Ac-LDL uptake and lectin binding.Abdominal aorta was obtained from 1 to 2 month old Sprague-Dawley rats.Vascular SMCs were cultured by tissue explant method and identified byα-SM-actin immunofluorescence.In transwell co-culture system, the confluent EPCs located in the upper chamber and SMCs were seeded on the lower cham-ber.The experiments were divided into passage 3 SMCs group (P3), passage 4 SMCs group (P4), passage 4 SMCs co-culture with EPCs derived from young rats group (P4YE) and passage 4 SMCs co-culture with EPCs derived from aged rats group (P4AE).The protein expression ofα-SM-actin and osteopontin was detected by Western blotting.[3H]-TdR incor-poration assay was used to determine the proliferation.SMC migration was analyzed by scratch wound healing assay.RE-SULTS:Compared with P3 group,α-SM-actin expression in P4 group significantly decreased and osteopontin protein ex-pression obviously increased, whereas no significant change was found in P4YE group.Compared with P4 group, confluent EPCs derived from young and aged rats both markedly increased α-SM-actin and decreased osteopontin expression in P4 SMCs.Compared with aged rat-derived EPCs, young rat-derived EPCs were more effectively to induce a delayed SMC phe-notype transition (from contractile phenotype to a synthetic phenotype), and to inhibit SMC proliferation and migration. CONCLUSION:Co-culture of confluent EPC induces a delayed vascular SMC phenotype transition and inhibits SMCs pro-liferation and migration.Young rat derived EPCs are more effective to induce a delayed vascular SMC phenotype transition and has stronger inhibitory effects on SMCs proliferation and migration compared with that derived from aged rats.
