中南大学学报（医学版）
中南大學學報（醫學版）
중남대학학보（의학판）
JOURNAL OF CENTRAL SOUTH UNIVERSITY (MEDICAL SCIENCES)
2010年
1期
52-62
,共11页
方立%陈美芳%余国龙%肖智林%陈晓彬%谢秀梅
方立%陳美芳%餘國龍%肖智林%陳曉彬%謝秀梅
방립%진미방%여국룡%초지림%진효빈%사수매
内皮祖细胞%血管平滑肌细胞%增殖%细胞周期
內皮祖細胞%血管平滑肌細胞%增殖%細胞週期
내피조세포%혈관평활기세포%증식%세포주기
endothelial progenitor cell%vascular smooth muscle cell%proliferation%cell cycle
目的:观察内皮祖细胞(endothelial progenitor cell,EPCs)对血管平滑肌细胞(vascular smooth muscle cell,VSMCs)增殖的影响.方法:采用6%羟乙基淀粉沉降红细胞和密度梯度离心法分离人脐血单个核细胞,EGM-2细胞培养基进行培养,诱导单个核细胞贴壁并向EPCs分化;采用荧光显微镜双染色、流式细胞术鉴定EPCs,间接免疫荧光检测VSMCs收缩表型标志物α-SM-actin和calponin的表达;Transwell培养板建立早期EPCs和大鼠VSMCs共培养模式,以20%胎牛血清刺激VSMCs增殖,分别共培养6,12,24,48,72 h后收集VSMCs,采用BrdU标记法、蛋白定量、流式细胞术分析VSMCs DNA合成能力、细胞总蛋白含量以及细胞周期进程.结果:从脐血单个核细胞成功培养了EPCs.EPCs和大鼠VSMCs共培养12,24,48,72 h后,VSMCs DNA合成能力、细胞总蛋白含量均较对照组明显降低(P＜0.05),其中以48 h最明显;流式细胞术显示,共培养组VSMCs细胞周期中S期细胞所占百分率较对照组显著降低(P＜0.05),而细胞周期中G_1期细胞所占百分率均相应高于对照组(P＜0.05),其中均以48 h最明显.结论:早期EPCs能够抑制VSMCs增殖.
目的:觀察內皮祖細胞(endothelial progenitor cell,EPCs)對血管平滑肌細胞(vascular smooth muscle cell,VSMCs)增殖的影響.方法:採用6%羥乙基澱粉沉降紅細胞和密度梯度離心法分離人臍血單箇覈細胞,EGM-2細胞培養基進行培養,誘導單箇覈細胞貼壁併嚮EPCs分化;採用熒光顯微鏡雙染色、流式細胞術鑒定EPCs,間接免疫熒光檢測VSMCs收縮錶型標誌物α-SM-actin和calponin的錶達;Transwell培養闆建立早期EPCs和大鼠VSMCs共培養模式,以20%胎牛血清刺激VSMCs增殖,分彆共培養6,12,24,48,72 h後收集VSMCs,採用BrdU標記法、蛋白定量、流式細胞術分析VSMCs DNA閤成能力、細胞總蛋白含量以及細胞週期進程.結果:從臍血單箇覈細胞成功培養瞭EPCs.EPCs和大鼠VSMCs共培養12,24,48,72 h後,VSMCs DNA閤成能力、細胞總蛋白含量均較對照組明顯降低(P＜0.05),其中以48 h最明顯;流式細胞術顯示,共培養組VSMCs細胞週期中S期細胞所佔百分率較對照組顯著降低(P＜0.05),而細胞週期中G_1期細胞所佔百分率均相應高于對照組(P＜0.05),其中均以48 h最明顯.結論:早期EPCs能夠抑製VSMCs增殖.
목적:관찰내피조세포(endothelial progenitor cell,EPCs)대혈관평활기세포(vascular smooth muscle cell,VSMCs)증식적영향.방법:채용6%간을기정분침강홍세포화밀도제도리심법분리인제혈단개핵세포,EGM-2세포배양기진행배양,유도단개핵세포첩벽병향EPCs분화;채용형광현미경쌍염색、류식세포술감정EPCs,간접면역형광검측VSMCs수축표형표지물α-SM-actin화calponin적표체;Transwell배양판건립조기EPCs화대서VSMCs공배양모식,이20%태우혈청자격VSMCs증식,분별공배양6,12,24,48,72 h후수집VSMCs,채용BrdU표기법、단백정량、류식세포술분석VSMCs DNA합성능력、세포총단백함량이급세포주기진정.결과:종제혈단개핵세포성공배양료EPCs.EPCs화대서VSMCs공배양12,24,48,72 h후,VSMCs DNA합성능력、세포총단백함량균교대조조명현강저(P＜0.05),기중이48 h최명현;류식세포술현시,공배양조VSMCs세포주기중S기세포소점백분솔교대조조현저강저(P＜0.05),이세포주기중G_1기세포소점백분솔균상응고우대조조(P＜0.05),기중균이48 h최명현.결론:조기EPCs능구억제VSMCs증식.
Objective To explore the effect of endothelial progenitor cells (EPCs) on the proliferation of co-cultured rat vascular smooth muscle cells(VSMCs). Methods Mononuclear cells were isolated from fresh cord blood by 6% hydroxyethyl starch (HES) and density gradient centrifugation. Isolated mononuclear cells were cultured in EGM-2 medium supplemented with 20% fetal bovine serum (FBS), VEGF,bFGF and other growth factors. Biological features of EPCs were observed at different time points, and EPCs were identified by morphology, fluorescence double-staining and flow cytometry. Indirect immunofluorescence was performed to analyze the expression of α-SM-actin, calponin of VSMCs special antigen. Co-culture system of EPCs and VSMCs was established by transwell permeable support. FBS (20%) was used to stimulate the proliferation of VSMCs. In a VSMCs/EPCs co-culture system, the DNA synthesis ability, total protein level and cell cycle of VSMCs were determined by BrdU marking method,protein quantitation and flow cytometry after co-culture for 6, 12, 24,48 and 72 h. Results After co-culture for 12, 24, 48, and 72 h, the DNA synthesis ability and total protein level of VSMCs significantly decreased compared with the control group(P＜0.05). Flow cytometry showed that the percentage of S phase of VSMCs in VSMCs/EPCs co-cultured group significantly decreased and the percentage of G_1 phase increased markedly compared with the control group(P＜0.05). The maximal inhibitory effect was observed at 48 h. Conclusion Early EPCs could inhibit the proliferation of VSMCs.
