中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2014年
5期
347-351
,共5页
国桓%杨汀%王望%刘杰%孙然%王军%王辰
國桓%楊汀%王望%劉傑%孫然%王軍%王辰
국환%양정%왕망%류걸%손연%왕군%왕신
肺动脉平滑肌细胞%血小板源性生长因子%增殖%迁移
肺動脈平滑肌細胞%血小闆源性生長因子%增殖%遷移
폐동맥평활기세포%혈소판원성생장인자%증식%천이
Pulmonary artery smooth muscle cells%Platelet-derived growth factor%Proliferation%Migration
目的 分离培养并鉴定大鼠不同分支级别肺动脉平滑肌细胞(PASMC),观察其对血小板源性生长因子(PDGF)反应性的差异,探讨相同刺激因素下,不同级别肺血管病理改变不同的细胞学机制.方法 分离SD大鼠肺动脉并按解剖学分支分为3组:大动脉组,包括肺动脉干及肺内一级主干;中动脉组,指肺内主干二、三级分支;小动脉组,包括肺内动脉四级及以上分支.分离培养不同分支PASMC并用平滑肌细胞抗体进行免疫荧光染色鉴定.给予PDGF刺激,四甲基偶氮唑蓝(MTT)比色法检测细胞活性;Western blot方法测定增殖细胞核抗原表达水平变化以反映细胞增殖能力;伤口愈合实验检测细胞迁移能力的变化.结果 不同分支PASMC在光学显微镜下观察无明显差别;免疫荧光染色结果显示,三种常用的平滑肌细胞标志物即平滑肌肌动蛋白(α-SMA)、钙结合蛋白(S100A4)和平滑肌调宁蛋白(calponin)抗体染色均呈阳性,各组间荧光强度无明显差异.正常培养条件下3组细胞增殖率分别为(137.0±12.1)％、(166.1±23.6)％、(156.8±2.5)％,Western blot检测PCNA表达灰度值分别为(0.41±0.10)、(0.55±0.18)和(0.48±0.28),迁移到空白区域的细胞个数分别为(49±14)、(68±13)和(71±7),组间比较差异无统计学意义(P＞0.05).给予10 μg/LPDGF干预48 h后,中、小动脉组PASMC的增殖率分别为(215.7±17.8)％、(195.2±6.6)％,均高于大动脉组[(167.0±14.4)％,均P＜0.01],干预后3组细胞内PCNA蛋白表达灰度值分别为(0.93±0.11)、(1.21 ±0.14)和(0.62±0.03),迁移到空白区域的细胞个数分别为(194 ±46)、(171 ±16)和(68±21),中、小肺动脉组与大动脉组相比差异均有统计学意义(均P＜0.01).结论 尽管不同分支PASMC通常无明显差异,但其增殖与迁移能力的改变在PDGF刺激下根据血管级别而有所不同.提示在相同病理条件下,不同级别肺动脉的病理改变可能与平滑肌细胞本身的反应性不同有关.
目的 分離培養併鑒定大鼠不同分支級彆肺動脈平滑肌細胞(PASMC),觀察其對血小闆源性生長因子(PDGF)反應性的差異,探討相同刺激因素下,不同級彆肺血管病理改變不同的細胞學機製.方法 分離SD大鼠肺動脈併按解剖學分支分為3組:大動脈組,包括肺動脈榦及肺內一級主榦;中動脈組,指肺內主榦二、三級分支;小動脈組,包括肺內動脈四級及以上分支.分離培養不同分支PASMC併用平滑肌細胞抗體進行免疫熒光染色鑒定.給予PDGF刺激,四甲基偶氮唑藍(MTT)比色法檢測細胞活性;Western blot方法測定增殖細胞覈抗原錶達水平變化以反映細胞增殖能力;傷口愈閤實驗檢測細胞遷移能力的變化.結果 不同分支PASMC在光學顯微鏡下觀察無明顯差彆;免疫熒光染色結果顯示,三種常用的平滑肌細胞標誌物即平滑肌肌動蛋白(α-SMA)、鈣結閤蛋白(S100A4)和平滑肌調寧蛋白(calponin)抗體染色均呈暘性,各組間熒光彊度無明顯差異.正常培養條件下3組細胞增殖率分彆為(137.0±12.1)％、(166.1±23.6)％、(156.8±2.5)％,Western blot檢測PCNA錶達灰度值分彆為(0.41±0.10)、(0.55±0.18)和(0.48±0.28),遷移到空白區域的細胞箇數分彆為(49±14)、(68±13)和(71±7),組間比較差異無統計學意義(P＞0.05).給予10 μg/LPDGF榦預48 h後,中、小動脈組PASMC的增殖率分彆為(215.7±17.8)％、(195.2±6.6)％,均高于大動脈組[(167.0±14.4)％,均P＜0.01],榦預後3組細胞內PCNA蛋白錶達灰度值分彆為(0.93±0.11)、(1.21 ±0.14)和(0.62±0.03),遷移到空白區域的細胞箇數分彆為(194 ±46)、(171 ±16)和(68±21),中、小肺動脈組與大動脈組相比差異均有統計學意義(均P＜0.01).結論 儘管不同分支PASMC通常無明顯差異,但其增殖與遷移能力的改變在PDGF刺激下根據血管級彆而有所不同.提示在相同病理條件下,不同級彆肺動脈的病理改變可能與平滑肌細胞本身的反應性不同有關.
목적 분리배양병감정대서불동분지급별폐동맥평활기세포(PASMC),관찰기대혈소판원성생장인자(PDGF)반응성적차이,탐토상동자격인소하,불동급별폐혈관병리개변불동적세포학궤제.방법 분리SD대서폐동맥병안해부학분지분위3조:대동맥조,포괄폐동맥간급폐내일급주간;중동맥조,지폐내주간이、삼급분지;소동맥조,포괄폐내동맥사급급이상분지.분리배양불동분지PASMC병용평활기세포항체진행면역형광염색감정.급여PDGF자격,사갑기우담서람(MTT)비색법검측세포활성;Western blot방법측정증식세포핵항원표체수평변화이반영세포증식능력;상구유합실험검측세포천이능력적변화.결과 불동분지PASMC재광학현미경하관찰무명현차별;면역형광염색결과현시,삼충상용적평활기세포표지물즉평활기기동단백(α-SMA)、개결합단백(S100A4)화평활기조저단백(calponin)항체염색균정양성,각조간형광강도무명현차이.정상배양조건하3조세포증식솔분별위(137.0±12.1)％、(166.1±23.6)％、(156.8±2.5)％,Western blot검측PCNA표체회도치분별위(0.41±0.10)、(0.55±0.18)화(0.48±0.28),천이도공백구역적세포개수분별위(49±14)、(68±13)화(71±7),조간비교차이무통계학의의(P＞0.05).급여10 μg/LPDGF간예48 h후,중、소동맥조PASMC적증식솔분별위(215.7±17.8)％、(195.2±6.6)％,균고우대동맥조[(167.0±14.4)％,균P＜0.01],간예후3조세포내PCNA단백표체회도치분별위(0.93±0.11)、(1.21 ±0.14)화(0.62±0.03),천이도공백구역적세포개수분별위(194 ±46)、(171 ±16)화(68±21),중、소폐동맥조여대동맥조상비차이균유통계학의의(균P＜0.01).결론 진관불동분지PASMC통상무명현차이,단기증식여천이능력적개변재PDGF자격하근거혈관급별이유소불동.제시재상동병리조건하,불동급별폐동맥적병리개변가능여평활기세포본신적반응성불동유관.
Objective To explore the functional responses of normal rat pulmonary artery smooth muscle cells (PASMCs) from different orders of pulmonary artery to the platelet-derived growth factor (PDGF).Methods The pulmonary artery branches were gently isolated from Sprague-Dawley rats (250-350 g) and eventually cut into three groups according to the vascular grading:the proximal pulmonary arteries,including pulmonary trunk and the first order branches of intrapulmonary arteries; the middle pulmonary arteries,including the second to third orders of intrapulmonary arteries; the distal pulmonary arteries,including the forth to fifth orders of intrapulmonary arteries.PASMCs from the three groups of vessels were isolated and cultured in DMEM.Cells were identified by immunofluorescence-stained smooth muscle cell markers α-SMA,S100A4 and Calponin antibody.Cell proliferation was quantified by using 0.5 g/L methyl thiazolyl tetrazolium (MTT) assay.Western blot was used for detecting the protein expression level of proliferating cell nuclear antigen (PCNA) in PASMCs.Wound healing test was employed for cell migration assay.Results All of the three groups of cells positively expressed α-SMA,S100A4 and Calponin marker and no obvious differences were found in the appearance among the three groups.Under control condition (cultured in DMEM + 2％ FBS),three groups of PASMCs showed similar cell proliferation rate (137.0 ± 12.1) ％,(166.1 ± 23.6) ％,(156.8 ± 2.5) ％.The expression level of PCNA by Western blot was (0.42 ±0.10),(0.55 ±0.18),(0.48 ±0.28),respectively.The numbers of cells migrating to blank areas of the three groups were (49 ± 14),(68 ± 13) and (71 ±7).The above results were not significantly different.While after incubation with PDGF (10 μg/L,48 h),the proliferation rate of middle and distal groups were (215.7 ± 17.8) ％ and (195.2 ± 6.6) ％,higher than that of the proximal group (167.0 ± 14.4) ％,(P ＜ 0.01).In middle and distal groups,the expression level of PCNA were (0.93 ± 0.11) and (1.21 ± 0.14) respectively,which were higher than that in the proximal group (0.62 ± 0.03),(P ＜0.001).The number of cells migrating to blank areas of the middle and distal groups were (194 ± 46),(171 ± 16),and exhibited increased migration ability compared to the proximal group (67 ± 21),(P ＜ 0.01).Conclusion These results suggest that PASMCs from different orders of pulmonary arteries have different responses to PDGF stimulation.This will help to understand the possible mechanism for that under same pathophysiological stimulus,different orders of pulmonary arteries may have different responses.
