目的 研究移植内皮祖细胞(endothelial progenitor cell,EPC)对新生大鼠高氧肺损伤的影响及其机制. 方法 从4周龄Sprague-Dawyley大鼠骨髓中培养获取EPC并鉴定.另取新生Sprague-Dawley仔鼠60只,生后室温下适应性饲养24 h后,随机分为空气组、高氧组、移植组和Nω-硝基-L-精氨酸甲酯(Nω-nitro-L-argininemethylester,L-NAME)干预组(简称干预组),每组15只.空气和高氧组分别在空气和85％高氧中饲养28d.移植组在85％高氧中暴露28d,其中在第21天尾静脉注射EPC 1×10 5个.干预组在移植组的基础上,自第21天开始连续腹腔注射L-NAME至第28天,每日剂量为20 mg/kg.第28天处死所有仔鼠,留取血标本,采用流式细胞技术检测CD34+细胞,酶联免疫吸附方法检测血清血管内皮细胞生长因子(vescular endothelial growth factor,VEGF).同时留取肺组织标本,观察辐射状肺泡计数和肺微血管计数、免疫荧光方法观察移植EPC在肺内的定植情况,实时荧光定量聚合酶链反应和蛋白质印迹技术检测肺组织中VEGF、VEGF受体(vescular endothelial growth factor receptor,VEGFR)2和内皮源性一氧化氮合酶(endothelial nitric oxide synthase,eNOS)的表达,并用硝酸还原酶法检测肺组织中一氧化氮的表达.采用单因素方差分析和Bonferroni方法进行统计学分析. 结果 (1)培养所得细胞具有典型的EPC形态改变;结合异硫氢基荧光素标记荆豆凝集素-1并摄取Dil荧光标记的乙酰化低密度脂蛋白的双阳性细胞约占总细胞数的85％;培养所得细胞中CD34+细胞含量为68.2％～72.4％.(2)空气组、高氧组、移植组和干预组仔鼠外周血CD34+细胞数量分别为(1.91±0.34)％、(1.06±0.10)％、(1.47±0.06)％和(0.77±0.11)％(F=32.710,P=0.000),高氧组低于空气组,移植组高于高氧组,干预组又低于移植组(P值均＜ 0.05).4组仔鼠血清VEGF水平分别为(7.90±2.72)、(6.38±0.72)、(14.00±1.66)和(11.70±1.91) pg/ml(F=22.809,P=0.000),移植组高于高氧组(P＜0.05).(3)移植的EPC在肺部主要定植于内皮下和肺泡间质.干预组定植的EPC明显少于移植组[分别为(16.95±0.50)个/视野与(10.70±0.47)个/视野,t=17.820,P=0.000].(4)4组仔鼠辐射状肺泡计数和肺组织微血管密度差异均有统计学意义(F值分别为859.580和211.150,P值均=0.000),其中高氧组RAC和微血管密度均小于空气组(分别为7.98±0.23与13.12±0.20,3.98±0.42与9.50±0.22,P值均＜0.05),移植组微血管密度为5.40±0.41,大于高氧组的3.98±0.42(P＜0.05).(5)高氧组肺组织VEGF mRNA表达量为0.23±0.16,低于空气组的1.05±0.33;移植组为0.69±0.09,高于高氧组;干预组为0.31±0.08,低于移植组(P值均＜0.05).高氧组肺组织VEGF蛋白表达低于空气组(分别为0.52±0.01与0.82±0.01),移植组为0.58±0.05,高于高氧组(P值均＜0.05).高氧组肺组织VEGFR2 mRNA表达低于空气组(分别为0.35±0.13与1.07±0.45,P＜0.05).高氧组肺组织eNOS mRNA表达低于空气组(分别为0.46±0.10与1.05±0.36,P＜0.05),eNOS蛋白表达亦低于空气组(分别为0.32±0.01与0.51±0.03),而移植组(0.86±0.02)高于高氧组(P值均＜ 0.05). 结论 外周静脉移植的EPC可以定植于肺组织中,改善高氧损伤的肺泡和肺血管发育,可能与上调肺组织eNOS和VEGF的表达有关.
目的 研究移植內皮祖細胞(endothelial progenitor cell,EPC)對新生大鼠高氧肺損傷的影響及其機製. 方法 從4週齡Sprague-Dawyley大鼠骨髓中培養穫取EPC併鑒定.另取新生Sprague-Dawley仔鼠60隻,生後室溫下適應性飼養24 h後,隨機分為空氣組、高氧組、移植組和Nω-硝基-L-精氨痠甲酯(Nω-nitro-L-argininemethylester,L-NAME)榦預組(簡稱榦預組),每組15隻.空氣和高氧組分彆在空氣和85％高氧中飼養28d.移植組在85％高氧中暴露28d,其中在第21天尾靜脈註射EPC 1×10 5箇.榦預組在移植組的基礎上,自第21天開始連續腹腔註射L-NAME至第28天,每日劑量為20 mg/kg.第28天處死所有仔鼠,留取血標本,採用流式細胞技術檢測CD34+細胞,酶聯免疫吸附方法檢測血清血管內皮細胞生長因子(vescular endothelial growth factor,VEGF).同時留取肺組織標本,觀察輻射狀肺泡計數和肺微血管計數、免疫熒光方法觀察移植EPC在肺內的定植情況,實時熒光定量聚閤酶鏈反應和蛋白質印跡技術檢測肺組織中VEGF、VEGF受體(vescular endothelial growth factor receptor,VEGFR)2和內皮源性一氧化氮閤酶(endothelial nitric oxide synthase,eNOS)的錶達,併用硝痠還原酶法檢測肺組織中一氧化氮的錶達.採用單因素方差分析和Bonferroni方法進行統計學分析. 結果 (1)培養所得細胞具有典型的EPC形態改變;結閤異硫氫基熒光素標記荊豆凝集素-1併攝取Dil熒光標記的乙酰化低密度脂蛋白的雙暘性細胞約佔總細胞數的85％;培養所得細胞中CD34+細胞含量為68.2％～72.4％.(2)空氣組、高氧組、移植組和榦預組仔鼠外週血CD34+細胞數量分彆為(1.91±0.34)％、(1.06±0.10)％、(1.47±0.06)％和(0.77±0.11)％(F=32.710,P=0.000),高氧組低于空氣組,移植組高于高氧組,榦預組又低于移植組(P值均＜ 0.05).4組仔鼠血清VEGF水平分彆為(7.90±2.72)、(6.38±0.72)、(14.00±1.66)和(11.70±1.91) pg/ml(F=22.809,P=0.000),移植組高于高氧組(P＜0.05).(3)移植的EPC在肺部主要定植于內皮下和肺泡間質.榦預組定植的EPC明顯少于移植組[分彆為(16.95±0.50)箇/視野與(10.70±0.47)箇/視野,t=17.820,P=0.000].(4)4組仔鼠輻射狀肺泡計數和肺組織微血管密度差異均有統計學意義(F值分彆為859.580和211.150,P值均=0.000),其中高氧組RAC和微血管密度均小于空氣組(分彆為7.98±0.23與13.12±0.20,3.98±0.42與9.50±0.22,P值均＜0.05),移植組微血管密度為5.40±0.41,大于高氧組的3.98±0.42(P＜0.05).(5)高氧組肺組織VEGF mRNA錶達量為0.23±0.16,低于空氣組的1.05±0.33;移植組為0.69±0.09,高于高氧組;榦預組為0.31±0.08,低于移植組(P值均＜0.05).高氧組肺組織VEGF蛋白錶達低于空氣組(分彆為0.52±0.01與0.82±0.01),移植組為0.58±0.05,高于高氧組(P值均＜0.05).高氧組肺組織VEGFR2 mRNA錶達低于空氣組(分彆為0.35±0.13與1.07±0.45,P＜0.05).高氧組肺組織eNOS mRNA錶達低于空氣組(分彆為0.46±0.10與1.05±0.36,P＜0.05),eNOS蛋白錶達亦低于空氣組(分彆為0.32±0.01與0.51±0.03),而移植組(0.86±0.02)高于高氧組(P值均＜ 0.05). 結論 外週靜脈移植的EPC可以定植于肺組織中,改善高氧損傷的肺泡和肺血管髮育,可能與上調肺組織eNOS和VEGF的錶達有關.
목적 연구이식내피조세포(endothelial progenitor cell,EPC)대신생대서고양폐손상적영향급기궤제. 방법 종4주령Sprague-Dawyley대서골수중배양획취EPC병감정.령취신생Sprague-Dawley자서60지,생후실온하괄응성사양24 h후,수궤분위공기조、고양조、이식조화Nω-초기-L-정안산갑지(Nω-nitro-L-argininemethylester,L-NAME)간예조(간칭간예조),매조15지.공기화고양조분별재공기화85％고양중사양28d.이식조재85％고양중폭로28d,기중재제21천미정맥주사EPC 1×10 5개.간예조재이식조적기출상,자제21천개시련속복강주사L-NAME지제28천,매일제량위20 mg/kg.제28천처사소유자서,류취혈표본,채용류식세포기술검측CD34+세포,매련면역흡부방법검측혈청혈관내피세포생장인자(vescular endothelial growth factor,VEGF).동시류취폐조직표본,관찰복사상폐포계수화폐미혈관계수、면역형광방법관찰이식EPC재폐내적정식정황,실시형광정량취합매련반응화단백질인적기술검측폐조직중VEGF、VEGF수체(vescular endothelial growth factor receptor,VEGFR)2화내피원성일양화담합매(endothelial nitric oxide synthase,eNOS)적표체,병용초산환원매법검측폐조직중일양화담적표체.채용단인소방차분석화Bonferroni방법진행통계학분석. 결과 (1)배양소득세포구유전형적EPC형태개변;결합이류경기형광소표기형두응집소-1병섭취Dil형광표기적을선화저밀도지단백적쌍양성세포약점총세포수적85％;배양소득세포중CD34+세포함량위68.2％～72.4％.(2)공기조、고양조、이식조화간예조자서외주혈CD34+세포수량분별위(1.91±0.34)％、(1.06±0.10)％、(1.47±0.06)％화(0.77±0.11)％(F=32.710,P=0.000),고양조저우공기조,이식조고우고양조,간예조우저우이식조(P치균＜ 0.05).4조자서혈청VEGF수평분별위(7.90±2.72)、(6.38±0.72)、(14.00±1.66)화(11.70±1.91) pg/ml(F=22.809,P=0.000),이식조고우고양조(P＜0.05).(3)이식적EPC재폐부주요정식우내피하화폐포간질.간예조정식적EPC명현소우이식조[분별위(16.95±0.50)개/시야여(10.70±0.47)개/시야,t=17.820,P=0.000].(4)4조자서복사상폐포계수화폐조직미혈관밀도차이균유통계학의의(F치분별위859.580화211.150,P치균=0.000),기중고양조RAC화미혈관밀도균소우공기조(분별위7.98±0.23여13.12±0.20,3.98±0.42여9.50±0.22,P치균＜0.05),이식조미혈관밀도위5.40±0.41,대우고양조적3.98±0.42(P＜0.05).(5)고양조폐조직VEGF mRNA표체량위0.23±0.16,저우공기조적1.05±0.33;이식조위0.69±0.09,고우고양조;간예조위0.31±0.08,저우이식조(P치균＜0.05).고양조폐조직VEGF단백표체저우공기조(분별위0.52±0.01여0.82±0.01),이식조위0.58±0.05,고우고양조(P치균＜0.05).고양조폐조직VEGFR2 mRNA표체저우공기조(분별위0.35±0.13여1.07±0.45,P＜0.05).고양조폐조직eNOS mRNA표체저우공기조(분별위0.46±0.10여1.05±0.36,P＜0.05),eNOS단백표체역저우공기조(분별위0.32±0.01여0.51±0.03),이이식조(0.86±0.02)고우고양조(P치균＜ 0.05). 결론 외주정맥이식적EPC가이정식우폐조직중,개선고양손상적폐포화폐혈관발육,가능여상조폐조직eNOS화VEGF적표체유관.
Objective To study the effect of transplanted endothelial progenitor cell (EPC) on hyperoxia-induced lung injury in neonatal rats.Methods Rat bone marrow mononuclear cells were cultured in endothelial cell growth medium to obtain EPCs,which were identified by morphology,phagocytosis and CD34+ analyses.Sixty neonatal Sprague-Dawley rats were allowed to acclimate in room air for 24 h after birth,and were then divided into four groups (15 per group),including the air group,the hyperoxia group,the EPCs transplantation group and the N ω-nitro-L-arginine methyl ester (L-NAME) intervention group.Neoborn rats in the Air and Hyperoxia groups were fed in the room air or hyperoxia (85％ oxygen) for 28 days.For rats in transplantation group were exposed continuously to hyperoxia for 28 days,and got an EPC (1 × 105 cells) injection on the 21st day.Rats in Intervention group were exposed continuously to hyperoxia for 28 days,got an EPC (1 × 105 cells) injection on the 21st day,and a daily injection of L-NAME from day 21 to day 28,with a daily dose of 20 mg/kg.Levels of circulating CD34+ cells and serum VEGF expression were detected.Specimens from lung tissues were analyzed by immunohistochemistry or immunofluorescence.The expression of vascular endothelial growth factor (VEGF),VEGF receptor 2 (VEGFR2) and eNOS were detected by realtime polymerase chain reaction and Western-blotting.NO production were detected by nitrate reductase assay.One way ANOVA and Bonferroni test were used for statistical analysis.Results (1) The cultured cells had a typical cobblestone appearance; double positive cell binding of fluorescein Ulex Europaeus agglutinin-1 and uptake of Dil-labeled acetylated low density lipoprotein accounted for approximately 85％ of the total number of cells.CD34+ cells accounted for 68.2％-72.4％ of total cultured cells.(2) Circulating CD34+ cells in the air group,hyperoxia group,EPC transplantation group and L NAME intervention group were (1.91 ± 0.34)％,(1.06 ± 0.10)％,(1.47 ± 0.06)％ and (0.77 ± 0.11)％ (F=32.710,P=0.000).The number of circulating CD34+ cells in the hyperoxia group was lower than the air group,in the EPC transplantation group the number of these cells was higher than the hyperoxia group,and in the L-NAME intervention group the number of these cells was lower than that in the EPC transplantation group,and the differences between these two groups were statistically significant (P ＜ 0.05,respectively).Serum VEGF in the four groups was (7.90±2.72),(6.38±0.72),(14.00± 1.66) and (11.70± 1.91) pg/ml,respectively.The difference between the four groups was statistically significant (F=22.809,P=0.000),and serum VEGF in the EPC transplantation group was higher than that in the hyperoxia group (P ＜ 0.05).(3) Transplanted EPCs could engraft in pulmonary vascular endothelium and alveolar interstitium,and L-NAME intervention significantly reduced the engraftment of EPCs in the lungs (10.7±0.47 / field vs 16.95±0.5 /field,t=17.820,P=0.000).(4) There were significant differences in the radial alveolar count (RAC) and number of microvessels between the four groups (F=859.580 or 211.150,P=0.000,respectively).RAC and the number of microvessels in the hyperoxia group were less than those in the air group (7.98±0.23 vs 13.12±0.20,3.98±0.42 vs 9.50±0.22,P ＜ 0.05,respectively).The number of microvessels in the EPC transplantation group was 5.40±0.41,being higher than that in the hyperoxia group (P＜0.05).(5) VEGF mRNA in lungs in the hyperoxia group was lower than that in the air group (0.23 ± 0.16 vs 1.05 ± 0.33,P ＜ 0.05); in the EPC transplantation group,VEGF mRNA was higher than that in the hyperoxia group (0.69 ± 0.09 vs 0.23 ± 0.16,P ＜ 0.05); and in the L-NAME intervention group,VEGF mRNA was lower than that in the EPC transplantation group (0.31 ±0.08 vs 0.69±0.09,P ＜ 0.05).VEGF protein in the lungs in the hyperoxia group was lower than the air group (0.52±0.01 vs 0.82±0.01,P ＜ 0.05),and was higher in the EPC transplantation group than the hyperoxia group (0.58±0.05 vs 0.52±2501,P ＜ 0.05).VEGFR2 mRNA in the hyperoxia group was lower than the air group (0.35±0.13 vs 1.07±0.45,P ＜ 0.05).eNOS mRNA in the hyperoxia group was lower than the air group (0.46±0.10 vs 1.05±0.36,P ＜ 0.05).eNOS protein in the hyperoxia group was lower than the air group (0.32±0.01 vs 0.51 ±0.03,P ＜ 0.05),and was higher in the EPC transplantation group than the hyperoxia group (0.86±0.02 vs 0.32±0.01,P ＜ 0.05).Conclusion Transplanted EPC can engraft in the lung tissue,improving alveolar and pulmonary vascular development,which may be associated with upregulation of the expression of eNOS and VEGF in lung.
