背景:有研究显示大鼠皮下注射粒细胞集落刺激因子后,外周血CD34+细胞明显增高.并发现运用粒细胞集落刺激因子不仅动员了骨髓干细胞,而且加速损伤血管段的再内皮化,抑制了新生内膜增生.目的:观察重组人粒细胞集落刺激因子对大鼠损伤动脉的再内皮化及抑制新生内膜增生作用.设计:随机对照动物实验.单位:南京医科大学附属南京第一医院.材料:实验于2005-12/2006-04于南京市第一医院完成.选用40只雄性SD大鼠,8～12周龄SPF级,体质量200～250 g,购于国家啮齿类实验动物种子中心上海分中心.重组人粒细胞集落刺激因子购于山东齐鲁制药有限公司,2F动脉取栓导管购于Edwards Lifesciences公司,CD34单克隆抗体、CD45单克隆抗体购于联科生物有限公司.方法:用Excel软件随机将大鼠分成治疗组及对照组,每组20只.治疗组给予皮下注射重组人粒细胞集落刺激因子(100 μg/kg),1次/d,共8 d.对照组皮下注射等量生理盐水,1次/d,共8 d.治疗5 d后,腹腔注射麻醉大鼠,颈外动脉做一切口,将2F动脉取栓导管插入至左颈总动脉分叉处,注入200 μL空气使球囊扩张,然后回拉球囊至肩胛舌骨肌处,回抽空气后重新送回导管,反复3次后拔出导管,结扎颈外动脉.①治疗前和治疗5 d后,所有大鼠均取1 mL静脉血进行白细胞和CD34+细胞计数.②球囊损伤后14和28 d,每组分别麻醉后处死大鼠10只,取左颈总动脉.每组各5只用于暴露动脉内膜腔,应用图形分析软件计算再内皮化面积,再内皮化面积=未被伊文氏蓝着色区域/损伤总面积;每组另有5只,取左颈总动脉,取血管横切面,经HE染色后用图像分析软件计算新生内膜与中膜比(I/M),评估新生内膜增生程度.③采用免疫组化方法检测血管内膜CD31+与vWF+(Ⅷ因子)细胞,推测损伤后内皮修复程度.主要观察指标:①大鼠外周血白细胞及CD34+细胞表达.②球囊损伤后血管再内皮化程度.③新生内膜增生程度(新生内膜与中膜比).④血管内膜CD31+与vWF+(Ⅷ因子)细胞检测结果.结果:纳入SD大鼠40只均进入结果分析.①外周血白细胞及CD34+细胞表达:治疗5 d后,治疗组大鼠白细胞总数高于对照组[(27.60±2.45)×109 L-1,(10.11±1.81)×109 L-1,P＜0.01],CD34+细胞数量高于对照组(38.31×107 L-1,3.14×107 L-1,P＜0.01).②血管再内皮化程度:球囊损伤后14和28 d,治疗组再内皮化面积分别为(68.3±8.3)%,(97.6±4.1)%,高于对照组[(33.8±6.3)%,(76.1±5.2)%,P＜0.01].③新生内膜增生程度:球囊损伤后14,28 d,治疗组新生内膜与中膜比分别为0.39±0.11,0.45±0.09,均低于对照组(0.87±0.15,1.26±0.16,P＜0.01),治疗组血管新生内膜增生程度被显著抑制.④血管内膜CD31+与vWF+(Ⅷ因子)细胞检测:球囊损伤后28 d,治疗组血管内膜被接近于连续完整的CD31+与vWF+细胞覆盖,对照组血管内膜仅见零星、分散的CD31+与vWF+细胞.结论:重组人粒细胞集落刺激因子可促进大鼠血管损伤后外周血中CD34+细胞表达及血管内膜再内皮化,抑制新生内膜增生.
揹景:有研究顯示大鼠皮下註射粒細胞集落刺激因子後,外週血CD34+細胞明顯增高.併髮現運用粒細胞集落刺激因子不僅動員瞭骨髓榦細胞,而且加速損傷血管段的再內皮化,抑製瞭新生內膜增生.目的:觀察重組人粒細胞集落刺激因子對大鼠損傷動脈的再內皮化及抑製新生內膜增生作用.設計:隨機對照動物實驗.單位:南京醫科大學附屬南京第一醫院.材料:實驗于2005-12/2006-04于南京市第一醫院完成.選用40隻雄性SD大鼠,8～12週齡SPF級,體質量200～250 g,購于國傢齧齒類實驗動物種子中心上海分中心.重組人粒細胞集落刺激因子購于山東齊魯製藥有限公司,2F動脈取栓導管購于Edwards Lifesciences公司,CD34單剋隆抗體、CD45單剋隆抗體購于聯科生物有限公司.方法:用Excel軟件隨機將大鼠分成治療組及對照組,每組20隻.治療組給予皮下註射重組人粒細胞集落刺激因子(100 μg/kg),1次/d,共8 d.對照組皮下註射等量生理鹽水,1次/d,共8 d.治療5 d後,腹腔註射痳醉大鼠,頸外動脈做一切口,將2F動脈取栓導管插入至左頸總動脈分扠處,註入200 μL空氣使毬囊擴張,然後迴拉毬囊至肩胛舌骨肌處,迴抽空氣後重新送迴導管,反複3次後拔齣導管,結扎頸外動脈.①治療前和治療5 d後,所有大鼠均取1 mL靜脈血進行白細胞和CD34+細胞計數.②毬囊損傷後14和28 d,每組分彆痳醉後處死大鼠10隻,取左頸總動脈.每組各5隻用于暴露動脈內膜腔,應用圖形分析軟件計算再內皮化麵積,再內皮化麵積=未被伊文氏藍著色區域/損傷總麵積;每組另有5隻,取左頸總動脈,取血管橫切麵,經HE染色後用圖像分析軟件計算新生內膜與中膜比(I/M),評估新生內膜增生程度.③採用免疫組化方法檢測血管內膜CD31+與vWF+(Ⅷ因子)細胞,推測損傷後內皮脩複程度.主要觀察指標:①大鼠外週血白細胞及CD34+細胞錶達.②毬囊損傷後血管再內皮化程度.③新生內膜增生程度(新生內膜與中膜比).④血管內膜CD31+與vWF+(Ⅷ因子)細胞檢測結果.結果:納入SD大鼠40隻均進入結果分析.①外週血白細胞及CD34+細胞錶達:治療5 d後,治療組大鼠白細胞總數高于對照組[(27.60±2.45)×109 L-1,(10.11±1.81)×109 L-1,P＜0.01],CD34+細胞數量高于對照組(38.31×107 L-1,3.14×107 L-1,P＜0.01).②血管再內皮化程度:毬囊損傷後14和28 d,治療組再內皮化麵積分彆為(68.3±8.3)%,(97.6±4.1)%,高于對照組[(33.8±6.3)%,(76.1±5.2)%,P＜0.01].③新生內膜增生程度:毬囊損傷後14,28 d,治療組新生內膜與中膜比分彆為0.39±0.11,0.45±0.09,均低于對照組(0.87±0.15,1.26±0.16,P＜0.01),治療組血管新生內膜增生程度被顯著抑製.④血管內膜CD31+與vWF+(Ⅷ因子)細胞檢測:毬囊損傷後28 d,治療組血管內膜被接近于連續完整的CD31+與vWF+細胞覆蓋,對照組血管內膜僅見零星、分散的CD31+與vWF+細胞.結論:重組人粒細胞集落刺激因子可促進大鼠血管損傷後外週血中CD34+細胞錶達及血管內膜再內皮化,抑製新生內膜增生.
배경:유연구현시대서피하주사립세포집락자격인자후,외주혈CD34+세포명현증고.병발현운용립세포집락자격인자불부동원료골수간세포,이차가속손상혈관단적재내피화,억제료신생내막증생.목적:관찰중조인립세포집락자격인자대대서손상동맥적재내피화급억제신생내막증생작용.설계:수궤대조동물실험.단위:남경의과대학부속남경제일의원.재료:실험우2005-12/2006-04우남경시제일의원완성.선용40지웅성SD대서,8～12주령SPF급,체질량200～250 g,구우국가교치류실험동물충자중심상해분중심.중조인립세포집락자격인자구우산동제로제약유한공사,2F동맥취전도관구우Edwards Lifesciences공사,CD34단극륭항체、CD45단극륭항체구우련과생물유한공사.방법:용Excel연건수궤장대서분성치료조급대조조,매조20지.치료조급여피하주사중조인립세포집락자격인자(100 μg/kg),1차/d,공8 d.대조조피하주사등량생리염수,1차/d,공8 d.치료5 d후,복강주사마취대서,경외동맥주일절구,장2F동맥취전도관삽입지좌경총동맥분차처,주입200 μL공기사구낭확장,연후회랍구낭지견갑설골기처,회추공기후중신송회도관,반복3차후발출도관,결찰경외동맥.①치료전화치료5 d후,소유대서균취1 mL정맥혈진행백세포화CD34+세포계수.②구낭손상후14화28 d,매조분별마취후처사대서10지,취좌경총동맥.매조각5지용우폭로동맥내막강,응용도형분석연건계산재내피화면적,재내피화면적=미피이문씨람착색구역/손상총면적;매조령유5지,취좌경총동맥,취혈관횡절면,경HE염색후용도상분석연건계산신생내막여중막비(I/M),평고신생내막증생정도.③채용면역조화방법검측혈관내막CD31+여vWF+(Ⅷ인자)세포,추측손상후내피수복정도.주요관찰지표:①대서외주혈백세포급CD34+세포표체.②구낭손상후혈관재내피화정도.③신생내막증생정도(신생내막여중막비).④혈관내막CD31+여vWF+(Ⅷ인자)세포검측결과.결과:납입SD대서40지균진입결과분석.①외주혈백세포급CD34+세포표체:치료5 d후,치료조대서백세포총수고우대조조[(27.60±2.45)×109 L-1,(10.11±1.81)×109 L-1,P＜0.01],CD34+세포수량고우대조조(38.31×107 L-1,3.14×107 L-1,P＜0.01).②혈관재내피화정도:구낭손상후14화28 d,치료조재내피화면적분별위(68.3±8.3)%,(97.6±4.1)%,고우대조조[(33.8±6.3)%,(76.1±5.2)%,P＜0.01].③신생내막증생정도:구낭손상후14,28 d,치료조신생내막여중막비분별위0.39±0.11,0.45±0.09,균저우대조조(0.87±0.15,1.26±0.16,P＜0.01),치료조혈관신생내막증생정도피현저억제.④혈관내막CD31+여vWF+(Ⅷ인자)세포검측:구낭손상후28 d,치료조혈관내막피접근우련속완정적CD31+여vWF+세포복개,대조조혈관내막부견령성、분산적CD31+여vWF+세포.결론:중조인립세포집락자격인자가촉진대서혈관손상후외주혈중CD34+세포표체급혈관내막재내피화,억제신생내막증생.
BACKGROUND:It has been reported that treatment with granulocyte-colony stimulating factor (G-CSF) increases the abundance of circulating CD34+ cells in rats. Data from the study, more important, suggested that mobilized by G-CSF may enhance rapid reendothelization and reduce neointimal formation after vascular injury.OBJECTIVE: To evaluate whether BM-derived CD34+ cells could enhance rapid reendothelization and reduce neointimal formation after balloon-injured carotid artery in an intact rat model.DESIGN: Randomized control animal study.SETTING: Nanjing First Hospital Affiliated to Nanjing Medical University.MATERIALS: The experiment was carried out in Nanjing First Hospital from December 2005 to April 2006. A total of 40 male Sprague-Dawley rats, weighing 200-250 g and of SPF grade, were purchased from National Rodent Laboratory Animal Resources, Shanghai Branch. The recombinant human G-CSF was purchased from Qilu Pharmaceutical. The 2F Fogarty arterial embolectomy catheters were purchased from Edwards Lifesciences. Anti-human CD34 and anti-human CD45 were purchased from Multi Sciences.METHODS: SD rats were divided randomly into treated group (n =20) and control group (n =20). Subcutaneous injection of recombinant human G-CSF (100 μg/kg/day) once daily for 8 days for treated group. Control group as treated with subcutaneous injection of saline. Five days after initiation of G-CSF treatment or saline, the rats were anesthetized by intraperitoneal injection with ketamine. The left common carotid artery was exposed through a midline incision of the ventral side of the neck. A 2F Fogarty arterial embolectomy catheter was inserted through the external carotid artery,inflated with 200 μL air, and passed 3 times along the length of the segment, which was defined proximally by the carotid bifurcation and distally by the edge of the omohyoid muscle. After removal of the catheter, the proximal ligature of the external carotid artery was tied off. ① An average of 1 mL venous blood per rat was collected for enumeration of the white blood wells (WBCs) and CD34+ cells before and 5 days after initiating G-CSF or saline treatment. ② Ten rats in each group were killed with overdose ketamine at 14 and 28 days after balloon injury and left common carotid arteries were harvested. The luminal surface of carotid arteries (n =5, each group) was exposed to calculate the reendothelialized area, which was manually traced with software (Image ProPlus). Reendothelialized area = non-stained with Evans blue area/the total area of balloon-injuried. The cross sections of carotid arteries (n =5, each group) were stained with hematoxylin and eosin (HE) and calculated intima-to-media area ratio (I/M) with software (Image ProPlus) to assess the extent of neointimal thickening. ③ To evaluate the extent of reendothelialization of arteries injury, sections were stained with CD31 and vWF by immunohistochemistry analysis.MAIN OUTCOME MEASURES: ① The number of WBCs and CD34+ cells; ② the extent of reendothelialization of arteries injury; ③ the extent of neointimal hyperplasia (I/M); ④ CD31 + and vWF+ endothelial cells.RESULTS: A total of 40 rats were involved in the final analysis. ① The number of WBCs and CD34+ cells: After 5 days of treatment, the number of WBCs in the treated rats increased more than 2.7-fold compared with control group [(27.60±2.45) ×109 L-1, (10.11±1.81) ×109 L-1, P ＜ 0.01], CD34+ cells increased more than 12.2-fold compared with control group (38.31×107 L-1, 3.14×107 L-1, P ＜ 0.01). ② The extent of reendothelialization: At 14 and 28 days after balloon injury,carotid artery of reendothelialization in the treated group were (68.3±8.3)％ and (97.6±4.1)％, superior than the control group (33.8±6.3)％ and (76.1±5.2)％ (P ＜ 0.01). ③ The extent of neointimal hyperplasia: At 14 and 28 days after balloon injury, the neointima-media (I/M) ratios in the treated rats were 0.39±0.11 and 0.45±0.09, less than the control group 0.87±0.15,1.26±0.16 (P ＜ 0.01). A highly significant inhibition of neointimal hyperplasia was observed in the treated group. ④ CD31+ and vWF+ endothelial cells: At 28 days after injury, sections from G-CSF treated group showed almost complete and continuous monolayer of CD31 and vWF positive cells.In contrast, a patchy and interrupted CD31 and vWF positive cells were found lining the lumen of control group.CONCLUSION:Treatment with G-CSF significantly increases the number of CD34+ cells and accelerates the rate of reendothelialization of injured vessels, leading to marked inhibition of neointimal formation after vascular injury in rats.
