CAJ | 학술논문

目的探讨人受体活性修饰蛋白-1 (hRAMP1)基因修饰的间充质干细胞(MSCs)移植对兔颈动脉粥样硬化并球囊成形术后血管平滑肌细胞(VSMCs)增殖和凋亡的影响. 方法密度梯度离心法加贴壁培养法获得MSCs,并以携带hRAMP1腺病毒和空腺病毒转染MSCs获得hRAMP1基因修饰的MSCs(hRAMP1-MSCs)和空病毒修饰的MSCs(Ad-MSCs).建立动脉粥样硬化狭窄并球囊损伤血管成形术的兔模型,数字抽签随机分为hRAMP1-MSCs组、Ad-MSCs组和对照组,模型建立成功后经球囊局部移植MSCs或PBS,细胞移植后7d,检测MSCs归巢和分化情况,28d检测血管损伤局部RAMP1表达,测量损伤血管新生内膜/中膜面积;检测增殖细胞核抗原(PCNA)的表达和细胞凋亡. 结果细胞移植后7d,hRAMP1-MSCs组和MSCs组损伤血管增生内膜均有CD31和增强型绿色荧光蛋白(EGFP)分布;细胞移植后28d,RAMP1-MSCs组RAMP1基因表达较Ad-MSCs组和对照组增加,63.0±4.9比28.3±2.5和27.2±7.2,差异有统计学意义(均P＜0.05),而Ad-MSCs组和对照组间RAMP1基因表达差异无统计学意义(P＞0.05);细胞移植后28d,在各组增生内膜细胞中α平滑肌肌动蛋白(α-SMA)表达均阳性,而hRAMP1-MSCs组内膜厚度低于Ad-MSCs组和对照组(P＜0.05),Ad-MSCs组亦低于对照组(P＜0.05),同时hRAMP1-MSCs组增殖细胞核抗原(PCNA)增殖率也低于Ad MSCs组和对照组(均P＜0.05),而Ad-MSCs组的增值率亦低于对照组(P＜0.05);细胞移植后28 d hRAMP1-MSCs组凋亡率最高,对照组凋亡率最低(均P＜0.05). 结论基因修饰后的干细胞治疗可能更有效地抑制内膜增生,从而降低血管成形术后再狭窄的发生.
목적 탐토인수체활성수식단백-1 (hRAMP1)기인수식적간충질간세포(MSCs)이식대토경동맥죽양경화병구낭성형술후혈관평활기세포(VSMCs)증식화조망적영향. 방법 밀도제도리심법가첩벽배양법획득MSCs,병이휴대hRAMP1선병독화공선병독전염MSCs획득hRAMP1기인수식적MSCs(hRAMP1-MSCs)화공병독수식적MSCs(Ad-MSCs).건립동맥죽양경화협착병구낭손상혈관성형술적토모형,수자추첨수궤분위hRAMP1-MSCs조、Ad-MSCs조화대조조,모형건립성공후경구낭국부이식MSCs혹PBS,세포이식후7d,검측MSCs귀소화분화정황,28d검측혈관손상국부RAMP1표체,측량손상혈관신생내막/중막면적;검측증식세포핵항원(PCNA)적표체화세포조망. 결과 세포이식후7d,hRAMP1-MSCs조화MSCs조손상혈관증생내막균유CD31화증강형록색형광단백(EGFP)분포;세포이식후28d,RAMP1-MSCs조RAMP1기인표체교Ad-MSCs조화대조조증가,63.0±4.9비28.3±2.5화27.2±7.2,차이유통계학의의(균P＜0.05),이Ad-MSCs조화대조조간RAMP1기인표체차이무통계학의의(P＞0.05);세포이식후28d,재각조증생내막세포중α평활기기동단백(α-SMA)표체균양성,이hRAMP1-MSCs조내막후도저우Ad-MSCs조화대조조(P＜0.05),Ad-MSCs조역저우대조조(P＜0.05),동시hRAMP1-MSCs조증식세포핵항원(PCNA)증식솔야저우Ad MSCs조화대조조(균P＜0.05),이Ad-MSCs조적증치솔역저우대조조(P＜0.05);세포이식후28 d hRAMP1-MSCs조조망솔최고,대조조조망솔최저(균P＜0.05). 결론 기인수식후적간세포치료가능경유효지억제내막증생,종이강저혈관성형술후재협착적발생.
Objective To explore the effect of transplantation of mesenchymal stem cells (MSCs) transfected with the human receptor activity modifying protein 1 (hRAMP1) gene on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) after carotid balloon angioplasty was performed in rabbits with carotid atherosclerosis.Methods Density gradient centrifugation and adherent culture were carried out to obtain MSCs,which were then transinfected with an adenovirus vector carrying the hRAMP1 gene or an empty adenovirus vector.A rabbit model of atherosclerotic stenosis and balloon angioplasty was successfully established.Results were randomly divided into three groups:the hRAMP1-MSCs group,theadipose-derived MSCs (Ad-MSCs) group and the control group.MSCs were transinfected with Ad-EGFP-hRAMP1,Ad-EGFP or PBS by transplantation into the injured carotid arteries.Homing and differentiation were assessed with MSCs harvested at 7 d.With MSCs collected at 28 d,Western blotting was used to measure the expression of the hRAMP1 target gene in the carotid artery; the neointima and media area in the injured carotid arteries were estimated; carotid artery morphology was examined with H&E staining; and the proliferation and apoptosis of VSMCs were determined by immunohistochemistry and TUNEL.Results The expression of CD31 and EGFP was found in proliferating neointima lesions at 7d in the hRAMP1-MSCs group and the Ad-MSCs group.At 28d of MSC transplantation,the level of RAMP1 significantly increased in the hRAMP1-MSCs group,compared with the Ad-MSCs and control groups [(63.0±4.9) vs.(28.3±2.5) and (27.2±7.2),all P＜0.05],but there was no differencein the RAMP1 level between the Ad MSCs group and the control group (P＞0.05).Positive expression of the α-smooth muscle antibody (α-SMA) was found in all three groups at 28 d of MSC transplantation.The thickness of the hyperplastic neointima significantly decreased in the hRAMP1-MSCs group,compared with the other two groups (P＜0.05),and was lower in the Ad-MSCs group than in the control group (P＜0.05).The expression of proliferating cell nuclear antigen (PCNA) was lower in the hRAMP1-MSCs group than in the Ad-MSCs and control groups at 28d of MSC transplantation (P ＜0.05),while the PCNA level was lower in the Ad-MSCs group than in the control group (P＜ 0.05).The VSMC apoptosis rate significantly increased in the hRAMP1-MSCs group,compared with the Ad MSCs and control groups (P＜0.05),and was the lowest in the control group (P＜0.05).Conclusions Gene-modified stem cell therapy can effectively inhibit vascular intimal hyperplasia,thereby reducing restenosis after angioplasty.