干细胞移植对兔后肢动脉血管成形术后血清血管内皮生长因子及转化生长因子β1水平的影响及意义

Effects of stem cell transplantation on the serum VEGF and TGF-beta1 levels in the intracavitary angioplasty of rabbits hind limb artery

目的探讨干细胞移植防治血管成形术后再狭窄的可能机制。方法 2014年12月至2015年2月,新西兰大白兔18只随机分入对照组(A组,n=6)、球囊损伤组(B组,n=6)及球囊损伤+人脐血干细胞移植(HUCBSC)组(C组,n=6)。采用Ficoll密度梯度离心法分离制备人脐血单个核细胞悬液。A组只分离左侧后肢动脉标记后闭合伤口;C组行动脉分离后夹闭动脉两端,穿刺成功以3 mm×40 mm球囊进行局部扩张并间断牵拉球囊,每次30 s,共3次,成功制备动脉缺血损伤模型后,经导管注入脐血干细胞悬液0.5 ml于后肢动脉损伤局部,缓慢撤出球囊,恢复血供;B组经同样方法介入操作后,注入等体积的磷酸盐缓冲液(PBS缓冲液);术毕均予逐层缝合关闭伤口。分别于术前、术后7 d及术后14 d经耳中央动脉各采血3 ml,分离血清,采用ELISA法分别测定3组血管内皮生长因子(VEGF)及转化生长因子-β1(TGF-β1)水平;实验结束时留取局部血管组织进行HE染色,观察移植前后血管病理变化表现。组内比较采用配对样本t检验,组间比较采用均数方差分析及双变量回归与相关分析进行统计学处理。结果 (1)术前A组、B组及C组VEGF水平比较差异无统计学意义(F=0.000,P=0.978);术后7 d B组较A组明显增高(t=2.286,P=0.045),C组较A组及B组均明显增高(t=6.196,P=0.000;t=2.336,P=0.042);术后14 d B组与A组比较无明显差异(t=1.294,P=0.225),C组较A组及B组仍显著增高(t=6.738,P=0.000;t=2.641,P=0.038)。(2)术前A组、B组及C组TGF-β1水平比较组间差异无统计学意义(F=0.003,P=0.997);术后7 d B组较A组明显增高(t=9.052,P=0.000),C组较A组增高,但与B组比较明显减低,差异均有统计学意义(t=2.675,P=0.023;t=3.104,P=0.011);术后14 d B组与A组比较仍增高(t=3.443,P=0.006),而C组与B组比较显著降低(t=2.932,P=0.020),较A组增高,但差异无统计学意义(t=1.262,P=0.236);(3)球囊损伤及干细胞移植后不同时间点血清VEGF与TGF-β1水平变化呈负相关关系(r=-0.539,P=0.000);(4)术后14 d A组内膜连续完整,管腔正常,B组血管内膜均有不同程度增生且不连续,弹力层断裂,管腔变窄,C组内膜增生较B组明显减轻,管腔狭窄不明显。结论介入治疗后血清VEGF和TGF-β1水平均增高,HUCBSC后血清VEGF和TGF-β1水平变化呈负相关关系,两者共同参与促进受损内皮功能修复及改善血管壁重塑过程,是干细胞移植防治血管成形术后再狭窄的重要原因之一。

Objective This study investigated the effects of human cord blood stem cell transplantation（HCBSCT） on serum vascular endothelial growth factor（VEGF） and transforming growth factor beta 1（TGF-β1） levels and the pathological changes of blood vessel of rabbit after after balloon injury of hind limb artery, and explored the mechanism of the prevention of postangioplasty restenosis by HCBSCT. Methods 18 healthy New Zealand white rabbits were divided into control group（group A, n = 6）, Balloon injury group（ group B, n = 6） and Balloon injury ＋ HCBSCT group（group C, n = 6） randomly. Ficoll density gradient centrifugation was used to isolate mononuclear cells from human cord blood, and the concentration of the cells was adjusted, then kept at-80℃ for use. Every rabbits＇ left hind limb artery was exposed and labeled in aseptic operation after general anesthesia. Rabbitd in group A were directly sutured after the femoral artery was labeled. In group C vessels were mobiled, and clipped with clamps at the both ends to block the blood. Then the vessels were expanded with a3 mm×40 mm balloon for 3 times, 30 sec each time to induce arterial ischemic damage. After intra-catheter injection of 0.5 ml umbilical cord blood stem cell suspension in the injuried hind limbs artery, the balloon was pulled out. Blood supply was restored after 20 min ischemia. Rabits in Group B were treated in the same way except that phosphate buffer solution（PBS） of the volume was given insteat of stem cells. Blood（3 ml） was collected from central ear artery before operation and at 7 days and 14 days postoperatively for the measurement of VEGF and TGF-β1 by enzyme-linked immunosorbent adsorption assay（ELISA）. At the end of the experiment, the imjured vascular tissue was stained with HE and the pathological changes before and after transplantation were observed. Paired t-test, variance analysis LSD multiple comparison, bivariate regression and correlation analysis were used for statistical analysis. Results（1） The VEGF levels in preoperative group A, group B and group C were no significantly difference（F = 0.000, P = 0.978）; 7d after operation the VEGF level of group B was significantly higher than that of group A（t = 2.286, P = 0.045）, the VEGF level in group C was higher than both groups A and B（t = 6.196, P = 0.000; t = 2.336, P = 0.042）; VEGF levels were similar between group B and group at 14 days（t = 1.294, P = 0.225）; VEGF level was higher in group C than those in group A and group B（t = 6.738, P = 0.000; t = 2.641, P = 0.038）.（2） Preoperative TGF-beta1 levels were similar among the three groups（F = 0.003, P = 0.997）; At 7 day, TGF-betas level of group B was significantly higher than that of group A（t = 9.052, P = 0.000）. TGF-betas level of C group was higher than that of group A, but lower than that of group B（t = 2.675, P = 0.023; t = 3.104, P = 0.011）; At 14 day group B had higher TGF-beta1 level than group A（t = 3.443, P = 0.006）, and C（t = 2.932, P = 0.020）; TGF-beta1 levels were similar between group A and group B（t = 1.262, P = 0.236）.（3） The level of VEGF was negatively correlated with that of TGF-beta1（r =-0.539, P = 0.000）.（4） At 14 days, rabbits in group A had intact intima and lumen, while vascular intima hyperplasia and disrupted elastic layer was seen in group B, with narrowing lumen. Compared with group B, intimal hyperplasia and lumen stenosis of group C was significantly less obvious. Conclusion After interventional treatment the levels of serum VEGF and TGF-beta1 increase. In rabbits receving HCBSCT, the level of VEGF and TGF-beta1 was negatively correlated, which may contribute to improve endothelial function and improve vascular remodeling, and decrease postangioplasty restenosis.