脂肪来源干细胞促进放射损伤骨骼肌血管生成的实验研究

The experimental study of adipose-derived stem cells promoting vascular growth in irradiated skeletal muscle

目的 观察新西兰兔脂肪来源干细胞（adipose-derived stem cells,ASCs）植入放射损伤骨骼肌后的微血管密度改变,分析组织中血管内皮生长因子（VEGF）和碱性成纤维生长因子（bFGF）的变化,探讨ASCs移植对兔放射损伤骨骼肌血管生成的影响，方法 64只实验动物单侧臀部予9 MeV电子线一次性照射80 Gy,采用随机数字表法分为ASCs组和磷酸缓盐冲溶液（PBS）组,每组为32只，照射后24 h,ASCs组和PBS组照射侧分别肌肉注射1 ml含5×10^7个ASCs的细胞悬液及PBS，免疫组织化学法检测照射后1、4、8和26周骨骼肌组织中CD31阳性的微血管、VEGF和bFGF的表达,蛋白印迹法检测骨骼肌组织中VEGF、bFGF蛋白表达,ELISA法检测ASCs在条件培养基中分泌的VEGF、bFGF浓度,结果 ASCs组受照射骨骼肌组织中CD31染色阳性的微血管密度较PBS组在照射后4、8和26周增高（t=8.14、7.44、18.58,P＜0.05）.ASCs组受照射骨骼肌组织VEGF、bFGF的累计光密度值（IOD）较PBS组在照射后1、4、8和26周增高（VEGF：t=3.13、4.66、2.19、6.79,P＜0.05;bFGF：t =3.14、3.84、4.28、3.61,P＜0.05）,ASCs组VEGF蛋白的表达水平较PBS组在照射后1、4、8和26周增高（t=4.28、5.02、4.24、4.56,P＜0.05）,ASCs组bFGF蛋白的表达水平较PBS组在照射后4、8和26周增高（=3.36、3.16、5.77,P＜0.05）,ASCs在放射损伤肌肉匀浆上清条件培养基中较在正常肌肉匀浆上清条件培养基中分泌的VEGF浓度在48、72和96 h增高（t=2.81,9.96,4.75,P＜0.05）,分泌的bFGF浓度在72和96 h增高（t=3.80,4.33,P＜0.05），结论 ASCs能上调骨骼肌放射损伤组织VEGF和bFGF分泌、增加新生血管形成,可能是修复骨骼肌放射损伤的部分机制。

Objective To investigate the mechanisms in the function of adipose-derived stem cells （ASCs） in angiogenesis of the radiation-induced skeletal muscle injury.Methods Totally 64 New Zealand rabbits were randomly divided into ASCs and PBS group （each with 32） after a monodose irradiation of 80 Gy by electron beam.The irradiated buttocks were injected with 5 ×10^7 allogeneic ASCs labeled with CM-Dil suspended in 1 ml PBS or 1 ml PBS respectively via local intramuscular 24 h after irradiation.Immunohistochemical staining was performed to evaluate the expression of VEGF,bFGF and CD31 in the muscular tissues.Western blot was performed to evaluate the expression of VEGF and bFGF protein in the muscular tissues.ELISA was used to test VEGF and bFGF secreted by ASCs in conditioned medium.Results After irradiation,the density of microvessel positively stained with CD31 showed remarkable increased after ASCs implantation at 4,8 and 26 weeks compared with PBS group,respectively （t =8.14,7.44,18.58,P 〈 0.05）.The integrated optimal density （IOD） of VEGF and bFGF was all significantly higher than PBS group at 1,4,8,and 26 weeks on each section of ASCs group,respectively （VEGF：t=3.13,4.66,2.19,6.79,P 〈0.05; bFGF：t =3.14,3.84,4.28,3.61,P 〈0.05）.Moreover,the expression levels of VEGF protein which detected using western blot increased at 1,4,8 and 26 weeks in the ASCs groups compared with those of the PBS group （t =4.28,5.02,4.24,4.56,P 〈 0.05）.The expression levels of bFGF protein detected using Western blot showed increase at 4,8,and 26 weeks in the ASCs groups compared with PBS group （t =3.36,3.16,5.77,P 〈 0.05）.The expression levels of VEGF secreted by ASCs in irradiated muscle supernatant were higher than those secreted by ASCs in normal muscle supernatant at 48,72,and 96 h,respectively （t =2.81,9.96,4.75,P 〈 0.05）.The expression levels of bFGF secreted by ASCs in irradiated muscle supernatant were higher than those in normal muscle supernatant at 72 h and 96 h,respectively （t =3.80,4.33,P 〈 0.05）.Conclusions ASCs transplantation could up-regulate the expressions of VEGF and bFGF,and promote the angiogenesis of the irradiated muscle tissues,which may be partly associated with the repair of radiationinduced skeletal muscle injury.