静脉注射移植ADSCs对兔早期激素性股骨头坏死的干预作用及其机制

Interventional effect and mechanism of intravenous injection of ADSCs on early steroid-induced femoral head necrosis in rabbits

目的观察静脉注射移植脂肪源性干细胞(简称脂肪干细胞)对兔早期激素性股骨头坏死的干预作用,并探讨其可能的机制。方法于3只新西兰大白兔颈背部脂肪组织中分离脂肪干细胞,取第3代脂肪干细胞制成细胞悬液。取42只新西兰大白兔,采用马血清联合激素法建立兔早期激素性股骨头坏死模型,从造模成功的兔中选取36只,随机分为模型组、局部注射组及静脉注射组,每组12只。局部注射组行左髋关节穿刺并注射5 mL脂肪干细胞,静脉注射组兔左耳缘静脉注射5 mL脂肪干细胞,模型组不作任何处理。干预后第4周、第8周,记录局部注射组及静脉注射组感染情况。各组随机取6只兔,处死后取左侧股骨头。比较其股骨头X线片及组织病理学表现,采用实时荧光定量PCR法检测股骨头组织骨形态蛋白2(BMP-2)mRNA相对表达量,采用免疫组化法检测股骨头组织血管内皮生长因子(VEGF)蛋白表达。结果局部注射组1只于注射脂肪干细胞10天,左髋部出现局部红肿、皮温高,至注射2周后消退,余兔注射部位无感染情况。静脉注射组局部均未出现感染表现。干预后第4周:模型组股骨头形态呈"蘑菇头"样改变,骨小梁变细,空骨陷窝增多,造血组织减少。局部注射组、静脉注射组骨小梁稀疏,空骨陷窝数量均少于模型组,造血组织减少,脂肪细胞稍增多,两组股骨头X线片及组织病理学表现无明显差异。干预后第8周:模型组股骨头变扁,空骨陷窝较前增多,部分骨小梁断裂,造血组织明显减少。局部注射组、静脉注射组股骨头无明显塌陷,可见少量空骨陷窝,两组股骨头X线片及组织病理学表现无明显差异。干预后第4周、第8周,局部注射组、静脉注射组股骨头组织BMP-2 mRNA相对表达量及VEGF蛋白相对表达量均高于模型组(P均<0.05),而局部注射组与静脉注射组股骨头组织BMP-2 mRNA相对表达量及VEGF蛋白相对表达量比较差异均无统计学意义(P均>0.05)。结论静脉注射移植脂肪干细胞对兔早期激素性股骨头坏死的干预效果与股骨头局部注射移植脂肪干细胞效果相当;提高股骨头组织BMP-2及VEGF表达是其可能的作用机制。

Objective To observe the intervention effect of intravenously transplanted adipose-derived stem cells(ADSCs)on early steroid-induced femoral head necrosis in rabbits and to explore its possible mechanism. Methods ADSCs was isolated from adipose tissues of neck of 3 New Zealand white rabbits,and ADSCs of the the third generation were used to make cell suspension.Forty-two healthy New Zealand white rabbits were selected to establish models of early SANFH by using horse serum combined with hormones.After X-ray and MRI examination,we confirmed the success of modeling,thirty-six rabbits were randomly selected from the successful rabbits and then were divided into the model group,the local injection group,and the intravenous injection group,with 12 rabbits in each.In the local injection group,5 mL ADSCs were injected into the left hip joint,and 5 mL ADSCs were injected into the left ear vein of the intravenous injection group.No treatment was done in the model group.At the 4th week and the 8th week after the intervention,6 rabbits were randomly selected from each group.After the sacrifice,the left femoral heads were taken and the femoral head X-ray film and histopathological findings were compared.The relative expression of bone morphogenetic protein 2(BMP-2)mRNA in the femoral head tissues was detected by real-time fluorescent quantitative PCR.The expression of vascular endothelial growth factor(VEGF)protein in the femoral head tissues was detected by immunohistochemistry.Results In the local injection group,local infection was observed in one rabbit after injection of ADSCs,but no infection was observed in the intravenous group.At the 4th week after the intervention,the model group,the local injection group,and the intravenous injection group showed a decrease in the density of the femoral head and a cystic change,and the shape of the femoral head was irregular.The trabecular bone of the model group was unclear,and the shape of the femoral head changed like a mushroom head;the trabecular bone became thinner,the empty bone sag increased,and the hematopoietic tissue decreased.In the local injection group and the intravenous injection group,the trabecular bone was sparse,and the empty bone lacuna was seen,but the number was less than that of the model group,the hematopoietic tissues were reduced,and the fat cells were slightly increased.At the 8th week after the intervention,the cystic changes in the model group were aggravated,the femoral head was flattened,the empty bone sag was increased,some trabecular bone was broken,and the hematopoietic tissue was significantly reduced.In the local injection group and the intravenous injection group,the shape of the femoral head was not smooth,and there was no obvious collapse.A small amount of empty bone lacuna was observed.At the 4th week and the 8th week after intervention,the relative expression of BMP-2 mRNA and VEGF protein in the local injection group and the intravenous injection group was higher than that in the model group(P<0.05).There was no significant difference in the expression of BMP-2 mRNA and VEGF protein between the local injection group and the intravenous injection group(P>0.05).Conclusion The effect of intravenous injection of ADSCs on early steroid-induced femoral head necrosis in rabbits is comparable to that of local injection of ADSCs in the femoral head,and improving the expression of BMP-2 and VEGF in femoral head may be the possible mechanism.