同种异体脂肪干细胞移植促进大鼠骨骼肌功能的恢复

Transplantation of allogeneic adipose-derived mesenchymal stem cells promotes the recovery of skeletal muscle function

背景:超过骨骼肌自我再生能力的广泛损伤可导致不可逆的纤维化、瘢痕形成和功能丧失。以往研究证实,脂肪来源干细胞可用于各种肌组织的再生,其对骨骼肌急性损伤后功能恢复的影响鲜有报道。目的:观察同种异体脂肪干细胞移植对骨骼肌急性损伤大鼠骨骼肌再生与功能恢复的作用。方法:从10只SD大鼠皮下脂肪组织中分离脂肪间充质干细胞,进行流式细胞术鉴定和多向分化能力鉴定。将72只SD大鼠随机分为正常对照组、模型组、胶原蛋白组和脂肪间充质干细胞移植组,后3组建立腓肠肌急性钝挫伤模型,胶原蛋白组分5个注射点在腓肠肌肌腹注射共1 mL大鼠胶原蛋白Ⅰ,脂肪干细胞移植组分5个注射点在腓肠肌肌腹注射含1×10~6个脂肪干细胞的1mL大鼠胶原蛋白Ⅰ。移植后7,14,28d,苏木精-伊红染色观察腓肠肌结构并测量腓肠肌纤维横截面积;用张力传感器测量腓肠肌等长收缩肌力;称量腓肠肌湿质量并计算其与体质量比值;Western blot检测腓肠肌组织中Pax7、MyoG、MyoD的表达。结果与结论:①大鼠脂肪干细胞表达CD29和CD90,不表达CD31和CD34,具有成脂、成骨和成软骨分化能力;②在移植后28d,脂肪干细胞移植组大鼠腓肠肌损伤部位可观察到新生的骨骼肌纤维,4组大鼠腓肠肌损伤部位均未观察到明显的纤维增生;③在移植后28 d,脂肪干细胞组腓肠肌湿质量及其与体质量比值均高于模型组和胶原蛋白组(P<0.01),脂肪干细胞移植组腓肠肌中肌纤维横截面积大于模型组和胶原蛋白组(P <0.01);④移植后7,14,28 d,模型组、胶原蛋白组、脂肪干细胞移植组腓肠肌等长收缩肌力均低于正常对照组(P <0.01),移植后28 d,脂肪干细胞移植组腓肠肌等长收缩肌力高于模型组和胶原蛋白组(P <0.01);⑤移植后14 d,脂肪干细胞移植组腓肠肌组织中Pax7和MyoD的表达高于模型组和胶原蛋白组(P <0.05),移植后28 d,脂肪干细胞移植组腓肠肌组织中MyoD和MyoG的表达高于模型组和胶原蛋白组(P <0.05);⑥结果表明,脂肪干细胞移植到急性受损骨骼肌中能促进肌纤维的再生和功能恢复。

BACKGROUND:An extensive damage beyond the ability of skeletal muscle to regenerate itself can lead to irreversible fibrosis,scarring and dysfunction.Previous studies have shown that adipose-derived mesenchymal stem cells can be used for the regeneration of various muscle tissues.The effect of stem cells on functional recovery after acute skeletal muscle injury has rarely been reported.OBJECTIVE:To study the effect of allogeneic adipose-derived mesenchymal stem cells transplantation on skeletal muscle regeneration and functional recovery in rats with acute skeletal muscle injury.METHODS:Adipose-derived mesenchymal stem cells were isolated from subcutaneous adipose tissues of 10 Sprague-Dawley rats and identified by flow cytometry for multidirectional differentiation.Seventy-two Sprague-Dawley rats were randomly divided into normal control group,model group,collagen group and adipose-derived mesenchymal stem cells transplantation group.A rat model of acute blunt injury of the gastrocnemius muscle was established in the latter three groups.Then,1 mL of type collagenⅠalone or containing 1×10~6 adipose-derived mesenchymal stem cells was injected into the gastrocnemius muscle in the collagen and adipose-derived mesenchymal stem cells transplantation groups,respectively,and there were five injection sites per rat.Hematoxylin-eosin staining was used to observe the structure of gastrocnemius muscle and measure the cross-sectional area of gastrocnemius muscle fiber 7,14 and 28 days after transplantation.The isometric contraction force of gastrocnemius muscle was measured.The wet weight of gastrocnemius muscle was measured and the ratio of wet weight to body weight was calculated.The expressions of Pax7,MyoG and MyoD in gastrocnemius muscle tissue were detected by western blot assay.RESULTS AND CONCLUSION:Isolated rat adipose-derived mesenchymal stem cells were positive for CD29 and CD90,and negative for CD31 and CD34.These cells had the ability of adipogenesis,osteogenesis and chondrogenesis.Neonatal skeletal muscle fibers were observed in the injured site of the gastrocnemius muscles at 28 days after adipose-derived mesenchymal stem cells transplantation,and no fibrogenesis was observed in the gastrocnemius muscles of the four groups.At 28 days after transplantation,the wet weight of gastrocnemius muscle and its ratio to body weight in the adipose-derived mesenchymal stem cells transplantation group were higher than those in the model group and collagen group(P<0.01),and the cross-sectional area of muscle fibers in the gastrocnemius muscle in adipose-derived mesenchymal stem cells transplantation group was larger than that in the model group and collagen group(P<0.05).At 7,14,and 28 days after transplantation,the isometric contractile muscle strength of the gastrocnemius muscle in the model group,collagen group and adipose-derived mesenchymal stem cells transplantation group were lower than that in the normal control group(P<0.01).At 28 days after transplantation,the isometric contractile muscle strength of the gastrocnemius muscle in the adipose-derived mesenchymal stem cells transplantation group was higher than that in the model group and collagen group(P<0.01).The expressions of Pax7 and MyoD in the gastrocnemius muscle of adipose-derived mesenchymal stem cells transplantation group were higher than those of the model group and collagen group at 14 days after transplantation(P<0.05).The expressions of MyoD and MyoG in the gastrocnemius muscle of adipose-derived mesenchymal stem cells transplantation group was higher than that of model group and collagen group at 28 days after transplantation(P<0.05).To conclude,adipose-derived mesenchymal stem cells transplantation for acute skeletal muscle injury can promote the regeneration of muscle fibers and functional recovery.