人羊水来源干细胞参与小鼠损伤肌肉修复的实验研究

目的研究人羊水来源干细胞(human amniotic fluid colony derived stem cells,hAFCSCs)是否参与小鼠肌肉损伤修复过程,探讨应用hAFCSCs治疗肌肉损伤的方法及可行性。方法 B超引导下穿刺抽取人孕中期羊水,体外分离、培养得到hAFCSCs,取第6～8代细胞备用,同时提取总RNA行RT-PCR鉴定干细胞相关基因。取16只6～8周龄Nod/Scid小鼠,体重20～24 g,利用心肌毒素联合X线照射建立双侧胫骨前肌肌肉损伤模型,右侧胫骨前肌内注射hAFCSCs(3.3×107个/mL)30μL作为实验组,左侧胫骨前肌同法注射等量完全培养液(含15%FBS、18%Chang B、2%Chang C、1%青链霉素、1%L-谷氨酰胺的α-MEM培养基)作为对照组。细胞移植术后2、4周各处死小鼠8只,取材行肝细胞生长因子受体(c-Met)、成肌调节因子(Myf-5)、层粘连蛋白(Laminin)、结蛋白(Desmin)与人特异性细胞核有丝分裂器(NuMa)组织免疫双重荧光染色观察。结果原代hAFCSCs培养5～7 d后可见细胞克隆形成;8～10 d后可挑取细胞形态均一的克隆进行稳定传代,6～8代后可获得足量的干细胞。RT-PCR检测示所获得hAFCSCs体外扩增培养至第6代仍表达干细胞相关基因。细胞移植术后2周免疫双重荧光染色示,实验组胫骨前肌内检测到NuMa表达,未检测到hAFCSCs表达肌源性细胞相关表型;术后4周,实验组胫骨前肌内检测到NuMa表达,部分细胞同时表达NuMa和c-Met、Myf-5,部分肌纤维同时表达NuMa和Desmin、Laminin。术后2、4周,对照组胫骨前肌均未检测到NuMa表达。结论 hAFCSCs体外培养后移植到小鼠肌肉损伤部位,可参与损伤肌肉的修复过程。

Involvement of human amniotic fluid colony derived stem cells in regeneration of mouse injured muscle

Objective To study whether human amniotic fluid colony derived stem cells(hAFCSCs) are involved in regeneration of injured muscles in mice and to investigate the method and feasibility of hAFCSCs-based cytotherapy in the treatment of injured muscles.Methods Human second-trimester amniotic fluid was collected through ultrasound-guided amniocentesis,hAFCSCs were isolated from second-trimester amniotic fluid and cultured,and the cells at 6th-8th passages were spared.The mRNA was extracted to identify the stem ...