人羊水集落衍生干细胞参与小鼠损伤肌肉的再生

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

作者信息

Ma Xiaorong, Zhang Shengli, Shang Yafeng, Gao Tongbin, Wang Xue, Chen Fang, Zhou Junmei

机构信息

Central Laboratory, Shanghai Children's Hospital Affiliated to Shanghai Jiaotong University, Shanghai, 200040, P.R.China.

出版信息

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2011 Jul;25(7):842-7.

PMID:21818952

Abstract

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

s 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 cell related genes by RT-PCR. The muscular injury model of bilateral tibialis anterior muscle was established by cardiotoxin and X-ray irradiation in 16 Nod/Scid mice (aged 6-8 weeks, and weighing 20-24 g). The hAFCSCs (3.3 x 1(0)7/mL, 3 microIL) were injected into the right injured tibialis anterior muscles as the experimental group, while the same volume of complete medium (lphaa-MEM containing 15%FBS, 18%Chang B, 2%Chang C, 1% penicillin-streptomycin, and 1% L-glutamine) was injected into the left injured tibialis anterior muscles as the control group. At 2 and 4 weeks after cell transplantation, the immunofluorescence staining of tibialis anterior muscles was performed to detect hepatocyte growth factor receptor (c-Met), myogenic regulatory factor (Myf-5), Laminin, Desmin, and human specific nuclear mitotic apparatus protein (NuMa).

RESULTS

s The clone formation was observed at 5-7 days of primary hAFCSCs culture; after 8-10 days, the clones with homogeneous morphology were selected for subculture. Adequate stem cells were available after 6th-8th subculture. RT-PCR analysis showed that hAFCSCs expressed mRNA of the stem cell related genes. The immunofluorescence double-staining showed that NuMa expressed in tibialis anterior muscles of the experimental group and no myogenic phenotype expressed at 2 weeks after cell transplantation, and that single cell co-expressed NuMa and c-Met or Myf-5 at 4 weeks after cell transplantation. In some myofibers, NuMa and Laminin or Desmin were also co-expressed. No NuMa positive hAFCSCs were detected in the control group at 2 and 4 weeks after cell transplantation.

CONCLUSION

n hAFCSCs can participate in the regeneration of injured mouse muscle.

摘要

目的

研究人羊水来源的集落干细胞（hAFCSCs）是否参与小鼠损伤肌肉的再生，并探讨基于hAFCSCs的细胞治疗在治疗损伤肌肉中的方法和可行性。

方法

通过超声引导下羊膜穿刺术收集人孕中期羊水，从孕中期羊水中分离并培养hAFCSCs，保留第6 - 8代细胞。提取mRNA，通过RT-PCR鉴定干细胞相关基因。对16只Nod/Scid小鼠（6 - 8周龄，体重20 - 24 g）采用心脏毒素和X射线照射建立双侧胫前肌肌肉损伤模型。将hAFCSCs（3.3×10⁷/mL，3 μL）注射到右侧损伤的胫前肌作为实验组，而将相同体积的完全培养基（含15%胎牛血清、18%Chang B、2%Chang C、1%青霉素 - 链霉素和1% L - 谷氨酰胺的α - MEM）注射到左侧损伤的胫前肌作为对照组。细胞移植后2周和4周，对胫前肌进行免疫荧光染色，检测肝细胞生长因子受体（c - Met）、生肌调节因子（Myf - 5）、层粘连蛋白、结蛋白和人特异性核有丝分裂器蛋白（NuMa）。

结果

原代hAFCSCs培养5 - 7天观察到克隆形成；8 - 10天后，选择形态均匀的克隆进行传代培养。第6 - 8代传代培养后可获得足够的干细胞。RT-PCR分析表明hAFCSCs表达干细胞相关基因的mRNA。免疫荧光双染显示，实验组胫前肌中NuMa表达，细胞移植后2周无生肌表型表达，细胞移植后4周单个细胞共表达NuMa和c - Met或Myf - 5。在一些肌纤维中，NuMa也与层粘连蛋白或结蛋白共表达。细胞移植后2周和4周，对照组未检测到NuMa阳性的hAFCSCs。