人头颈部骨骼肌干细胞的分离培养及生长特性研究

Isolation, culture and growth characteristics of human muscle stem cells

目的体外建立人骨骼肌干细胞的纯化及鉴定方法，并对所分离干细胞的生物学特性进行分析研究。方法分离人头颈部正常骨骼肌组织，采用无血清培养基，悬浮状态培养技术得到人骨骼肌干细胞，体外扩增并观察生长特性。细胞免疫荧光染色和反转录聚合酶链反应（RT—PCR）技术鉴定标记物的表达情况，单细胞克隆观察单个细胞生长特性。将分离来的细胞分别转移至采用成平滑肌、成脂、成骨诱导培养基内，观察细胞的生长分化特性，并通过特异性染色予以鉴定。结果悬浮培养条件下，倒置相差显微镜观察示骨骼肌干细胞在培养基内不断增殖形成细胞球。免疫荧光染色示细胞球表达卫星细胞的标记物Pax7及ALDH1，贴壁生长扩增传代后表达肌原细胞标记物Myod及Desmin；RT—PCR检测示表达干细胞标记物0ct3／4，Nanog，Sox2和Pax7；单克隆形成实验显示单个细胞可在悬浮状态下形成新的细胞克隆。骨骼肌细胞在体外可以诱导分化为平滑肌、骨和脂肪细胞。结论在体外可以成功分离、纯化和扩增人骨骼肌干细胞，其具有干细胞的自我更新和多向分化潜能。

Objective To establish the methods for purification, culture, and identification of adult human skeletal muscle stem cells in vitro and to explore the biological properties of the cells. Methods Muscle stem cells were obtained by reformed enzymatic digestion of muscle tissue from the consenting donors and cultured in serum-free medium. The morphology was inspected by an inverted phase contrast microscope. Phenotypic characteristics of the cells and expression of cell-specific markers were determined using reverse transcription-polymerase chain reaction （RT-PCR） and immunocytochemistry. The growth of single cells in suspension culture was observed and recorded continuously. The cells were analyzed for their muhi-lineage differentiation potential into osteoblastic, adipocyte, and smooth muscle cell lineages. Results Primary cultured human skeletal muscle stem cells proliferated and formed the big spheres when cultured with serum free medium. Immunofluorescence staining displayed Pax7 and ALDH1 positive expression in the cell spheres. Furthermore, Myod and Desmin showed positive expression in the monolayer cells derived from the spheres. The gene expressions of Oct3/4, Nanog, Sox2 and Pax7 in the cells were determined by RT- PCR. The cell clones formed from single cells grew well. In addition, they were capable of spontaneous differentiation into myotubes in differentiation medium and into other mesodermal cell lineages in induction medium. Conclusions Human muscle stem ceils with properties of self-renewal capacity and multidifferentiation could be successfully isolated and expanded in vitro.