Myf-5在脂肪细胞成肌诱导分化中的作用及其机制

Autologous adipose tissue has become an ideal donor for the muscle transdifferentiation because of its efficiency and easy accessibility. Myogenic regulatory factor Myf-5 is an important molecule involved in skeletal muscle cells during growth and development. Studies have confirmed that adipose derived stem cells and dedifferentiated fat cells, which are isolated from mature adipocytes using the ceiling culture method,both possess myogenic potentials.By in vitro tissue culture system,our group has successfully made adipocytes induced differentiation from a part of the cells in rat pedicled adipose tissue to skeletal muscle-like cells.Our previous study also show that a certain proportion of Myf-5 positive stem cells can be isolated and identified from human subcutaneous adipose tissues.The role and the mechanisms of Myf-5 and Myf-5 positive fat stem cells in fat tissues differentiating into myogenic cells remain to be elucidated. We hypothesize that Myf-5 positive cells and Myf-5 molecules can promote dedifferentiated fat cells being induced into muscle cells, and expression regulation of Myf-5 molecules will contribute to the regulation of adipose tissue into muscle differentiation rate and efficiency. In this study, we will separate Myf-5 positive adipose derived stem cells subpopulations using flowcytometry,and study their muscular differentiation potential.And then the effects of modulating the expression of Myf-5 on fat cell phenotype in myogenic differentiation process will be studied.At the same time,the signal transduction mechanisms involved in the myogenic differentiation process of co-cultured dedifferentiated fat cells with adipose derived stem cells will be observed. The research results will reveal the mechanism of differentiation of cells derived from fat tissues into skeletal muscle cells, and help to seek a key target in myogenic differentiation,which will improve the myogenic differentiation speed and efficiency induced from cells derived from fat tissues.

自体脂肪组织是肌肉转化，修复骨骼肌缺损的理想供体。成肌调节因子Myf-5是骨骼肌细胞生长发育过程中的重要分子。已有研究证实，脂肪干细胞和去分化的脂肪细胞都具有成肌潜能。本课题组已成功诱导脂肪组织分化成肌，并在脂肪干细胞中鉴定出一定比例的Myf-5阳性细胞群。Myf-5以及Myf-5阳性的脂肪干细胞群在脂肪组织来源细胞成肌诱导分化中的作用及其机制尚待阐明。我们推测Myf-5分子的表达可以促进去分化的脂肪细胞诱导成肌，调节Myf-5分子的表达量将有助于调控脂肪组织成肌诱导分化的效率。本课题拟采用流式细胞仪分选脂肪干细胞中Myf-5阳性的细胞亚群，研究该群细胞的成肌潜能；明确干预myf-5的表达在成肌诱导分化过程中对脂肪细胞表型的影响；同时观察相关的信号转导机制在脂肪源性干细胞与去分化的脂肪细胞共培养成肌转化中的作用。探究脂肪组织来源的细胞成肌分化的机制和规律，寻求干预靶点，促进脂肪的成肌转化。

本课题着眼于研究来源于脂肪组织的细胞，包括脂肪干细胞（ADSCs）、去分化的脂肪细胞（DFAT）、成熟的脂肪细胞（MAs）3种主要细胞的成肌分化能力，主要通过细胞及其转分化功能实验试图寻找提高脂肪组织诱导成肌效率的方法。由于在前期工作中发现血管基质片段（SVF）中约半数以上的细胞Myf-5分子表达阳性，结合已有的关于小鼠脂肪干细胞成肌分化的文献报道，提出的研究假说认为Myf-5阳性的干细胞亚群在脂肪组织的成肌诱导过程中起到重要的作用。本研究成功分离培养和鉴定了脂肪组织来源的两种具有干细胞特性的细胞ADSCs和DFAT，通过体外诱导实验发现DFAT比ADSCs的成脂、成软骨以及成肌能力更强，而ADSCs比DFAT的成骨能力更强。通过免疫荧光染色和western blot证实myf-5、myod等肌细胞的分子标记在ADSCs、DFAT和MAs中均有较广泛的、一定量的基础表达。免疫组化染色结果证实Myf-5广泛表达于脂肪组织中，生肌诱导液体外灌注培养人源脂肪组织3周，小部分脂肪组织可以转分化为类肌样组织。进一步的细胞诱导成肌实验证实，在本课题的诱导条件下DFAT的成肌能力比ADSCs更强。经与骨骼肌细胞直接共培养，均可以增强ADSCs和DFAT诱导成肌的效率。为了研究这两种细胞不同的成肌信号转导途径，用IGF-1对其进行了干预，结果表明尽管IGF-1作用都能够促进Akt的磷酸化，但是对于ADSCs的促进作用强于DFAT，并且其对促进ADSCs成肌的作用更明显，而DFAT对成肌诱导液本身的成肌作用较IGF-1更敏感。提示了在相同的诱导环境下，ADSCs和DFAT可能具有不同的成肌途径。尽管本课题的工作未能如预期分离到干细胞的亚群，但是对于脂肪组织来源的细胞成肌分化潜能及其机制进行了有益的探索，证实了体外灌注培养人源的脂肪组织可以获得少量的类肌样组织，发现了DFAT比ADSCs具有较强的成肌诱导优势，尽管其信号途径可能不完全相同，但是与骨骼肌细胞的共培养均有助于促进这两种细胞的成肌分化。