Primary Study Of The Cell Dedifferentiation During The Healing Process Of Injured Skeletal Muscle

The contribution of the stem cell is well known and accepted, because of the two important biology features:multidifferentiation and self-renewal. However, obtaining embryonic stem cell is a traumatic operation and also limited by ethics and moral norm, while the role of mature cell dedifferentiation is always disputed by researchers. The behavior of cell dedifferentiation is the reflection of orgnism protection and adaptability during the long-term evolutionary process, which can be controled and finite proliferation. Due to the ability of cell dedifferentiation, the newt has powerful regeneration capability. This theory is widespreadly approved. However, the local and whole surroundings have an effect on the regeneration that would lead to restricted ability of cell dedifferentiation for mammalian. Therefore, the function of cell dedifferentiation of wound healing in mammlian couldn't be sufficiently considered. During the process of wound healing in mammlian,how do the cell dedifferentiation changed in histomorphology? Is there any relationship between dedifferentiation and cell apoptosis? We established the damaged model of skeletal muscle, then observed the histomorphology change and analysed the relationship between dedifferentiation and cell apoptosis. Owing to the two important biology features:multipotency and self-renewal, the research of stem cell is very popular. But the harvesting of embryo-stem cell is traumatic operation and also wrapped by ethnic and moral norm.A new chemical material called Reversine developed by Chen could induce mature cell dedifferentiation to similar stem cell. Induced by different materials, the similar stem cell could become osteoblast and adipocyte. Scientists have expored the mechanism of action by Reversine. However, it is still to be solved that cell growth during the process of cell dedifferentiation treated by Reversine. We established the C2C12 myoblast cell line in vitro to research the proliferation and differentiation treated by Reversine. It will provide a experimental reference for wound healing of damaged mammalian tissue in the field of regeneration.To observe the histological and morphologic change of damaged skeletal muscle; To sutdy the cell growth of C2C12 myoblasts induced by Reversine.1 The model of hind limb damaged gastrocnemius of Sprague-Dawley rats was made. The opened wounds were sealed for up to 27 days.1.1 Morphologic change of damaged skeletal muscle was observed by transmission electron microscope (TEM).1.2 The histologica change of damaged skeletal muscle was observed by HE staining and immunity fluorescence.2 C2C12 cell lines were treated with 1μM Reversine for 12 h and 24 h respectively.2.1 Then flow cytometry analysis was used to detect the apoptosis of the three groups with the C2C12 myoblast stained by Annexin-V/PI.2.2 C2C12 myoblast cell was divided into five groups:A group was control group, B group was 1μM Reversine added only for 7 d, C group was bone induction factor added only for 7 d, D group was 1μM Reversine induced by 12 h and then bone induction factor added only for 7 d, E group was 1μM Reversine induced by 12 h and then 1μM Reversine combined with bone induction factor added for 7 d. The morphological change of C2C12 myoblast cell was observed by light microscope. The expression of Inhibitor of differentiation (Id2), muscle regulatory factor (Myogenin) and myogenic factor (Desmin) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in five groups.1 TEM showed that muscle fiber was fibronolysis and primitive cell was appeared after damaged.2 HE staining showed that Compared with control group, cells were disarranged and cellular nucleus were massive aggregated in damaged skeletal muscle group. The damaged tissue also had larger shape than controls. The apocyte changed into monocyte, which illustrate the monocyte came from muscle fiber and then proceeded dedifferentiation in order to play the role of reparative regeneration.3 Our result indicated that AIF expression was higher in treated group than controls.Interestingly, the over-expression of AIF was almost appeared in the cellular nucleus. Cell autophagy induced by AIF advanced the process of cell immature.Meanwhile AIF may provid some kind of signaling molecule for cell dedifferentiation in order to play the role of reparative regeneration.4 Compared with A group, there was significant apoptosis of C2C12 cells in C group with 1μM Reversine treated by 24 h, while no significant apoptosis in B group with 1μM Reversine treated by 12 h.5 Compared with D group, cell proliferation was significantly inhibited in E group and H group, cell was proliferation and gradual fusion in F group and the G group.6 Compared to control group, the expression of Id2 was significant enhanced with 1μM Reversine 12h treated, which indicated that the cell reproductive activity was significant enhanced. Simultaneously, the expression of Myogenin and Desmin was obviously weakened, which manifest Tolerance cell treated with Reversine differentiation ability was descended and cell growth was inhibited.1 The cell dedifferentiation phenomenon may exist during the process of wound healing of damaged muscle.2 Reversine significantly inhibits the C2C12 myoblasts proliferation and differentiation effect during the process of dedifferentiation induced by Reversine. The expression of some specific myogenic transcription factors: Myogenin and Desmin was also significantly inhibited.