| Chapter 1:Isolation,primary culture,and characterizing the immunofluorescence of muscle satellite cellsObjective:To identify the morphological changes of extraocular muscle satellite cells(ESC)in vitro.To compare the expression of immunofluorescene of ESC with that of skeletal muscle satellite cells (SSC).Methods:To isolate and culture ESC in vitro by muscle block enzyme digestion.Light microscopy was used to monitor the morphological changes of the cells during the period of their growth.And to compare immunofluorescene characters of ESC with that of SSC, which are the marker of myotube(Mosin heavy chain-neonatal,MyHC-n), myogenic markers(Desmin and Myogenin,F5D),marker of myoblast (α-smooth muscle actin,α-SMA),marker of striated muscle(light meromyosin,MF20),and marker of extraocular muscle(myosin heavy chain-extraocular specific,N2.261).Results:ESC and SSC had the same appearance on the morphological changes while growing in vitro.They came out from first day after seeding the muscle fibers.The cells adhered to the matrix in 1 week,expanded,and fused to each other.Cell mass also found after 2 weeks,which located nearby one of the residual independent myofibril and myotube formed radially on it(MyHC-n were stained positive). 3-dimensional meshwork formed shortly after myotube merged,which showedα-SMA positive.Both ESC and SSC stained as positive results in those antibodies,such as Desmin,Myogenin,α-SMA,and MF20.It was only differed in N2.261 staining between ESC and SSC,while ESC+ and SSC-.Conclusion:ESC has been isolated and cultured in vitro successfully. The morphological and immuno-fluorescene characters are intact features of ESC just as SSC are.Both of the two myogenic cells are able to be self-expanded and differentiated to form myofibril,they have same features of regeneration and myogenic potential in vitro. Chapter 2:Establishing laceration model of extraocular muscle in rabbit and treating those rabbits by transplanting autologous adult stem cellsObjective:To establish laceration model of extraocular muscle in rabbits and find its nature of wound healing.To evaluate the effect of auto-transplantation by adult stem cell on the process of wound healing in this model.Methods:Established the model of wound healing in eye by lacerating the extraocular muscle.The muscle samples were harvested, frozen,sliced and stained by Masson's trichrome staining.Imaging processing(using software of ImageProPlus,IPP)was used to quantify the process of natural healing.4 targets were analyzed.They were: muscle(M,collagen(C),non-collagen(NC)and nuclei(N).Bone marrow mesenchymal stem cells(MSC)were isolated,cultured and expanded in vitro by a putative method.ESC and SSC were labeled by Cell tracker CM-DiI,and transplanted and traced the destiny of transplanted cells in the traumatic area.Both ESC and SSC transplantation were performed to the wound area 1 week after trauma.5 groups were set up as two treatment groups(MSC and SSC transplantation)and three control groups,which were:negative control group 1 and 2,positive control group.The morphological changes were recorded by Masson's staining and analyzed by IPP. Results:Laceration accelerated the formation of collagen in early stage of wound healing process.Apparent fibrosis showed up just one week after being suffered laceration.Diffused fibrosis and scar emerged dramatically after two weeks.Number of nuclei reached its peak at day 14.Inflammatory cells and fibrosis developed heavier than ever.CM-DiI labeled cells could fuse to adjacent muscles from first day on and stayed in site at least for 14 days.Positive stained muscle fibers were found abounded in the wound area at day 14.Cell transplantation resulted of consistent muscle area at different time points of all groups.In comparison of treatment groups and positive control groups,the later got significant higher amount of non-muscle connective tissue(including collagen and non-collagen part)statistically(P＜0.05),while they showed the same level of increasing amount of non-muscle tissue(P＞0.05). Non-muscle tissue proliferated dramatically after 2 weeks of transplantation while compared with earlier stages(P＜0.05)in all groups. Number of nuclei fluctuated within these 2 weeks,which showed low level at day 7 and high level at day 3 or 14.SSC and MSC played similar way in benefiting the mechanical trauma of extraocular muscle.Conclusions:Laceration could lead to degeneration of both muscle and the surrounding connective tissue,which act as fibrosis and scar formation.Both MSC and SSC has good consistent with traumatic muscle in ocular environment.And they could slow down the development of fibrosis and scar formation,and also benefit the process of wound healing of extraocular muscle in the way of upgrading the regeneration and downgrading the fibrosis.
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