Combined Gene Therapy With Human Interferon-γ Gene And Human Insulin-like Growth Factor-1 Gene For Skeletal Muscle Injury In The Mouse

On the basis of the research that exogenous hIGF-1 and myoblasts with hIGF-1 gene could promote muscle regeneration and mildly inhibit muscle fibrosis,we would emphasize about the effects of hIFNγon muscle fibrosis.After confirming the anti-fibrosis for skeletal muscle by exogenous hIFNγ,we constructed a recombinant plasmid pEGFP-C2-hIFNγand transfected C2C12 myoblasts.Following analyzing the expression and activity of hIFNγin transfected cells in vitro,the myoblasts with hIFNγgene or with hIGF-1 gene were combined to be transplanted into injured skeletal muscle.Then the survival potential and expression of different transfected cells were observed,and their effects for repair of injured skeletal muscle were explored.PartⅠCombined Injection with Exogenous hIFNγand hIGF-1 for Acute Skeletal Muscle Contusion in the MouseObjective To observe the effects of hIFNγor/and hIGF-1 on regeneration and fibrosis of skeletal muscle after acute contusion.Methods A standard contusion model was reproduced at the right gastrocnemius in 64 male mice of 7-12 weeks.All the mice were randomly divided into 4 groups,such as group A(injection with hIFNγ),group B(injection with hIGF-1),group C(injection with hIGF-1 and hIFNγ),and group D(injection with physiological saline).All injections were introduced on day 10 after injury at local injured gastrocnemius with different interventions.On day 7,14,28,42(eg,the day before intervention,and 4d, 18d,32d and intervention) following contusion,the local injured gastrocnemius were harvested of 4 mice from each group.Then the expression of MHC-Ⅱb and vimentin was detected by fluorescent quantitation PCR and immunofluorescence cytochemistry technology.The data were analyzed by one-way ANOVA using a statistical software package program(SPSS10.0),and were tested.The results were considered to be significant at p values＜0.05. Results(1)At the time following intervention,the expression of MHC-Ⅱb mRNA and protein in local injured muscle of group B and group C were significantly higher than those of group A and D;(2)After intervention,the expression of vimentin mRNA and protein in local injured muscle of group A,group B,and group C were more inhibited than those of group D.It was more significant for the inhibition of vimentin expression in group A and group C.Conclusions(1) It was indicated that hIGF-1 could enhance muscle regeneration,and inhibit fibrosis to some extent,by injection into the injured skeletal muscle following acute contusion;(2) It was identified that hIFNγinjected into injured muscle had the effect of anti-fibrosis,what is more significant than that of hIGF-1;(3)Combined injection with hIGF-1 and hIFNγcould improve muscle regeneration and inhibit fibrosis simultaneously,and promot the injured muscle healing.PartⅡExpression and Activity Detection of hIFNγProtein Following Construction and Transfection of pEGFP-C2-hIFNγinto C2C12 MyoblastsObjective To construct and transfect recombinant plasmid of pEGFP-C2-hIFNγinto the C2C12 myoblasts,and to observe the expression of hIFN gene and the activity of hIFNγprotein in the C2C12 myoblasts.Methods(1) with the technology of gene rearrangement,hIFNγgene was cloned into pEGFP-C2 plasmid;then its correctness was evaluated by the means of restriction enzyme analysis and sequencing;(2) pEGFP-C2-hIFNγwas transfected into myoblasts C2C12 with liposome mediated transfection;(3) After 24h,the transient expression of EGFP in the transfected C2C12 cells was observed under fluorescence microscope;(3) After transfection,the positive cell clones selected with G418 were culturing for 4 weeks;the hIFNγgene expression of transfected C2C12 cells were tested by RT-PCR and Western Blot analysis;(4) The WI-38 fibroblasts treated with TGFβ-1 were cultured with myoblasts with or without hIFNγgene,then theα-SMA expression of WI-38 cells were analyzed by fluorescent quantitation PCR and immunofluorescence cytochemistry technology.Results(1) Correct construction of pEGFP-C2-hIFNγwas identified by methods of restriction enzyme analysis and nucleotide;(2) Green fluorescence was emitted from transfected cells under fluorescent microscope after 24h;(3) the hIFNγexpression in myoblasts C2C12 transfected with pEGFP-C2-hγIFN was detected by RT-PCR and western blot analysis;(4) theα-SMA expression of WI-38 cells treated with TGFβ-1 were inhibited by transfected myoblasts with hIFNγgene.Conclusions(1) The pEGFP-C2-hIFNγ,a eukaryotic expression plasmid of hIFNγ, gene,has been constructed.And the mouse myoblasts C2C12 could be transfected with the recombinant plasmid with hIFNγ;(2) The transfected C2C12 myoblasts could express hIFNγwith activity stablely.PartⅢSurvival and Expression of Myoblasts with hIFNγGene or hIGF-1 Gene Following Transplantation into Injured Skeletal Muscle in the MouseObjective To observe the survival and expression of transfected myoblasts with hIFNγgene or with hIGF-1 gene following combined transplantation into injured skeletal muscle.Methods Acute skeletal muscle contusion models were produced on right gastrocnemius in 60 male mice of 7-12 weeks,and another 4 normal mice as control. The 60 mice were divided into 5 groups randomly as group A(injection with C2C12 cells transfected hIFNγgene),group B(injection with C2C 12 cells transfected hIGF-1 gene),group C(combined injection with C2C12 cells transfected hIFNγgene and C2C12 cells transfected hIGF-1 gene),group D(injection with blank C2C12 cells), group E(injection with physiological saline).The intervention was introduced on the 10^th day following injury by different injection into the injured muscle.The injured right gastrocnemius were harvested from 4 mice in every group on day 4,18,and 32 following intervention,which were tested by BrdU staining to evaluate cells surviving, and analyzed the expression of hIFNγand hIGF-1 by fluorescent quantitation PCR and immunofluorescence cytochemistry technology.Results(1)The BrdU staining in group A,B,D,and D were positive,and no difference was found among groups;(2) hIFNγexpression were found only in the group A and C without difference;(3) hIGF-1 expression were found only in the group B and C without difference.Conclusions(1) The transfected myoblasts and the blank myoblasts could survive for a period in the injured muscle following transplantation.It indicated that transfection had no influence on cells survival potential;(2)The Myoblasts carried with hIFNγgene or hIGF-1 gene could express hIFNγor hIGF-1 respectively in the injured muscle;(3) There were no difference about cells survival and expression following combined transplantation of transfected cells with hIFNγor hIGF-1 gene.PartⅣCombined Transplantaion of Myoblasts with hIFNγGene and Myoblasts with hIGF-1 Gene for Acute Skeletal Muscle Contusion in the MouseObjective To observe the effects of combined transplantation of myoblasts with hIFNγgene and myoblasts with hIGF-1 gene on acute skeletal muscle contusion in the mouse.Methods Acute skeletal muscle contusion models were produced on right gastrocnemius in 80 male mice of 7-12 weeks.The 80 mice were divided into 5 groups randomLy as group A(injection with C2C12 cells transfected with hIFNγgene),group B(injection with C2C12 cells transfected with hIGF-1 gene),group C(combined injection with C2C12 cells transfected with hIFNγgene and C2C12 cells transfected with hIGF-1 gene),group D(injection with blank C2C12 cells),group E(injection with physiological saline).The intervention was introduced on the 10^th day following injury by different injection into the injured muscle.The injured right gastrocnemius were harvested from 4 mice in every group on day 7,14,28,and 42 following injury(eg,the day before intervention,and 4d,18d,32d after intervention),which were tested about expression of MHC-Ⅱb,α-SMA and vimentin by fluorescent quantitation PCR and immunofluorescence cytochemistry technology.Results(1) After intervention,the expression of vimentin in the group A and group C was significantly lower than that of group B,D,and E.the vimentin expression of group B was lower than that of group D,and E on the day 28 and day 42.It was similar of vimentin expression between group A and C,or group D and E.(2) After intervention,the expression ofα-SMA in the group A and group C was significantly lower than that of group B,D,and E.theα-SMA expression of group B was lower than that of group D,and E on the day 28 and day 42.It was similar ofα-SMA expression between group A and C,or group D and E.(3) Following intervention,the expression of MHC-Ⅱb in the group B and group C was significantly higher than that of group A, D,and E.It was similar of MHC-Ⅱb expression between group B and C,or among group A,D and E.Conclusion(1) Local injection of C2C 12 myoblasts with hIFNγgene could inhibit the expression of vimentin andα-SMA significantly,what indicated its function of anti-fibrosis;(2) Local injection of C2C 12 myoblasts with hIGF-1 gene could promote skeletal muscle regeneration and inhibit fibrosis moderately,evidenced by its effects of significant enhancement of the MHC-Ⅱb expression and mild inhibition of the expression of vimentin andα-SMA.(3) It was indicated that combined injection of myoblasts with hIFNγgene or hIGF-1 gene would promote skeletal muscle repair by stimulation of muscle regeneration and anti-fibrosis.