| Objective:The formation of prosthetic capsular contractures associated with Staphylococcus epidermidis infection is a major cause of failure in breast implant reconstruction.Through in vitro and in vivo studies,it is clear that Staphylococcus epidermidis biofilm combined with prosthetic stimulation promotes the phenotypic transformation of fibroblasts to myofibroblasts.To explore its possible molecular mechanism and provide help for clinical prevention and treatment of capsular contracture.Methods:1.The screening of key factors of fibroblast transforming to myofibroblast caused by the stimulation with the Staphylococcus epidermidis biofilm1.1 Detect the expression of the marker protein α-SMA and mRNA of fibroblast transformation to myofibroblast:(1)Imitate the infectious environment caused by Staphylococcus epidermidis biofilm after the prosthesis is implanted to build the conditioned medium:Co-culture the normal culture medium and sterile silica gel(Sil)for 24h,and then filter the bacteria to obtain the conditioned medium MA.Co-culture DMEM,sterile silica gel(Sil),and Staphylococcus epidermidis biofilm-forming negative strain ATCC12228 for 24h and then filter the bacteria to obtain the conditioned medium MB.Co-culture DMEM,sterile silica gel(Sil),and Staphylococcus epidermidis biofilm-forming negative strain ATCC35984 for 24h,and then filter the bacteria to obtain the conditioned medium MC;(2)Imitate the infectious environment caused by Staphylococcus epidermidis biofilm after the prosthesis is implanted:Mark the Co-culture of different conditioned medium,silica gel,and fibroblast as experiment groups(MA+Sil group,MB+Sil group,MC+Sil group),mark the fibroblast cultured on DMEM as the control group(NC);(3)Detect the expression of α-SMA and mRNA of the experiment groups and the control group through immunofluorescence and qPCR detection.1.2.Screen the phenotypic modulation-related genes:screen the phenotypic modulation-related key candidate genes through bioinformatics analysis and conduct transcription-level fibroblast sequencing in the experimental and control groups.1.3.Screen the target gene through qPCR sequencing results.2.The effect and mechanism of Spata13 participating in TGF-β signaling pathway in fibroblast transforming to myofibroblast2.1.Knockdown Spata13 to affect the phenotypic modulation of fibroblast:knockdown Spata13 in the experiment groups and control group,and then compare the fibroblast proliferation,the content of HYP,and the expression of α-SMA with when not knocking down Spata13.2.2.Suppress the TGF-β signaling pathway to affect the phenotypic modulation way of fibroblast:compare the fibroblast proliferation,the content of HYP,and the expression of α-SMA in the experiment groups and control group between when the TGFβRI inhibitor is used and not used.2.3.The recovery experiment of Spata13 regulating the phenotypic modulation of fibroblast by participating in TGF-β signaling pathway:add TGF-β1 when Spata13 is knocked down and Spata13 is overexpressed when TGFβRI inhibitor is added,and then detect the fibroblast proliferation,the content of HYP,and the expression ofα-SMA.2.4.Verify the regulation method of Spata13 participating in TGF-β signaling pathway by a positive feedback loop:(1)conduct Luciferase gene report experiment to verify the methods of Smad3 regulating Spata13 at the transcription level;(2)explore the location of Spata13 and TGFβR in cells under the stimulation with TGF-β1 through Fluorescence confocal method;(3)conduct IP experiment to detect the interaction of nodal molecules in the TGF-β signaling transduction when adding TGF-β1;3.The interventional explore of TGFβRI inhibitor effect on the capsular contracture of prosthesis-implantation rats3.1.The method of building the rat implant Staphylococcus epidermidis infection model:implant the silicon sheet under the breast of rats,and simultaneously inject 0.5ml of DMEM into the rats of a and b group,0.5ml of ATCC12228 bacterial fluid into c and d groups,and 0.5ml of ATCC35984 bacterial fluid into e and f group respectively.3.2.inject TGFβRI inhibitor to detect the way of capsular contracture:administrator TGFβRI inhibitor to b,d and f group,and normal administrator saline to a,b and c group and then evaluate the thickness of the contractured capsule and collagen content of the capsule through HE staining,Masson staining,and evaluate the expression of α-SMA and Spata13 in the capsule through immunohistochemistry.Results:1.The screening of key factors of fibroblast transforming to myofibroblast caused by the stimulation with the Staphylococcus epidermidis biofilm1.1.The increase of α-SMA expression and mRNA in the conditioned medium combining silica gel stimulating fibroblast group(MA+Sil group,MB+Sil group,and MC+Sil group)is statistically significant(P<0.05)compared to normal culture group(NC group);1.2.Bioinformatics results show that Spata13 is the significantly changed related gene in the phenotypic modulation of fibroblast.1.3.qPCR results:the level of mRNA of Spata13 in MA+Sil group,MB+Sil group,and MC+Sil group is higher and is statistically significant compared to that in NC group(P<0.001).2.The effect and mechanism of Spata13 participating in TGF-η signaling pathway in fibroblast transforming to myofibroblast2.1.The expression of Spata13 mediates the phenotypic modulation of fibroblast:compare the parameters in NC group,MA+Sil group,MB+Sil group,and MC+Sil group when knocking down and not knocking down Spata13.The results show that the expression of α-SMA and Collagen Ⅰ in the NC group is not statistically significant,and the expression in other groups is decreased(P<0.05);the HYP content and fibroblast proliferation are suppressed(P<0.05).2.2.Use TGFβRI inhibitor to suppress the phenotypic modulation of fibroblast:compare the parameters when giving and not giving TGFβRI inhibitor in NC group,MA+Sil group,MB+Sil group,and MC+Sil group.The results show that the expression of Spata13 andα-SMA is suppressed in every group(P<0.05),and the HYP content and fibroblast proliferation are suppressed(P<0.05).2.3.Determine that Spata13 regulates the phenotypic modulation of fibroblast through participating in TGF-β signaling pathway:(1)HYP content is increased and fibroblast proliferation is more rapid in TGF-β1-added group compared with NC group(P<0.01)and the expression of Spata 13,α-SMA and p-Smad3 is increased(P<0.05).HYP content isn’t increased significantly,and fibroblast proliferation isn’t rapid in the TGF-β1-added group when knocking down Spata13 than in the NC group.The expression of Spata13,α-SMA,and p-Smad3 is not changed significantly.(2)the HYP content,the speed of fibroblast proliferation,and the expression of α-SMA and p-Smad3 in overexpression pCDH-Spata13 group is increased compared to pCDH group(P<0.05);the HYP content,fibroblast proliferation,and the expression of Spata13,α-SMA and p-Smad3 in overexpression pCDH-Spata13 group when giving TGFβRI inhibitor isn’t increased significantly compared to pCDH group.2.4.Spata 13 takes part in the regulation of the TGF-β signaling pathway by a positive feedback loop:(1)Luciferase gene Report experiment results:after the overexpression of Smad3,the activity of wild type promoter of Spata13 is upregulated significantly(P<0.01),while the activity of the mutant promoter of Spata13 isn’t upregulated;(2)under the stimulation with TGF-β1,the location consistency of Spata13 and TGFβR in cells is higher consistently;(3)the stimulation with TGF-β1 induces the increase of the interaction of TGFβR and Smad3,while under the stimulation with TGF-β1 and the knockdown of Spata13 at the same time,the Smad3 binding to TGFβR is decreased significantly.3.The interventional explore of TGFβRI inhibitor effect on the capsular contracture of prosthesis-implantation rats3.1.Build the animal model successfully:5 rats(10 breasts)took part in the model building in each group.All the rats were survived.The number of breasts in each successfully building group is 7 in a group,7 in b group,8 in c group,7 in d group,7 in e group,and 7 in f group.3.2.the thickness of the rats’ capsule,collagen content,the expression of α-SMA and Spata13 in the TGFβRI inhibitor group is increased compared with those in the control group(P<0.05).Conclusion:1.The stimulation with Staphylococcus epidermidis biofilm combined with silica gel can promote the transformation of fibroblast to myofibroblast.Staphylococcus epidermidis infection may be an important factor for the capsular contracture of breast prosthesis;2.Under stimulation with Staphylococcus epidermidis biofilm combined with silica gel,Spata13 takes part in TGF-β signaling pathway regulating the phenotypic modulation of fibroblast to myofibroblast.It may be a mechanism of bacteria biofilm stimulating breast prosthesis;3.TGFβRI inhibitor can relieve the capsular contracture of the rat prosthesis induced by the stimulation with Staphylococcus epidermidis and reduce fibroblast’s phenotypic modulation.TGFβRI inhibitor may be the potential medicine for the treatment of capsular contracture of breast prosthesis. |
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