| Objective: This research is intended to explore the role of shallow thermal injury in prostatic wound healing after thulium laser resection of the prostate(Tm LRP).Furthermore,the mechanism of the activation stroma cells which induced by shallow thermal injury could release exosome accelerate prostatic epithelial cell transform into mesenchymal cell.Additional,we establish beagles,Tm LRP model and explore the role of shallow thermal injury in prostatic wound healing after Tm LRP.Consequently,we uncover the role of shallow thermal injury of Tm LRP induced epithelial cells and mesenchymal cells cross-talk in the prostatic wound healing.Methods: 1.Cell shallow thermal injury model was established by water bath and CCK-8 assay was used to detect cell survival rates in order to choose the optimal temperature and time,meanwhile,flow cytometry(FCM)and protein immunoblotting was used to check the effectiveness of cell shallow thermal model.2.CCK-8 assay,scratched wound healing assay,flow cytometry,transwell assay,EDU staining assay and immunoblotting assay was used to detect the proliferation,migration and cell cycle distribution of shallow thermal injury induced WPMY-1 cells to BPH-1 cells by indirect cell co-culture model.3.Immunoblotting and immunofluorescence(IF)was applied to determine the epithelium to mesenchymal transtion(EMT)level of BPH-1 which induced by shallow thermal injury WPMY-1 visa indirect co-culture cell model.Thereby,we try to explain shallow thermal injury induced stroma cells may secret some proteins or exosome promote EMT of BPH-1 and then influence prostatic wound healing.Antibody array was used to detect the supernatant ingredient of the shallow thermal injury induced WPMY-1 and analysis the cytokines or proteins of shallow thermal injury induced WPMY-1 supernatant.Meanwhile,the supernatant exosome of shallow thermal injury induced WPMY-1 was isolated.The isolated exosomes were used to stimulate shallow thermal injury BPH-1,signal pathway PCR-array was used to detect proliferation,EMT and wound healing associated signal pathway.Immunoblotting assay was used to detect the change of proteins of such signal pathway.4.The beagles prostatic Tm LRP was established and was randomly divided into control,oral Gefitinib and oral GW4869 group.HE staining,immunohistochemistry(IHC)and immunofluorescence(IF)was used to observe the re-epithelialization of prostatic wound edge and the proliferation,trans-differentiation and migration of basal progenitor cells of three groups.Enzyme-linked immunosorbent assay(ELISA)and immunofluorescence assay was used to detect the infiltration of M1/M2 type macrophage and different level of IL6,IL12,TNF-α as well as IL-10,TGF-β,the marker of M1/M2 type macrophage and matrix metalloproteinases MMP2,MMP9 was also detected by immunoblotting assay.Results: 1.The shallow thermal injury cell model was successfully established,and CCK-8 assay was used to choose the optimal temperature and time for WPMY-1 and BPH-1 cells according to cell survival viability.The WPMY-1 and BPH-1 cells under such condition were damaged rather than death directly.The FCM assay showed that the WPMY-1 and BPH-1 cells were arrested in G2 cell cycle and showed high apoptosis rate.Protein level of cell cycle associated protein cyclin B1,chk1 and p21 was up-regulated,meanwhile,heat injury associated protein heat shock protein HSP70,HSP90 and cell apoptosis associated protein PARP was significantly up-regulated.2.The shallow thermal injury BPH-1 cells were divided into three groups,non-co-culture group(control group),indirect co-culture with WPMY-1 group(WPMY-1 group),indirect co-culture with shallow thermal injury WPMY-1 group(WPMY-1-HI group),among these groups the BPH-1 cells of WPMY-1-HI group showed enhanced cell proliferation ability and lower cell apoptosis rates,cyclin B1,chk-1 and p21 was down-regulated;among these three group BPH-1 cells of WPMY-1-HI group showed an increased cell migration ability and possessed obvious mesenchymal character,the western blot and q RT-PCR assay showed that the epithelium associated cell marker E-cadherin was significantly down-regulated whereas mesenchymal cell associated marker N-cadherin,Vimentin,Snail was significantly up-regulated,namely these cells went through EMT.3.The supernatant of shallow thermal injury WPMY-1 was collected and applied to antibody array analysis,results showed that the Epidermal Growth Factor Receptor(EGFR)was significantly up-regulated.We collected the supernatant of shallow thermal injury WPMY-1 and isolated the exosomes,the result showed that EGFR was up-regulated same as antibody array.The shallow thermal injury WPMY-1 derived exosomes were used to stimulate BPH-1 cells and PCR array was used to show the change of cell proliferation,wound healing and EMT associated signaling pathway NF-k B was significantly activated.Immunoblotting assay was used to detect the proteins of NF-k B pathway,result showed that the p-NK-k B,p-IKKa/b,p-ikb-a was significantly up-regulated.And the cell proliferation,migration and EMT process was prohibited by inhibitor of NF-k B signaling pathway.4.The beagles,Tm LRP model were established and randomly divided into three groups,control group,oral Gefitinib and oral GW4869 group.The HE staining results showed that the speed of prostatic urethra wound healing was significantly slower in Gefitinib and GW4869 than in control group;the EGFR expression of the prostatic urethra wound edge was significantly down-regulated as well as Ki-67,which implied that down-regulated EGFR expression may regulated prostatic wound healing speed.The immunofluorescence assay showed that the p63/uroplakin-3 double positive urothelium cells of Gefitinib group and GW4869 group was significantly decreased than control group which implied that EGFR could regulate p63 positive urothelium cells proliferation,trans-differentiation and migration.The ELISA assay showed that the M1 type macrophage secreted cytokines IL-6,IL-12 and TNF-α was increased in Gefitinib and GW4869 than control group whereas the M2 type macrophage secreted cytokines IL-10 and TGF-β was decreased in Gefitinib and GW4869 group than control group.The immunofluorescence assay showed the M1 type macrophage marker CD86 was higher in Gefitinib and GW4869 group than in control group whereas the M2 type macrophage marker CD206 was lower in Gefitinib and GW4869 group than in control group,which implied that reduced EGFR expression could promote M1 macrophage infiltration and inhibit M2 macrophage infiltration.Additionally,the expression of matrix metalloproteinases MMP2,MMP9 was also decreased in Gefitinib group,these results demonstrated that decreased EGFR expression could significantly enhance M1 type macrophage infiltration and weaken M2 type macrophage infiltration and prolong the wound healing time.Conclusion: 1.In cell shallow thermal injury co-culture model,BPH-1 cells of WPMY-1-HI group showed enhanced cell viability and went through EMT in order to protect the wound surface temporary.2.The shallow thermal injury WPMY-1 released exosome containing EGFR which passed the wound healing signal to BPH-1 and help to complete wound surface protect and wound healing.3.The EGFR-NF-k B signaling pathway play key role in prostatic wound protection and wound healing.4.The beagle dogs which oral administration Gefitinib and GW4869 showed decreased wound healing speed as well as lower proliferation,trans-differentiation and migration ability of basal progenitor cells.Meanwhile,the macrophage polarization of wound surface in Gefitinib and GW4869 group was also influenced showed that increased M1 type macrophage with secreted cytokines IL6,IL12,TNF-α which prolong the wound healing and decreased M2 type macrophage with secreted cytokines IL10,TGF-β which shorten the wound healing.From these results demonstrated above we can draw a conclusion that the shallow thermal injury which Tm LRP induced could promote stroma cell release exosome containing EGFR sequentially enhanced stroma cell and epithelial cell cross-talk which may protect wound surface and accelerate wound healing process. |
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