| BackgroundKeloid is the overhealing of wound by pathological fibrosis,which is a persistent tumor like hyperplasia exceeding the wound edge and invading adjacent tissues with intense pain and unbearable itching.It can not degenerate by itself and relapse easily even after operation,which also can cause deformity in appearance and dysfunction,seriously affecting patients' physical and mental health and became one of the most difficult issues in plastic surgery field.Although various studies have been conducted,the pathogenesis of keloids is still unclear.It is generally thought that keloids are relatively lack of oxygen while the inflammatory response and high metabolic formation of keloids makes the hypoxia situation worse.Therefore,we speculated that anoxia plays an important role in the development and progression of keloid.Hypoxia-inducible factor 1(HIF-1?)is the key factor of mediating cell anoxic stress response and regulating the biological behaviors of anoxic cells.The HIF-1? is a heterodimer mainly composed by functional subunit HIF-1? and constructional subunit HIF-1?.As the central control factor of anoxic response,HIF-1? expression is strictly regulated by oxygen concentration.Under normoxic conditions,HIF-1? protein can be rapidly degenerated by ubiquitin-proteasome system.Under hypoxia,the expression of HIF-la protein was increased by hypoxia,and the target gene expression was activated by binding to the specific DNA sequence of the target gene,which caused a series of adaptive responses to hypoxia.Various researches showed that HIF-1? can directly or indirectly regulat hundreds of adaptive anoxic genes expression and play an important role in the generation of tumor and vessel,cell energy metabolism,wound healing,inflammatory reaction,fibrosis disease,tumor radiotherapy and chemotherapy.At present,there are few studies on anoxia and signal pathway mechanism of HIF-1? both at home and abroad.Based on the biological characteristics of keloid and similar characteristics of malignant tumor,this study draws on the research result of the relationship of HIF-la with the biological change of keloid at the molecular level based on our previous work achievement,identify the function and mechanism of HIF-1? and anoxic signal pathway in the formation of keloid to provide evidence for its clinic treatment and HIF-1 targeted drugs development.Objective1.Compare the HIF-la expression in keloid tissue and normal skin tissue;2.Compare the HIF-la expression in fibrocytes and normal skin tissue of keloid fibroblasts cultured in vitro;3.Identify the efficacy of HIF-la under anoxic conditions,and the effects of anoxic pathway on the proliferation,cycle and apoptosis of keloid fibroblast,investigate molecule mechanism for the effects and estimate whether HIF-la can regulate keloid;4.Identify the effects and mechanism of TGF-?/Smads pathway,TLR4/MyD88/NF?B pathway and ERK pathway in the formation of keloid regulated by HIF-1?.5.The study of Transplant-keloid-animal model demonstrated that HIF-1??inhibitor(2-methoxyestradiol,2-ME)reduced the volume of transplanted keloid.Methods1.The first part.Detect the HIF-la expression difference of keloid tissue and normal skin tissue by RT-PCR,Western Blot.Cultivate fibroblastoma of keloid and normal skin in vitro and detect the HIF-1? expression difference of fibrocytes in keloid and normal skin tissue respectively by immunofluorescence,RT-PCR and Western Blot.Construct anoxic model with mixed gas by 1%oxygen,5%carbon dioxide and 94%nitrogen.Detect HIF-la expression difference of the fibroblast under anoxic conditions by Western Blot.2.The second part.Silencing the expression of HIF-ladepends on RNA interference technology,and Divide cells into normoxia group,anoxic group,con-siRNA group and HIF-la siRNA group.Detect the proliferation,cycle and apoptosis of cells in the above four groups by CCK8 reagent and Flow cytometry respectively;3.The third part.Test the expression difference of HIF-la and TGF-?/Smad signal pathway,TLR-MyD88-NF-icB pathway and ERK1/2 of cells in the above four groups by Western Blot and the differential expressions of TGF-?1,CTGF,IL-6 and IL-8 in the cell medias of the above four groups by ELISA.Then retest the KFs cells to detect if there is any difference of pathway after NF-?B inhibitor is added.4.The forth part.After transplanting the keloid tissue into the BALB/C nude mice successfully,give continuous intraperitoneal injection of HIF1?a inhibitor(2-methoxy estradiol,2-ME)for 21 days.Then,general observation of keloid and histological analysis of keloid were conducted.Research Results1.First Part In keloid tissues,HIF-la was widely distributed in its epidermal basal layer and a small amount of stratum spinosum cells and it mainly existed in cytoplasm surrounding cell nucleus while in normal skin fibroblasts,HIF-1? was barely observed.The transcription level and protein level of HIF-la in keloid tissues and KFs were both higher than that in normal skin tissues and NFs,P<0.05;In normoxia condition,there is little HIF-la KFs,which is mainly distributed in cytoplasm,while in hypoxic,HIF-la is highly expressed in KFs,in both cytoplasm and nuclei.2.Second Part the expression of HIF-la in KFs in hypoxic condition was significantly higher than in normoxia one;in the hypoxic group,the proliferation speed of KFs was evidently higher than that in normoxia condition;G1 stage cell percentage is similar and the percentage of apoptosis cells was similar.While siRNA-HIF-1?infected KFs cells,the expression of HIF-la in KFs cells in hypoxic group are decresed,besides,the proliferation speed of KFs was significantly slowed down;Gl stage cell percentage is significantly increased and the percentage of apoptosis cells was also significantly increased;NC group was similar to the hypoxic group in cell proliferation,circle and apoptosis and there was no significant difference.3.Third Part TGF-?? receptor,phosphorylated Smad2/3,Smad4 and TLR-4,MyD88,NF-?B and ERK1/2 level and content of TGF-?1,CTGF,VEGF,IL-6,IL-8 in the supernate of this group were all evidently higher than that in normoxia group(P<0.05).when HIF-1? gene were silenced,the expression of the listed protein in KFs from hypoxic group was evidently lowered(P<0.05).NF-?B inhibitor and block the TLR4/Myd88/NF-?B pathway,which blocked TGF-?/smad pathway as a follow-up effect,making the expression of downstream molecules such as TGF-??CTGF,VEGF all lowered.4.Forth Part An inhibitory effect of HIF-1? inhibitor on keloids were observed in vivo.The keloid volume was significantly less than that of the control group and the blank group after 2 weeks of intraperitoneal injection of HIF-la inhibitor,which x mainly inhibited the growth of keloid fibroblasts.Masson Staining showed that the keloid tissue staining of HIF-la in inhibitor group was significantly less than that of control group and blank group.Conclusion1.HIF-1 level in Keloid tissues and its fibroblast is evidently higher than that in the normal skin tissue and fibroblasts.In hypoxic condition,HIF-la protein level in KFs is significantly higher than that in normoxia condition.2.In hypoxic condition,HIF-la can enhance the proliferation activity of KFs and reduce its apoptosis level.3.In hypoxic condition,HIF-la can activate TGF-?/Smad signaling pathway and the expression of downstream TGF-?,GTGF and VEGF;activate TLR4-MyD88-NF-?B signaling pathway and the expression of downstream IL-6;activate ERK1/2 and the expression of downstream IL-8 and promote the formation of keloid.Besides,the TLR4-MyD88-NF-Kb pathway indicated by HIF-1? in KFs cells may have some association with TGF-?/Smad pathway know as crosstalk work.4.After 21 days of intraperitoneal injection of HIF-la inhibitor(2-ME2)into nude mice,the volume of keloid tissue was significantly decreased and the fibroblasts were also significantly decreased. |
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