| Background and objectiveKeloids are defined as an abnormal scar that grows beyond the boundary of the original site of a skin injury and keloids do not subside,characterized by excess accumulation of disproportionate extracelluar matrix and fibroblast proliferation.Their biological behaviour is similar to that of tumor. The recurrence rate is high after operation. But there are no strategies available for keloid therapy. At the same time, the mechanisms of the keloid's occurance are unknown. There is no effective method to treat it on clinic.lt has been the clinical tough problem and researching focal point in plastic surgery.Two isoforms of mammalian sphingosine kinase (SphK1 and SphK2) have been cloned and characterized. SphK1 is implicated in the regulation of cell proliferation and antiapoptotic processes, SphK2 cause apoptosis and growth arrest phenotypes. TGF-βis one of important factors leading to keloid production, increasing the expression of SphK1 in fibroblasts of human skin and lung. Little research has been done on the expression of SphK1 and SphK2 in human keloid fibroblasts and the relation between SphK1 and TGF-P1.In this study, we detect the expression and function of sphingosine kinase SphK1 and SphK2 in human keloid fibroblasts, infer the effect of SphK1 and SphK2 in keloid pathogenesis and apoptosis.At the same time,we observe the relation between SphK1 and TGF-β1, indicate that SphK1 is involved in TGF-βsignal transduction pathway.Methods1. Specimens of keloid and surrounding normal skin were collected from 12 patients with keloid during operation. Primary fibroblasts were isolated, cultured, and randomly divided into 3 groups: normal skin group, keloid group, and keloid with transforming growth factor (TGF)-β1 group cultured with TGF-β1 for 48h.2. Immunofluorescence technique was used to detect the location of SphK1 and SphK2 protein.3. Total RNA was extracted with Trizol from fibroblasts of 3 groups, then reverse transcribed into cDNA using the Reverse Transcription Kit, Real-time PCR was used to measure the mRNA expression levels of SphK1 and SphK2.4. Total protein was extracted with RIPA lysate from fibroblasts of 3 groups, protein concentration was detected by BCA Protein Quantitative Kit, Western blotting was used to measure the protein expression levels of SphK1 and SphK2.Results1. In normal skin group.keloid group and keloid with TGF-P1 group,SphK1 protein was localized primarily in the nuclei of the fibroblasts, and SphK2 protein was detected both in the cytoplasm and nuclei in the 3 groups.2.The mRNA levels of SphK1 in the keloid group were 0.0608±0.0190,significantly higher than these of the normal skin group (0.0383±0.0147,P<0.05), but significantly lower than these of the keloid fibroblasts with TGF-p1 group(0.0790±0.0280, P<0.05). There was not significant differences in the SphK2 mRNA level among these 3 groups (P> 0.05). The mRNA levels of SphK1 in these 3 groups were significantly higher than the mRNA levels of SphK2(P<0.05). SphK1/SphK2 in the keloid group were 20.2037±3.2254,significantly higher than these of the normal skin group (10.5784±1.9007,P<0.01),but significantly lower than these of the keloid fibroblasts with TGF-β1 group(22.7297±3.3306,P<0.05) 3.The protein levels of SphK1 in the keloid group were 0.8308±0.1093,significantly higher than these of the normal skin group (0.6800±0.1126,P<0.05),but significantly lower than these of the keloid fibroblasts with TGF-β1 group(1.4267±0.1938,P<0.01). There was not significant differences in the SphK2 protein level among these 3 groups (P> 0.05). The protein levels of SphK1 in these 3 groups were significantly higher than the protein levels of SphK2(P<0.05). SphK1/SphK2 in the keloid group were 11.4413±0.5012,significantly higher than these of the normal skin group (8.8747±0.6927,P<0.01),but significantly lower than these of the keloid fibroblasts with TGF-β1 group(18.4230±0.7713,P<0.01).Conclusions1. In normal skin group,keloid group and keloid with TGF-β1 group,SphK1 protein was localized primarily in the nuclei of the fibroblasts, and SphK2 protein was detected both in the cytoplasm and nuclei in the 3 groups.2.There are significant differences in the SphK1 expression in normal skin fibroblasts and human keloid fibroblasts,mRNA and protein expression of SphK1 increase significantly in human keloid fibroblasts,SphK1 promotes human keloid fibroblasts proliferation,SphK1 plays a leading role in keloid pathogenesis; There was not significant differences in the SphK2 mRNA level among these 3 groups. SphK2 cause apoptosis and growth arrest phenotypes, SphK2 can not promote keloid fibroblasts apoptosis efficiently.The disequilibrium of SphK1 and SphK2 plays a leading role in keloid pathogenesis.3. SphK1/SphK2 in the keloid group were significantly higher than these of the normal skin group, it may be concerned with keloid fibroblasts proliferation, indexing keloid fibroblasts proliferation.4.The SphK1 mRNA and protein levels are increased by TGF-β1 stimulation in keloid fibroblasts,TGF-β1 promotes SphK1 expression, perhaps indicating that SphK1 is involved in TGF-βsignal transduction pathway.
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