CAJ | 학술논문

目的验证人瘢痕疙瘩成纤维细胞中钙/钙调蛋白依耐性丝氨酸蛋白激酶(calcium/calmodulin dependent serine protein kinase,CASK)/分化抑制因子1(inhibitors of differentiation 1,Id1)通路的存在及意义.方法通过免疫荧光激光共聚焦证实瘢痕疙瘩和正常皮肤成纤维细胞中CASK和Id1蛋白表达和定位;RT-PCR和Western-blot分析瘢痕疙瘩和正常皮肤成纤维细胞中CASK和Idl的表达和差异;通过免疫沉淀验证瘢痕疙瘩成纤维细胞中CASK和Idl蛋白的天然结合.结果正常情况下体外培养的瘢痕疙瘩和正常皮肤成纤维细胞中均存在CASK和Id1蛋白的表达,CASK和Id1主要分布在成纤维细胞胞浆和胞核中;RT-PCR结果表明瘢痕疙瘩组CASKmRNA的表达量为0.658±0.024,低于正常对照组的I.076±0.008(t=11.159,P＜0.05);Id1的表达量为0.497±0.014,高于正常对照组的0.307±0.017(t=15.148,P＜0.05);Western-blot结果表明瘢痕疙瘩组CASK蛋白的表达量为0.057±0.006,低于正常对照组的0.168±0.012(t=13.524,P ＜0.05);Idl的表达量为0.812±0.035,高于正常对照组的0.368±0.031(t=16.356,P＜0.05);免疫沉淀结果显示,CASK沉淀物中能检测到Id1,Id1沉淀物中能检测到CASK,CASK和Id1在瘢痕疙瘩成纤维细胞中存在天然结合.结论可能存在CASK/Id1信号通路参与了瘢痕疙瘩成纤维细胞的增殖,这与瘢痕疙瘩的发生存在一定联系.
목적 험증인반흔흘탑성섬유세포중개/개조단백의내성사안산단백격매(calcium/calmodulin dependent serine protein kinase,CASK)/분화억제인자1(inhibitors of differentiation 1,Id1)통로적존재급의의.방법 통과면역형광격광공취초증실반흔흘탑화정상피부성섬유세포중CASK화Id1단백표체화정위;RT-PCR화Western-blot분석반흔흘탑화정상피부성섬유세포중CASK화Idl적표체화차이;통과면역침정험증반흔흘탑성섬유세포중CASK화Idl단백적천연결합.결과 정상정황하체외배양적반흔흘탑화정상피부성섬유세포중균존재CASK화Id1단백적표체,CASK화Id1주요분포재성섬유세포포장화포핵중;RT-PCR결과표명반흔흘탑조CASKmRNA적표체량위0.658±0.024,저우정상대조조적I.076±0.008(t=11.159,P＜0.05);Id1적표체량위0.497±0.014,고우정상대조조적0.307±0.017(t=15.148,P＜0.05);Western-blot결과표명반흔흘탑조CASK단백적표체량위0.057±0.006,저우정상대조조적0.168±0.012(t=13.524,P ＜0.05);Idl적표체량위0.812±0.035,고우정상대조조적0.368±0.031(t=16.356,P＜0.05);면역침정결과현시,CASK침정물중능검측도Id1,Id1침정물중능검측도CASK,CASK화Id1재반흔흘탑성섬유세포중존재천연결합.결론 가능존재CASK/Id1신호통로삼여료반흔흘탑성섬유세포적증식,저여반흔흘탑적발생존재일정련계.
Objective To verify the existence and significance of calcium/calmodulin dependent serine protein kinase/inhibitors of differentiation 1 (CASK/Id1) pathway in fibroblasts of human keloid.Methods Immunofluorescence laser was used to confirm CASK and Id1 protein expression and localization in fibroblasts of the keloid and normal skin.RT-PCR and Western-blot were adopted to analysis the CASK and Id1 expression and differences between keloid and normal skin fibroblasts.The natural combination of CASK and Id1 protein of keloid fibroblasts was tested by immunoprecipitation.Results CASK and Id1 protein expression were both found in fibroblast cells of keloid and normal skin under normal circumstances.Most of CASK and Id1 were distributed in the cytoplasm and nucleus of fibroblasts.The results of RT-PCR showed that the expression of CASK mRNA in the keloid group was 0.658 ± 0.024,which was lower than that in the normal control group(1.076 ± 0.008,t =11.159,P ＜ 0.05).The expression of Id1 mRNA was 0.497 ± 0.014,which was higher than that in the normal control group (0.307 ± 0.017,t =15.148,P ＜0.05).The results of Western-blot showed that the expression level for CASK protein in the keloid group was 0.057 ± 0.006,which was lower than that in the normal control group (0.168 ± 0.012,t =13.524,P ＜ 0.05) ; the expression of Id1 protein was 0.812 ± 0.035,which was higher than that in the normal control group(0.368 ± 0.031,t =16.356,P ＜ 0.05).The results of immunoprecipitation showed that Id1 could be detected in the CASK precipitate,while CASK also could be detected in the Id1 precipitate.There was a natural binding of CASK and Id1 in keloid fibroblasts.Conclusion CASK/Id1 signal pathway may be existed and involved in the proliferation of keloid fibroblasts,which is related with the occurrence of keloid.