M2型丙酮酸激酶对瘢痕疙瘩成纤维细胞功能的影响

The Effect of Pyruvate Kinase M2 on the Function of Keloid Fibroblasts

目的探索M2型丙酮酸激酶(Pyruvate Kinase M2,PKM2)对瘢痕疙瘩成纤维细胞增殖、糖酵解以及胶原合成的影响。方法应用免疫组化染色检测正常皮肤组织和瘢痕疙瘩组织中PKM2及增殖相关标记物(Ki67)的表达;应用Real-time PCR检测正常皮肤来源成纤维细胞和瘢痕疙瘩来源成纤维细胞中PKM2及纤维化指标(COL1A1、COL1A2、COL3A1)的表达;应用Western-blot检测正常皮肤来源成纤维细胞和瘢痕疙瘩来源成纤维细胞中PKM2的表达;应用Seahorse XF技术检测正常皮肤来源成纤维细胞和瘢痕疙瘩来源成纤维细胞的糖酵解功能。使用PKM2抑制剂处理瘢痕疙瘩成纤维细胞后,应用CCK-8、Ed U标记方法检测细胞增殖的变化,Seahorse XF技术检测细胞糖酵解功能的变化,Western-blot检测细胞中纤维化指标(Ⅰ型胶原、Ⅲ型胶原)的表达变化。结果 PKM2、增殖相关标记物(Ki67)在瘢痕疙瘩组织中的表达较正常皮肤组织中明显升高;瘢痕疙瘩成纤维细胞中的PKM2、Ⅰ型胶原和Ⅲ型胶原表达水平均显著高于正常皮肤成纤维细胞;与正常皮肤成纤维细胞相比,瘢痕疙瘩成纤维细胞的糖酵解功能显著上调。PKM2抑制剂处理后,瘢痕疙瘩成纤维细胞增殖能力明显受到抑制、糖酵解功能显著下调、Ⅰ型胶原纤维表达显著减少。结论瘢痕疙瘩成纤维细胞高表达PKM2,抑制PKM2可能是临床治疗瘢痕疙瘩的新途径,值得进一步深入研究。

Objective To explore the effect of PKM2 on proliferation,collagen synthesis,glycolysis of keloid fibroblasts(KFs).Methods Immunohistochemical staining was used to detect the expressions of PKM2 and proliferation-related marker(Ki67)in normal skin and keloid tissues.Real-time PCR was used to detect the expressions of PKM2 and fibrosis indexes(COL1A1,COL1A2 and COL3A1)in normal skin-derived fibroblasts and keloid derived fibroblasts.Western-blot was used to detect PKM2 expression in normal skin fibroblasts and keloid fibroblasts.Seahorse XF was used to detect glycolytic function of normal skin fibroblasts and keloid fibroblasts.After PKM2 inhibitor was used to treat keloid fibroblasts,changes in cell proliferation were detected by CCK-8 and EdU markers,changes in cell glycolysis function were detected by Seahorse XF,and changes in the expression of fibrosis indicators(typeⅠand typeⅢcollagen)in keloid fibroblasts were detected by Western-blot.Results The expressions of PKM2 and proliferation-related markers(Ki67)in keloid tissues were significantly higher than that in normal skin tissues.The expression levels of PKM2,collagenⅠand collagenⅢin keloid fibroblasts were significantly higher than that in normal skin fibroblasts.Compared with normal skin fibroblasts,glycolytic function of keloid fibroblasts was significantly upregulated.After PKM2 inhibitor treatment,the proliferation ability of keloid fibroblasts was significantly inhibited,the glycolytic function was significantly down-regulated,and the expression of typeⅠcollagen was significantly reduced.Conclusion Keloid fibroblasts demonstrate increased expression of PKM2.Inhibition of PKM2 may be a new way for clinical treatment of keloid,which deserve further stydy.