剪接因子SRSF2对瘢痕疙瘩成纤维细胞生物学特性的作用研究

Effect of SRSF2 on the biological characteristics of keloid fibroblasts

目的探究剪接因子SRSF2对瘢痕疙瘩成纤维细胞生物学特性的影响,为瘢痕疙瘩发病机制的研究提供新的思路。方法正常皮肤标本来源于中国医学科学院整形外科医院8例植皮手术患者多余的皮肤组织,年龄8~53岁;瘢痕疙瘩组织标本来源于12例同一医院行手术治疗的瘢痕疙瘩患者,年龄18~24岁。检测正常皮肤和瘢痕疙瘩组织中SRSF2的表达;体外培养瘢痕疙瘩成纤维细胞,检测TGF-β1刺激与细胞中SRSF2表达的关系;利用慢病毒载体下调细胞内SRSF2表达后,检测细胞状态,并从mRNA及蛋白层面分析细胞外基质、TGF-β1分泌表达水平。结果SRSF2在瘢痕疙瘩组织中表达明显高于正常皮肤组织;TGF-β1可促进瘢痕疙瘩成纤维细胞SRSF2表达;敲减SRSF2后,瘢痕疙瘩成纤维细胞凋亡率增高,细胞周期阻滞于G0/G1期,且Cyclin E1表达下降,另外COL3A1、FN1 mRNA表达降低,TGF-β1表达及分泌减少。结论抑制瘢痕疙瘩成纤维细胞中SRSF2的表达,细胞生长受限,胶原表达降低,TGF-β1分泌减少,提示SRSF2可作为瘢痕疙瘩治疗新靶点。

Objective This study aims to explore the influence of the serine and arginine rich splicing factor 2 (SRSF2) on the biological characteristics of keloid fibroblast, by comparing the expression levels of SRSF2 in normal skin and keloid, with the purpose to provide a new method to study the pathogenesis of keloid. Methods Samples of normal skin were derived from excess tissue of skin grafts, collected from 8 patients, aged 8-53 years old, while the specimens of keloid were from 12 keloid patients, aged 18-24 years old. All the patients were admitted in the Plastic Surgery Hospital, Chinese Academy of Medical Sciences. The expression of SRSF2 was assessed by immunohistochemistry and immunofluorescent staining in both normal and keloid tissue. Keloid fibroblasts were cultured in vitro to detect the relationship between TGF-β1 stimulation and SRSF2 expression. After constructing the lentiviral sh-RNA-expression vector, targeting SRSF2 and infecting keloid fibroblast, the apoptosis and proliferation of cells were analyzed by the MuseTM Cell Analyzer. The expression of CyclinE1 was analyzed by real-time PCR and western blot, and the secretion and expression of extracellular matrix and TGF-β1 were detected by real-time PCR and ELISA.The software SPSS 21.0 was used to do the statistical analysis. Results The expression of SRSF2 was significantly higher in the keloid samples than in the normal skin samples. The percentage of SRSF2 positive cells in keloid was (77.04±4.37)%, while the percentage of SRSF2 positive cells in normal skin was (25.10±1.24)%. TGF-β1 promotes the expression of SRSF2 to (159.73±17.03)% times in keloid fibroblasts. After the SRSF2 knock-out, the apoptosis rate of the keloid fibroblasts increased, its cell cycle arrest was observed at the G0/G1 phase, and the expression of Cyclin E1 decreased. Apoptotic cells in control group was about (18.83±1.24)%, while (25.81±7.09)% and (26.71±6.14)% were in the two knock-out groups respectively. Cells in G0/G1 phase was about (58.97±1.73)% in control group, while (63.95±2.07)% and (64.65±3.23)% were in the two knock-out groups respectively. Compared to the control group, the expression of Cyclin E1 mRNA in the two knock-out groups were (31.60±6.81)% and (33.01±11.39)% respectively. Additionally, the expression of COL3A1 FN1 mRNA decreased, and the expression and secretion of TGF-β1 was declined. Compared to the control group, the expression of COL3A1 mRNA in the two knock-out groups were (64.90±23.71)% and (67.97±13.50)%, the expression of FN1 mRNA in the two knock-out groups were (59.10±8.11)% and (70.70±18.26)%, the expression of TGFB1 mRNA in the two knock-out groups were (53.37±15.51)% and (67.53±19.33)% respectively. The concentration of TGF-β1 in the medium of control group was (115.60±18.17)pg/ml, while (75.35±12.25) pg/ml and (72.06±14.66) pg/ml were in the two knock-out groups respectively. Conclusions The expression of SRSF2 increased in keloid. The inhibition of SRSF2 in keloid fibroblast resulted in the growth restriction and apoptosis of cells, and decreased the secretion of extracellular matrix and TGF-β1, which providing a new insight into keloid pathogenesis, and suggesting that SRSF2 could be a new therapeutic target for keloid conditions.