反义寡核苷酸对人瘢痕疙瘩成纤维细胞结缔组织生长因子基因表达和胶原合成的作用

Effects of antisense oligonucleotides on the expression of connective tissue growth factor gene and on the collagen synthesis in the cultured human keloid fibroblasts

目的 研究结缔组织生长因子 (CTGF)在人瘢痕疙瘩发病机制中的作用。 方法 体外分离、培养人瘢痕疙瘩成纤维细胞 (HKF)。在脂质体介导下 ,将异硫氰酸荧光素标记的硫代磷酸化CTGF反义寡核苷酸 (ASODN)转染至HKF中 ,设为CTGFASODN治疗组 (AT组 ) ,同时设脂质体对照组 (LC组 ,仅加入脂质体 )和空白对照组 (C组 ,不加脂质体和ASODN)。于荧光显微镜下观察CT GFASODN在各组细胞中的分布 ;通过逆转录聚合酶链式反应检测细胞中CTGFmRNA指数 (RI);采用3 H 脯氨酸掺入法检测细胞的胶原合成量。 结果 转染后 12h,AT组细胞胞浆内可见大量荧光 ,LC、C组HKF内无此现象。AT组转染后 4 8hRI值为 0 .12± 0 .6 2 ,明显低于LC组值 0 .5 1± 0 .18及C组值 0 .5 4± 0 .35 (P <0.0 1)。AT组的3 H 脯氨酸掺入率为 10 8.96± 79.0 5 ,较LC组值 171.2 4± 14 .99及C组值 16 5 .38± 4 3.6 1低 (P <0.0 1)。 结论 CTGFASODN能够抑制体外培养的HKF中CTGF基因表达和胶原合成 ,表明CTGF在人瘢痕疙瘩纤维化进程中发挥了一定作用。

Objective To study the role of connective tissue growth factor (CTGF) in the pathogenesis of human keloid. Methods Human keloid fibroblasts (HKF) were isolated from human keloid and cultured in vitro. The cells were then divided into 3 groups according to different processing, i.e. ASODN treatment (AT), in which phosphorothioate CTGF antisense oligonucleotides (ASODN) labeled by fluorescent isothiocyananate were transfected into the HKFs by liposome; liposome control (LC, with liposome only); control groups (without liposome or ASODN). The distribution of CTGF ASODN in all groups of cells was observed under fluorescent microscope. The CTGF mRNA index (RI)of HKF was assessed by reverse transcription polymerase chain reaction method (RT-PCR). The collagen synthesis of HKF was assessed by 3H-proline incorporation method. Results A large amount of fluorescence could be observed in the cytoplasm of HKFs in AT 12 hours after transfection, but not in LC and C groups. The CTGF mRNA index of HKF in AT group 48 hours after transfection was significantly lower than that in LC and C groups(0.12± 0.62 vs 0.51±0.18 vs 0.54±0.35, P< 0.01). The 3H-proline incorporation rate in AT group (108.96±79.05) was lower than that in LC and C groups ( P<0.01). Conclusion The expression of CTGF gene and collagen synthesis of the cultured HKF could be inhibited by CTGF ASODN, implying that CTGF played a role in the development of excessive fibrosis of human keloid.