创伤修复中白细胞蛋白酶抑制因子和中性粒细胞趋化因子差异表达的研究

Differential expression of secretory leukocyte protease inhibitor and cytokine-induced neutrophil chemoattractant-2 in the process of wound repair

目的研究真皮间充质干细胞(dermal mesenchymal stem cells, dMSCs)参与创伤修复中白细胞蛋白酶抑制因子(secretory leukocyte protease inhibitor, SLPI)和中性粒细胞趋化因子(cytokine-induced neutrophil chemoattractant-2, CINC-2)的差异表达,并探讨其相关功能。方法采用前期建立的技术分离、纯化并扩增dMSCs,通过RT-PCR检测创伤修复时SLPI和CINC-2在dMSCs中表达,通过3H-TdR掺入实验研究SLPI和dMSCs有丝分裂的关系,利用Boyden趋化小室研究dMSCs对中性粒细胞的趋化作用。结果分离的大鼠dMSCs呈梭形,在体外显示多向分化潜能。RT-PCR提示伤口液刺激24 h后dMSCs中SLPI,CINC-2表达升高,同时检测到在全层切割伤后1 d大鼠皮肤组织中SLPI和CINC-2均为高表达,3H-TdR掺入实验提示SLPI能够促进dMSCs的有丝分裂,趋化小室实验显示dMSCs对中性粒细胞有趋化作用,加入中和抗体后趋化作用减弱。结论创伤后SLPI,CINC-2在dMSCs中表达升高,其表达升高可能同dMSCs的增殖和趋化有关。

Objective To study the differences in gene expression of secretory leukocyte protease inhibitor （SLPI） and cytokine-induced neutrophil chemoattractant-2 （CINC-2） in dermal mesenchymal stem cells （dMSCs） during trauma. Methods dMSCs were routinely isolated from male neonatal Wistar rats aged one day, purified and expanded by the technique established in the prophase. Two pieces of sponges were implanted into two 7-centimeter-long incisions on the back of Wistar rats aged 3 months. The wound fluid extracted from the sponges was used to stimulate the dMSCs. The skins around the wound were excised and grinded. Then SLPI and CINC-2 were detected in dMSCs with or without wound fluid stimulation and the rats skin tissues of normal controls or the wounded by RT-PCR. Mitogenic responses of dMSCs to SLPI were examined via the incorporation of ^3H-TdR into DNA. ＂Chemotactic activity of dMSCs to neutrophil was evaluated by using a 24-well microchmotaxis Boyden chamber. Results Most of the neonatal dMSCs were spindle-shaped cells and exhibited multi-directional differentiation potential in vitro. In RT-PCR and experiment, SLPI and CINC-2 both increased in the stimulated dMSCs and expressed strongly in the rat skin 1 day after injury. A concentration-dependent proliferation promotion was demonstrated in dMSCs incubated for 24 h in the presence of SLPI. Neutrophil experiment suggested that CINC-2 was involved in promoting the migration of neutrophil to dMSCs. Conclusion SLPI and CINC-2 were up-regulated in the process of wound repair. They might play important roles in the proliferation and chemotaxis of dMSCs.