重组胸腺素β4调节创伤皮肤层粘连蛋白5表达的研究

EXPRESSION OF LAMININ 5 IN RAT SKIN WOUNDS REGULATED BY RECOMBINANT THYMOSIN β4

目的探讨重组胸腺素β4(thymosinβ4,Tβ4)调节层粘连蛋白5(laminin5,LN-5)表达促进创伤愈合的机制。方法成年雄性SD大鼠60只,体重200～250g,采用直径8mm自制打孔器于脊柱两侧脊肋角处各制备1个全层皮肤缺损模型。根据伤口用药的不同,随机分为实验组(45只)和对照组(15只),实验组再根据用药剂量不同分为低、中、高剂量组,每组15只。实验组于模型制备后即刻、每天7:00、19:00分别予每个伤口表面滴加50μLTβ4,低、中、高剂量组Tβ4浓度分别为2、6、18μg/50μL,对照组同法滴加等量PBS液(pH7.2),共滴加7d。造模后观察大鼠大体情况,术后2、4、7d观察大鼠伤口愈合情况及取材行免疫组织化学观察。结果除高剂量组1只大鼠于术后7d死亡,余均存活至实验完成。术后7d,中剂量组伤口明显缩小,多数伤口与创缘皮肤衔接良好。术后2d,对照组和低、中、高剂量组伤口愈合率分别为7.67%±5.46%、29.01%±7.43%、26.54%±11.49%、10.39%±3.96%;术后4d分别为28.16%±13.76%、37.99%±13.05%、42.00%±9.56%、39.58%±12.74%;术后7d分别为59.08%±19.02%、64.15%±17.92%、77.39%±8.45%、69.78±8.45%。术后2d,低、中、高剂量组伤口愈合率与对照组比较,差异均有统计学意义(P<0.05)。术后2d,各组均可见较多LN-5阳性表达,对照组与低剂量组阳性表达强(P<0.05),中剂量组阳性表达最少;术后4d,中剂量组阳性表达最强(P<0.05);术后7d,中剂量组仍有较强阳性表达(P<0.01)。结论重组Tβ4早期抑制LN-5表达,有利于细胞的增殖、分化,中晚期上调LN-5表达,改善基质环境,促进表皮细胞迁移和创伤愈合。

Objective To explore the mechanism of recombinant thymosin β4 （Tβ4） accelerating skin wound healing in rats by regulating laminin 5 expression. Methods Two full thickness 8 mm punch wounds were made at the costovertebral angle on dorsal surface of each adult male rats weighing 200-250 g. Sixty rats were randomized into the control group （n=15） and the experimental group （n=45）, which was subdivided into low, medium and high dose groups （n=15）. Tβ4 was applied topically at 2, 6, 18 μg in 50 μL PBS for every 12 hours after model making in the experimental group. The identical amounts of phosphate buffered saline was applied in the control group. Wound healing was observed after model making and immunohistochemical observation was conducted 2, 4 and 7 days after operation. Results Seven days after operation, wound contracted obviously and most of the wounds connected well with the margin. In the control group, low dose group, medium dose group and high dose group, the wound healing rate were 7.67% ± 5.46%, 29.01% ± 7.43%, 26.54% ± 11.49% and 10.39% ± 3.96% respectively 2 days after operation; 28.16% ± 13.76%, 37.99% ± 13.05%, 42.00%± 9.56% and 39.58%± 12.74% respectively 4 days after operation; 59.08%± 19.02%, 64.15% ± 17.92%, 77.39%± 8.45% and 69.78% ±8.45% respectively 7 days after operation. At 2 days after operation, significant differences were notified in healing rats between 3 sub-experimental groups and the control group （P 〈 0.05）. Immunohistochemistry staining showed that there was a little more positive expression of laminin 5 2 days after operation that beneficial to promote the proliferation and differentiation of cell in every group, including positive cells and ECM. But in medium group there was fewer expression, only at the borderline and bottom of the wound, while the expression significantly increased 4 days after operation （P 〈 0.05） and there was a relative high expression 7 days after operation （P 〈 0.01）. Conclusion Tβ4 can inhibit the expression oflaminin 5 early, and then up-regulate laminin 5 expression to moderate the reformation of ECM, promote the migration of epidemic cell and accelerate skin wound healing.