凝血酶肽类对创面愈合及表皮细胞增生与移行的作用研究

Effects of thrombin peptides on wound healing the proliferation and migration Of normal human epidermal keratinocyte (NHEK)

目的探讨新合成的凝血酶受体激活肽 ＴＰ５０８对创面愈合及表皮细胞增生与移行的作用。方法ＳＤ大鼠 １８只，麻醉下在每一大鼠背部作 ４个直径 １.５ ｃｍ圆形皮肤切除创面。 ＴＰ５０８治疗组 ９只大鼠，每 １创面用 ＴＰ５０８ ０.１μｇ（４０ｍｌ）治疗。对照组９只大鼠用４０μｌ等渗盐水处理。用 ＪＡＶＡ Ｊａｎｄｅｌ和 ＩＭＡＧＥ ＰＲＯ图形处理软件计算创面面积。人表皮细胞ＮＨＥＫ９４５增生采用ＭＴＴ测定法和细胞计数仪直接计数法。细胞移行实验采用４８孔 Ｂｏｙｄｅｎ细胞移行测定板进行，移行至滤膜下表面的细胞数以斑点密度表示。结果术后 ７ ｄ和 １４ ｄ，ＴＰ５０８处理组创面面积分别为盐水组的７３．７％和４５．４％（Ｐ＜０．０１）。凝血酶、ＴＰ５０８显著促进表皮细胞增生。培养液组细胞移行斑点密度为 ７６．６±１３．８，添加 １μｇ／ｍｌ凝血酶和 １０ μｇ／ｍｌ ＴＰ５０８，其斑点密度分别增至 １０４．４±１２．２和１０９．４±１４．６。结论凝血酶及ＴＰ５０８对大鼠皮肤切除创面愈合及人表皮细胞增生与移行均有显著作用，后者在一定程度上解释了ＴＰ５０８促进创面愈合的机理。

Objective To define the effects of throwbin peptides on wound healing and NHEK proliferation and migration. Methods A wound model was made with four 1.5 cm circular full thickness dermal excisions on the back of each Sprague-Dawley rat. 0.1μg (40 μl) TP508 was applied to each circular excisional wound in 9 rats, the other 9 received saline only. Wound area was calculated with JAVA Jandel and IMAGE PRO software. NHEK945 proliferation was assessed by MTT assay and direct cell count with a Coulter Counter. Cell migration was determined by 48-well Boyden Chamber. Cells migrated onto the lower surface of the filter were assessed by a ChemiImager 4000 Image Analyzer and. expressed as spot density. Results Wound area in rats treated with TP508 was 73.7% and 45.4% of saline control on day 7 and 14, respectively. NHEK945 proliferation was accelerated after adding thrombin and TP508. The spot density of migrated cells was 76. 7± 13. 8 in medium alone. After adding 1μg/ml of thrombin and 10μg/ml of TP508. the spot density was 104. 4 ±12. 2 and 109.4±14. 6, respectively. Conclusion Results of this study suggest that both thrombin and TP508 hare significant actions on wound healing and NHEK proliferation and migration, which is important in wound repair.