原代培养法分离培养人皮肤瘢痕成纤维细胞,抗波形蛋白抗体免疫细胞化学SP法鉴定所获细胞为间质来源。采用MTT法观察成纤维细胞增殖情况并计算增殖抑制率、TUNEL法检测细胞凋亡及凋亡指数。于兔耳腹侧皮肤制备创面,建立瘢痕模型,术后28d,瘢痕形成基本稳定时始采用自制的水蛭索外用膏剂对兔耳瘢痕模型进行治疗,每天3次,连续用药28d。术后56d处死动物,耳部瘢痕取材,多聚甲醛固定,行HE染色及VG染色。光镜观察瘢痕区形态学改变,检测瘢痕组织的增生指数、成纤维细胞数密度及胶原纤维面积密度。水蛭素能抑制体外培养的皮肤瘢痕成纤维细胞的生长、促进凋亡,局部外用含水蛭素的膏剂可以抑制动物实验性皮肤瘢痕的增生。
Fibroblasts were isolated from human scar tissue and identified by vimentin immunohistochemistry staining. MTI" assay was used to examine the proliferation of the fibroblasts, then the inhibition rate was calculated. Apoptosis and its index in each group were determined by TUNEL assay. Full-thickness wounds down to cartilage were created in the ventral side ( both sides) of ears of New Zealand rabbit,6 wounds for each ear. Twenty-eight days after the wounds initi- ated, hirudin Ointment was applied to the hypertrophic scars three times a day for 28 days. 56 days after ear skin wounds created,the rabbits were scarified,and scars were harvested and stained with H&E and VG staining. Hypertrophy index (HI), numerical density of fibroblasts on area (NA) and area density of collagen fibers on area (AA) were measured and calculated under microscope. The results showed that hirudin can inhibit the proliferation of fibroblasts in human skin scars. Hirudin ointment had practical potentialities in treatment and prevention of animal skin scar hyperplasia.