595nm Vbeam激光对兔耳增生性瘢痕成纤维细胞增殖的影响

Influence of 595 nm Vbeam laser on proliferation of fibroblasts in the hyperplastic scars in rabbits' ears

目的:观察595nmVbeam激光照射对增生性瘢痕动物模型伤口愈合中成纤维细胞增殖的抑制作用。方法:实验于2004-10/2005-06在哈尔滨医科大学附属第二医院激光美容中心完成。成年大耳白兔20只,建立兔耳腹侧面增生性瘢痕模型,上皮化后切取一组瘢痕8处,然后将瘢痕随机分为激光治疗组和对照组,激光治疗组在创面上皮化后按一定参数进行激光照射,开始时每周照射1次,1个月后改为每两周照射1次,共两个月。对照组不进行治疗。在上皮化后1,2,4,6,8周分别切除两组增生性瘢痕各8处,每次取材前用游标卡尺测量瘢痕厚度,对切取组织进行苏木精-伊红和Masson染色,并对上皮化后4周组织进行电镜观察,对上皮化和上皮化后4周组织进行增殖细胞核抗原蛋白测定,对比研究在瘢痕形成过程中595nmVbeam激光照射对兔耳增生性瘢痕成纤维细胞的影响,观察成纤维细胞超微结构的改变,检测增殖细胞核抗原蛋白表达变化。结果:实验大耳白兔20只全部进入结果分析。兔耳增生性瘢痕经595nmVbeam激光照射后,按不同时间段取材与对照组比较,①瘢痕厚度明显变薄:创面上皮化后即出现增生块高出于皮面,且不断增厚,在上皮化后4周时最高,以后逐渐下降。激光照射(即上皮化后)2,4,6周后激光治疗组瘢痕增生厚度显著小于对照组激光治疗组:(1.95±0.08),(2.16±0.09),(1.53±0.08)mm;对照组:(2.04±0.09),(2.37±0.11),(1.68±0.09)mm,P<0.05。②苏木精-伊红染色显示不同时间取材的兔耳增生性瘢痕组织对照组真皮层明显增厚,为周围正常真皮厚度的三四倍。而激光治疗组真皮层较对照组变薄。Masson染色显示瘢痕胶原纤维呈蓝色,对照组可见蓝染较深的胶原纤维,外形粗大,排列杂乱无章。而经激光照射的瘢痕虽然也可见蓝染的胶原纤维,但与对照组比较蓝染变浅且纤细,胶原排列也趋于一致。③高倍镜下成纤维细胞计数在上皮化后随时间逐渐增多,在上皮化后4周时达到高峰,以后又下降,但在上皮化后1,2,4,6,8周激光治疗组成纤维细胞数量与对照组比较未见明显减少(P>0.05)。④透射电镜观察显示经595nmVbeam激光照射4周后成纤维细胞核变小、畸变,胞质中细胞器减少,细胞功能不活跃,线粒体空泡变性,细胞周围的胶原呈溶解状态。⑤增殖细胞核抗原蛋白表达激光治疗组较对照组明显减弱(增殖细胞核抗原阳性细胞反应率上皮化后为18.33%,上皮化后4周,激光治疗组为41.93%,对照组为64.26%,P<0.05)。结论:595nmVbeam激光照射可抑制兔耳增生性瘢痕成纤维细胞的增殖过程,应用595nmVbeam激光预防和治疗瘢痕可行。

AIM： To investigate the inhibitory effect of 595nm Vbeam laser irradiation on proliferation of fibroblasts in the hyperplastic scarring models. METHODS： The experiment was accomplished in the Laser Cosmetic Center, Second Affiliated Hospital, Harbin Medical University between October 2004 and June 2005. A total of 20 adult flap-eared rabbits were used to establish the models of hyperplastic scars in ventral side of rabbits＇ ears. A group of 8 scars after re-epithelization were cut out, and then the scars were classified randomly into laser treatment group and the control group. Those in the laser treatment group were treated every week with 595nm Vbeam laser at proper parameter after re-epithelization, and then every 2 weeks after a month. The total treatment period was 2 months. Those in the control group were not treated, The thickness of the scars was measured by vernier caliper before the 8 scars of each group were taken at the 1^st, 2^nd, 4^th, 6^th and 8^th weeks after re-epithelization; the scar tissues were observed by hematoxylin-eosin （HE） and Masson staining; the ultrastrncture of fibroblasts was observed by transmission electron microscopy at 4 weeks after re-epithelization, as well as the expression of proliferation cell nuclear antigen （PCNA） protein was detected at re-epithelization and 4 weeks after re-epithelization. The influence of 595 nm Vbeam laser irradiation on fibroblasts was studied comparatively in the process of forming hypertrophic scars in rabbits＇ ears： The change of uhrastrncture of fibroblast was observed, and the change of PCNA protein was detected. RESULTS： All of the 20 flap-eared rabbits were entered the result analysis. After 595nm Vbeam laser irradiation, the material was taken on different time and compared with those in the control group. ①The thickness of hypertrophic scars was obviously rower： The hyperplastic plaques were higher than the skin surface after re-epithelization and grew higher gradually until the 4^th week and then declined. The thickness of the hyperplastic scars was lower obviously in the laser treatment group at the 2^nd, 4^th and 6^th weeks after re-epeithelization than these of the control group [laser treatment group： （1.95±0.08）, （2.16±0.09）, （1.53±0.08） mm; control group： （2.04±0.09）, （2.37±0.11）, （1.68±0.09） mm,P〈 0.05]. ②HE staining showed that dermis layer of the scar tissue was incrassate in the control group, which was 3-4 times than normal, but it was thinner markedly in the laser treatment group as compared with the control group. Massen staining showed that collagen fibers were stained blue. They were thick with disorderly arranged and stained deeply in the control group, while much fine with orderly arranged and stained slightly in the laser treatment group. ③The number of fibroblast increased gradually after re-epithelization and reached the peak at the 4^th week and then decreased. But there were no significantly decrease of the fibroblast counting in the laser treatment group as compared with the control group at the 1^st, 2^nd, 4^th, 6^th and 8^th after re-epithelization （P 〉 0.05 ）. ④Transmission electron microscopy showed that the nuclear of fibroblast became small and aberrance, organelle decreased in the cytoplasm, the function of the cell became fal-lowed, mitochondria vacuolized, as well as the collagen around the cell dissolved after 4 weeks, 595 nm Vbeam laser irradiation. ⑤The expression of PCNA was obviously weaker in the laser treatment group than that in the control group （the positive rate of PCNA was 18.33% after re-epithelization, and 4 weeks after re-epithelization it was 41.93% in the laser treatment group while 64.26% in the control group, P 〈 0.05）. CONCLUSION： 595 nm Vbeam laser irradiation can inhibit the proliferation of fibroblasts in hyperplastic scars of rabbits ＇ ears and has significant effect on preventing and treating hyperplastic scars.