点阵激光联合脉冲染料激光加多点微量注射曲安奈德对兔耳增生性瘢痕的影响

Experimental study of the effects of fractional laser and pulsed dye laser combined with multi-point triamcinolone injection on hypertrophic scar in rabbit ears

目的探讨点阵激光联合脉冲染料激光加多点微量注射曲安奈德治疗兔耳增生性瘢痕的机制。方法10只新西兰大白兔,在每只兔双耳腹侧各制作4个增生性瘢痕模型,共80个。采用简单随机抽样法分成4组:对照组、激光联合组、激光联合多点微量注射组、单纯传统注射组,每组20个瘢痕模型。对照组不给予处理;激光联合组:首先用脉冲染料激光治疗,接着同期采用点阵激光治疗;激光联合多点微量注射组:先用脉冲染料激光及点阵激光治疗,后即刻瘢痕内进行多点微量注射曲安奈德;单纯传统注射组瘢痕内注射曲安奈德。观察并记录4组增生性瘢痕的大体外观,分别在治疗后7 d、2个月切取增生性瘢痕组织,采用HE染色观察增生性瘢痕的组织学变化;Masson染色观察胶原表达情况,并测量胶原容积分数;CD31染色观察微血管数量的变化,并测量微血管密度。通过单因素方差分析(one-way ANOVA)检验组间差异,以及LSD posthoc检验进行组间对比,对胶原容积分数和微血管密度进行Pearson相关系数分析。结果治疗后2个月,组织学结果提示激光联合组、激光联合多点微量注射组较对照组瘢痕厚度显著变薄,质地软化,红色逐渐变淡,和周围皮肤的颜色逐渐趋近。单纯传统注射组较对照组瘢痕的厚度变薄,质地变软,色泽变淡。激光联合多点微量注射组较激光联合组、单纯传统注射组瘢痕的厚度更薄。Masson染色结果显示,各组增生性瘢痕组织中胶原容积分数表达均有减少。各组第7天、2个月时胶原容积分数比较,激光联合组、激光联合多点微量注射组、单纯传统注射组差异有统计学意义(P<0.05),与对照组差异无统计学意义(P>0.05);进一步对治疗后2个月时各组间胶原容积分数进行比较,激光联合多点微量注射组<激光联合组<单纯传统注射组,组间差异均有统计学意义(P<0.05)。CD31染色观察:治疗后,各组增生性瘢痕组织中微血管密度均降低。各组第7天、2个月时进行微血管密度比较,激光联合组、激光联合多点微量注射组、单纯传统注射组间比较,差异有统计学意义(P<0.05),而对照组无统计学意义(P>0.05);治疗后2个月时各组间微血管密度比较,激光联合多点微量注射组<激光联合组<单纯传统注射组,但激光联合多点微量注射组与激光联合组比较差异无统计学意义(P>0.05),激光联合多点微量注射组、激光联合组分别与单纯传统注射组比较差异有统计学意义(P<0.05)。Pearson相关系数分析结果为r=0.972,可认为胶原含量与微血管数量之间呈正相关。结论采用点阵激光联合脉冲染料激光加多点微量注射曲安奈德治疗兔耳增生性瘢痕,能够均匀降低成纤维细胞数量,促进胶原密度、排列的正常化趋势,降低胶原容积分数和微血管密度,疗效优于点阵激光联合脉冲染料激光治疗和单纯传统曲安奈德注射治疗。增生性瘢痕中胶原含量和微血管密度呈正相关,抑制微血管的增生,可以提高瘢痕治疗效果。

Objective To explore the therapeutic mechanism of fractional laser combined with pulsed dye laser and multi-point microinjection of triamcinolone in the treatment of hypertrophic scars in rabbit ears.Methods In this study,80 hypertrophic scar models of rabbit ears were made and randomly divided into 4 groups:control group,laser combined group,laser combined multi-point microinjection group,simple traditional injection group,20 scar models in each group.The control group was not given treatment;the laser combined group was treated with pulsed dye laser first,followed by fractional laser at the same time;the laser combined with multi-point microinjection group was treated with pulsed dye laser and fractional laser first,followed by multi-point microinjection of triamcinolone in the scar immediately after laser treatment;the traditional injection group was injected with triamcinolone in the scar.The gross appearance of hypertrophic scars was observed and recorded in 4 groups.At 7 days and 2 months after treatment,the hypertrophic scars were cut and the histological study were performed by HE staining;the expression of collagen was observed by Masson staining and the volume fraction of collagen was measured;the number of micro-vessels was observed by CD31 staining and the micro-vessel density was measured.One way ANOVA was used to show the difference between groups,LSD posthoc test was used to compare between groups,and Pearson correlation coefficient was used to analyze collagen volume fraction and micro-vessel density.Results Two months after the treatment,the histological results showed that the scar thickness of the laser combined group and the laser combined with multi-point microinjection group was significantly thinner than that of the control group,the texture of the scar was softened,the red color gradually became lighter,and approached to the color of the surrounding skin.Compared with the control group,the scar in the traditional injection group was thinner,softer and lighter.The scar thickness of laser combined with multi-point microinjection group was thinner than that of laser combined group and simple traditional injection group.Masson staining was used to calculate the collagen volume fraction(CVF):the expression of CVF in hypertrophic scar tissue of each group was reduced.The CVF values of each group at the 7th day and the 2nd month were compared.There was statistically significant difference between the laser combined group,the laser combined multi-point microinjection group and the simple traditional injection group(P<0.05),but there was no statistical difference between the control group(P>0.05).The CVF value of each group was further compared at 2 months after treatment,the laser combined multi-point microinjection group<laser combined group<simple traditional injection group,and there was a significant difference between the groups(P<0.05).CD31 staining observation:after treatment,micro-vessel density in each group of hypertrophic scar tissue decreased.The micro-vessel density(MVD)was compared at the 7th and 2nd month in each group.There was statistical difference in laser combined group,laser combined multi-point microinjection group and simple traditional injection group(P<0.05),but there was no statistical difference in the control group(P>0.05).2 months after treatment,the MVD values of each group were compared with the result as the laser combined with multi-point microinjection group<laser combined group<simple traditional injection group,but there was no statistical difference between the laser combined with multi-point microinjection group and the laser combined group(P>0.05).There was statistical difference between the laser combined with multi-point microinjection group and the laser combined group and the simple traditional injection group(P<0.05).The Pearson correlation coefficient was r=0.972,which indicated that there was a positive correlation between the collagen content and the number of micro-vessels.Conclusions The use of fractional laser combined with pulsed dye laser and multi-point microinjection of triamcinolone in the treatment of hypertrophic scars in rabbit ears can evenly reduce the number of fibroblasts,promote the normalization of collagen density and arrangement,and reduce collagen volume fraction and micro-vessel density.The curative effect is better than fractional laser combined with pulse dye laser therapy and simple traditional triamcinolone injection therapy.There is a positive correlation between collagen content and micro-vessel density in hypertrophic scars.Simultaneously inhibiting the proliferating blood vessels can improve the therapeutic effectiveness.