乳糜化脂肪对移植于裸鼠背部皮下的人增生性瘢痕影响的实验研究

目的评价乳糜化脂肪对移植于裸鼠背部皮下人增生性瘢痕的影响。 方法选取18只BALB/c裸鼠；收集解放军总医院第四医学中心烧伤整形科行腹部、大腿外侧部脂肪负压抽吸手术获取的脂肪颗粒细胞及面、颈部手术切除的增生性瘢痕组织；脂肪颗粒细胞经过纳米转换头等操作处理为乳糜化脂肪；无菌条件下将增生性瘢痕组织切割成多块小样本；用眼科剪于各个裸鼠背部各作4个皮肤切口，稍做游离后将前述人增生性瘢痕组织块埋植入切口内，制备增生性瘢痕裸鼠动物模型。18只裸鼠按照随机数字表法分为3组，对照组，曲安奈德组和乳糜化脂肪组，每组6只裸鼠，24块瘢痕组织。移植后1周，对照组于每块移植瘢痕组织块内注射0.9%氯化钠溶液(0.05 mL/g)；曲安奈德组注射曲安奈德注射液(0.05 mL/g)；乳糜化脂肪组注射乳糜化脂肪(0.05 mL/g)；之后每周各注射1次。移植后4周取材，比较移植前人增生性瘢痕组织块重量、移植后4周人增生性瘢痕组织块重量变化百分比；行苏木精-伊红染色和Masson染色，生物显微镜下观察组织形态学变化；比较Masson染色半定量分析阳性面积(天蓝色)占整体视野总面积的百分比。行免疫组织化学染色检测核心蛋白聚糖表达。对数据行单因素方差分析和Dunnett-t检验。 结果移植前，3组人增生性瘢痕组织块重量比较，差异无统计学意义(F＝26.230，P＝0.119)，移植后4周，乳糜化脂肪组、曲安奈德组、对照组人增生性瘢痕组织块重量变化百分比分别为(73.2±15.9)%、(79.5±8.6)%、(87.0±14.9)%，比较差异有统计学意义(F＝16.680，P＝0.042)，乳糜化脂肪组分别与对照组、曲安奈德组比较，差异均有统计学意义(t＝14.130、1.228，P＝0.010、0.099)。组织学观察可见，苏木精-伊红染色与Masson染色整体变化趋势相符：对照组真皮层可见大量胶原纤维及纤维细胞，胶原纤维排列紊乱；曲安奈德组真皮层见粗大胶原纤维，连续性及整齐度可；乳糜化脂肪组真皮层胶原纤维数量少且排列规律。Masson染色半定量分析：乳糜化脂肪组、曲安奈德组、对照组阳性面积(天蓝色)占整体视野总面积的百分比分别为(74.3±13.5)%、(80.9±9.0)%、(91.2±4.5)%，3组比较差异有统计学意义(F＝1.708，P＝0.025)，乳糜化脂肪组分别与曲安奈德组、对照组比较，差异均有统计学意义(t＝13.130、7.806，P＝0.027、0.019)。增生性瘢痕中核心蛋白聚糖免疫组织化学分析结果：光学显微镜下可见曲安奈德组、乳糜化脂肪组显示出核心蛋白聚糖阳性染色结果，且乳糜化脂肪组染色强于曲安奈德组。对照组极其低地表达点、斑片状核心蛋白聚糖染色。乳糜化脂肪组、曲安奈德组、对照组核心蛋白聚糖表达量分别为0.021±0.010、0.025±0.012、0.088±0.058，3组比较差异有统计学意义(F＝32.160，P＝0.034)，乳糜化脂肪组分别与曲安奈德组、对照组比较，差异均有统计学意义(t＝8.016、1.224，P＝0.048，0.027)。 结论乳糜化脂肪注射至人增生性瘢痕可降低成纤维细胞密度和数量，促进胶原排列、数量及形状正常化，改善胶原堆积等组织学特征。

Experimental study on the effect of chyle fat on human hypertrophic scar transplanted under the back of nude mice

ObjectiveTo evaluate the effect of chyle fat on hypertrophic scar of human transplanted under the back of nude mice. MethodsEighteen BALB/c nude mice were selected. Fat granule cells were collected from the abdominal and later thigh fat suction operations and hypertrophic scar tissue excised from the face and neck in the Department of Burns and Plastic Surgery, Fourth Medical Center of PLA General Hospital. Fat granule cells were processed into nano-transformed heads to make chyle fat. Each human hypertrophic scar tissue were cut into small pieces. Four skin incisions were made on the back of each nude mice with scissors. These specimens were implanted into the backs of the mice with 4 pieces per nude mouse. Eighteen nude mice were divided into 3 groups: control group, triamcinolone acetonide group and chyle fat group according to the random number table method, 6 nude mice and 24 scar tissues per group. One week after transplantation, the control group was injected with 0.9% sodium chloride solution into each transplanted scar tissue(0.05 mL/g); triamcinolone acetonide was injected into the triamcinolone acetonide group (0.05 mL/g); chyle fat group was injected with chyle fat (0.05 mL/g); and then injected once a week. Comparison of pre-transplant hypertrophic scar tissue weight and percentage change in weight of hypertrophic scar tissue 4 weeks after transplantation was calculated. After hematoxylin-eosin staining and Masson staining, histomorphological changes under biological microscope were observed. Masson staining semi-quantitative analysis of positive area (sky blue) as a percentage of total field of view was compared. Immunohistochemical staining was performed to detect the expression of decorin. Data were processed with One-way ANOVA and Dunnett-t test. ResultsBefore transplantation, there were no significant differences in the weight of hypertrophic scar tissue among the 3 groups (F=26.230, P=0.119). At 4 weeks after transplantation, the percentage change of weight of hypertrophic scar tissue in the chyle fat group, triamcinolone acetonide group and control group was (73.2±15.9)%, (79.5±8.6)%, (87.0±14.9)%, respectively. The difference was statistically significant (F=16.680, P=0.042). The percentage change of weight of hypertrophic scar tissue in the chyle fat group was compared with the control group and triamcinolone acetonide group, the differences were statistically significant (t=14.130, 1.228, P=0.010, 0.099). Histological observations showed that hematoxylin-eosin staining was consistent with the overall trend of Masson staining. The results of hematoxylin-eosin staining were: large amount of collagen fibers and fibroblasts and disorder of collagen fibers were seen in the dermis of the control group; the dermis of the triamcinolone acetonide group were found in coarse collagen fibers, and the continuity and uniformity were generally; the number of collagen fibers in the dermis of the chyle fat group was small and arranged. Semi-quantitative analysis of Masson staining: the percentages of the positive area(sky blue) of the chyle fat group, triamcinolone acetonide group were accounted for (74.3±13.5)%, (80.9±9.0)%, (91.2±4.5)%, respectively. Comparative differences were statistically significant (F=1.708, P=0.025). The chyle fat group was significantly different from the triamcinolone acetonide group and the control group (t=13.130, 7.806; P=0.027, 0.019). Immunohistochemical analysis of decorin in hypertrophic scar: under the optical microscope, the triamcinolone acetonide group and the chyle fat group showed positive results of decorin, and the chyle fat group staining was stronger than that of the triamcinolone acetonide group. However, the control group showed extremely low expression point and patchy decorin staining. The expression levels of decorin in the chyle fat group, triamcinolone acetonide group and control group were 0.021±0.010, 0.025±0.012 and 0.088±0.058, respectively. The difference was statistically significant (F=32.160, P=0.034). The expression levels of decorin in the chyle fat group was compared with the triamcinolone acetonide group and the control group, there were statistically significant (t=8.016, 1.224; P=0.048, 0.027). ConclusionsInjecting chyle fat into hypertrophic scar can reduce the density and quantity of fibroblasts, promote the normalization of collagen arrangement, quantity and shape, and improve histological features such as collagen accumulation.