Effects of recombinant human metallothionein-Ⅲ combined with wound dressing on wound healing of full-thickness skin defects in mice
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摘要:
目的 探讨重组人金属硫蛋白Ⅲ（rh-MT-Ⅲ）联合创面敷料在小鼠全层皮肤缺损创面愈合中的作用。 方法 该研究为实验研究。取24只6周龄美国癌症研究所小鼠，雌雄各半，在每只小鼠背部制备2个对称的圆形全层皮肤缺损创面。将小鼠按性别、体重分层随机分为在创面涂布相应的溶液的生理盐水组、敷料组、rh-MT-Ⅲ组和在创面涂布rh-MT-Ⅲ和创面敷料的混合液的联合治疗组，每组6只小鼠。伤后1~7 d，每天观察所有小鼠的活动、饮食和毛发生长变化，记录小鼠体重、创面面积并计算相对创面面积百分比。伤后7 d，取小鼠创面组织，行苏木精-伊红染色观察新生肉芽组织情况，行Masson染色观察胶原纤维形成情况，行免疫荧光染色检测细胞增殖情况（以Ki67相对荧光强度表示）和细胞凋亡情况［以原位末端标记（TUNEL）相对荧光强度表示］。以上实验样本数均为6。 结果 伤后7 d内，4组小鼠均无活动、饮食或毛发生长异常。伤后1~7 d，4组小鼠体重总体比较，差异均无统计学意义（P>0.05）。联合治疗组小鼠伤后2、3、4、5、6、7 d相对创面面积百分比均明显低于生理盐水组、rh-MT-Ⅲ组（P<0.05），伤后3、4、5、6、7 d相对创面面积百分比均明显低于敷料组（P<0.05）。敷料组小鼠伤后6、7 d及rh-MT-Ⅲ组小鼠伤后7 d相对创面面积百分比均明显低于生理盐水组（P<0.05）。伤后7 d，联合治疗组小鼠创面组织中可见大量毛细血管和成纤维细胞，且创面上缘新生上皮组织生长优于其他3组；联合治疗组小鼠创面组织中胶原纤维致密程度较其他3组更高且排列更为有序。伤后7 d，敷料组、rh-MT-Ⅲ组、联合治疗组小鼠创面组织中Ki67相对荧光强度分别为（289±35）%、（197±17）%、（389±56）%，均明显高于生理盐水组的（100±15）%（P<0.05），联合治疗组小鼠创面组织中Ki67相对荧光强度明显高于敷料组、rh-MT-Ⅲ组（P值均<0.05）。伤后7 d，敷料组、rh-MT-Ⅲ组、联合治疗组小鼠创面组织中TUNEL相对荧光强度为（55.5±5.7）%、（66.7±8.0）%、（20.0±2.2）%，均明显低于生理盐水组的（100.0±12.9）%（P<0.05），联合治疗组小鼠创面组织中TUNEL相对荧光强度明显低于敷料组、rh-MT-Ⅲ组（P值均<0.05）。 结论 rh-MT-Ⅲ可协同创面敷料促进创面周围肉芽组织生长以及胶原沉积，还可提高细胞增殖活力，减少细胞凋亡，通过促进创缘皮肤再上皮化，从而加速小鼠全层皮肤缺损创面愈合。 Abstract:Objective To investigate the effects of recombinant human metallothionein-Ⅲ (rh-MT-Ⅲ) combined with wound dressing on wound healing of full-thickness skin defects in mice. Methods This study was an experimental study. Twenty-four half male and half female 6 weeks old Institute of Cancer Research mice were taken, and two symmetrical round full-thickness skin defect wounds were prepared on the back of each mouse. The mice were stratified and randomly divided into normal saline group, dressing group, rh-MT-Ⅲgroup (applying the corresponding solution on the wounds), and combined treatment group (applying a mixture of rh-MT-Ⅲ and wound dressing on the wounds) according to sex and body weight, with 6 mice in each group. From 1 to 7 d after injury, all mice were observed daily for changes in activity, diet, and fur growth, their body weights and wound areas were recorded, and the relative wound area percentages were calculated. On 7 d after injury, the wound tissue of mice was taken for hematoxylin-eosin staining to observe the newborn granulation tissue, for Masson staining to observe collagen fiber formation, and for immunofluorescence staining to detect cell proliferation (denoted as Ki67 relative fluorescence intensity) and cell apoptosis (denoted as TdT-mediated dUTP nick end labeling (TUNEL) relative fluorescence intensity). The sample size in the above experiments was 6. Results There were no abnormalities in activity, diet, or fur growth in the 4 groups of mice within 7 d after injury. There were no statistically significant differences in the overall comparison of the body weights of mice in the 4 groups from 1 to 7 d after injury (P>0.05). The relative wound area percentages of mice in combined treatment group were significantly lower than those in normal saline group and rh-MT-Ⅲ group on 2, 3, 4, 5, 6, and 7 d after injury (P<0.05), and the relative wound area percentages of mice in combined treatment group were significantly lower than those in dressing group on 3, 4, 5, 6, and 7 d after injury (P<0.05). The relative wound area percentages of mice in dressing group on 6 and 7 d after injury and in rh-MT-Ⅲ group on 7 d after injury were significantly lower than those in normal saline group (P<0.05). On 7 d after injury, a large number of capillaries and fibroblasts could be seen in wound tissue of mice in combined treatment group, and the growth of new epithelial tissue at the upper edge of the wound was better than that of the other three groups; the collagen fibers in the wound tissue of mice in combined treatment group had higher degree of density and arrangement in a more orderly manner than those of the other three groups. On 7 d after injury, the Ki67 relative fluorescence intensity in the wound tissue of mice in dressing group, rh-MT-Ⅲ group, and combined treatment group was (289±35)%, (197±17)%, and (389±56)%, which was significantly higher than (100±15)% in normal saline group, respectively (P<0.05), and the Ki67 relative fluorescence intensity in the wound tissue of mice in combined treatment group was significantly higher than that in dressing group and rh-MT-Ⅲ group, respectively (with both P values <0.05). On 7 d after injury, the TUNEL relative fluorescence intensity in the wound tissue of mice in dressing group, rh-MT-Ⅲ group, and combined treatment group was (55.5±5.7)%, (66.7±8.0)%, and (20.0±2.2)%, which was significantly lower than (100.0±12.9)% in normal saline group, respectively (P<0.05), and the TUNEL relative fluorescence intensity in the wound tissue of mice in combined treatment group was significantly lower than that in dressing group and rh-MT-Ⅲ group, respectively (with both P values <0.05). Conclusions rh-MT-Ⅲ combined with wound dressing can promote the growth of granulation tissue around the wound as well as collagen deposition, increase the cell proliferation vitality, reduce cell apoptosis, and promote the re-epithelialization of skin at the edge of the wounds, thus accelerating the healing of full-thickness skin defect wounds in mice. -
Key words:
- Wounds and injuries /
- Metallothionein /
- Biological dressings /
- Cell proliferation /
- Apoptosis /
- Wound repair
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参考文献
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1 4组全层皮肤缺损小鼠伤后各时间点体重比较（样本数为6，
注：生理盐水组、重组人金属硫蛋白Ⅲ（rh-MT-Ⅲ）组、敷料组创面分别涂布相应的溶液，联合治疗组创面涂布rh-MT-Ⅲ和创面敷料的混合液；时间因素主效应，F=4.39，P=0.003；处理因素主效应，F=0.78，P=0.518；二者交互作用，F=0.41，P=0.957；伤后1、2、3、4、5、6、7 d，4组小鼠体重总体比较，差异均无统计学意义（F值分别为0.60、0.32、1.06、1.18、0.94、0.69、0.18，P值分别为0.623、0.809、0.390、0.343、0.442、0.568、0.911）
2 4组全层皮肤缺损小鼠伤后各时间点创面愈合情况。2A、2B、2C、2D.分别为生理盐水组伤后1、3、5、7 d创面情况；2E、2F、2G、2H.分别为敷料组伤后1、3、5、7 d创面情况；2I、2J、2K、2L.分别为rh-MT-Ⅲ组伤后1、3、5、7 d创面情况；2M、2N、2O、2P.分别为联合治疗组伤后1、3、5、7 d创面情况，联合治疗组创面愈合速度快于其他3组
注：生理盐水组、重组人金属硫蛋白Ⅲ（rh-MT-Ⅲ）组、敷料组创面分别涂布相应的溶液，联合治疗组创面涂布rh-MT-Ⅲ和创面敷料的混合液
3 4组全层皮肤缺损小鼠伤后7 d创面组织病理学变化情况。3A、3B、3C、3D.分别为生理盐水组、敷料组、rh-MT-Ⅲ组及联合治疗组创面组织病理学变化情况，图3D新生肉芽组织、毛细血管和成纤维细胞（Fb）情况优于图3A、3B、3C 苏木精-伊红×100；3E、3F、3G、3H.分别为图3A、3B、3C、3D中方框中图形放大图，图3H毛细血管和Fb数量明显多于图3E、3F、3G 苏木精-伊红×400
注：生理盐水组、重组人金属硫蛋白Ⅲ（rh-MT-Ⅲ）组、敷料组创面分别涂布相应的溶液，联合治疗组创面涂布rh-MT-Ⅲ和创面敷料的混合液；黄色箭头指示成纤维细胞，红色箭头指示毛细血管
4 4组全层皮肤缺损小鼠伤后7 d创面组织中胶原纤维形成情况。4A、4B、4C、4D.分别为生理盐水组、敷料组、rh-MT-Ⅲ组及联合治疗组胶原纤维形成情况，图4B、4C、4D胶原纤维沉积优于图4A Masson×100；4E、4F、4G、4H.分别为图4A、4B、4C、4D中方框中图形放大图，图4H胶原纤维致密程度优于图4E、4F、4G Masson×400
注：生理盐水组、敷料组、重组人金属硫蛋白Ⅲ（rh-MT-Ⅲ）组创面分别涂布相应的溶液，联合治疗组创面涂布rh-MT-Ⅲ和创面敷料的混合液；黄色箭头指示胶原纤维，红色箭头指示纤维蛋白
5 4组全层皮肤缺损小鼠伤后7 d创面组织中Ki67荧光强度 Ki67-4'，6-二脒基-2-苯基吲哚×100。5A、5B、5C、5D.分别为生理盐水组、敷料组、rh-MT-Ⅲ组、联合治疗组Ki67荧光强度，图5B、5C、5D中Ki67荧光强度明显高于图5A
注：生理盐水组、重组人金属硫蛋白Ⅲ（rh-MT-Ⅲ）组、敷料组创面分别涂布相应的溶液，联合治疗组创面涂布rh-MT-Ⅲ和创面敷料的混合液；细胞核染色为蓝色，Ki67阳性染色为红色；以Ki67相对荧光强度表示细胞增殖情况
6 4组全层皮肤缺损小鼠伤后7 d创面组织中TUNEL荧光强度 TUNEL-4'，6-二脒基-2-苯基吲哚×100。6A、6B、6C、6D.分别为生理盐水组、敷料组、rh-MT-Ⅲ组、联合治疗组TUNEL荧光强度，图6B、6C、6D中TUNEL荧光强度明显低于图6A
注：生理盐水组、重组人金属硫蛋白Ⅲ（rh-MT-Ⅲ）组、敷料组创面分别涂布相应的溶液，联合治疗组创面涂布rh-MT-Ⅲ和创面敷料的混合液；细胞核染色为蓝色，原位末端标记（TUNEL）阳性染色为绿色；以TUNEL相对荧光强度表示细胞凋亡情况
表1 4组全层皮肤缺损小鼠伤后各时间点相对创面面积百分比比较（%，
表1. Comparison of relative wound area percentages in four groups of mice with full-thickness skin defects at various time points after injury
组别 创面数（个） 1 d 2 d 3 d 4 d 5 d 6 d 7 d 生理盐水组 6 100.0±12.7 81.4±5.7 63.2±7.6 47.7±5.8 39.2±4.5 35.7±4.1 33.4±2.7 敷料组 6 100.0±8.8 75.2±8.1 55.5±9.8 47.6±6.2 37.2±2.3 28.7±4.2a 23.1±1.8a rh-MT-Ⅲ组 6 100.0±11.2 80.8±8.6 60.4±4.2 51.5±4.4 38.8±4.7 32.6±5.0 27.7±1.2a 联合治疗组 6 100.0±5.0 66.8±9.7ab 40.9±3.2abc 30.9±1.8abc 25.5±3.2abc 20.0±2.1abc 14.8±1.9abc F值 <0.01 4.10 13.20 21.36 17.61 17.54 93.68 P值 >0.999 0.020 <0.001 <0.001 <0.001 <0.001 <0.001 注：生理盐水组、重组人金属硫蛋白Ⅲ（rh-MT-Ⅲ）组、敷料组创面分别涂布相应的溶液，联合治疗组创面涂布rh-MT-Ⅲ和创面敷料的混合液；时间因素主效应，F=563.49，P<0.001；处理因素主效应，F=24.09，P<0.001；二者交互作用，F=2.40，P=0.003；与生理盐水组相比，aP<0.05；与rh-MT-Ⅲ组相比，bP<0.05；与敷料组相比，cP<0.05 
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