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摘要:
目的 探讨猪真皮深层组织错位损伤对皮肤纤维化的影响，以期为烧伤瘢痕治疗提供一定的理论依据。 方法 采用实验研究方法。取6只2个月龄雌性杜洛克猪，将背部右侧切取中厚皮片和真皮深层组织片并原位回植的15个术区纳入真皮原位回植组，将背部左侧切取中厚皮片和真皮深层组织片并将真皮深层组织片放在脂肪层下的15个术区纳入真皮错位组。观察2组伤后7、14、21 d术区毛发生长情况及伤后14 d横断面结构。伤后7、14、21 d，测量并比较2组术区皮肤厚度（表皮到脂肪上缘的距离）、真皮厚度（表皮下缘到脂肪上缘的距离，不包括真皮和脂肪间的纤维组织厚度）、真皮-脂肪界面（真皮原位回植组为真皮深层下缘至脂肪上缘、真皮错位组为真皮浅层下缘至脂肪上缘）的纤维组织厚度，测量真皮原位回植组术区真皮切割界面（真皮浅层下缘至真皮深层上缘）纤维组织厚度并与真皮-脂肪界面纤维组织厚度进行比较；行天狼猩红染色，观测并比较2组术区真皮-脂肪界面及真皮原位回植组术区真皮切割界面和真皮-脂肪界面Ⅰ、Ⅲ型胶原蛋白含量；行免疫组织化学染色观察2组术区增殖细胞核抗原（PCNA）、转化生长因子β1（TGF-β1）、成纤维细胞生长因子2（FGF-2）、肝细胞生长因子（HGF）的阳性表达。样本数均为6。对数据行独立样本t检验。 结果 伤后7、14、21 d，真皮原位回植组术区毛发均较真皮错位组浓密。伤后14 d，真皮错位组术区皮肤横断面呈“三明治”样结构，真皮原位回植组术区皮肤横断面结构正常。伤后7、14、21 d，真皮错位组术区皮肤厚度分别为（4 234±186）、（4 688±360）、（4 548±360）μm，与真皮原位回植组的（4 425±156）、（4 714±141）、（4 310±473）μm均相近（P>0.05）；真皮错位组术区真皮厚度均较真皮原位回植组明显变薄（t值分别为-9.73、-15.85、-15.41，P<0.01）；真皮错位组术区真皮-脂肪界面纤维组织厚度均较真皮原位回植组明显增厚（t值分别为14.48、20.58、15.67，P<0.01）；真皮原位回植组术区真皮-脂肪界面和真皮切割界面纤维组织厚度均无明显差异（P>0.05）。伤后7、14、21 d，真皮错位组术区真皮-脂肪界面Ⅲ型胶原蛋白含量均较真皮原位回植组明显增加（t值分别为2.65、0.61、7.39，P<0.05或P<0.01），而2组术区真皮-脂肪界面Ⅰ型胶原蛋白含量均无明显差异（P>0.05）；真皮原位回植组术区真皮-脂肪界面与真皮切割界面Ⅰ、Ⅲ型胶原蛋白含量均无明显差异（P>0.05）。伤后7、14、21 d，真皮错位组术区真皮浅层和脂肪组织中均有PCNA、TGF-β1、FGF-2、HGF阳性表达，真皮原位回植组术区真皮浅层、真皮深层和脂肪层均有PCNA、TGF-β1、FGF-2、HGF阳性表达。 结论 猪真皮组织固有厚度不足是引起纤维化的关键因素，纤维化的生物学目的旨在“补偿”皮肤的固有厚度，脂肪组织也可能是影响皮肤纤维化修复的重要组成部分。 Abstract:Objective To explore the effect of deep dermal tissue dislocation injury on skin fibrosis in pig, in order to provide some theoretical basis for burn scar treatment. Methods The experimental research method was applied. Six 2-month-old female Duroc pigs were taken. Fifteen operative areas on the right dorsum of pigs on which medium-thick skin grafts and deep dermal tissue slices were cut and re-implanted were included into dermal in situ reimplantation group, and fifteen operative areas on the left dorsum of pigs on which medium-thick skin grafts and deep dermal tissue slices were cut and the deep dermal tissue slice was placed under the fat layer were included into the dermal dislocation group. The hair growth in the operative areas on post-injury day (PID) 7, 14, and 21 and the cross-sectional structure on PID 14 were observed in the two groups. On PID 7, 14, and 21, the skin thickness (the distance from the epidermis to the upper edge of the fat), the dermal thickness (the distance from the lower edge of the epidermis to the upper edge of the fat, excluding the fibrotic tissue thickness between the dermis and the fat), and the fibrosis tissue thickness of the dermis-fat interface (from the lower edge of the deep dermis to the upper edge of the fat in dermal in situ reimplantation group and from the lower edge of the superficial dermis to the upper edge of the fat in dermal dislocation group) in the operative areas were measured and compared between the two groups; the fibrotic tissue thickness at the dermal cutting interface (from the lower edge of the superficial dermis to the upper edge of the deep dermis) in the operative areas in dermal in situ reimplantation group was measured and compared with the fibrotic tissue thickness at the dermal-fat interface. Sirius red staining was performed to observe and compare the type Ⅰ and Ⅲ collagen content in the dermal-fat interface in the operative areas between the 2 groups and between the dermal cutting interface and dermal-fat interface in the operative areas in dermal in situ reimplantation group. Immunohistochemical staining was performed to observe the positive expressions of proliferating cell nuclear antigen (PCNA), transforming growth factor β1 (TGF-β1), fibroblast growth factor 2 (FGF-2), and hepatocyte growth factor (HGF) in the operative areas in the two groups. The sample number was 6. Data were statistically analyzed with independent sample t test. Results On PID 7, 14, and 21, the hairs in the operative areas in dermal in situ reimplantation group were denser than those in dermal dislocation group. On PID 14, the skin cross section in the operative areas in dermal dislocation group showed a "sandwich"-like structure, while the skin cross section in the operative areas in dermal in situ reimplantation group had normal structure. On PID 7, 14, and 21, the skin thickness in the operative areas in dermal dislocation group was (4 234±186), (4 688±360), and (4 548±360) μm, respectively, which was close to (4 425±156), (4 714±141), and (4 310±473) μm in dermal in situ reimplantation group (P>0.05); the dermal thickness in the operative areas in dermal dislocation group was significantly thinner than that in dermal in situ reimplantation group (with t values of -9.73, -15.85, and -15.41, respectively, P<0.01); the fibrotic tissue thickness at the dermal-fat interface in the operative areas in dermal dislocation group was significantly thicker than that in dermal in situ reimplantation group (with t values of 14.48, 20.58, and 15.67, respectively, P<0.01); there was no statistically significant difference between the fibrotic tissue thickness at the dermal-fat interface and the dermal cutting interface in the operative areas in dermal in situ reimplantation group (P>0.05). On PID 7, 14, 21, the type Ⅲ collagen content in the dermal-fat interface in the operative areas in dermal dislocation group was increased significantly compared with that in dermal in situ replantation group (with t values of 2.65, 0.61, and 7.39, respectively, P<0.05 or P<0.01), whereas there were no statistically significant differences in the type Ⅰ collagen content at the dermal-fat interface in the operative areas between the 2 groups (P>0.05) and the type Ⅰ and Ⅲ collagen content between the dermal-fat interface and the dermal cutting interface in the operative areas in dermal in situ reimplantation group (P>0.05). On PID 7, 14, and 21, PCNA, TGF-β1, FGF-2, and HGF were positively expressed in the superficial dermis and adipose tissue in the operative areas in dermal dislocation group, while PCNA, TGF-β1, FGF-2, and HGF were positively expressed in the superficial dermis, deep dermis, and adipose tissue in the operative areas in dermal in situ reimplantation group. Conclusions Inadequate intrinsic thickness of dermal tissue is the key factor causing fibrosis, and the biological purpose of fibrosis is to "compensate" the intrinsic thickness of the skin. Besides, adipose tissue may also be an important component of fibrotic skin repair. -
Key words:
- Skin /
- Cicatrix /
- Fibrosis /
- Adipose tissue /
- Deep dermal tissue /
- Cutting injury
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参考文献
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1 猪背部2组术区真皮深层组织损伤模型设计。1A.真皮原位回植组模型设计示意图；1B.真皮错位组模型设计示意图，将真皮深层组织片放在脂肪层下；1C.中厚皮片及真皮深层组织片切开实物图
注：蓝色箭头指示中厚皮片，黄色箭头指示真皮深层组织片，红色箭头指示脂肪组织
2 猪背部2组术区真皮深层组织损伤后各时间点毛发生长情况。2A、2B、2C.分别为真皮错位组伤后7、14、21 d；2D、2E、2F.分别为真皮原位回植组伤后7、14、21 d，术区毛发分别较图2A、2B、2C浓密
3 猪背部2组术区真皮深层组织损伤后14 d皮肤组织横断面结构。3A.真皮错位组呈“三明治”样结构，蓝色箭头指示真皮浅层组织，黄色箭头指示脂肪组织，红色箭头指示真皮深层组织；3B.真皮原位回植组皮肤组织结构正常，蓝色箭头指示真皮浅层组织，红色箭头指示脂肪组织
4 猪背部2组术区真皮深层组织损伤后14 d术区各层次组织厚度 苏木精-伊红×200。4A、4B.分别为真皮错位组和真皮原位回植组，图4A真皮厚度（表皮下缘到脂肪上缘的距离，黑色箭头长度）较图4B明显变薄，图4A皮肤厚度（表皮到脂肪上缘的距离，红色箭头长度）与图4B无明显差异，图4A真皮-脂肪界面（真皮浅层下缘至脂肪上缘）的纤维组织厚度（蓝色箭头长度）较图4B明显增加；图4B中真皮切割界面（真皮浅层下缘到真皮深层上缘）纤维组织厚度（黄色箭头长度）和真皮-脂肪界面无明显差异
5 猪背部2组术区真皮深层组织损伤后14 d Ⅰ、Ⅲ型胶原蛋白含量 青石蓝-天狼猩红×200。5A.真皮错位组真皮浅层下缘至脂肪上缘有大量Ⅲ型胶原蛋白、少量的Ⅰ型胶原蛋白；5B.真皮原位回植组真皮浅层下缘至真皮深层上缘可见大量Ⅲ型胶原蛋白，少量Ⅰ型胶原蛋白；5C.真皮原位回植组真皮深层下缘至脂肪上缘Ⅰ、Ⅲ型胶原蛋白均与图5B相近，Ⅱ型胶原蛋白较图5A明显减少，Ⅰ型胶原蛋白与图5A相近
注：观察区域为2条虚线中间区域；Ⅲ型胶原蛋白染绿色，Ⅰ型胶原蛋白染红色
6 2组猪背部真皮深层组织损伤后14 d真皮和脂肪组织细胞增殖及促纤维化相关细胞因子表达情况 二氨基联苯胺-苏木精×100。6A、6B.分别为真皮错位组真皮和脂肪组织均可见PCNA表达；6C、6D.分别为真皮原位回植组真皮和脂肪组织均可见PCNA表达；6E、6F.分别为真皮错位组真皮和脂肪组织均可见TGF-β1表达；6G、6H.分别为真皮原位回植组真皮和脂肪组织均可见TGF-β1表达；6I、6J.分别为真皮错位组真皮和脂肪组织均可见FGF-2表达；6K、6L.分别为真皮原位回植组真皮和脂肪组织均可见FGF-2表达；6M、6N.分别为真皮错位组真皮和脂肪组织均可见HGF表达；6O、6P.分别为真皮原位回植组真皮和脂肪组织均可见HGF表达
注：细胞增殖及促纤维化相关细胞因子阳性表达均为棕色；PCNA为增殖细胞核抗原，TGF-β1为转化生长因子β1，FGF-2为成纤维细胞生长因子2，HGF为肝细胞生长因子
表1 猪背部2组术区真皮深层组织损伤后各时间点皮肤各层组织厚度比较（μm，
组别 样本数 7 d 14 d 21 d 真皮厚度 皮肤厚度 真皮-脂肪界面纤维组织厚度 真皮厚度 皮肤厚度 真皮-脂肪界面纤维组织厚度 真皮厚度 皮肤厚度 真皮-脂肪界面纤维组织厚度 真皮错位组 6 1 793±148 4 234±186 937±120 1 674±138 4 688±360 1 700±105 1 995±211 4 548±360 1 688±198 真皮原位回植组 6 2 933±246 4 425±156 193±38 3 650±272 4 714±141 411±74 4 145±269 4 310±473 319±91 t值 -9.73 -1.92 14.48 -15.85 -0.13 20.58 -15.41 0.98 15.67 P值 <0.001 0.084 <0.001 <0.001 0.874 <0.001 <0.001 0.349 <0.001 注：真皮厚度指表皮下缘到脂肪上缘的距离，但不包括真皮和脂肪间的纤维组织厚度，皮肤厚度指表皮到脂肪上缘的距离，真皮原位回植组真皮-脂肪界面厚度为真皮深层下缘至脂肪上缘，真皮错位组真皮-脂肪界面厚度为真皮浅层下缘至脂肪上缘 
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表2 真皮原位回植组猪背部术区真皮深层组织损伤后各时间点真皮切割界面和真皮-脂肪界面纤维组织厚度比较（μm，
位置 样本数 7 d 14 d 21 d 真皮切割界面 6 190±34 349±25 389±33 真皮-脂肪界面 6 193±39 361±20 402±29 t值 -0.16 -0.91 -0.69 p值 0.875 0.384 0.505 注：真皮切割界面为真皮浅层下缘至真皮深层上缘，真皮-脂肪界面为真皮深层下缘至脂肪上缘
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表3 猪背部2组术区真皮深层组织损伤后各时间点真皮-脂肪界面Ⅰ型和Ⅲ型胶原蛋白含量比较（
组别 样本数 7 d 14 d 21 d Ⅰ型胶原蛋白 Ⅲ型胶原蛋白 Ⅰ型胶原蛋白 Ⅲ型胶原蛋白 Ⅰ型胶原蛋白 Ⅲ型胶原蛋白 真皮错位组 6 706±120 1 289±333 837±39 1 197±309 1 112±127 1 931±306 真皮原位回植组 6 788±41 613±120 841±84 360±32 1 001±57 1 001±57 t值 -1.80 2.65 -0.11 0.61 1.96 7.39 P值 0.142 0.024 0.916 <0.001 0.078 <0.001 注：真皮原位回植组真皮-脂肪界面为真皮深层下缘至脂肪上缘，真皮错位组真皮-脂肪界面为真皮浅层下缘到脂肪上缘
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表4 真皮原位回植组背部术区真皮深层组织损伤后各时间点真皮-脂肪界面与真皮切割界面Ⅰ型和Ⅲ型胶原蛋白含量比较（
位置 样本数 7 d 14 d 21 d Ⅰ型胶原蛋白 Ⅲ型胶原蛋白 Ⅰ型胶原蛋白 Ⅲ型胶原蛋白 Ⅰ型胶原蛋白 Ⅲ型胶原蛋白 真皮切割界面 6 745±44 632±87 988±176 1 070±88 1 146±121 1 006±74 真皮-脂肪界面 6 788±41 613±120 1 052±53 1 046±82 1 001±157 1 001±57 t值 -1.80 0.31 -0.85 0.50 2.65 0.14 P值 0.103 0.762 0.414 0.633 0.068 0.889 注：真皮切割界面为真皮浅层下缘至真皮深层上缘，真皮-脂肪界面为真皮深层下缘至脂肪上缘
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