摘要
目的探讨自体脂肪源性基质血管成分（SVF）局部移植对兔耳增生性瘢痕（HS）形成的影响及机制。方法取24只新西兰大耳白兔，于每只兔左耳腹侧面造成4个直径为1 cm的全层皮肤缺损创面，制作兔耳HS模型，观察创面上皮化情况及局部组织增生情况，记录创面愈合（完全上皮化）时间和HS形成时间。按随机数字表法将24只兔分为SVF组、单纯DMEM组和单纯HS组，每组8只兔、32个创面。于伤后25 d（创面完全上皮化后），将0.2 mL含有CM-Dil标记的自体SVF的低糖DMEM培养液注射于SVF组兔HS，单纯DMEM组兔HS同前注射等量低糖DMEM培养液，每5天注射1次，共3次；单纯HS组兔HS不予任何处理。伤后40 d，取各组兔耳HS，行苏木素-伊红染色观察组织学形态，行Van Gieson染色观察胶原排列情况，荧光显微镜下观察HS中CM-Dil标记的SVF情况，实时荧光定量反转录PCR法和蛋白质印迹法分别检测HS中转化生长因子β1（TGF-β1）、Smad3及Samd7的mRNA和蛋白表达情况。对数据行单因素方差分析、Tukey检验。结果（1）兔耳创面完全上皮化时间为伤后（20.0±2.0）d；伤后25 d，HS形成。伤后40 d，单纯DMEM组及单纯HS组兔耳HS仍保持增生状态，SVF组兔耳HS体积缩小、变平，质地变软，色泽稍变浅。（2）伤后40 d，与单纯DMEM组和单纯HS组比较，SVF组兔耳HS内上皮脚样结构较多，炎性细胞数量较少。SVF组兔耳HS胶原排列相对较规则，胶原之间间隙更大。（3）伤后40 d，SVF组兔耳HS仍可见CM-Dil标记的SVF。（4）伤后40 d，与单纯DMEM组和单纯HS组比较，SVF组兔耳HS中TGF-β1和Smad3的mRNA表达量明显下调（P〈0.05），Smad7的mRNA表达量明显上调（P〈0.05）；单纯DMEM组与单纯HS组兔耳HS中TGF-β1、Smad3、Smad7的mRNA表达量无明显差异（P〉0.05）。（5）伤后40 d，与单纯DMEM组（0.74±0.03、0.73±0.10、0.54±0.09）和单纯HS组（0.72±0.08、0.71±0.12、0.53±0.06）比较，SVF组兔耳HS中TGF-β1和Smad3的蛋白表达量（0.57±0.06、0.42±0.09）明显下调（P〈0.05），Smad7的蛋白表达量（0.71±0.05）明显上调（P〈0.05）；单纯DMEM组与单纯HS组兔耳HS中的TGF-β1、Smad3、Smad7的蛋白表达量无明显差异（P〉0.05）。结论兔HS形成早期瘢痕内移植自体SVF可抑制HS形成，其机制可能与TGF-β1/Smad信号通路有关。
ObjectiveTo explore the effects of local transplantation of autologous adipose-derived stromal vascular fraction （SVF） on the hyperplastic scar （HS） formation in rabbit ears and the mechanism.MethodsTwenty-four New Zealand white rabbits were used to reproduce HSs by making four full-thickness skin defect wounds with a diameter of 1 cm on the ventral surface of left ear of each rabbit. Wound epithelization and local-tissue proliferation were observed, and wound healing （complete epithelization） time and formation time of HS were recorded. The 24 rabbits were divided into SVF group, pure DMEM group, and pure HS group according to the random number table, with 8 rabbits and 32 wounds in each group. On post injury day （PID） 25 （after the complete epithelization of wounds）, 0.2 mL of low glucose DMEM medium containing CM-Dil labeled autologous SVF was injected into HSs of rabbits in SVF group, while the same amount of low glucose DMEM medium was injected into HSs of rabbits in pure DMEM group. The frequency of injection was once every 5 days, totally for 3 times. HSs of rabbits in pure HS group did not receive any treatment. On PID 40, HSs of rabbits′ ears in each group were harvested, then the histological form was observed by hematoxylin and eosin staining, the arrangement of collagen in HS was observed by Van Gieson staining, the distribution of CM-Dil-labeled SVF in the HS was observed with fluorescence microscope, and the mRNA expression and the protein expression of transforming growth factor β1 （TGF-β1）, Smad3, and Smad7 in HS were determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively. Data were processed with one-way analysis of variance and Tukey test.Results（1） Complete epithelization time of wounds of rabbits′ ears was （20.0±2.0） d post injury, and HSs were formed on PID 25. On PID 40, HSs of rabbits′ ears in pure DMEM group and pure HS group were still in hyperplasia, while those in SVF group became smaller, flat, soft, and light colored. （2） On PID 40, compared with those in pure DMEM group and pure HS group, the number of epithelium foot like structures was more and the amount of inflammatory cells was less. The collagen of HSs of rabbits′ ears in SVF group was arranged more regularly with broader gap between collagens. （3） On PID 40, CM-Dil-labeled SVF could still be observed in the HSs of rabbits′ ears in SVF group. （4） On PID 40, compared with those in pure DMEM group and pure HS group, the mRNA expressions of TGF-β1 and Smad3 in the HSs of rabbits′ ears in SVF group were significantly down-regulated （P〈0.05）, while the mRNA expression of Smad7 was significantly up-regulated （P〈0.05）. There were no significant differences in the mRNA expressions of TGF-β1, Smad3, and Smad7 in the HSs of rabbits′ ears between pure DMEM group and pure HS group （P〉0.05）. （5） On PID 40, compared with those in pure DMEM group （0.74±0.03, 0.73±0.10, 0.54±0.09） and pure HS group （0.72±0.08, 0.71±0.12, 0.53±0.06）, the protein expressions of TGF-β1 and Smad3 in the HSs of rabbits′ ears in SVF group （0.57±0.06, 0.42±0.09） were significantly down-regulated （P〈0.05）, while the protein expression of Smad7 （0.71±0.05） was significantly up-regulated （P〈0.05）. The protein expressions of TGF-β1, Smad3, and Smad7 in the HSs of rabbits′ ears in pure DMEM group and pure HS group were close （P〉0.05）.ConclusionsAutologous SVF transplantation can inhibit the formation of HS in the early stage of scar formation of rabbit, the mechanism may be related to the TGF-β1/Smad signaling pathway.
作者
邓呈亮
李修权
刘志远
姚远镇
魏在荣
王达利
Deng ChengLiang;Li Xiuquan;Liu Zhiyuan;Yao Yuanzheng;Wei Zairong;Wang Dali(Department of Burns and Plastic Surgery,the Affiliated Hospital of Zunyi Medical College,Zunyi 563003,Chin)
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2018年第8期542-548,共7页
Chinese Journal of Burns
基金
国家自然科学基金（81060157）
作者简介
通信作者：王达利，Email：daliwangzy@sina．com
