吴昌炎·，房志强2，江宝华3，陈石海1

(1．广西医科大学附属第一医院整形美容科广西南宁5 30000；2．龙岩华美罗桥医院整形科；3．江苏省昆山市时光医疗美容医院)

[摘要]目的：探讨自体表皮细胞悬液在修复皮肤组织缺损创面的作用。方法：在2 o只大鼠身上分别取邮票大小自体全厚皮片进行消化，分离成单个表皮细胞的悬液；经传代培养后，使用自体表皮细胞悬液分别对2 0只大鼠背部表皮皮肤缺损创面进行覆盖移植，术后观察移植物成活率和植皮区收缩率，同时取移植物进行组织学观察；并与对照组2 o只自体愈合创面进行相互比较。结果：表皮细胞悬液移植后1周渐融合成片，伤1：2愈合；经6周后，伤口平整，轻度瘢痕愈合。而对照组在6周时伤口不平整，呈瘢痕愈合，色素沉着明显。结论：在表皮缺损创面上应用体外培养的自体表皮细胞悬液移植修复可达到重构皮肤的目的。

[关键词]移植：组织缺损；表皮细胞悬液；细胞培养；皮肤重建

[中图分类号]R622

[文献标识码]A

[文章编号]1 008—645 5(2012)12—2201- 03

Application of the Suspension of epidermal cell cultured in tissue defect

WU Chang—yanl，FANG Zhi—qian92,JIANG Bao—hua3,CHEN Shi—hall

(1．Department of Plastic Surgery，Fi rst Affiliated Hospital of Guangxi Medical University，Nanning 530021，Guangxi，China；2 Department of Plastic Surgery，Longyan Huamei Luoqiao Hospital；3 Department of Plastic Surgery，Kunshan Shiguang

Cosmetology Hospital)

Abstract：

Objective To repair the tissue defect by the Suspension of epidermal cell．

Methods a stamp—size of normal skin was harvested in forty rats and epidermal cells suspension was made th rough decomposing，digesting，culturing and re——digesting in lab．Then it Was implanted to the skin defects made on the dorsa of rats．Shrinkage of the graft survival rate was observed after grafting and samples were taken to make routine paraffin sections for microscopica||y examination，then compared with autogenous healing．

Results After Suspension of epidermal cell was cultured for one week．1he wounds healed gradually The surface of healing wounds was smooth with a slight scar three to six weaks later．But the surface of healing wounds of control group was uneven，scar healing and pigmentation was obvious

Conculsion In the skin defect On the application Of in vitro culture wound autologous skin celI suspension liquid transDlantatiOn to repair lhe purpose of lhe reconstruction of the skin．

Key words：transDIantatiOn：tissue defect；suspension Of epidermal cell；cellular culture；reconstruction Of human skin.

黄韶斌 1 胡志成 1 张逸 2 唐冰 1 王鹏 1 徐海琳 1 王志勇 1 董云先 1 程璞 1 荣燕超 1 吴军 3 朱家源 1

1 中山大学附属第一医院烧伤科，广州 510080；

2 南通大学附属医院烧伤整形外科226001；

3 深圳大学第一附属医院烧伤整形科 518037

通信作者：朱家源，Email：该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。

【摘要】

目的 探讨异体表皮干细胞（ESC）对裸鼠全层皮肤缺损创面移植异体全厚皮成活的影响及其机制。方法 采用实验研究方法。采用酶消化法从 1 只 4 周龄雄性 BALB/c-NU 裸鼠（品系、鼠龄与性别下同）中获取培养 7 d 呈铺路石状原代 ESC，其第 3 代细胞经流式细胞仪鉴定阳性表达ESC 标志物 CD44 且阴性表达 CD45，经免疫荧光法鉴定阳性表达 ESC 标志物 p63 与整合素 6α 且阴性表达 CD71。取对数生长期的第 3 代 ESC 进行后续实验。取 26 只裸鼠，采用随机数字表法平均分为磷酸盐缓冲液（PBS）组和 ESC 组，在每只裸鼠背部制备全层皮肤缺损创面后，2 组创面分别喷涂等体积的 PBS、ESC，创面上均移植从另外 4 只裸鼠背部切取制备的全厚皮。分别取 2 组 10 只裸鼠，观察术后0（即刻）、3、7、14、21 d 创面愈合与皮片存活情况并计算术后 3、7、14、21 d 皮片存活比和皮片收缩率（样本数为各时间点存活皮片数），采用激光散斑血流成像仪检测术后 3、7、14 d 皮片的血流灌注情况并计算各时间点 ESC 组与 PBS 组裸鼠皮片血流灌注比（样本数为各时间点 2 组配对均存活皮片对数）；分别取 2 组剩余 3 只裸鼠，取术后 7 d 皮片组织，分别采用实时荧光定量反转录 PCR 法和蛋白质印迹法检测组织中肿瘤坏死因子 α（TNF-α）、白细胞介素 8（IL-8）、IL-10、Ⅰ型胶原、Ⅲ型胶原与基质金属蛋白酶 9（MMP-9）的 mRNA 和蛋白表达。对数据行 Log-rank 检验、重复测量方差分析、单因素方差分析、独立样本 t 检验与 Bonferroni 校正。

结果 以术后 0 d 情况为参照，2 组裸鼠术后 3、7、14、21 d 创面逐渐愈合，皮片收缩情况逐渐明显，其中 PBS 组裸鼠创面收缩愈合情况较 ESC 组明显。术后3 d，ESC 组 1 只裸鼠皮片移植失败，PBS 组 3 只裸鼠皮片移植失败；术后 7 d，PBS 组又有 1 只裸鼠皮片移植失败。2 组裸鼠术后 3、7、14、21 d 皮片存活比相近（P>0.05）。术后 3、7、14、21 d，ESC 组裸鼠皮片收缩率分别为（9.2±0.4）%、（19.7±1.2）%、（53.6±3.5）%、（62.2±5.1）%，显著低于 PBS 组的（11.0±0.9）%、（47.8±2.8）%、（86.1±7.1）%、（89.7±9.0）%（t=5.719、26.650、11.940、7.617，P<0.01）。术后 3、7、14 d，2 组裸鼠皮片均有血流灌注信号；ESC 组与 PBS 组裸鼠皮片血流灌注比均值均大于 1，3 个时间点总体比较，差异无统计学意义（P>0.05）。术后 7 d，与 PBS 组比较，ESC 组裸鼠皮片组织中 TNF-α、IL-8、Ⅰ型胶 原 和 Ⅲ 型 胶 原 的 mRNA（t=2.823、2.934、2.845、2.860，P<0.05）和 蛋 白 表 达 均 明 显 降 低 ，IL-10 和MMP-9 的 mRNA（t=3.877、2.916，P<0.05）和蛋白表达均明显升高。

结论 异体 ESC 可减轻裸鼠全层皮肤缺损创面移植异体全厚皮片收缩，促进移植皮片与创面之间新生血管的形成，减轻炎症反应，降低胶原蛋白表达，促进 MMP-9 的表达，从而提高移植皮片的成活质量。

【关键词】 干细胞移植； 皮肤移植； 伤口愈合； 表皮干细胞

基金项目：国家自然科学基金面上项目（81871565）；广东省科技计划（2016B090916001）；广东省基 础 与 应 用 基 础 研 究 基 金（2019A1515012208）；中 山 大 学 临 床 医 学 研 究 5010 计 划（2013001、2018003）；中山大学高校基本科研业务费（19ykpy66）

      Effects and mechanisms of allogeneic epidermal stem cells on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds Huang Shaobin1, Hu Zhicheng1, Zhang Yi2, Tang Bing1, Wang Peng1, Xu Hailin1, Wang Zhiyong1, Dong Yunxian1, Cheng Pu1, Rong Yanchao1, Wu Jun3, Zhu Jiayuan1

1 Department of Burns, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China

2 Department of Burns and Plastic Surgery, Affiliated Hospital of Nantong University, Nantong 226001, China ;;

3 Department of Burns and Plastic Surgery, the First Affiliated Hospital of Shenzhen University, Shenzhen 518037, China Corresponding author: Zhu Jiayuan,

Email: 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。

【Abstract】

      Objective To investigate the effects and mechanisms of allogeneic epidermal stem cells (ESCs) on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds. Methods Experimental research methods were applied. Primary ESCs that appeared paving stone-like after being cultured for 7 d were obtained by enzymatic digestion method from one 4-week-old male BALB/c-NU nude mouse (the same strain, age, and sex below). The cells of third passage were identified by flow cytometry to positively express ESC marker CD44 and negatively express CD45, meanwhile, the positive expression of ESC markers of p63 and integrin 6α, and negative expression of CD71 were identified by immunofluorescence method. The ESCs of third passage in the logarithmic growth phase were used for the following experiments. Twenty-six nude mice were equally divided into phosphate buffered saline (PBS) group and ESCs group according to the random number table. A full -thickness skin defect wound was made on the back of each nude mouse, and then the wounds of the two groups were sprayed with equal volumes of PBS and ESCs, respectively. The wounds were transplanted with full -thickness skin grafts cut from the backs of 4 other nude mice. Each ten nude mice from the two groups were selected, the wound healing and skin survival on post surgery day (PSD) 0 (immediately), 3, 7, 14, and 21 were observed, and the survival ratio and shrinkage rate of skin grafts on PSD 3, 7, 14, and 21 were calculated (the number of sample was the number of surviving skin grafts at each time point); the blood perfusion in the skin grafts on PSD 3, 7, and 14 was detected by the laser speckle blood flow imager, and the blood flow ratio of nude mice skin grafts in ESCs group to PBS group at each time point was calculated (the number of sample was the pair number of surviving skin grafts in group pairing at each time point). The skin graft tissue of each 3 nude mice remained in the two groups were collected on PSD 7, and the mRNA expressions and protein expressions of tumor necrosis factor α (TNF-α), interleukin 8 (IL-8), IL-10, type Ⅰ collagen, type Ⅲ collagen, and matrix metalloproteinase 9 (MMP-9) in the tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Data were statistically analyzed with Log-rank test, analysis of variance for repeated measurement , one-way analysis of variance , independent sample t test, and Bonferroni correction. Results Taking the condition on PSD 0 as a reference, the wounds of nude mice in the two groups healed gradually on PSD 3, 7, 14, and 21, and the shrinkage of skin grafts was gradually obvious. Among them, the shrinkage healing of wound of nude mice in PBS group was more significant than that in ESCs group. On PSD 3, the skin graft of 1 nude mouse failed in ESCs group, while the skin graft of 3 nude mice failed in PBS group. On PSD 7, the skin graft of another nude mouse failed in PBS group. The survival ratio of skin grafts of nude mice in the two groups was similar on PSD 3, 7, 14, and 21 (P>0.05).On PSD 3, 7, 14, and 21, the shrinkage rates of skin grafts of nude mice in ESCs group were (9.2±0.4)% , (19.7±1.2)% , (53.6±3.5)% , and (62.2±5.1)% , respectively, which was significantly lower than (11.0±0.9)% , (47.8±2.8)% , (86.1±7.1)% , and (89.7±9.0)% in PBS group (t=5.719, 26.650, 11.940, 7.617, P<0.01). On PSD 3, 7, and 14, blood perfusion signals were observed in the skin grafts of nude mice in the two groups. The average blood perfusion ratios of the skin grafts of nude mice in ESCs group to PBS group were greater than 1, and there was no statistically significant difference in the overall comparison of 3 time points (P>0.05). On PSD 7, compared with those of PBS group, the mRNA and protein expressions of TNF-α, IL-8, type Ⅰ collagen, and type Ⅲ collagen in the skin graft tissue of nude mice in ESCs group were significantly reduced, while the mRNA and protein expressions of IL-10 and MMP-9 in the skin graft tissue of nude mice in ESCs group were significantly increased (in mRNA comparison, t=2.823, 2.934, 2.845, 2.860, 3.877, 2.916, P<0.05). Conclusions Allogeneic ESCs can reduce the shrinkage of allogeneic full-thickness skin grafts transplanted on full-thickness skin defect wounds in nude mice, promote the formation of new blood vessels between the skin graft and the wound , reduce inflammation and collagen protein expression, and promote the expression of MMP -9, thus improving the survival quality of skin grafts.

【Key words】 Stem cell transplantation; Skin transplantation; Wound healing; Epidermal stem cells

Fund program : General Program of National Natural Science Foundation of China (81871565); Science and Technology Planning Project of Guangdong Province of China (2016B090916001); Foundation for Basic and Applied Basic Research of Guangdong Province of China (2019A1515012208); Sun Yat-sen University Clinical Research 5010 Program (2013001, 2018003); Fundamental Scientific Research of Sun Yat-sen University (19ykpy66)