异体表皮干细胞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

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

doi:10.3760/cma.j.cn501120-20200704-00339

异体表皮干细胞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

doi: 10.3760/cma.j.cn501120-20200704-00339

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

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

详细信息

通讯作者:
朱家源，Email：zhujiay@mail.sysu.edu.cn

计量
- 文章访问数: 148
- HTML全文浏览量: 31
- PDF下载量: 17
- 被引次数: 0
出版历程
- 收稿日期: 2020-07-04

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

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

Funds:

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

More Information

Corresponding author: Zhu Jiayuan, Email: zhujiay@mail.sysu.edu.cn

摘要

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。
- 干细胞移植 /
- 皮肤移植 /
- 伤口愈合 /
- 表皮干细胞
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.
- Stem cell transplantation /
- Skin transplantation /
- Wound healing /
- Epidermal stem cells
所有作者均声明不存在利益冲突

·读者·作者·编者·

本刊2022年重点号专栏征稿启事

敬请大家浏览并关注本刊2022年各期重点选题，欢迎您针对有意向的选题内容积极投稿。若稿件通过编委会专家组评审，将有机会被纳入当期重点号专栏刊发。欢迎大家积极参与，感谢您的支持！

征稿要求：原创性论著，字数5 000字左右（需附中英文摘要及关键词），至少于当期专栏出刊前4个月投稿。

投稿途径：登录本刊官网www.zhsszz.org→点击左侧“在线投稿”注册投稿即可，投稿时请务必在题目中注明投第几期重点选题。

HTML全文

1 从裸鼠皮肤分离的原代表皮干细胞培养各时间点的形态倒置相差显微镜×200。1A.培养3 d，细胞形成克隆群；1B.培养7 d，细胞连接成片状，呈铺路石样

下载: 全尺寸图片幻灯片

2 免疫荧光法检测裸鼠第3代表皮干细胞培养2 d相应标志物表达 Alexa Fluor 488-Alexa Fluor 594-4',6-二脒基-2-苯基吲哚，图中标尺为10 μm。2A、2B、2C、2D.分别为细胞核染色、p63染色、整合素6α染色、细胞核及p63与整合素6α染色重叠图片，p63与整合素6α均呈阳性表达；2E、2F、2G、2H.分别为细胞核染色、CD71染色、整合素6α染色、细胞核及CD71与整合素6α染色重叠图片，CD71呈阴性表达，整合素6α呈阳性表达

注：细胞核阳性染色为蓝色，p63与CD71阳性染色为红色，整合素6α阳性染色为绿色

下载: 全尺寸图片幻灯片

3 2组裸鼠全层皮肤缺损创面移植异体全厚皮术后各时间点创面大体观察。3A、3B、3C、3D、3E.分别为磷酸盐缓冲液组术后0、3、7、14、21 d创面情况，创面逐渐缩小，收缩愈合明显；3F、3G、3H、3I、3J.分别为表皮干细胞组术后0、3、7、14、21 d创面情况，收缩愈合较不明显，图3F、3G分别与图3A、3B相近，图3H、3I、3J收缩情况分别弱于图3C、3D、3E

下载: 全尺寸图片幻灯片

4 2组各10只裸鼠全层皮肤缺损创面移植异体全厚皮术后各时间点皮片存活比

注：红色为表皮干细胞（ESC）组，蓝色为磷酸盐缓冲液（PBS）组；术后3 d，ESC组1只裸鼠皮片坏死，PBS组3只裸鼠皮片坏死

下载: 全尺寸图片幻灯片

5 2组裸鼠全层皮肤缺损创面移植异体全厚皮术后各时间点皮片血流灌注情况。5A、5B、5C.分别为磷酸盐缓冲液组术后3、7、14 d皮片血流灌注情况，各时间点均有血流灌注；5D、5E、5F.分别为表皮干细胞组术后3、7、14 d皮片血流灌注情况，分别较图5A、5B、5C血流灌注明显

注：红色代表血流灌注多，黄色次之，蓝色代表血流灌注少

下载: 全尺寸图片幻灯片

6 蛋白质印迹法检测2组裸鼠全层皮肤缺损创面移植异体全厚皮术后7 d皮片组织中炎症因子的蛋白表达

注：TNF-α为肿瘤坏死因子α，IL为白细胞介素，GAPDH为3-磷酸甘油醛脱氢酶；1为磷酸盐缓冲液组，2为表皮干细胞组

下载: 全尺寸图片幻灯片

7 蛋白质印迹法检测2组裸鼠全层皮肤缺损创面移植异体全厚皮术后7 d皮片组织中胶原蛋白相关因子的蛋白表达

注：MMP-9为基质金属蛋白酶9，GAPDH为3-磷酸甘油醛脱氢酶；1为磷酸盐缓冲液组，2为表皮干细胞组

下载: 全尺寸图片幻灯片

表1 实时荧光定量反转录PCR法检测裸鼠皮片组织炎症因子及胶原蛋白相关因子mRNA表达的引物序列及产物大小

基因名称	引物序列（5′→3′）	产物大小（bp）
肿瘤坏死因子α	上游：CATCCGTTCTCTACCCAGCC	221
肿瘤坏死因子α	下游：AATTCTGAGCCCGGAGTTGG	221
白细胞介素8	上游：TCCACTCCCAGCATCGTAGA	128
白细胞介素8	下游：CAGAGTAAAGGGCGGGTCAG	128
白细胞介素10	上游：CTTCGGCCCAGTGAAGAGTT	112
白细胞介素10	下游：TGGAGCTTGCTAAAGGCACT	112
基质金属蛋白酶9	上游：CGCCAACTATGACCAGGATA	224
基质金属蛋白酶9	下游：GTTGCCCCCCCAGTTACAGT	224
Ⅰ型胶原	上游：GTACATCAGCCCAAACCCCA	221
Ⅰ型胶原	下游：CAGGATCGGAACCTTCGCTT	221
Ⅲ型胶原	上游：ATATGTGTCTGCGACTCGGG	208
Ⅲ型胶原	下游：GGGCAGTCTAGTGGCTCATC	208
3-磷酸甘油醛脱氢酶	上游：AGACAGCCGCATCTTCTTGT	141
3-磷酸甘油醛脱氢酶	下游：TGATGGCAACAATGTCCACT	141

下载: 导出CSV

表2 2组裸鼠全层皮肤缺损创面移植异体全厚皮术后各时间点皮片收缩率（%，x¯±s）

组别	3 d	7 d	14 d	21 d
ESC组	9.2±0.4	19.7±1.2	53.6±3.5	62.2±5.1
PBS组	11.0±0.9	47.8±2.8	86.1±7.1	89.7±9.0
t值	5.719	26.650	11.940	7.617
P值	<0.001	<0.001	<0.001	<0.001
注：表皮干细胞（ESC）组术后各时间点样本数均为9，磷酸盐缓冲液（PBS）组术后3 d样本数为7、术后7~21 d样本数均为6；处理因素主效应，F=388.99，P<0.001；时间因素主效应，F=741.51，P<0.001；两者交互作用，F=37.49，P<0.001

下载: 导出CSV

2022年1期	烧伤缺血缺氧性损害与休克的防治	组稿专家：申传安
2022年2期	烧伤后炎症与免疫	组稿专家：孙炳伟、贺伟峰
2022年3期	烧伤感染、脓毒症	组稿专家：姚咏明、袁志强
2022年4期	扩张术与瘢痕修复	组稿专家：马显杰
2022年5期	烧伤后脏器功能损害	组稿专家：郇京宁
2022年6期	特殊原因创面（冻伤、自身免疫病创面等）	组稿专家：于家傲
2022年7期	生长因子调控创面修复	组稿专家：肖健
2022年8期	烧伤营养	组稿专家：韩春茂
2022年9期	瘢痕的光电治疗	组稿专家：章一新
2022年10期	生物材料在创面修复中的应用	组稿专家：罗高兴
2022年11期	创面修复中的细胞与干细胞治疗	组稿专家：史春梦
2022年12期	烧伤康复	组稿专家：谢卫国

下载: 导出CSV

参考文献(33)

[1]	AkitaS.Wound repair and regeneration: mechanisms, signaling[J].Int J Mol Sci,2019,20(24):6328.DOI: 10.3390/ijms20246328.
[2]	WerdinF,TenenhausM,RennekampffHO.Chronic wound care[J].Lancet,2008,372(9653):1860-1862.DOI: 10.1016/S0140-6736(08)61793-6.
[3]	SorgH,TilkornDJ,HagerS,et al.Skin wound healing: an update on the current knowledge and concepts[J].Eur Surg Res,2017,58(1/2):81-94.DOI: 10.1159/000454919.
[4]	KaplaniK,KoutsiS,ArmenisV,et al.Wound healing related agents: ongoing research and perspectives[J].Adv Drug Deliv Rev,2018,129:242-253.DOI: 10.1016/j.addr.2018.02.007.
[5]	AndreassiA,BilenchiR,BiagioliM,et al.Classification and pathophysiology of skin grafts[J].Clin Dermatol,2005,23(4):332-337.DOI: 10.1016/j.clindermatol.2004.07.024.
[6]	ConverseJM,SmahelJ,BallantyneDLJr,et al.Inosculation of vessels of skin graft and host bed: a fortuitous encounter[J].Br J Plast Surg,1975,28(4):274-282.DOI: 10.1016/0007-1226(75)90031-4.
[7]	GkotsouliasE.Split thickness skin graft of the foot and ankle bolstered with negative pressure wound therapy in a diabetic population: the results of a retrospective review and review of the literature[J].Foot Ankle Spec,2020,13(5):383-391.DOI: 10.1177/1938640019863267.
[8]	BoyceST,SimpsonPS,RiemanMT,et al.Randomized, paired-site comparison of autologous engineered skin substitutes and split-thickness skin graft for closure of extensive, full-thickness burns[J].J Burn Care Res,2017,38(2):61-70.DOI: 10.1097/BCR.0000000000000401.
[9]	TakabayashiY,IshiharaM,KuwabaraM,et al.Improved survival of full-thickness skin graft with low-molecular weight heparin-protamine micro/nanoparticles including platelet-rich plasma[J].Ann Plast Surg,2017,78(5):562-568.DOI: 10.1097/SAP.0000000000001051.
[10]	NicaO,PopaDG,GrecuAF,et al.Histological aspects of full-thickness skin grafts augmented with platelet-rich fibrin in rat model[J].Rom J Morphol Embryol,2019,60(2):581-588.
[11]	BehrB,KoSH,WongVW,et al.Stem cells[J].Plast Reconstr Surg,2010,126(4):1163-1171.DOI: 10.1097/PRS.0b013e3181ea42bb.
[12]	KiranaS,StratmannB,PranteC,et al.Autologous stem cell therapy in the treatment of limb ischaemia induced chronic tissue ulcers of diabetic foot patients[J].Int J Clin Pract,2012,66(4):384-393.DOI: 10.1111/j.1742-1241.2011.02886.x.
[13]	MarandaEL,Rodriguez-MenocalL,BadiavasEV.Role of mesenchymal stem cells in dermal repair in burns and diabetic wounds[J].Curr Stem Cell Res Ther,2017,12(1):61-70.DOI: 10.2174/1574888x11666160714115926.
[14]	KølleSF,Fischer-NielsenA,MathiasenAB,et al.Enrichment of autologous fat grafts with ex-vivo expanded adipose tissue-derived stem cells for graft survival: a randomised placebo-controlled trial[J].Lancet,2013,382(9898):1113-1120.DOI: 10.1016/S0140-6736(13)61410-5.
[15]	TengM,HuangYS,ZhangHS.Application of stems cells in wound healing--an update[J].Wound Repair Regen,2014,22(2):151-160.DOI: 10.1111/wrr.12152.
[16]	ChenCY,RaoSS,RenL,et al.Exosomal DMBT1 from human urine-derived stem cells facilitates diabetic wound repair by promoting angiogenesis[J].Theranostics,2018,8(6):1607-1623.DOI: 10.7150/thno.22958.
[17]	LiZ,MaitzP.Cell therapy for severe burn wound healing[J/OL].Burns Trauma,2018,6:13[2020-07-04].http://burnstrauma.biomedcentral.com/track/pdf/ 10.1186/s41038-018-0117-0.DOI: 10.1186/s41038-018-0117-0.
[18]	ShawkyLM,El BanaEA,MorsiAA.Stem cells and metformin synergistically promote healing in experimentally induced cutaneous wound injury in diabetic rats[J].Folia Histochem Cytobiol,2019,57(3):127-138.DOI: 10.5603/FHC.a2019.0014.
[19]	WangP,HuZC,CaoXL,et al.Fibronectin precoating wound bed enhances the therapeutic effects of autologous epidermal basal cell suspension for full-thickness wounds by improving epidermal stem cells' utilization[J].Stem Cell Res Ther,2019,10(1):154.DOI: 10.1186/s13287-019-1236-7.
[20]	HuangSB,HuZC,WangP,et al.Rat epidermal stem cells promote the angiogenesis of full-thickness wounds[J].Stem Cell Res Ther,2020,11(1):344.DOI: 10.1186/s13287-020-01844-y.
[21]	HarrisJE,DhupaS.Treatment of degloving injuries with autogenous full thickness mesh scrotal free grafts[J].Vet Comp Orthop Traumatol,2008,21(4):378-381.DOI: 10.3415/vcot-07-04-0029.
[22]	ZografouA,TsigrisC,PapadopoulosO,et al.Improvement of skin-graft survival after autologous transplantation of adipose-derived stem cells in rats[J].J Plast Reconstr Aesthet Surg,2011,64(12):1647-1656.DOI: 10.1016/j.bjps.2011.07.009.
[23]	XiaoSE,LiuZY,YaoYZ,et al.Diabetic human adipose-derived stem cells accelerate pressure ulcer healing by inducing angiogenesis and neurogenesis[J].Stem Cells Dev,2019,28(5):319-328.DOI: 10.1089/scd.2018.0245.
[24]	PhinneyDG,PittengerMF.Concise review: MSC-derived exosomes for cell-free therapy[J].Stem Cells,2017,35(4):851-858.DOI: 10.1002/stem.2575.
[25]	LeeDE,AyoubN,AgrawalDK.Mesenchymal stem cells and cutaneous wound healing: novel methods to increase cell delivery and therapeutic efficacy[J].Stem Cell Res Ther,2016,7:37.DOI: 10.1186/s13287-016-0303-6.
[26]	RustadKC,WongVW,SorkinM,et al.Enhancement of mesenchymal stem cell angiogenic capacity and stemness by a biomimetic hydrogel scaffold[J].Biomaterials,2012,33(1):80-90.DOI: 10.1016/j.biomaterials.2011.09.041.
[27]	BadiavasEV,FalangaV.Treatment of chronic wounds with bone marrow-derived cells[J].Arch Dermatol,2003,139(4):510-516.DOI: 10.1001/archderm.139.4.510.
[28]	ZhugeY,RegueiroMM,TianRX,et al.The effect of estrogen on diabetic wound healing is mediated through increasing the function of various bone marrow-derived progenitor cells[J].J Vasc Surg,2018,68(6S): S127-135.DOI: 10.1016/j.jvs.2018.04.069.
[29]	MesaKR,KawaguchiK,CockburnK,et al.Homeostatic epidermal stem cell self-renewal is driven by local differentiation[J].Cell Stem Cell,2018,23(5):677-686.e4.DOI: 10.1016/j.stem.2018.09.005.
[30]	D'ArcangeloD,TinaburriL,DellambraE.The role of p16INK4a pathway in human epidermal stem cell self-renewal, aging and cancer[J].Int J Mol Sci,2017,18(7):1591.DOI: 10.3390/ijms18071591.
[31]	JacksonCJ,TønsethKA,UtheimTP.Cultured epidermal stem cells in regenerative medicine[J].Stem Cell Res Ther,2017,8(1):155.DOI: 10.1186/s13287-017-0587-1.
[32]	HuZ,GuoD,LiuP,et al.Randomized clinical trial of autologous skin cell suspension for accelerating re-epithelialization of split-thickness donor sites[J].Br J Surg,2017,104(7):836-842.DOI: 10.1002/bjs.10508.
[33]	HuZC,ChenD,GuoD,et al.Randomized clinical trial of autologous skin cell suspension combined with skin grafting for chronic wounds[J].Br J Surg,2015,102(2):e117-123.DOI: 10.1002/bjs.9688.