Effects of porcine acellular dermal matrix combined with human epidermal stem cells on wound healing of full-thickness skin defect in nude mice
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摘要:
目的 探讨复合人表皮干细胞（ESC）的猪脱细胞真皮基质（ADM）对裸鼠全层皮肤缺损创面愈合的影响。 方法 采用实验研究方法。采用数码相机拍照观察猪ADM形态，采用苏木精-伊红（HE）染色观测猪ADM中细胞残留，采用扫描电子显微镜观察猪ADM表面结构，采用红外光谱仪分析猪ADM二级结构，采用动态光散射粒度分析仪分析猪ADM粒径，采用纳米粒度电位仪分析猪ADM电位。采用倒置荧光显微镜观察猪ADM在培养基中放置30 min、1 d、5 d的形态（样本数为2）；采用随机数字表法（分组方法下同）将猪ADM分为5 min组、10 min组、20 min组、30 min组、60 min组、120 min组，常温静置相应时间，称量法计算吸水量（样本数为3）；将Swiss小鼠胚胎成纤维细胞（Fb）分为仅有培养基的空白对照组和另加入相应终质量浓度的ADM浸提液的50.0 g/L ADM浸提液组、37.5 g/L ADM浸提液组、25.0 g/L ADM浸提液组、12.5 g/L ADM浸提液组、6.5 g/L ADM浸提液组，分别于培养24、48、72 h，采用细胞计数试剂盒8法检测细胞增殖率并进行细胞毒性分级（样本数为5）；将1只6周龄雄性SD大鼠红细胞分为生理盐水组、超纯水组及添加相应终质量浓度猪ADM浸提液的5 mg/mL ADM浸提液组、10 mg/mL ADM浸提液组、15 mg/mL ADM浸提液组，于反应3 h，采用酶标仪检测血红蛋白的吸光度值代表猪ADM血液相容性（样本数为3）。从陆军军医大学（第三军医大学）第一附属医院泌尿外科2020年7月收治的1名6岁健康男童包皮环切术后废弃包皮中分离培养ESC并采用流式细胞术鉴定。混合培养法构建复合ESC的猪ADM（以下简称ESC/ADM）颗粒，培养3 d后采用HE染色和激光扫描共聚焦显微镜观察ESC/ADM复合效果。将36只7~8周龄雄性无胸腺裸鼠分成单纯磷酸盐缓冲液（PBS）组、单纯ADM组、单纯ESC组、ESC/ADM组，每组9只，并建立全层皮肤缺损创面模型，伤后即刻，创面均一次性采用相应试剂处理。分别于伤后1、7、11、15 d，观察创面愈合情况并计算创面愈合率（样本数为3）；伤后7 d，行HE染色观察创面上皮化情况并测量未上皮化长度（此处及以下样本数均为4）；伤后11 d，行Masson染色观察创面胶原沉积和真皮化情况并计算创面切面真皮面积，行免疫荧光染色并计算创面表达ESC特异性标志物CD49f的细胞数，行实时荧光定量反转录PCR检测创面移植ESC的3-磷酸甘油醛脱氢酶（GAPDH）的mRNA表达。对数据行独立样本 t检验、单因素方差分析、重复测量方差分析、LSD- t检验。 结果 猪ADM为白色微粒状，内部无细胞存在，由网状结构组成，结构排列无序，表面粗糙；猪ADM分别在1 659、1 549、1 239 cm -1波数处出现吸收峰；猪ADM在溶液中主要粒径分布在500~700 nm；猪ADM表面带负电荷。放置30 min、1 d、5 d，猪ADM均形态相对稳定；放置30~120 min猪ADM吸水量保持相对高水平。6.5 g/L ADM浸提液组、12.5 g/L ADM浸提液组、25.0 g/L ADM浸提液组小鼠胚胎Fb在培养24 h细胞毒性分级为1级，其余各组各时间点细胞毒性分级均为0级。反应3 h，超纯水组红细胞中血红蛋白的吸光度值明显高于生理盐水组和15 mg/mL ADM浸提液组（ t值分别为8.14、7.96， P<0.01）。第4代细胞常规培养3 d形态呈鹅卵石样且低表达CD71和高表达CD49f的被鉴定为ESC。猪ADM颗粒上有ESC附着、生长。伤后1 d，单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组裸鼠创面大小基本一致。伤后7、11、15 d，各组裸鼠创面收缩，其中单纯ADM组、单纯ESC组、ESC/ADM组裸鼠创面收缩明显。伤后7 d，单纯ESC组、ESC/ADM组裸鼠创面愈合率明显高于单纯PBS组（ t值分别为2.83、4.72， P<0.05或 P<0.01）；伤后11 d，ESC/ADM组裸鼠创面愈合率明显高于单纯PBS组（ t=4.86， P<0.01）；伤后15 d，单纯ADM组、单纯ESC组、ESC/ADM组裸鼠创面愈合率均明显高于单纯PBS组（ t值分别为2.71、2.90、3.23， P<0.05）。伤后7 d，单纯ADM组、单纯ESC组、ESC/ADM组裸鼠创面未上皮化长度分别为（816±85）、（635±66）、（163±32）μm，明显短于单纯PBS组的（1 199±43）μm（ t值分别为5.69、10.19、27.54， P<0.01）。伤后11 d，单纯ADM组、单纯ESC组、ESC/ADM组裸鼠创面切面真皮面积明显大于单纯PBS组（ t值分别为27.14、5.29、15.90， P<0.01）；单纯ADM组和ESC/ADM组裸鼠创面的胶原生成比单纯PBS组明显，单纯ESC组和单纯PBS组相近。伤后11 d，单纯ESC组和ESC/ADM组裸鼠创面中CD49f表达阳性数量分别为（135±7）、（185±15）个，GAPDH的mRNA表达阳性；单纯PBS组、单纯ADM组裸鼠创面均无CD49f、GAPDH的mRNA表达。 结论 ESC/ADM颗粒能够促进裸鼠全层皮肤缺损创面愈合，这可能与其提高了ESC移植后的存活率和ADM促进了真皮结构重排、血管新生有关。 Abstract:Objective To explore the effects of porcine acellular dermal matrix (ADM) combined with human epidermal stem cells (ESCs) on wound healing of full-thickness skin defect in nude mice. Methods The morphology of porcine ADM was analyzed by photograph of digital camera, the cell residues in porcine ADM were observed by hematoxylin-eosin (HE) staining, the surface structure of porcine ADM was observed by scanning electron microscope, the secondary structure of porcine ADM was analyzed by infrared spectrometer, the porcine ADM particle size was analyzed by dynamic light scattering particle size analyzer, and the porcine ADM potential was analyzed by nano-particle size potentiometer. The morphology of porcine ADM was observed by inverted fluorescence microscope when it was placed in culture medium for 30 min, 1 d, and 5 d ( n=2). The porcine ADM was divided into 5 min group, 10 min group, 20 min group, 30 min group, 60 min group, and 120 min group according to the random number table (the same grouping method below) in static state at normal temperature for the corresponding time to calculate the water absorption by weighing method ( n=3). Swiss white mouse embryonic fibroblasts (Fbs) were divided into blank control group (culture medium only), and 50.0 g/L ADM extract group, 37.5 g/L ADM extract group, 25.0 g/L ADM extract group, 12.5 g/L ADM extract group, and 6.5 g/L ADM extract group which were added with the corresponding final concentrations of ADM extract respectively. At post culture hour (PCH) 24, 48, and 72, the cell survival rate was detected by cell counting kit 8 and the cytotoxicity was graded ( n=5). The erythrocytes of a 6-week-old male Sprague-Dawley male rat were divided into normal saline group, ultra-pure water group, and 5 mg/mL ADM extract group, 10 mg/mL ADM extract group, and 15 mg/mL ADM extract group which were treated with the corresponding final concentrations of porcine ADM extract respectively. After reaction for 3 h, the absorbance value of hemoglobin was detected by microplate reader to represent the blood compatibility of porcine ADM ( n=3). ESCs were isolated and cultured from the discarded prepuce of a 6-year-old healthy boy who was treated in the Department of Urology of the First Affiliated Hospital of Army Medical University (the Third Military Medical University) in July 2020, and then identified by flow cytometry. The porcine ADM particles of composite ESC (hereinafter referred to as ESC/ADM) were constructed by mixed culture. After 3 days of culture, the composite effect of ESC/ADM was observed by HE staining and laser scanning confocal microscope. Thirty-six 7-8-week-old male non-thymic nude mice were divided into phosphate buffer solution (PBS) alone group, ADM alone group, ESC alone group, and ESC/ADM group, with 9 mice in each group, and the wound model of full-thickness skin defect was established. Immediately after injury, the wounds were treated with the corresponding reagents at one time. On post injury day (PID) 1, 7, 11, and 15, the wound healing was observed and the wound healing rate was counted ( n=3). On PID 7, the epithelialization of wounds was observed by HE staining and the length of un-epithelialized wound was measured (with this and the following sample numbers of 4). On PID 11, the dermal area and collagen deposition of wounds were observed by Masson staining and the dermal area of wound section was calculated, the number of cells expressing CD49f, a specific marker of ESC, was calculated with immunofluorescence staining, the mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in ESC after wound transplantation was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Data were statistically analyzed with independent sample t test, one-way analysis of variance, analysis of variance for repeated measurement, and least significant difference t test. Results The porcine ADM was white particles and composed of reticular structure, with no cells inside, disordered structure, and rough surface. The absorption peak of porcine ADM appeared at the wave numbers of 1 659, 1 549, and 1 239 cm -1, respectively. The main particle size distribution of porcine ADM in solution was 500 to 700 nm, with negative charge on the surface. The morphology of porcine ADM in static state at 30 min and on 1 and 5 d was relatively stable. The water absorption of porcine ADM remained relatively high level in static state from 30 min to 120 min. The cytotoxicity of mouse embryonic Fbs in 6.5 g/L ADM extract group, 12.5 g/L ADM extract group, and 25.0 g/L ADM extract group was grade 1 at PCH 24, and the cytotoxicity of the other groups was 0 grade at each time point. After reaction for 3 h, the absorbance value of hemoglobin of erythrocytes in ultra-pure water group was significantly higher than the values in normal saline group and 15 mg/mL ADM extract group (with t values of 8.14 and 7.96, respectively, P<0.01). After 3 days of culture, the cells of the fourth passage showed pebble-like morphology, with low expression of CD71 and high expression of CD49f, which were identified as ESCs. There was ESC attachment and growth on porcine ADM particles. On PID 1, the wound sizes of nude mice were almost the same in PBS alone group, ADM alone group, ESC alone group, and ESC/ADM group. On PID 7, 11, and 15, the wound contraction of nude mice in each group was observed, especially in ADM alone group, ESC alone group, and ESC/ADM group. On PID 7, the wound healing rates of nude mice in ESC alone group and ESC/ADM group were significantly higher than the rate in PBS alone group (with t values of 2.83 and 4.72 respectively, P<0.05 or P<0.01). On PID 11, the wound healing rate of nude mice in ESC/ADM group was significantly higher than that in PBS alone group ( t=4.86, P<0.01). On PID 15, the wound healing rates of nude mice in ADM alone group, ESC alone group, and ESC/ADM group were significantly higher than the rate in PBS alone group (with t values of 2.71, 2.90, and 3.23 respectively, P<0.05). On PID 7, the length of un-epithelialized wound of nude mice in ADM alone group, ESC alone group, and ESC/ADM group was (816±85), (635±66), and (163±32) μm, respectively, which were significantly shorter than (1 199±43) μm in PBS alone group (with t values of 5.69, 10.19, and 27.54 respectively, P<0.01). On PID 11, the dermal areas of wound section of nude mice in ADM alone group, ESC alone group, and ESC/ADM group were significantly larger than the area in PBS alone group (with t values of 27.14, 5.29, and 15.90 respectively, P<0.01); the collagen production of nude mice in ADM alone group and ESC/ADM group was more obvious than that in PBS alone group, and the collagen production of nude mice in ESC alone group and PBS alone group was similar. On PID 11, in the wounds of nude mice in ESC alone group and ESC/ADM group, the cells with positive expression of CD49f were respectively 135±7 and 185±15, and the mRNA expressions of GAPDH were positive; while there were no expressions of CD49f nor mRNA of GAPDH in the wounds of nude mice in PBS alone group and ADM alone group. Conclusions ESC/ADM particles can promote the wound healing of full-thickness skin defects in nude mice, which may be related to the improved survival rate of ESCs after transplantation and the promotion of dermal structure rearrangement and angiogenesis by ADM. -
Key words:
- Wound healing /
- Stem cell research /
- Skin /
- Epidermal stem cells /
- Composite matrix
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参考文献
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1 猪脱细胞真皮基质（ADM）物理特性分析结果。1A.数码相机下猪ADM呈白色；1B.猪ADM中无细胞成分存在 苏木精-伊红×200，图中标尺为200 μm；1C.猪ADM表面呈细丝状 扫描电子显微镜×1 000，图中标尺为2 μm；1D.红外光谱仪分析显示猪ADM在1 659、1 549、1 239 cm -1波数处出现吸收峰；1E.动态光散射粒度分析仪分析显示，猪ADM粒径主要为500~700 nm；1F.纳米粒度电位仪分析显示，猪ADM表面带负电荷
注：图1D中横坐标a、b、c、d、e、f、g、h分别为波数4 000、3 500、2 500、2 000、1 500、1 000、500 cm-1;图1E为经lg处理的数据形成的描记图，坐标轴数据为未经lg处理的原始数据
2 猪脱细胞真皮基质（ADM）在角质形成细胞无血清培养基中放置各时间点形态 倒置荧光显微镜×40，图中标尺为500 μm。2A.放置30 min，猪ADM形态不规则，呈网状；2B、2C.分别为放置1、5 d，猪ADM大小和形态与图2A相近
3 复合人表皮干细胞（ESC）的猪脱细胞真皮基质（ADM）颗粒构建结果。3A.第4代细胞呈多角形、细胞核大，鹅卵石样排列 倒置荧光显微镜×200，图中标尺为200 μm；3B.表达CD71 -CD49f +的细胞比例约97%；3C.培养3 d，ADM中有ESC生长 苏木精-伊红×200，图中标尺为200 μm；3D.培养3 d，ADM上有ESC生长 激光扫描共聚焦显微镜×200，图中标尺为200 μm
注：图3B中Q1示CD71+CD49f-细胞，Q2示CD71+CD49f+细胞，Q3示CD71-CD49f+细胞，Q4示CD71-CD49f-细胞；图3C中红色箭头示ESC；图3D中绿色荧光示ESC
4 4组裸鼠伤后各时间点全层皮肤缺损创面愈合情况。4A、4B、4C、4D.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组伤后1 d创面情况，大小均与参照卡片相近；4E、4F、4G、4H.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组伤后7 d创面情况，图4F、4G、4H创面较图4E明显缩小；4I、4J、4K、4L.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组伤后15 d创面情况，图4J、4K、4L创面较图4I明显缩小
注：图中浅蓝色卡片为参照物，直径6 mm；PBS为磷酸盐缓冲液、ADM为脱细胞真皮基质、ESC为表皮干细胞;ESC/ADM为复合ESC的ADM
5 4组裸鼠伤后7 d全层皮肤缺损创面上皮化情况和伤后11 d创面胶原沉积和切面真皮化情况。5A、5B、5C、5D.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组创面上皮化情况，其中图5B、5C、5D上皮化较图5A明显更快，苏木精-伊红×200，图中标尺为200 μm。5E、5F、5G、5H.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组伤后11 d创面胶原沉积和切面真皮化情况，图5F和5H创面胶原沉积较图5E多，图5F、5G、5H创面切面真皮面积较图5E大，Masson×200，图中标尺为200 μm
注：红色箭头示创面上皮化位置，黄色虚线区域为创面切面真皮面积；PBS为磷酸盐缓冲液、ADM为脱细胞真皮基质、ESC为表皮干细胞;ESC/ADM为复合ESC的ADM
6 免疫荧光法检测4组裸鼠伤后11 d全层皮肤缺损创面处ESC特异性标志物CD49f表达情况。6A、6B、6C、6D.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组，红框为各组创面上皮化位置 花青素3-4',6-二脒基-2-苯基吲哚×40，图中标尺为200 μm；6E、6F、6G、6H.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组，图6E、6F中无CD49f表达，图6G、6H中CD49f表达阳性 花青素3-4',6-二脒基-2-苯基吲哚×200，图中标尺为50 μm
注：ESC为表皮干细胞、PBS为磷酸盐缓冲液、ADM为脱细胞真皮基质，ESC/ADM为复合ESC的ADM；第2行各图分别为正上方图中红框处放大图，黄色箭头示CD49f表达阳性
表1 6组小鼠胚胎成纤维细胞培养各时间点增殖率比较（%，
组别 样本数 24 h 48 h 72 h 空白对照组 15 100 100 100 6.5 g/L ADM 浸提液组 15 97.7±3.4 110.8±2.8 101.7±2.5 12.5 g/L ADM 浸提液组 15 99.7±0.8 148.3±2.8 135.1±2.8 25.0 g/L ADM 浸提液组 15 97.3±4.2 163.3±1.6 168.7±2.8 37.5 g/L ADM 浸提液组 15 110.2±4.3 171.9±1.4 181.3±3.9 50.0 g/L ADM 浸提液组 15 117.0±3.2 184.8±5.1 189.4±2.5 F值 13.31 299.20 430.10 P值 <0.001 <0.001 <0.001 t 1值 0.93 5.48 0.96 P 1值 0.401 0.005 0.391 t 2值 0.59 24.15 17.44 P 2值 0.585 <0.001 <0.001 t 3值 0.90 54.37 34.17 P 3值 0.418 <0.001 <0.001 t 4值 3.37 72.68 29.64 P 4值 0.028 <0.001 <0.001 t 5值 7.53 23.36 51.16 P 5值 0.002 <0.001 <0.001 注：各组各时间点样本数为5；处理因素主效应， F=106.10， P<0.001；时间因素主效应， F=63.87， P<0.001；两者交互作用， F=8.44， P<0.001； F值、 P值为各时间点组间总体比较所得； t 1值、 P 1值 ，t 2值 、P 2值 ，t 3值 、P 3值， t 4值、 P 4值， t 5值 、P 5值分别为6.5 g/L ADM浸提液组、12.5 g/L ADM浸提液组、25.0 g/L ADM浸提液组、37.5 g/L ADM浸提液组、50.0 g/L ADM浸提液组与空白对照组各时间点比较所得 
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表2 4组裸鼠伤后各时间点全层皮肤缺损创面愈合率比较（%，
组别 创面数（个） 7 d 11 d 15 d 单纯PBS组 18 31.3±4.8 47.9±5.3 67.0±6.4 单纯ADM组 18 38.0±5.5 54.2±6.5 77.6±2.4 单纯ESC组 18 44.8±6.7 59.0±7.6 79.1±3.4 ESC/ADM组 18 51.4±5.6 69.8±5.7 81.2±4.2 F值 6.97 6.39 6.39 P值 0.006 0.008 0.008 t 1值 1.57 1.30 2.71 P 1值 0.167 0.242 0.035 t 2值 2.83 2.06 2.90 P 2值 0.030 0.085 0.027 t 3值 4.72 4.86 3.23 P 3值 0.003 0.003 0.018 注：PBS为磷酸盐缓冲液、ADM为脱细胞真皮基质、ESC为表皮干细胞;ESC/ADM为复合ESC的ADM；各组各时间点创面数为6个；处理因素主效应， F=877.40， P<0.001；时间因素主效应， F=506.20， P<0.001；两者交互作用， F=84.90， P=0.611； F值、 P值为各时间点组间总体比较所得； t 1值、 P 1值， t 2值、 P 2值， t 3值、 P 3值分别为单纯ADM组、单纯ESC组、ESC/ADM组与单纯PBS组各时间点比较所得
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表3 4组裸鼠全层皮肤缺损创面伤后7 d未上皮化长度和伤后11 d创面切面真皮面积（
组别 样本数 未上皮化长度（μm） 创面切面真皮 面积（mm 2） 单纯PBS组 4 1 199±43 0.86±0.05 单纯ADM组 4 816±85 2.70±0.08 单纯ESC组 4 635±66 1.38±0.13 ESC/ADM组 4 163±32 2.57±0.14 F值 103.40 138.50 P值 <0.001 <0.001 t 1值 5.69 27.14 P 1值 0.005 <0.001 t 2值 10.19 5.29 P 2值 0.001 0.006 t 3值 27.54 15.90 P 3值 <0.001 <0.001 注：PBS为磷酸盐缓冲液、ADM为脱细胞真皮基质、ESC为表皮干细胞;ESC/ADM为复合ESC的ADM； F值、 P值为各指标4组间总体比较所得； t 1值、 P 1值， t 2值 、P 2值， t 3值 、P 3值分别为单纯ADM组、单纯ESC组、ESC/ADM组与单纯PBS组比较所得
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