MEBT／MEBO对慢性难愈合创面组织中CK15表达水平的影响

单云龙 岑小宁 包崇婵 卓臣义 唐习强 韦 骋 王雪玲 左远娟 唐乾利

慢性难愈合创面是指治疗1个月以上仍未愈合也无明显愈合倾向的创面［1］。近年来，众多文献显示，皮肤再生医疗技术 （moist exposed burn therapy／moist exposed burn ointment， MEBT／MEBO）在慢性难愈合创面的治疗中取得了良好的临床疗效［2-4］，但具体作用机制尚未完全明确。部分研究发现，细胞角蛋白 （cytokeratin，CK）15可通过影响表皮干细胞 （epidermal stem cells，ESCs）的增殖而影响创面的修复［5］，故笔者于本研究中动态监测了MEBT／MEBO对大鼠慢性难愈合创面组织中CK15表达水平的影响，以期进一步探讨MEBT／MEBO在慢性难愈合创面修复中的部分分子生物学作用机制。

Chronic refractory wounds are wounds that do not heal and show no obvious tendency to heal［1］in more than one month.In recent years， some literature have shown that MEBT／MEBO has achieved good clinical efficacy in the treatment of chronic refracto⁃ry wounds［2-4］， but its specific mechanism of action is not entirely clear⁃cut.Some studies have found that cytokeratin （CK） 15 can accelerate wound repair by influencing the proliferation of epider⁃mal stem cells （ESCs）［5］.Therefore， in this study， the author monitored in real time the influence of MEBT／MEBO on the ex⁃pression of CK15 in chronic refractory wound tissues of rats so as to further explore the molecular mechanism of MEBT／MEBO in the repair of chronic refractory wounds.

1 实验材料

1.1 实验动物

SPF级健康成年雄性Wistar大鼠90只，均来源于长沙市天勤生物技术有限公司，体重200～250 g，饲养于右江民族医学院动物实验中心SPF级动物室，室内湿度50% ～70%，温度23～25℃，空气流通良好，卫生合格。本实验经右江民族医学院动物伦理委员会审批通过，符合动物实验的伦理学要求。

1.2 主要试剂

湿润烧伤膏 （moist exposed burn ointment，MEBO）：汕头市美宝制药有限公司生产；重组牛碱性成纤维细胞生长因子 （recombinant bovine basic fibroblast growth factor， rb⁃bFGF） 凝胶： 珠海亿胜生物制药有限公司生产；CK15一抗 （rab⁃bit anti⁃cytokeratin 15 antibody）： 美国 Affinity Bio⁃sciences公司生产； 内参一抗 （rabbit anti⁃beta⁃actin antibody）、辣根过氧化物酶标记山羊抗兔 IgG：北京中杉金桥生物技术有限公司生产；RNAlater Stabilization Solution：赛默飞世尔科技 （中国）有限公司生产；TIANScriptRT Kit、SuperReal PreMix Plus（SYBR Green） （FP205）： 天根生化科技 （北京）有限公司生产。

2 方法

2.1 实验分组与模型制备

将大鼠适应性喂养7 d后，按照随机数表法分为空白组、对照组、模型组、MEBO组与rb⁃bFGF组，每组18只，其中空白组大鼠只做背部备皮处理；对照组大鼠于水合氯醛腹腔注射麻醉及背部备皮处理后，在无菌条件下于备皮处做直径约15 mm深达深筋膜的全层皮肤缺损创面，建立急性创面模型；模型组、MEBO组和rb⁃bFGF组大鼠于水合氯醛腹腔注射麻醉及背部备皮处理后，在无菌条件下于备皮处做直径约15 mm深达深筋膜的全层皮肤缺损创面，并立即注射醋酸氢化可的松 （80 mg／kg），建立慢性难愈合创面模型［6-7］。

2.2 局部处理

造模完成后，空白组大鼠备皮处皮肤及对照组、模型组大鼠创面于5%碘伏消毒后，依次覆盖2层含有0.9%氯化钠注射液的湿纱布及2层无菌干纱布包扎固定，每天换药2次；MEBO组大鼠创面于5%碘伏消毒后，依次覆盖2层MEBO药纱 （1 cm2含有0.2 g MEBO）及2层无菌干纱布包扎固定，每天换药2次；rb⁃bFGF组大鼠创面于5%碘伏消毒后，依次覆盖2层 rb⁃bFGF药纱 （1 cm2含有 60 U rb⁃bFGF）及2层无菌干纱布包扎固定，每天换药2次。

1.Experiment materials

1.1.Experiment animals

Ninety SPF Wistar healthy male rats provided by Hubei Top⁃gene Biotechnology Co.， Ltd， weighing 200-250 g， raised in SPF animal rooms of the Animal Experiment Center of Youjiang Medical University for Nationalities were selected as subjects.Indoor humidity：50%-70%，temperature：23-25℃，with good excellent air circulation and good hygiene.This experiment was approved by the Animal Ethics Committee of Youjiang Medical University for Nationalities.

1.2.Main reagents

Moist Exposed Burn Ointment（MEBO）： produced by Shan⁃tou MEBO Pharmaceutical Co.， Ltd.； recombinant bovine basic fibroblast growth factor（rb⁃bFGF） gel： Zhuhai Essex Bio⁃pharma⁃ceutical Co.， Ltd.； CK15 primary antibody （rabbit anti⁃cytokera⁃tin 15 antibody）： produced by Affinity Biosciences， USA； rabbit anti⁃beta⁃actin antibody and HRP⁃labeled goat anti⁃rabbit IgG：produced by Beijing Zhongshan Jinqiao Biotechnology Co.， Ltd.；RNAlater Stabilization Solution： produced by Thermo Fisher Scien⁃tific （ China）； TIANScript RT Kit， SuperReal PreMix Plus（SYBR Green） （FP205）： produced by TIANGEN BIOTECH（Beijing） Co.， Ltd.

2.Methods

2.1.Grouping and model preparation

After being fed for 7 days to adapt surrounding environment，the rats were divided， according to the random number table， into blank group， control group， model group， MEBO group and rb⁃bFGF group， with 18 rats in each group.For rats in blank group，only back skin preparation was performed；for rats in control group，after intraperitoneal injection of chloral hydrate and back skin preparation， a full⁃thickness skin defect wound with a diame⁃ter of about 15 mm at the depth of fascia was made at skin prepara⁃tion site in aseptic condition to establish an acute wound model；for rats in model group， MEBO group， and rb⁃bFGF group， after intraperitoneal injection of chloral hydrate and back skin prepara⁃tion， a full⁃thickness skin defect wound with a diameter of about 15 mm at the depth of fascia was made at skin preparation site in aseptic condition， to which hydrocortisone acetate （80 mg／kg）was injected to establish an chronic refractory wound model［6-7］.

2.2.Local treatment

After models were established，the skin at the preparation site of rats in the blank group and the wounds of control group and model group were disinfected with 5%iodophor，then covered by two layers of wet gauze containing 0.9%sodium chloride injection， and band⁃aged up with two layers of dry sterile gauze in turn，and dressing change was performed twice a day； for MEBO group， after disinfec⁃tion with 5% iodophor，the wound was covered by two layers of MEBO gauze（0.2 g MEBO per 1 cm2）and bandaged up with two layers of sterile dry gauze in turn，and dressing change was performed twice a day； wounds of rb⁃bFGF group were disinfected with 5%iodophor， and then covered with two layers of rb⁃bFGF gauze （60 U rb⁃bFGF per 1 cm2） and bandaged up with two layers of dry sterile gauze in turn，and dressing change was performed twice a day.

2.3 标本采集

分别于治疗第3、7、14天，每组随机选取6只大鼠用脊椎脱臼法处死后，切取创缘0.5 cm范围内深达深筋膜下的创面及创周组织，并均分为2份，1份迅速置于冻存管后放在液氮罐中，并置于-80℃冰箱中冷冻保存，用于后期Western blotting技术检测；1份迅速置于注有RNA保存液的冻存管后放在液氮罐中，并置于-80℃冰箱中冷冻保存，用于后期 Real⁃time PCR技术检测。

2.4 Western blotting技术检测CK15蛋白表达水平

取备用标本进行液氮研磨后，加入蛋白裂解液充分裂解，提取总蛋白，并用BCA法测定样本蛋白浓度；将提取的蛋白上样依次置于80 V恒压下电泳30 min、120 V恒压下电泳60 min、300 mA恒流下电转60 min后，转移至PVDF膜上室温封闭120 min，并进行4℃恒温摇床；随后，一抗孵育过夜后洗膜，室温下孵育二抗60 min后再次洗膜；最后，X射线显影，晾干，拍照，Image J图像分析软件分析蛋白条带灰度值。

2.5 Real⁃time PCR技术检测CK15 mRNA表达水平

取备用标本采用Trizol法提取总RNA，并分别检测其在260 nm及280 nm波长的吸光度值，若A260与A280比值在1.8～2.0之间则视为RNA提取合格；按照TIANScript RT KIT试剂盒说明书以总RNA为模板逆转录合成cDNA；采用2⁃△△CT法应用荧光定量PCR仪进行相对定量分析，结果以待测基因与内参基因GAPDH的表达水平比值表示。其中CK15上游引物序列为 5′⁃AGGGGCAGGAGTGGGTT⁃3′， 下游引物序列 为 5′⁃CAGGCGGTCGTTGAGGT⁃3′； β⁃actin上游引物序列为5′⁃CCTAGACTTCGAGCAAGAG A⁃3′， 下游引物序列为5′⁃GGAAGGAAGGCTGG AAG⁃3′。

2.3.Specimen collection

On day 3， day 7and day 14 of treatment， 6 rats in each group were randomly selected and spinal dislocation was performed to kill them.Wound tissues，within 0.5 cm of the wound edge and deep to the fascia，were cut off and divided into two parts.One part was placed in a cryogenic vial.The cryogenic vial was put in a liquid ni⁃trogen tank，which was then stored in a refrigerator at the temperature of-80℃ for later Western blotting detection；the other part was placed in a cryogenic vial filled with RNA preservation solution as quickly as possible.The cryogenic vial was then put in a liquid nitro⁃gen tank，which was stored in a refrigerator at the temperature of-80 ℃ for later Real⁃time PCR detection.

2.4.Detection of CK15 expression level with Western blotting

Spare specimen was taken and ground with liquid nitrogen， pro⁃tein lysate was added to lyse it to the full， total protein was extracted，and the protein concentration of the specimen was measured with BCA Protein Assay； the extracted protein was loaded and run on electro⁃phoresis at 80 V constant pressure for 30 min， 120 V constant pres⁃sure for 60 min， 300 mA constant current for 60 min， then was trans⁃ferred to PVDF membrane and sealed for 120 min at room temperature and was then placed at 4℃.constant temperature shaker；the mem⁃brane was washed after overnight primary antibody incubation and washed again after secondary antibody incubation at room temperature for 60 min of electrotransformation； finally， it was developed with X⁃ray film， dryed， picture was taken， and the gray value of protein bands was analyzed with Image J software.

2.5.Detection of the expression levels of CK15 mRNA with real⁃time PCR

The total RNA was extracted with Trizol method from spare spec⁃imen，and its absorbance values at 260 nm and 280 nm wavelengths were measured respectively.The RNA extraction is considered as qualified if the ratio of A260 to A280 is between 1.8 and 2.0；cDNA was synthesized via reverse transcription using total RNA as template according to the instructions of TIANScript RT KIT； relative quantita⁃tive analysis was carried out with 2⁃△△CTmethod using quantitative fluorescence PCR，and the results were expressed as the ratio of the expression level of gene to be tested to reference gene GAPDH.The upstream primer sequence of CK15 is 5′⁃AGGGGCAGGAGTGTGGGT T⁃3′， the downstream primer sequence is 5′⁃CAGGCGGTCGTTGAGG T⁃3′； the upstream primer sequence of β⁃actin is 5′⁃CCTAGACTTCG AGCAAGAGA⁃3′， and the downstream primer sequence is 5′⁃GGAA GGAAGGCTGGAAG⁃3′.

2.6 统计学处理

采用SPSS 22.0统计软件对所得数据进行统计学分析，其中计量资料以均数±标准差（）表示，多个样本间比较采用单因素方差分析，且方差齐时使用LSD法、方差不齐时使用Games⁃Howell法；均以P＜0.05为差异具有统计学意义。

3 结果

3.1 CK15蛋白表达水平对比

治疗过程中，空白组大鼠皮肤组织中CK15蛋白表达水平无明显变化 （P＞0.05），其他各组大鼠创面组织中CK15蛋白表达水平均呈先降低后升高的趋势 （P均＜0.05）。治疗第3、7天，对照组、MEBO组、rb⁃bFGF组大鼠创面组织中CK15蛋白表达水平均显著低于模型组（P均＜0.05），而治疗第14天对照组、MEBO组、rb⁃bFGF组大鼠创面组织中CK15蛋白表达水平均显著高于模型组 （P均＜0.05），详见表1及图1。

2.6.Statistical analysis

SPSS 22.0 statistical software was used to conduct statistical analysis on the obtained data，where measurement data was expressed as mean ±standard deviation（x±s）.For comparison among multi⁃ple specimens， one⁃way ANOVA was used： LSD method for equal va⁃riances and Games⁃Howell method for unequal variances.P＜ 0.05 was considered statistically significant.

3.Results

3.1.Comparison of expression levels of CK15

During the treatment，no significant change was observed in the expression levels of CK15 in the skin tissues of the blank group（P＞0.05）and the expression levels of CK15 in the wound tissues of other groups showed a trend of decrease followed by increase（allP＜0.05）.On day 3 and day 7 of treatment， the expression levels of CK15 in the wound tissues of control group， MEBO group， and rb⁃bFGF group were significantly lower than those of model group （allP＜0.05） while on day 14 of treatment， the expression levels of CK15 in the wound tissues of rb⁃bFGF group was significantly higher than that of model group （allP＜0.05）.See Table 1 and Fig.1 for details.

表1 5组大鼠皮肤或创面组织中CK15蛋白表达水平对比 （）Table 1 Comparison of expression levels of CK15 in skin or wound tissues of all groups（）

表1 5组大鼠皮肤或创面组织中CK15蛋白表达水平对比 （）Table 1 Comparison of expression levels of CK15 in skin or wound tissues of all groups（）

注：各时间点5组大鼠皮肤或创面组织中CK15蛋白表达水平组内对比，其中与第3天对比，aP＜0.05，差异具有统计学意义；与第7天对比，bP＜0.05，差异具有统计学意义。各时间点5组大鼠皮肤或创面组织中CK15蛋白表达水平组间对比，其中与空白组对比，cP＜0.05，差异具有统计学意义；与对照组对比，dP＜0.05，差异具有统计学意义；与模型组对比，eP＜0.05，差异具有统计学意义Note：Expression levels of CK15 in skin or wound tissues of the five groups at each time point were compared within each group.Among them，com⁃parison with day 3（aP＜0.05） showed statistically significant difference；comparison with day 7（bP＜0.05）showed statistically significant difference.Expression levels of CK15 in the skin or wound tissues at each time point were compared between groups，in which comparison with blank group（cP＜0.05）showed statistically significant difference；comparison with control group（dP＜0.05） showed statistically significant difference；comparison with model group（eP＜0.05）showed statistically significant difference

组别Group鼠数 （只）Number of rats第3天Day 3第7天Day 7第14天Day 14 F值F value P值P value空白组Blank group 6 0.406±0.063 0.413±0.019 0.408±0.025 0.054 0.948对照组Control group 6 0.169±0.037c 0.091±0.006ac 0.349±0.016abc 192.766 0.000模型组Model group 6 0.272±0.031cd 0.154±0.004acd 0.280±0.022bcd 58.065 0.000 MEBO组MEBO group 6 0.184±0.026ce 0.079±0.002ace 0.357±0.046abce 126.888 0.000 rb⁃bFGF 组rb⁃bFGF group 6 0.197±0.049ce 0.083±0.002ace 0.364±0.026abe 118.244 0.000 F值F value 31.067 1062.935 15.533 - -P值P value 0.000 0.000 0.003 - -

图1 5组大鼠皮肤或创面组织中CK15蛋白表达条带图Fig.1 Histogram of expression levels of CK15 in skin or wound tissues of each group

3.2 CK15 mRNA表达水平对比

治疗过程中，空白组大鼠皮肤组织中CK15 mRNA表达水平无明显变化 （P＞0.05），其他各组大鼠创面组织中CK15 mRNA表达水平均呈先降低后升高的趋势 （P均＜0.05）。治疗第3、7天，对照组、MEBO组、rb⁃bFGF组大鼠创面组织中CK15 mRNA表达水平均显著低于模型组 （P均＜0.05），而治疗第14天对照组、MEBO组、rb⁃bFGF组大鼠创面组织中 CK15 mRNA表达水平均显著高于模型组 （P均＜0.05），详见表2。

3.2.Comparison of expression levels of CK15 mRNA

During the treatment，no significant change was observed in the expression levels of CK15 mRNA in skin tissues of blank group（P＞0.05）and the expression levels of CK15 mRNA in the wound tissues of other groups showed a trend of decrease first followed by increase（allP＜0.05 ）.On day 3 and day 7 of treatment， the expression levels of CK15 mRNA in the wound tissues of control group，MEBO group， and rb⁃bFGF group were significantly lower than those of model group （allP＜ 0.05） while on day 14 of treatment， expres⁃sion levels of CK15 mRNA in the wound tissues of rb⁃bFGF group was significantly higher than that of model group （allP＜0.05）.See Table 2 for details.

表2 5组大鼠皮肤或创面组织中CK15 mRNA表达水平对比 （）Table 2 Comparison of expression levels of CK15 mRNA in skin or wound tissues of each group（）

表2 5组大鼠皮肤或创面组织中CK15 mRNA表达水平对比 （）Table 2 Comparison of expression levels of CK15 mRNA in skin or wound tissues of each group（）

注：各时间点5组大鼠皮肤或创面组织中CK15 mRNA表达水平组内对比，其中与第3天对比，aP＜0.05，差异具有统计学意义；与第7天对比，bP＜0.05，差异具有统计学意义。各时间点5组大鼠皮肤或创面组织中CK15 mRNA表达水平组间对比，其中与空白组对比，cP＜0.05，差异具有统计学意义；与对照组对比，dP＜0.05，差异具有统计学意义；与模型组对比，eP＜0.05，差异具有统计学意义Note： Expression levels of CK15 mRNA in skin or wound tissues of the five groups at each time point were compared within each group.Among them，comparison with day 3 （aP ＜0.05） showed statistically significant difference； comparison with day 7 （bP ＜0.05） showed statistically significant differ⁃ence.Expression levels of CK15 mRNA in the skin or wound tissues of the five groups at each time point were compared between groups， in which compar⁃ison with blank group （cP＜0.05） showed statistically significant difference；comparison with control group（dP＜0.05） showed statistically significant difference；comparison with model group（eP＜0.05）showed statistically significant difference

组别Group鼠数 （只）Number of rats第3天Day 3第7天Day 7第14天Day 14 F值F value P值P value空白组Blank group 6 0.996±0.059 1.030±0.073 1.051±0.043 1.333 0.293对照组Control group 6 0.602±0.042c 0.278±0.020ac 0.871±0.024abc 580.869 0.000模型组Model group 6 0.759±0.027cd 0.419±0.037acd 0.785±0.025bcd 272.569 0.000 MEBO组MEBO group 6 0.600±0.042ce 0.307±0.027ace 0.936±0.026abcde 569.254 0.000 rb⁃bFGF 组rb⁃bFGF group 6 0.591±0.035ce 0.294±0.012ace 0.914±0.033abce 736.054 0.000 F值F value 103.112 381.668 59.668 - -P值P value 0.000 0.000 0.003 - -

4 讨论

皮肤具有强大的再生功能，以维持自我更新和创伤后的再生修复，且这种功能主要依赖位于表皮基底层的ESCs。生理状态下，ESCs处于相对静止状态，只有少量ESCs转化为短暂扩充细胞 （transit amplifying cell，TAC），继而分化为其他表皮细胞，以维持表皮的自我更新［8］；病理状态下，处于静止状态的 ESCs被激活并沿垂直方向经多次分化形成表皮 （垂直分化），同时沿水平方向不断增殖以填充受损组织、维持干细胞数量 （水平增殖），最终达到修复创面的目的［9-10］。部分研究显示，最早被发现于表皮基底层及毛囊凸起等部位的Ⅰ型角蛋白CK15作为中间丝蛋白，为ESCs提供了完整的蛋白骨架，赋予了ESCs额外的物理稳定性［11］，同时介导并增加了ESCs与细胞外基质 （extracellular matrix，ECM）的黏附作用，在创面愈合的水平增殖过程中发挥着关键作用［12-13］。

本研究中，笔者观察了近年来被广泛应用于慢性难愈合创面并取得显著效果的MEBT／MEBO对大鼠慢性难愈合创面组织中CK15表达的影响，以探索其作用机制。结果显示，（1）除空白组外，其他各组大鼠创面组织中CK15蛋白及CK15 mRNA表达水平均呈先降低后升高的趋势 （P均＜0.05）。可见，大鼠皮肤受损后，机体可通过降低CK15的表达而降低ESCs的稳定性及其与ECM的黏附性，进而促使ESCs大量增殖，以修复损伤创面［8-9］。 （2）治疗第3、7天，对照组、MEBO组、rb⁃bFGF组大鼠创面组织中CK15蛋白及CK15 mRNA表达水平均显著低于模型组 （P均＜0.05），而治疗第14天对照组、MEBO组、rb⁃bFGF组大鼠创面组织中CK15蛋白及CK15 mRNA表达水平均显著高于模型组 （P均＜0.05）。可见，大鼠皮肤受损后，创面应用MEBO及rb⁃bFGF均可显著降低创面组织中CK15的表达水平，从而加速创面愈合，且MEBO与rb⁃bFGF的治疗效果相当；至治疗第14天，对照组、MEBO组、rb⁃bFGF组大鼠创面趋于愈合，CK15蛋白及CK15 mRNA表达水平接近空白组，而模型组大鼠创面愈合速率明显低于其他各组，故其创面组织中CK15蛋白及CK15 mRNA表达水平仍低于正常水平。

综上所述，通过调控组织细胞中CK15的表达水平促进ESCs的增殖可能是MEBT／MEBO有效加快慢性难愈合创面愈合的部分作用机制，且其治疗效果与rb⁃bFGF相当，作用机制与rb⁃bFGF相似。另外，本研究只揭示了MEBT／MEBO促进ESCs水平增殖的作用，而其是否可以促进ESCs的垂直分化仍需进一步深入研究探讨。

4.Discussion

Skin has powerful regenerative capacity to maintain self⁃renewal and post⁃traumatic regenerative repair.This capability mainly attrib⁃utes to ESCs in the basal layer of epidermis.In physiological state，ESCs are at a relatively static state and only a small number of ESCs transform into transient amplifying cells（TAC）， which then differen⁃tiate into new epidermal cells to maintain self⁃renewal of epider⁃mis［8］； in pathological state， the static ESCs are activated and differ⁃entiate for many times in vertical direction to epidermis （vertical dif⁃ferentiation）.At the same time， they continuously proliferate in the horizontal direction to fill damaged tissues and maintain the number of stem cells （horizontal proliferation）， and finally achieving wound re⁃pair［9-10］.Some studies have shown that type I keratin CK15， which was first discovered in the epidermal basal layer and hair follicle bul⁃ges， serves as intermediate filament protein， providing a complete protein scaffold for ESCs and giving ESCs additional physical stabili⁃ty［11］.It also enhances the adhesion of ESCs to the extracellular ma⁃trix（ECM），thus playing a key role in promoting the horizontal pro⁃liferation［12-13］.

In recent years， MEBT／MEBO has been widely used in the repair of chronic refractory wounds and significant effect has been achieved.In this study， the author observed its influence on the ex⁃pression of CK15 in chronic refractory wound tissues of rats to explore its mechanism of action.The results showed that（1） except blank group，the expression levels of CK15 and CK15 mRNA in the wound tissue of other groups showed a trend of decrease first followed by in⁃crease（all P ＜0.05）.It can be concluded that after the skin is dam⁃aged， rat’s body can reduce the stability of ESCs and its adhesion to ECM by lowering the expression of CK15， thus promoting the prolifer⁃ation of ESCs to repair wounds［8-9］. （2） On day 3 and day 7 of treatment，the expression levels of CK15 and CK15 mRNA in the wound tissues of control group， MEBO group and rb⁃bFGF group were significantly lower than those of model group（all P＜0.05）while on day 14 of treatment，the expression levels of CK15 and CK15 mRNA in the wound tissue of control group， MEBO group and rb⁃bFGF group were significantly higher than those of model group（all P ＜0.05）.It can be concluded that after rat skin is damaged，applying either MEBO or rb⁃bFGF to the wound can significantly reduce the expres⁃sion levels of CK15， thereby accelerating wound healing， and the treatment effects of MEBO and rb⁃bFGF are comparable； on day 14 of treatment， the wounds of control group， MEBO group， and rb⁃bFGF group healed basically，the expression levels of CK15 and CK15 mRNA of the three groups were close to those of blank group，but the wound healing rate in the model group was significantly lower than that in other groups， that means， the expression levels of CK15 and CK15 mRNA in the wound tissues of model group were still lower than normal level.

In summary， MEBT／MEBO promotes the proliferation of ESCs by regulating the expression level of CK15 in tissue cells.This may constitute its mechanism of action in accelerating the healing of chron⁃ic refractory wounds， and its therapeutic effect and mechanism of ac⁃tion are similar to rb⁃bFGF.In addition， this study only reveals the role of MEBT／MEBO in promoting the horizontal proliferation of ESCs， and further research is needed to answer whether it can pro⁃mote the vertical differentiation of ESCs.

（收稿日期： 2019⁃10⁃15）