MEBT／MEBO對慢性難愈合創(chuàng)面組織中CK10表達水平的影響

舒清峰 陳端凱 唐乾利 單云龍 唐 強 岑小寧 卓臣義 馮 時

作者單位：533000 廣西 百色，右江民族醫(yī)學院／桂西高發(fā)病重點實驗室

近年來，大量臨床實踐及基礎研究顯示，皮膚再生醫(yī)療技術(shù) （moist exposed burn therapy／moist exposed burn ointment，MEBT／MEBO） 在慢性難愈合創(chuàng)面的治療中取得了卓越的成效［1－3］，且具體的作用機制成為了臨床研究的熱點。如徐榮祥等的研究顯示，MEBT／MEBO可激活創(chuàng)面組織內(nèi)的潛能再生細胞，并將其轉(zhuǎn)化為細胞角蛋白 （cytokeratin，CK）19陽性表達的干細胞，再在原位不斷增殖分化為正常表皮細胞，最終實現(xiàn)缺損創(chuàng)面的再上皮化和組織結(jié)構(gòu)重塑［4－8］。隨著研究的不斷深入，部分研究學者發(fā)現(xiàn)，CK10是表皮細胞分化成熟的標記分子之一，其表達水平的差異可體現(xiàn)創(chuàng)面的修復水平［9］。為此，筆者于本研究中動態(tài)監(jiān)測了經(jīng)不同方法處理后大鼠皮膚及創(chuàng)面組織內(nèi)CK10表達水平的變化情況，探討了MEBT／MEBO對表皮干細胞增殖分化的影響，以揭示MEBT／MEBO促進慢性難愈合創(chuàng)面修復的部分作用機制。

1 實驗材料

1.1 實驗動物

12周齡SPF級健康Wistar雄性大鼠90只［長沙市天勤生物技術(shù)有限公司提供，許可證號SCXK （湘） 2014?0011］，體重 （220 ±20） g，實驗過程中嚴格按照SPF級飼養(yǎng)環(huán)境飼養(yǎng)，濕度保持在60% ±10%，室溫保持在 （25±2）℃，通風通氣良好。本研究經(jīng)右江民族醫(yī)學院動物倫理委員會批準。

1.2 主要試劑

濕潤燒傷膏 （moist exposed burn ointment，MEBO）：汕頭市美寶制藥有限公司生產(chǎn)；重組牛堿性成纖維細胞生長因子 （recombinant bo?vine basic fibroblast growth factor，rb?bFGF） 凝膠：珠海億勝生物制藥有限公司生產(chǎn)；水合氯醛：泰興市豪申化工貿(mào)易有限公司生產(chǎn)；醋酸氫化可的松注射液：上海通用藥業(yè)股份有限公司生產(chǎn)；一抗稀釋液、二抗稀釋液：上海碧云天生物技術(shù)有限公司生產(chǎn)； Anti?CK10?Antibody：Abcam公司生產(chǎn)；辣根過氧化物酶標記山羊抗兔 IgG （100μL）、 β?actin 抗體 （100μL）： 北京中杉金橋生物技術(shù)有限公司生產(chǎn)；PVDF膜：Millpore公司生產(chǎn)。

2 方法

2.1 實驗分組與模型制備

將大鼠置于SPF級動物實驗室飼養(yǎng)1周并確定大鼠健康后，隨機分為空白組、急創(chuàng)組、慢創(chuàng)組、rb?bFGF組與MEBO組，每組18只，均選取脊柱兩側(cè)背部皮膚作為實驗區(qū)域，其中空白組大鼠僅對其背部皮膚進行面積約3.0 cm×3.0 cm的備皮處理；急創(chuàng)組大鼠于7%水合氯醛腹腔注射 （4 mL／kg）麻醉及備皮處理 （方法同空白組）后，使用直徑為1.5 cm的圓形印章進行標記，并在無菌環(huán)境下延標記線去除皮膚組織至筋膜層，建立急性皮膚組織缺損創(chuàng)面；慢創(chuàng)組、rb?bFGF組與 MEBO組大鼠于麻醉、備皮及去除皮膚組織 （方法均同急創(chuàng)組）后，立即注射醋酸氫化可的松注射液建立慢性難愈合創(chuàng)面模型［10］。

2.2 局部處理

模型制備成功后，空白組大鼠備皮處皮膚立即予以5%碘伏常規(guī)消毒3次，并依次覆蓋生理鹽水紗布及清潔敷料，膠帶固定，每天換藥2次；急創(chuàng)組與慢創(chuàng)組大鼠創(chuàng)面立即予以5%碘伏常規(guī)消毒3次，并依次覆蓋生理鹽水紗布及清潔敷料，膠帶固定，每天換藥2次；rb?bFGF組大鼠創(chuàng)面立即予以5%碘伏常規(guī)消毒3次，并均勻涂抹 rb?bFGF凝膠 （60 U／cm2）后依次覆蓋生理鹽水紗布及清潔敷料，膠帶固定，每天換藥2次；MEBO組大鼠創(chuàng)面立即予以5%碘伏常規(guī)消毒3次，并均勻涂抹MEBO（0.2 g／cm2）后依次覆蓋生理鹽水紗布及清潔敷料，膠帶固定，每天換藥2次。

2.3 標本采集

治療第3、7、14天，每組隨機選取6只大鼠于腹腔注射麻醉后，去除創(chuàng)面痂皮并于距離創(chuàng)面邊緣0.5 cm處切取創(chuàng)面組織 （空白組大鼠取相同部位正常皮膚組織）均分為兩份，其中一份立即置于裝有10%甲醛的EP管中固定，并于標本全部采集后行石蠟包埋保存；另一份立即置入EP管內(nèi)暫時存放在液氮中，并于標本全部采集后保存于－80℃冰箱內(nèi)。

2.4 組織學檢查

將石蠟包埋的組織切片后進行HE染色，并于電子顯微鏡下觀察組織內(nèi)炎性細胞與成纖維細胞的分布、新生血管的排列及皮膚附屬器的形成等情況。

2.5 Western blotting法檢測CK10表達水平

將冷凍組織置于放在冰上的裝有組織裂解液的EP管中充分裂解后提取總蛋白，檢測蛋白濃度，并使用蛋白緩沖液及組織裂解液的配比液高溫變性后分裝保存于－20℃冰箱中；將變性后的蛋白逐孔注入SDS?PAGE凝膠中，在100 V恒壓下電泳至Marker條帶均勻分開，并根據(jù)CK10分子量大小裁剪相應PVDF膜于250 mA恒流下行60～90 min轉(zhuǎn)膜處理；轉(zhuǎn)膜完成后，洗膜3次，在常溫下孵育一抗、二抗，并于抗體孵育后進行發(fā)光曝光處理，最后使用Image J軟件對蛋白灰度進行分析。每組大鼠標本重復檢測3次以上，最后取均值進行對比分析。

2.6 統(tǒng)計學處理

采用SPSS 22.0統(tǒng)計軟件對所得數(shù)據(jù)進行統(tǒng)計學分析，其中計量資料以均數(shù)±標準差（±s）表示，多個樣本均數(shù)比較采用單因素方差分析 （one?way ANOVA）， 并根據(jù)方差齊性檢驗結(jié)果采用LSD法或Games?Howell法進行組間對比，均以P＜0.05為差異具有統(tǒng)計學意義。

3 結(jié)果

3.1 組織學檢查結(jié)果

治療第3天，空白組大鼠皮膚組織與正常皮膚組織無明顯差異；急創(chuàng)組大鼠創(chuàng)面組織內(nèi)可見大量炎性細胞及少量成纖維細胞與新生毛細血管，組織水腫明顯；慢創(chuàng)組、rb?bFGF組及MEBO組大鼠創(chuàng)面組織內(nèi)可見大量炎性細胞及少量成纖維細胞，局部可見紅細胞溢出血管壁外，組織水腫較急創(chuàng)組更為明顯。

治療第14天，空白組大鼠皮膚組織與正常皮膚組織無明顯差異；急創(chuàng)組、rb?bFGF組及MEBO組大鼠創(chuàng)面基本愈合，組織內(nèi)可見排列整齊的新生毛細血管、完整的毛囊及皮脂腺等皮膚附屬器，組織結(jié)構(gòu)接近正常皮膚組織；慢創(chuàng)組大鼠創(chuàng)面明顯縮小，組織內(nèi)可見少量炎性細胞及大量成纖維細胞，部分成纖維細胞排列紊亂 （圖1）。

3.2 CK10表達水平

治療第3、7、14天，空白組大鼠皮膚組織內(nèi)CK10表達水平無明顯變化，P＞0.05，差異無統(tǒng)計學意義；急創(chuàng)組、慢創(chuàng)組、rb?bFGF組及MEBO組大鼠創(chuàng)面組織內(nèi)CK10表達水平均呈逐漸升高的趨勢，P均＜0.05，差異具有統(tǒng)計學意義 （表1，圖2－3）。

圖1 5組大鼠皮膚或創(chuàng)面組織HE染色典型圖 （×200）Fig.1 The typical HE staining results of rat skin or wound tissues in the five groups（×200）

治療第3天，5組大鼠皮膚或創(chuàng)面組織內(nèi)CK10表達水平對比，空白組＞急創(chuàng)組＝慢創(chuàng)組＝rb?bFGF組 ＝MEBO組，組間兩兩對比，除空白組分別與急創(chuàng)組、慢創(chuàng)組、rb?bFGF組及MEBO組對比，P均＜0.05，差異具有統(tǒng)計學意義外，其余各組組間兩兩對比，P均＞0.05，差異無統(tǒng)計學意義。治療第7天，5組大鼠皮膚或創(chuàng)面組織內(nèi)CK10表達水平對比，空白組＞急創(chuàng)組＝rb?bFGF組＝MEBO組＞慢創(chuàng)組，組間兩兩對比，除急創(chuàng)組、rb?bFGF組及MEBO組兩兩對比，P均＞0.05，差異無統(tǒng)計學意義外，其余各組組間兩兩對比，P均＜0.05，差異具有統(tǒng)計學意義。治療第14天，5組大鼠皮膚或創(chuàng)面組織內(nèi)CK10表達水平對比，空白組＝急創(chuàng)組＝rb?bFGF組＝MEBO組＞慢創(chuàng)組，組間兩兩對比，除空白組、急創(chuàng)組、rb?bFGF組及MEBO組分別與慢創(chuàng)組對比，P均＜0.05，差異具有統(tǒng)計學意義外，其余各組組間兩兩對比，P均＞0.05，差異無統(tǒng)計學意義 （表1，圖2－3）。

表1 5組大鼠皮膚或創(chuàng)面組織內(nèi)CK10表達水平對比 （±s）Table 1 Comparison of CK10 expression levels in rat skin or wound tissues among the five groups（±s）

表1 5組大鼠皮膚或創(chuàng)面組織內(nèi)CK10表達水平對比 （±s）Table 1 Comparison of CK10 expression levels in rat skin or wound tissues among the five groups（±s）

注：5組大鼠皮膚或創(chuàng)面組織內(nèi)CK10表達水平組內(nèi)對比，其中與第3天對比，aP＜0.05，差異具有統(tǒng)計學意義；與第7天對比，bP＜0.05，差異具有統(tǒng)計學意義。5組大鼠皮膚或創(chuàng)面組織內(nèi)CK10表達水平組間對比，其中與空白組對比，cP＜0.01，差異具有統(tǒng)計學意義；與急創(chuàng)組對比，dP＜0.05，差異具有統(tǒng)計學意義；與慢創(chuàng)組對比，eP＜0.05，差異具有統(tǒng)計學意義；與rb?bFGF組對比，fP＜0.05，差異具有統(tǒng)計學意義；與MEBO組對比，gP＜0.05，差異具有統(tǒng)計學意義Note： The expression levels of CK10 in rat skin or wound tissues were compared within each of the five groups，in which statistically significant differ?ences were observed in comparisons respectively with that on day 3 （aP ＜0.05） and with that on day 7 （bP ＜0.05）.The between?group comparisons of CK10 expression levels were done among the five groups，in which statistically significant differences were observed in comparisons with the blank group（cP ＜0.01），with the acute wound group（dP ＜0.05），with the chronic wound group（eP ＜0.05），with the rb?bFGF group（fP ＜0.05） and with the MEBO group（gP＜0.05）

組別Group鼠數(shù) （只）Number of rats （rat）第3天Day 3第7天Day 7第14天Day 14 F值F value P值P value空白組Blank group 6 1.14±0.03defg 1.12±0.05defg 1.15±0.05e 0.712 0.507急創(chuàng)組Acute wound group 6 0.58±0.03bc 0.93±0.07ace 1.20±0.04abe 235.103 0.000慢創(chuàng)組Chronic wound group 6 0.56±0.05bc 0.75±0.04acdfg 0.96±0.04abcdfg 126.461 0.000 rb?bFGF 組rb?bFGF group 6 0.59±0.04bc 0.88±0.06ace 1.17±0.04abe 222.623 0.000 MEBO組MEBO group 6 0.58±0.03bc 0.87±0.07ace 1.16±0.07abe 141.525 0.000 F值F value 279.706 31.114 22.550－ －P值P value 0.000 0.000 0.000－ －

圖2 Western blotting法檢測5組大鼠皮膚或創(chuàng)面組織內(nèi)CK10表達蛋白條帶圖Fig.2 CK10?expressed protein bands in rat skin or wound tissues in the five groups tested by Western blot?ting method

圖3 5組大鼠皮膚或創(chuàng)面組織內(nèi)CK10表達水平柱狀圖Fig.3 Histogram of CK10 expression levels in rat skin or wound tissues in the five groups

4 討論

表皮干細胞具有強大的增殖、分化能力，其在皮膚受到創(chuàng)傷時可被激活，并逐步分化為表皮及皮膚附屬器等組織結(jié)構(gòu)，最終實現(xiàn)缺損皮膚的再生修復，在創(chuàng)面修復過程中發(fā)揮著至關(guān)重要的作用［11－12］。研究顯示，表皮干細胞所生存的外界環(huán)境對表皮干細胞的增殖、分化影響巨大［13－15］，如創(chuàng)面涂抹 MEBO 后，表皮干細胞可因所處的外界環(huán)境發(fā)生改變而提高其增殖、分化能力，從而加速創(chuàng)面修復［16］。亦有研究顯示，CK10是表皮細胞增殖分化的標記性分子，其表達水平可隨著表皮干細胞的不斷分化、成熟而改變［9，17］。 因此，筆者為探討 MEBT／MEBO對表皮干細胞影響的部分作用機制，最終實現(xiàn)加快慢性難愈合創(chuàng)面修復的目的，于本研究中對比觀察了不同時間點經(jīng)不同方法處理后大鼠皮膚及創(chuàng)面組織中CK10表達水平的變化情況。

研究結(jié)果顯示，治療第3、7、14天，空白組大鼠皮膚組織內(nèi)CK10的表達水平無明顯變化，P＞0.05，差異無統(tǒng)計學意義，且鏡下觀組織形態(tài)與正常皮膚組織形態(tài)也無明顯差異，符合其表皮未予任何處理的特征；而急創(chuàng)組、慢創(chuàng)組、rb?bFGF組及MEBO組大鼠創(chuàng)面組織內(nèi)CK10的表達水平均逐漸升高，P均＜0.05，差異具有統(tǒng)計學意義，且鏡下觀組織形態(tài)也有明顯改變。治療第3天，空白組大鼠皮膚組織內(nèi)CK10的表達水平均較其他各組高，且P均＜0.05，差異具有統(tǒng)計學意義，而急創(chuàng)組、慢創(chuàng)組、rb?bFGF組及MEBO組的組織形態(tài)及CK10表達水平并無明顯差異，P均＞0.05，差異無統(tǒng)計學意義，表明正常表皮內(nèi)角化蛋白較為豐富，皮膚缺損后大量表皮細胞缺失，致使創(chuàng)面組織內(nèi)CK10的表達水平也隨之降低。治療第7天，空白組大鼠皮膚組織內(nèi)CK10的表達水平仍為最高，而急創(chuàng)組、慢創(chuàng)組、rb?bFGF組及MEBO組大鼠創(chuàng)面組織內(nèi)CK10表達水平呈現(xiàn)升高的趨勢，與第3天對比，P均＜0.05，差異具有統(tǒng)計學意義，與基底層細胞不表達CK10，隨著基底層干細胞不斷增殖、分化為基底上層細胞，CK10的表達水平也不斷升高［18］的研究結(jié)果一致；且急創(chuàng)組、rb?bFGF組及MEBO組的增長幅度均較慢創(chuàng)組明顯，P均＜0.05，差異具有統(tǒng)計學意義，說明慢創(chuàng)組大鼠創(chuàng)面修復速度較急創(chuàng)組慢，而rb?bFGF與MEBO對慢性難愈合創(chuàng)面均具有治療作用。治療第14天，除慢創(chuàng)組外，急創(chuàng)組、rb?bFGF組和MEBO組大鼠創(chuàng)面組織形態(tài)及CK10表達水平均接近空白組，P均＞0.05，差異無統(tǒng)計學意義，表明除慢創(chuàng)組外，急創(chuàng)組、rb?bFGF組和 MEBO組大鼠創(chuàng)面已修復至接近正常皮膚組織，且組間對比，P均＞0.05，差異無統(tǒng)計學意義，即MEBT／MEBO治療慢性難愈合創(chuàng)面的療效不亞于 rb?bFGF 凝膠。

綜上所述，CK10表達水平的變化規(guī)律可表明表皮細胞增殖、分化的狀況，MEBT／MEBO治療慢性難愈合創(chuàng)面的療效不亞于rb?bFGF凝膠，且通過對表皮干細胞外環(huán)境的影響促進表皮干細胞的增殖、分化可能是其加快創(chuàng)面修復的作用機制，仍需進一步深入研究考證。

In recent years，a large number of clinical practice and basic studies have shown that moist exposed burn therapy／moist exposed burn ointment（MEBT／MEBO） can realize significant curative effects in the treatment of chronic non?healing wounds［1－3］，and its action mechanism has become a focus of clinical research.As demonstrated in Rongxiang Xu，et al.，MEBT／MEBO can activate the potential re?generative cells in wound tissues and transform them into stem cells with positive expression of cytokeratin 19，and such stem cells cancontinuously proliferate and differentiate into normal epidermal cells in situ and eventually the damaged wounds can realize re?epithelial?ization and remodeling of tissue structure［4－8］.With the advances of research，some researchers found that CK10 is one of markers of epi?dermal cell differentiation and maturation，and its expression level may reflect the wound repair condition［9］.To this end，this study dy?namically monitored the changes of CK10 expression in rat skin and wound tissues managed with different approaches，and explored the influence of MEBT／MEBO on the proliferation and differentiation of epidermal stem cells，with the aim of revealing partially the action mechanism of MEBT／MEBO in promoting the repair of chronic non?healing wounds.

1.Experimental material

1.1.Experimental animals

Ninety SPF healthy Wistar male rats（provided by Changsha Tianqin Biotechnology Co.，Ltd.，License number SCXK （Xiang）2014?0011），aged 12 weeks old and weighing （220 ± 20） g，were selected as subjects.During the experiment course，the subjects were kept strictly in the SPF feeding environment，with good ventilation，humidity at 60% ±10%and room temperature at（25±2）℃.This study was approved by the Animal Ethics Committee of Youjiang Med?ical University for Nationalities.

1.2.Main agents

Moist exposed burn ointment（MEBO） is manufactured by Shantou MEBO Pharmaceutical Co.，Ltd.，recombinant bovine bas?ic fibroblast growth factor （rb?bFGF） gel by Zhuhai Essex Bio?Pharmaceutical company Limited，chloral hydrate by Taixing Haoshen Chemical Trading Co.，Ltd.，hydrocortisone acetate injection by Shanghai General Pharmaceutical Co.，Ltd.，primary antibody dilu?tion buffer and secondary antibody dilution buffer are from Shanghai Beyotime Biotechnology Co.，Ltd.，and Anti?CK10?Antibody from Abcam.Horseradish peroxidase?labeled goat anti?rabbit IgG （100μL）and β?actin antibody （100μL） are manufactured by Beijing ZSGB Biotechnology Co.，Ltd.，and PVDF membrane by Millpore.

2.Methods

2.1.Grouping and modelling

The subjects were fed in the SPF animal laboratory for one week and，after being identified as healthy，were randomly divided into blank group，acute wound group，chronic wound group，rb?bFGF group and MEBO group，18 rats each group.The back skin of bilat?eral spine was selected as the experimental area.In the blank group，the skin at an area of about 3.0 cm×3.0 cm was prepared on the back of rats.However，in the acute wound group，after intraperitone?al injection of 7%chloral hydrate （4 mL／kg） and skin preparation（same method as in the blank group），mark the prepared skin with acircular seal of about 1.5 cm in diameter，remove skin tissues to the fascial layer along the marked line under sterile condition to establish the skin defect wound.In the chronic wound group，rb?bFGF group and MEBO group，Hydrocortisone acetate injection was injected im?mediately after the same procedures of anesthetizing，preparing skin and removing skin tissues as in the acute wound group to establish chronic non?healing wound models［10］.

2.2.Local management of wounds

After successful model establishment，the prepared skin in the blank group was immediately given the routine disinfection with 5%iodophor for three times，followed by the successive covering of nor?mal saline soaked gauze and clean dressing，and the fixation with ad?hesive tape.The dressing was changed twice a day.The wounds in the acute wound group and chronic wound group were disinfected im?mediately with 5%iodophor for three times，and also covered with normal saline soaked gauze and clean dressing in turn，followed by the fixation with adhesive tape.The dressing change was performed twice a day.After the immediate routine disinfection with 5% io?dophor for three times，the wounds in the rb?bFGF group and MEBO group were respectively managed with rb?bFGF gel（60 U／cm2） and MEBO （0.2 g／cm2） before the successive covering of normal saline soaked gauze and clean dressing，and the fixation of adhesive tape，and the dressing was also changed twice a day in the two groups.

2.3.Sample collection

On day 3，7 and 14 of treatment，randomly select 6 rats in each group to undergo intraperitoneal anesthesia，and then remove the wound scab and take out wound tissues at 0.5 cm away from the wound edges（take out the normal skin tissues at the same location in the blank group）.Divide the wound tissues into two samples，of which one was fixed immediately in EP tube containing 10%formal?dehyde and then embedded in paraffin after complete sample collec?tion，and the other was immediately put into EP tube and temporarily stored in liquid nitrogen，and then stored in a refrigerator of－80℃after complete sample collection.

2.4.Histological examination

Do HE staining after the slicing of paraffin?embedded tissues，and observe，under electron microscope，the distribution of inflamma?tory cells and fibroblasts，the arrangement of new blood vessels and the formation of skin appendages in tissues.

2.5.Test the expression level of CK10 by Western blotting method

Fully decompose the frozen tissues in the EP tube containing tis?sue lysate on ice to extract the total protein，and detect the concentra?tion of the total protein.Subpackage and store the protein into a re?frigerator of－20℃ after high?temperature degeneration by using the composition of protein buffer and tissue lysate.Pour the degenerated protein into SDS?PAGE gel hole by hole，and then proceed with the electrophoresis at the constant voltage of 100 V till the Marker bandswere spread uniformly.Cut PVDF membrane into suitable size in ac?cordance with CK10 molecular weight，and transfer the protein onto a membrane for 60－90 min under the constant current of 250 mA.Af?ter this，the membrane was washed for three times，incubated with the primary antibody and secondary antibody at room temperature，followed by exposure development.And finally the protein shades of gray was analyzed using Image J.The samples in each group were de?tected repeatedly for more than three times and the obtained mean val?ues were compared and analyzed.

2.6.Statistical analysis

Statistical software SPSS 22.0 was used for statistical analysis，in which measurement data was expressed as mean±standard devia?tion（±s），the means of multiple samples were compared with the one?way ANOVA，and between?group comparisons were done with the LSD method or Games?Howell method according to the variance homo?geneity.P＜0.05 was considered statistically significant.

3.Results

3.1.Results of histological examination

On day 3 of treatment，there was no obvious difference between the skin tissues in the blank group and the normal rat skin tissues，while in the acute wound group，there was a large number of inflam?matory cells and a few fibroblasts and newly?born capillaries in wound tissues，and the tissues were markedly edematous.In the chronic wound group，rb?bFGF group and MEBO group，besides a large number of inflammatory cells and a few fibroblasts in the wound tissues，some red blood cells overflowing vascular wall can be seen in some locations and the tissue edema was more severe than the acute wound group.

On day 14 of treatment，the skin tissues in the blank group had no obvious difference from the normal skin tissues.The wounds in the acute wound group，rb?bFGF group and MEBO group were basically healed，well?arranged new capillaries and intact skin appendages in?cluding hair follicle and sebaceous glands were observable in tissues，and the tissue structure was similar to normal skin tissues.The wounds in the chronic wound group decreased obviously in size，few inflammatory cells and a large number of fibroblasts can be seen in tissues，and some fibroblasts were arranged disorderly （Fig.1）.

3.2.Expression level of CK10

On day 3，7 and 14 of treatment，no significant change was ob?served on the expression level of CK10 in rat skin tissues in the blank group，and there was no statistically significant difference，P＞0.05.In contrast，the expression of CK10 in rat skin tissues in the acute wound group，chronic wound group，rb?bFGF group and MEBO group all showed an increasing tendency，and the differences were statisti?cally significant，allP＜0.05 （Table 1，F(xiàn)ig.2－3）.

On day 3 of treatment，the comparison of CK expression levels among the five groups showed a pattern of the blank group＞the acute wound group＝chronic wound group＝rb?bFGF group＝MEBO group，

and the between?group pairwise comparisons all showed no statistically significant difference（P＞0.05） except the comparisons of the blank group with the other four groups （P＜0.05）.On day 7 of treatment，the comparison of CK10 expression levels among the five groups showed a pattern of the blank group ＞ the acute wound group ＝ rb?bFGF group ＝MEBO group ＞ chronic wound group，and the between?group pairwise comparisons all showed statistically significant differences（P＜0.05） except the pairwise comparisons between acute wound group，rb?bFGF group and MEBO group （allP＞0.05）.On day 14 of treatment，the comparison of CK10 expression levels among the five groups showed a pattern of the blank group＝the acute wound group＝rb?bFGF group ＝MEBO group ＞chronic wound group，and the between?group pairwise comparisons all showed no statistically significant difference（P＞0.05） except the comparisons of the chronic wound group with the other four groups（allP＜0.05） （Table 1，F(xiàn)ig.2－3）.

4.Discussion

Epidermal stem cells have strong ability of proliferation and dif?ferentiation，which can be activated when skin is injured to gradually differentiate into tissue structures of epidermis and skin appendages，and eventually realize the regenerative repair of injured skin，playing an important role in wound repair［11－12］.Studies showed that the liv?ing environment of epidermal stem cells exerts huge impacts on the proliferation and differentiation of epidermal stem cells［13－15］.For ex?ample，after the application of MEBO，with the change of the external environment of epidermal stem cells，their ability of proliferation and differentiation will be enhanced，facilitating the wound repair［16］.Moreover，some studies confirmed that CK10 is the molecule marker of epidermal cell proliferation and differentiation，and its expression level may change with the continuous differentiation and maturation of epi?dermal stem cells［9，17］.Therefore，with the aim of investigating the ac?tion mechanism of MEBT／MEBO on epidermal stem cells to realize the accelerated repair of chronic non?healing wounds，this study compared the changes of CK10 expression levels in rat skin and wound tissues at different time points after managed with different treatment approaches.

And the results showed that on day 3，7 and 14 of treatment，no significant change was observed on the expression level of CK10 in rat skin tissues in the blank group，and there was no statistically signifi?cant difference，P＞0.05.Microscope observation found that the tis?sue morphology has no significant difference from the normal skin tis?sues，being in line with the features of non?managed epidermis.In contrast，the expression of CK10 in rat wound tissues in the acute wound group，chronic wound group，rb?bFGF group and MEBO group all showed an increasing tendency，and the differences were statistical?ly significant（allP＜0.05）.The tissue morphology under microscopeobservation also presented marked changes.On day 3 of treatment，the expression level of CK10 in rat skin tissues in the blank group was higher than that in the other four groups and the differences were statis?tically significant，allP＜0.05.While no statistically significant differences was observed in the tissue morphology and CK expression levels among the acute wound group，chronic wound group，rb?bFGF group and MEBO group （allP＞0.05），which indicated that though the cytokeratin in normal epidermis was very abundant，with the dam?age of skin，plenty of epidermal cells will lose，subsequently resulting in the decrease of CK10 expression in wound tissues.On day 7 of treatment，the expression level of CK10 in the blank group was still highest，meanwhile the CK10 expression levels in wound tissues in the other four groups presented an increasing tendency，and there were sta?tistically significant differences as compared with that on day 3 of treat?ment（allP＜0.05），which was consistent with the study results that basal layer cells don’t express CK10 and the expression level of CK10 will increase continuously with the constant proliferation and differenti?ation of basal layer stem cells into cells above the basal layer［18］.How?ever，the increase extents in the acute wound group，rb?bFGF group and MEBO group were more significant than that in the chronic wound group （allP＜0.05），which indicated that the wound repair speed in the chronic wound group was slower than the acute wound group，and rb?bFGF gel and MEBO have the function of treating chronic non?healing wounds.On day 14 of treatment，except the chronic wound group，the tissue morphology and CK10 expression levels in the acute wound group，rb?bFGF group and MEBO group were close to that in the blank group，and the differences were not statistically significant（allP＞0.05），which demonstrated that except the chronic wound group，the rat wounds in the acute wound group，rb?bFGF group and MEBO group have basi?cally restored to the status of normal skin tissues，and their between?group comparisons showed no statistically significant differences（allP＞0.05），which indicated the curative effect of MEBT／MEBO in treating chronic non?healing wounds was not inferior to that of rb?bFGF gel.

To sum up，the change law of CK10 expression levels can reflect the condition of epidermal cell proliferation and differentiation，and the clinical efficacy of MEBT／MEBO in the treatment of chronic non?healing wounds is not inferior to that of rb?bFGF gel.Promoting the proliferation and differentiation of epidermal stem cells by changing the external living environment of epidermal stem cells may be the action mechanism of MEBT／MEBO in facilitating the restoration of chronic non?healing wounds，which，however，needs to be further studied in the future.

（收稿日期： 2018?12?28）