單云龍 岑小寧 包崇嬋 卓臣義 唐習(xí)強(qiáng) 韋 騁 王雪玲 左遠(yuǎn)娟 唐乾利

慢性難愈合創(chuàng)面是指治療1個(gè)月以上仍未愈合也無明顯愈合傾向的創(chuàng)面［1］。近年來，眾多文獻(xiàn)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)面的治療中取得了良好的臨床療效［2-4］，但具體作用機(jī)制尚未完全明確。部分研究發(fā)現(xiàn)，細(xì)胞角蛋白 （cytokeratin，CK）15可通過影響表皮干細(xì)胞 （epidermal stem cells，ESCs）的增殖而影響創(chuàng)面的修復(fù)［5］，故筆者于本研究中動(dòng)態(tài)監(jiān)測了MEBT／MEBO對(duì)大鼠慢性難愈合創(chuàng)面組織中CK15表達(dá)水平的影響，以期進(jìn)一步探討MEBT／MEBO在慢性難愈合創(chuàng)面修復(fù)中的部分分子生物學(xué)作用機(jī)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 實(shí)驗(yàn)材料

1.1 實(shí)驗(yàn)動(dòng)物

SPF級(jí)健康成年雄性Wistar大鼠90只，均來源于長沙市天勤生物技術(shù)有限公司，體重200～250 g，飼養(yǎng)于右江民族醫(yī)學(xué)院動(dòng)物實(shí)驗(yàn)中心SPF級(jí)動(dòng)物室，室內(nèi)濕度50% ～70%，溫度23～25℃，空氣流通良好，衛(wèi)生合格。本實(shí)驗(yàn)經(jīng)右江民族醫(yī)學(xué)院動(dòng)物倫理委員會(huì)審批通過，符合動(dòng)物實(shí)驗(yàn)的倫理學(xué)要求。

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(xì)胞生長因子 （recombinant bovine basic fibroblast growth factor， rb?bFGF） 凝膠： 珠海億勝生物制藥有限公司生產(chǎn)；CK15一抗 （rab?bit anti?cytokeratin 15 antibody）： 美國 Affinity Bio?sciences公司生產(chǎn)； 內(nèi)參一抗 （rabbit anti?beta?actin antibody）、辣根過氧化物酶標(biāo)記山羊抗兔 IgG：北京中杉金橋生物技術(shù)有限公司生產(chǎn)；RNAlater Stabilization Solution：賽默飛世爾科技 （中國）有限公司生產(chǎn)；TIANScriptRT Kit、SuperReal PreMix Plus（SYBR Green） （FP205）： 天根生化科技 （北京）有限公司生產(chǎn)。

2 方法

2.1 實(shí)驗(yàn)分組與模型制備

將大鼠適應(yīng)性喂養(yǎng)7 d后，按照隨機(jī)數(shù)表法分為空白組、對(duì)照組、模型組、MEBO組與rb?bFGF組，每組18只，其中空白組大鼠只做背部備皮處理；對(duì)照組大鼠于水合氯醛腹腔注射麻醉及背部備皮處理后，在無菌條件下于備皮處做直徑約15 mm深達(dá)深筋膜的全層皮膚缺損創(chuàng)面，建立急性創(chuàng)面模型；模型組、MEBO組和rb?bFGF組大鼠于水合氯醛腹腔注射麻醉及背部備皮處理后，在無菌條件下于備皮處做直徑約15 mm深達(dá)深筋膜的全層皮膚缺損創(chuàng)面，并立即注射醋酸氫化可的松 （80 mg／kg），建立慢性難愈合創(chuàng)面模型［6-7］。

2.2 局部處理

造模完成后，空白組大鼠備皮處皮膚及對(duì)照組、模型組大鼠創(chuàng)面于5%碘伏消毒后，依次覆蓋2層含有0.9%氯化鈉注射液的濕紗布及2層無菌干紗布包扎固定，每天換藥2次；MEBO組大鼠創(chuàng)面于5%碘伏消毒后，依次覆蓋2層MEBO藥紗 （1 cm2含有0.2 g MEBO）及2層無菌干紗布包扎固定，每天換藥2次；rb?bFGF組大鼠創(chuàng)面于5%碘伏消毒后，依次覆蓋2層 rb?bFGF藥紗 （1 cm2含有 60 U rb?bFGF）及2層無菌干紗布包扎固定，每天換藥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 標(biāo)本采集

分別于治療第3、7、14天，每組隨機(jī)選取6只大鼠用脊椎脫臼法處死后，切取創(chuàng)緣0.5 cm范圍內(nèi)深達(dá)深筋膜下的創(chuàng)面及創(chuàng)周組織，并均分為2份，1份迅速置于凍存管后放在液氮罐中，并置于-80℃冰箱中冷凍保存，用于后期Western blotting技術(shù)檢測；1份迅速置于注有RNA保存液的凍存管后放在液氮罐中，并置于-80℃冰箱中冷凍保存，用于后期 Real?time PCR技術(shù)檢測。

2.4 Western blotting技術(shù)檢測CK15蛋白表達(dá)水平

取備用標(biāo)本進(jìn)行液氮研磨后，加入蛋白裂解液充分裂解，提取總蛋白，并用BCA法測定樣本蛋白濃度；將提取的蛋白上樣依次置于80 V恒壓下電泳30 min、120 V恒壓下電泳60 min、300 mA恒流下電轉(zhuǎn)60 min后，轉(zhuǎn)移至PVDF膜上室溫封閉120 min，并進(jìn)行4℃恒溫?fù)u床；隨后，一抗孵育過夜后洗膜，室溫下孵育二抗60 min后再次洗膜；最后，X射線顯影，晾干，拍照，Image J圖像分析軟件分析蛋白條帶灰度值。

2.5 Real?time PCR技術(shù)檢測CK15 mRNA表達(dá)水平

取備用標(biāo)本采用Trizol法提取總RNA，并分別檢測其在260 nm及280 nm波長的吸光度值，若A260與A280比值在1.8～2.0之間則視為RNA提取合格；按照TIANScript RT KIT試劑盒說明書以總RNA為模板逆轉(zhuǎn)錄合成cDNA；采用2?△△CT法應(yīng)用熒光定量PCR儀進(jìn)行相對(duì)定量分析，結(jié)果以待測基因與內(nèi)參基因GAPDH的表達(dá)水平比值表示。其中CK15上游引物序列為 5′?AGGGGCAGGAGTGGGTT?3′， 下游引物序列 為 5′?CAGGCGGTCGTTGAGGT?3′； β?actin上游引物序列為5′?CCTAGACTTCGAGCAAGAG A?3′， 下游引物序列為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 統(tǒng)計(jì)學(xué)處理

采用SPSS 22.0統(tǒng)計(jì)軟件對(duì)所得數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析，其中計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差（）表示，多個(gè)樣本間比較采用單因素方差分析，且方差齊時(shí)使用LSD法、方差不齊時(shí)使用Games?Howell法；均以P＜0.05為差異具有統(tǒng)計(jì)學(xué)意義。

3 結(jié)果

3.1 CK15蛋白表達(dá)水平對(duì)比

治療過程中，空白組大鼠皮膚組織中CK15蛋白表達(dá)水平無明顯變化 （P＞0.05），其他各組大鼠創(chuàng)面組織中CK15蛋白表達(dá)水平均呈先降低后升高的趨勢 （P均＜0.05）。治療第3、7天，對(duì)照組、MEBO組、rb?bFGF組大鼠創(chuàng)面組織中CK15蛋白表達(dá)水平均顯著低于模型組（P均＜0.05），而治療第14天對(duì)照組、MEBO組、rb?bFGF組大鼠創(chuàng)面組織中CK15蛋白表達(dá)水平均顯著高于模型組 （P均＜0.05），詳見表1及圖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組大鼠皮膚或創(chuàng)面組織中CK15蛋白表達(dá)水平對(duì)比 （）Table 1 Comparison of expression levels of CK15 in skin or wound tissues of all groups（）

表1 5組大鼠皮膚或創(chuàng)面組織中CK15蛋白表達(dá)水平對(duì)比 （）Table 1 Comparison of expression levels of CK15 in skin or wound tissues of all groups（）

注：各時(shí)間點(diǎn)5組大鼠皮膚或創(chuàng)面組織中CK15蛋白表達(dá)水平組內(nèi)對(duì)比，其中與第3天對(duì)比，aP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與第7天對(duì)比，bP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義。各時(shí)間點(diǎn)5組大鼠皮膚或創(chuàng)面組織中CK15蛋白表達(dá)水平組間對(duì)比，其中與空白組對(duì)比，cP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與對(duì)照組對(duì)比，dP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與模型組對(duì)比，eP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義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

組別Group鼠數(shù) （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空白組Blank group 6 0.406±0.063 0.413±0.019 0.408±0.025 0.054 0.948對(duì)照組Control group 6 0.169±0.037c 0.091±0.006ac 0.349±0.016abc 192.766 0.000模型組Model group 6 0.272±0.031cd 0.154±0.004acd 0.280±0.022bcd 58.065 0.000 MEBO組MEBO group 6 0.184±0.026ce 0.079±0.002ace 0.357±0.046abce 126.888 0.000 rb?bFGF 組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 - -

圖1 5組大鼠皮膚或創(chuàng)面組織中CK15蛋白表達(dá)條帶圖Fig.1 Histogram of expression levels of CK15 in skin or wound tissues of each group

3.2 CK15 mRNA表達(dá)水平對(duì)比

治療過程中，空白組大鼠皮膚組織中CK15 mRNA表達(dá)水平無明顯變化 （P＞0.05），其他各組大鼠創(chuàng)面組織中CK15 mRNA表達(dá)水平均呈先降低后升高的趨勢 （P均＜0.05）。治療第3、7天，對(duì)照組、MEBO組、rb?bFGF組大鼠創(chuàng)面組織中CK15 mRNA表達(dá)水平均顯著低于模型組 （P均＜0.05），而治療第14天對(duì)照組、MEBO組、rb?bFGF組大鼠創(chuàng)面組織中 CK15 mRNA表達(dá)水平均顯著高于模型組 （P均＜0.05），詳見表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組大鼠皮膚或創(chuàng)面組織中CK15 mRNA表達(dá)水平對(duì)比 （）Table 2 Comparison of expression levels of CK15 mRNA in skin or wound tissues of each group（）

表2 5組大鼠皮膚或創(chuàng)面組織中CK15 mRNA表達(dá)水平對(duì)比 （）Table 2 Comparison of expression levels of CK15 mRNA in skin or wound tissues of each group（）

注：各時(shí)間點(diǎn)5組大鼠皮膚或創(chuàng)面組織中CK15 mRNA表達(dá)水平組內(nèi)對(duì)比，其中與第3天對(duì)比，aP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與第7天對(duì)比，bP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義。各時(shí)間點(diǎn)5組大鼠皮膚或創(chuàng)面組織中CK15 mRNA表達(dá)水平組間對(duì)比，其中與空白組對(duì)比，cP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與對(duì)照組對(duì)比，dP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與模型組對(duì)比，eP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義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

組別Group鼠數(shù) （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空白組Blank group 6 0.996±0.059 1.030±0.073 1.051±0.043 1.333 0.293對(duì)照組Control group 6 0.602±0.042c 0.278±0.020ac 0.871±0.024abc 580.869 0.000模型組Model group 6 0.759±0.027cd 0.419±0.037acd 0.785±0.025bcd 272.569 0.000 MEBO組MEBO group 6 0.600±0.042ce 0.307±0.027ace 0.936±0.026abcde 569.254 0.000 rb?bFGF 組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 討論

皮膚具有強(qiáng)大的再生功能，以維持自我更新和創(chuàng)傷后的再生修復(fù)，且這種功能主要依賴位于表皮基底層的ESCs。生理狀態(tài)下，ESCs處于相對(duì)靜止?fàn)顟B(tài)，只有少量ESCs轉(zhuǎn)化為短暫擴(kuò)充細(xì)胞 （transit amplifying cell，TAC），繼而分化為其他表皮細(xì)胞，以維持表皮的自我更新［8］；病理狀態(tài)下，處于靜止?fàn)顟B(tài)的 ESCs被激活并沿垂直方向經(jīng)多次分化形成表皮 （垂直分化），同時(shí)沿水平方向不斷增殖以填充受損組織、維持干細(xì)胞數(shù)量 （水平增殖），最終達(dá)到修復(fù)創(chuàng)面的目的［9-10］。部分研究顯示，最早被發(fā)現(xiàn)于表皮基底層及毛囊凸起等部位的Ⅰ型角蛋白CK15作為中間絲蛋白，為ESCs提供了完整的蛋白骨架，賦予了ESCs額外的物理穩(wěn)定性［11］，同時(shí)介導(dǎo)并增加了ESCs與細(xì)胞外基質(zhì) （extracellular matrix，ECM）的黏附作用，在創(chuàng)面愈合的水平增殖過程中發(fā)揮著關(guān)鍵作用［12-13］。

本研究中，筆者觀察了近年來被廣泛應(yīng)用于慢性難愈合創(chuàng)面并取得顯著效果的MEBT／MEBO對(duì)大鼠慢性難愈合創(chuàng)面組織中CK15表達(dá)的影響，以探索其作用機(jī)制。結(jié)果顯示，（1）除空白組外，其他各組大鼠創(chuàng)面組織中CK15蛋白及CK15 mRNA表達(dá)水平均呈先降低后升高的趨勢 （P均＜0.05）?？梢姡笫笃つw受損后，機(jī)體可通過降低CK15的表達(dá)而降低ESCs的穩(wěn)定性及其與ECM的黏附性，進(jìn)而促使ESCs大量增殖，以修復(fù)損傷創(chuàng)面［8-9］。 （2）治療第3、7天，對(duì)照組、MEBO組、rb?bFGF組大鼠創(chuàng)面組織中CK15蛋白及CK15 mRNA表達(dá)水平均顯著低于模型組 （P均＜0.05），而治療第14天對(duì)照組、MEBO組、rb?bFGF組大鼠創(chuàng)面組織中CK15蛋白及CK15 mRNA表達(dá)水平均顯著高于模型組 （P均＜0.05）?？梢姡笫笃つw受損后，創(chuàng)面應(yīng)用MEBO及rb?bFGF均可顯著降低創(chuàng)面組織中CK15的表達(dá)水平，從而加速創(chuàng)面愈合，且MEBO與rb?bFGF的治療效果相當(dāng)；至治療第14天，對(duì)照組、MEBO組、rb?bFGF組大鼠創(chuàng)面趨于愈合，CK15蛋白及CK15 mRNA表達(dá)水平接近空白組，而模型組大鼠創(chuàng)面愈合速率明顯低于其他各組，故其創(chuàng)面組織中CK15蛋白及CK15 mRNA表達(dá)水平仍低于正常水平。

綜上所述，通過調(diào)控組織細(xì)胞中CK15的表達(dá)水平促進(jìn)ESCs的增殖可能是MEBT／MEBO有效加快慢性難愈合創(chuàng)面愈合的部分作用機(jī)制，且其治療效果與rb?bFGF相當(dāng)，作用機(jī)制與rb?bFGF相似。另外，本研究只揭示了MEBT／MEBO促進(jìn)ESCs水平增殖的作用，而其是否可以促進(jìn)ESCs的垂直分化仍需進(jìn)一步深入研究探討。

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）