唐乾利 舒清峰 單云龍 李利青 唐 強(qiáng) 岑小寧 唐習(xí)強(qiáng) 許 彥 黃麗芳

作者單位：533000 廣西 百色，右江民族醫(yī)學(xué)院/桂西高發(fā)病防治重點(diǎn)實(shí)驗(yàn)室 （唐乾利，單云龍，李利青，唐強(qiáng)，岑小寧，唐習(xí)強(qiáng)）；570100 海南 ?？?，海南省腫瘤醫(yī)院普外科 （舒清峰）；530001 廣西 南寧，廣西中醫(yī)藥大學(xué)研究生院2018級(jí)中醫(yī)外科學(xué)專業(yè) （許彥，黃麗芳）

慢性難愈合創(chuàng)面通常是指規(guī)范治療1個(gè)月仍未正常愈合，也無(wú)愈合趨勢(shì)的創(chuàng)面［1］，發(fā)病機(jī)制十分復(fù)雜，目前尚未完全明確。部分研究指出，表皮干細(xì)胞在創(chuàng)面修復(fù)過(guò)程中發(fā)揮著關(guān)鍵作用，且細(xì)胞角蛋白 （cytokeratin，CK）在表皮干細(xì)胞參與創(chuàng)面修復(fù)過(guò)程中的作用顯著［2-3］。如，早在20年前徐榮祥教授［4］就證實(shí)了CK19型干細(xì)胞參與創(chuàng)面的再生修復(fù)，且其創(chuàng)立的皮膚再生醫(yī)療技術(shù) （moist exposed burn therapy/moist exposed burn ointment， MEBT/MEBO） 的核心藥物濕潤(rùn)燒傷膏 （moist exposed burn ointment，MEBO）能夠激活CK19型干細(xì)胞是該藥物促進(jìn)燒傷創(chuàng)面愈合的主要作用機(jī)制。近年來(lái)，唐乾利等［5-12］將MEBT/MEBO應(yīng)用于慢性難愈合創(chuàng)面的治療，并在開(kāi)展的大量臨床及實(shí)驗(yàn)研究中證實(shí)了其顯著的臨床療效。本研究為進(jìn)一步探討MEBT/MEBO促進(jìn)慢性難愈合創(chuàng)面修復(fù)的部分分子生物學(xué)作用機(jī)制，對(duì)比分析了MEBT/MEBO對(duì)大鼠慢性難愈合創(chuàng)面組織中CK14及CK19表達(dá)水平的影響。

Chronic refractory wounds usually refer to wounds that remain unhealed and show no tendency to heal after one month of standard treatment［1］， and the pathogenesis is very complicated and has not yet been figured out completely till now.Some studies have shown that epidermal stem cells play a key role in wound repair and cytokeratin（CK） is of significance in facilitating epidermal stem cell to repair wound［2-3］.As early as 20 years ago， Professor Rongxiang Xu［4］confirmed that CK19 stem cells were involved in the regenerative repair of wounds.MEBO， the core medicine of Professor Xu’s MEBT/MEBO （moist exposed burn therapy/moist exposed burn ointment），can activate CK19 stem cells，which is the main mechanism of action of MEBO to promote the healing of burn wounds.In recent years，Tang Qianli， et al.［5-12］have applied MEBT/MEBO in the treatment of chronic refractory wounds，and confirmed its remarkable clinical efficacy with a large number of clinical and experimental studies.This study was to further explore the molecular biology mechanisms of MEBT/MEBO in promoting chronic refractory wound healing by analyzing the influence of MEBT/MEBOon the expression levels of CK14 and CK19 in chronic refractory wound tissues of rats.

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物

健康12周齡SPF級(jí)Wistar雄性大鼠90只，體重 （220±20）g，均由長(zhǎng)沙市天勤生物技術(shù)有限公司提供，飼養(yǎng)環(huán)境及運(yùn)輸與實(shí)驗(yàn)過(guò)程嚴(yán)格按照SPF級(jí)要求執(zhí)行，環(huán)境濕度控制在 （60±10）%、溫度控制在 （25±2）℃，空氣流通良好，衛(wèi)生條件達(dá)標(biāo)。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)批準(zhǔn)，符合動(dòng)物實(shí)驗(yàn)的倫理學(xué)要求。

1.2 主要試劑

MEBO：汕頭市美寶制藥有限公司生產(chǎn)，國(guó)藥準(zhǔn)字Z20000004；重組牛堿性成纖維細(xì)胞生長(zhǎng)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)，國(guó)藥準(zhǔn)字S19991021；水合氯醛：泰興市豪申化工貿(mào)易有限公司生產(chǎn)，CAS登記號(hào)302-17-0；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)，國(guó)藥準(zhǔn)字H31021400；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)，貨號(hào)P0023A、P0265；Anti-CK14-Antibody：英國(guó) Abcam公司生產(chǎn)，貨號(hào)LS-C210317；辣根過(guò)氧化物酶標(biāo)記山羊抗兔 （100 μL）、 β-actin （100 μL）： 北京中杉金橋生物技術(shù)有限公司生產(chǎn)，貨號(hào)ZB-2301、 TA-09。

1.3 實(shí)驗(yàn)方法

1.Materials and methods

1.1 .Experimental animals

Ninety healthy 12-week-old SPF Wistar male rats，weighing（220±20） g， were provided by Changsha Tianqin Biotechnology Co.， Ltd.The raising condition， transportation and experimental process for the rats were strictly in line with SPF requirements：humidity at（60±10） %，temperature at（25±2） ℃，with good ventilation and the hygienic standard.This experiment was approved by the Animal Ethics Committee of Youjiang Medical University for Nationalities and met the ethical requirements of animal experiments.

1.2.Main reagents

Moist Exposed Burn Ointment（MEBO）： produced by Shantou MEBO Pharmaceutical Co.， Ltd.， China SFDA Approval No.：Z20000004；recombinant bovine basic fibroblast growth factor（rbbFGF） gel： Zhuhai Essex Bio-pharmaceutical Co.， Ltd.， China SFDA Approval No.： S19991021； chloral hydrate： Taixing Haoshen Chemical Trading Co.， Ltd.， CAS registry number： 302-17-0； hydrocortisone acetate injection： Shanghai General Pharmaceutical Co.，Ltd.， China SFDA Approval No.： H31021400； primary antibody diluent， secondary antibody diluent： Shanghai Beyotime， article numbers： P0023A， P0265； Anti-CK14-Antibody： Abcam， UK， article number： LS-C210317； horse radish peroxidase labeled goat antirabbit（100 μL）， β-actin （100 μL）： Beijing Zhongshan Jinqiao Biotechnology Co.， Ltd.， article number： ZB-2301， TA-09.

1.3 .Experiment methods

1.3.1.Grouping and modeling After being fed for one week in SPF animal lab， the rats were divided， according to the random number table， into blank group， acute wound group， chronic wound group，rb-bFGF group and MEBO group，with 18 rats in each group.For rats in the blank group， only back skin preparation was performed；for rats in the acute wound group，after intraperitoneal injection of 7%chloral hydrate （4 mL/kg）， skin preparation at the size of 3.0 cm ×3.0 cm was performed on the back，and then a full-thickness skin defect wound with a diameter of about 1.5 cm and extending fascia was made at the skin preparation site to establish an acute wound；for rats in the chronic wound group， rb-bFGF group， and MEBO group，in addition to what had been done on rats of the acute wound group， intramuscular injection of hydrocortisone acetate （80 mg/kg）was performed to establish chronic wound models［13］.

1.3.2 換藥方法 空白組大鼠備皮處皮膚及急創(chuàng)組與慢創(chuàng)組大鼠創(chuàng)面常規(guī)消毒3次后，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；rb-bFGF組大鼠創(chuàng)面常規(guī)消毒3次后，均勻涂抹 rb-bFGF凝膠 （60 U/cm2），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；MEBO組大鼠創(chuàng)面常規(guī)消毒3次后，均勻涂抹MEBO （0.2 g/cm2），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。

1.3.3 標(biāo)本采集 分別于局部處理第3、7、14天，每組隨機(jī)選取6只大鼠進(jìn)行腹腔注射麻醉后，于備皮處皮膚及去痂后的創(chuàng)面邊緣0.5 cm處切取創(chuàng)面組織并均分為2份，1份置于裝有10%甲醛的EP管中固定并進(jìn)行石蠟包埋；1份置于凍存管中存放于液氮中， -80℃冰箱內(nèi)保存?zhèn)溆谩?/p>

1.3.4 病理檢查 取石蠟包埋組織切片后行Masson染色，并于電子顯微鏡下觀察各組大鼠皮膚或創(chuàng)面組織中炎性細(xì)胞分布、成纖維細(xì)胞分布、新生血管排列及皮膚附屬器形成等組織形態(tài)學(xué)變化情況。

1.3.5 Western blotting技術(shù)檢測(cè)CK14及CK19蛋白表達(dá)水平 取冷凍組織用勻漿器磨碎后，于低溫環(huán)境中加入組織裂解液，提取總蛋白，并用紫外分光光度計(jì)測(cè)定總蛋白濃度，繪制蛋白濃度標(biāo)準(zhǔn)曲線；取蛋白上樣與SDS-PAGE蛋白上樣緩沖液混勻后在100℃下加熱5 min進(jìn)行蛋白變性，并置于-20℃冰箱中保存；SDS-PAGE凝膠制作后加入等體積蛋白上樣于100 V恒壓電泳、250 mA恒流轉(zhuǎn)膜、封閉后，置于4℃環(huán)境下孵育一抗過(guò)夜；一抗孵育后，PBST洗膜3次，然后加入二抗，室溫下孵育60 min；二抗孵育后，PBST洗膜3次，并曝光顯影；最后，通過(guò)Image J圖像分析軟件分析蛋白條帶灰度值。

C羅幼時(shí)生活在一個(gè)貧困的家庭，沒(méi)有足球，沒(méi)有球場(chǎng)，但他卻擁有最寶貴的財(cái)富——夢(mèng)想。所以，即使卑微，但他從不向命運(yùn)低頭。沒(méi)有球場(chǎng)，他就在街上踢球。因?yàn)樗膽褖?mèng)想，所以當(dāng)他被那些比他大的孩子一次次推倒時(shí)，他選擇了一次次站起來(lái)。后來(lái)，在面對(duì)人們的噓聲不斷時(shí)，他這樣告訴自已：“別在乎那些，按照自已的方式踢吧！噓聲不會(huì)改變你的比賽。”因?yàn)樗膽阎鴫?mèng)想。所以他是那樣執(zhí)著地站在球場(chǎng)上，終于，他成功了。是夢(mèng)想讓他四次捧起金球獎(jiǎng)獎(jiǎng)杯。因?yàn)閴?mèng)想之星始終在他的胸膛中閃耀，所以他的每一次崛起都令人驚艷。

1.3.2.Dressing Change The prepared skin in the blank group and the wounds in the acute wound group and chronic wound group were routinely disinfected 3 times，then covered with saline gauze and clean dressing in turn， which were bandaged up with tape， and dressing change was performed twice a day.After routine disinfection of the wound surface for 3 times，the wounds in the rb-bFGF group were managed with rb-bFGF gel（60 U/cm2） evenly， and then covered with saline gauze and clean dressing in turn，followed by tape fixation，and dressing change was performed twice a day.After 3 times of routine disinfection，the wounds in the MEBO group were evenly managed with MEBO （0.2 g/cm2）， and then covered with saline gauze and clean dressing sequentially， followed by tape fixation， and dressing change was also performed twice a day.

1.3.3.Specimen collection On day 3， 7 and 14 of treatment， 6 rats were randomly selected from each of the five groups to undergo intraperitoneal anaesthesia.And then，skin at the prepared site and wound tissues， 0.5 cm away from the wound edges， were cut off and divided into two parts.One part was placed in an EP tube with 10%formaldehyde to fix and then embedded with paraffin.The other part was put in a cryogenic vial，and then placed in liquid nitrogen and stored in a refrigerator at-80℃for later use.

1.3.4.Pathological examination Masson staining was performed after paraffin-embedded tissue slices were taken.Morphological changes， such as distribution of inflammatory cells and fibroblasts， the arrangement of new blood vessels and skin appendage formation in the skin or wound tissues of each group were observed under electron microscope.

1.3.5.Detection of CK14 and CK19 expression level with the Western blotting Frozen tissues were taken out and ground with a homogenizer， tissue lysate was added in a low temperature environment，total protein was extracted，and the protein concentration of the specimen was measured with ultraviolet spectrophotometer to draw standard curve of protein concentration.Protein loading buffer and SDS-PAGE protein loading buffer were mixed evenly and heated at 100℃for 5 min to denature the protein，and then were stored in the refrigerator at-20℃.After SDS-PAGE gel was made，equal volume of protein loading buffer was added，electrophoresis was conducted at 100 V constant pressure.The membrane was transferred at 250 mA constant current，sealed and placed at 4 ℃ for overnight primary antibody incubation.After the primary antibody incubation，the membrane was washed 3 times with PBST，and then the secondary antibody was added and incubated at room temperature for 60 min；after the secondary antibody incubation，the membrane was washed with PBST 3 times and then developed； finally， the gray value of protein bands was analyzed with Image J.

1.3.6 RT-PCR技術(shù)檢測(cè)CK14及CK19 mRNA轉(zhuǎn)錄水平 取備用組織樣本采用Trizol法提取RNA，并分別檢測(cè)其在260 nm及280 nm波長(zhǎng)的吸光度值，A260/A280值在1.8～2.0之間視為RNA提取合格；然后，嚴(yán)格按照TIANScript RT KIT試劑盒說(shuō)明書(shū)逆轉(zhuǎn)錄合成cDNA，并采用Real Time PCR反應(yīng)體系，以cDNA為模板進(jìn)行 PCR擴(kuò)增。引物序列：CK14上游為 5′-GCAGCCGTCAGTTCCTCCT-3′， 下 游 為 5′-CCCCACCATAGCCACTTCCAA-3′； CK19 上游為5′-GGTGGAAGTTTTAGTGGGGC-3′， 下游為 5′-CAAGTAGGAGGCGAGGCGAT-3′； β-actin 上 游為 5′-GGCACCACACCTTCTAC-3′， 下 游 為 5′-CTGGGTCATCTTTTCAC-3′。 采用 2-△△Ct法計(jì)算目的基因的相對(duì)表達(dá)量。

1.4 統(tǒng)計(jì)學(xué)處理

應(yīng)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，計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差（x ±s）表示，多個(gè)樣本間比較采用單因素方差分析 （方差齊采用LSD法，方差不齊采用Games-Howell法），均以P＜0.05為差異具有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 Masson染色結(jié)果分析

空白組大鼠各時(shí)間點(diǎn)皮膚組織細(xì)胞分布情況無(wú)明顯差異。局部處理第3天，急創(chuàng)組、慢創(chuàng)組、rb-bFGF組及MEBO組大鼠創(chuàng)面組織中均可見(jiàn)組織明顯水腫及炎性細(xì)胞廣泛浸潤(rùn)，且僅可見(jiàn)少量新生血管，4組間無(wú)明顯差異。局部處理第7天，急創(chuàng)組、rb-bFGF組及MEBO組大鼠創(chuàng)面組織形態(tài)學(xué)差異不明顯，均可見(jiàn)大量新生血管及部分不完整毛囊結(jié)構(gòu)，炎性細(xì)胞均明顯減少，而慢創(chuàng)組大鼠創(chuàng)面組織中新生血管相對(duì)較少，組織水腫及炎性細(xì)胞浸潤(rùn)情況無(wú)明顯改善，且未見(jiàn)皮膚附屬器形成。局部處理第14天，急創(chuàng)組、rb-bFGF組及MEBO組大鼠創(chuàng)面組織中均可見(jiàn)排列整齊的新生血管及毛囊、皮脂腺等皮膚附屬器，組織形態(tài)接近正常，而慢創(chuàng)組大鼠創(chuàng)面組織中仍可見(jiàn)少量炎性細(xì)胞浸潤(rùn)，部分區(qū)域可見(jiàn)大量成纖維細(xì)胞，逐漸形成瘢痕組織形態(tài) （圖1）。

1.3.6.Detection of CK14 and CK19 mRNA transcription levels with RT-PCR The RNA was extracted with Trizol method from the spare specimen，and its absorbance values at respective 260 nm and 280 nm wavelengths were measured.The RNA extraction was considered as qualified if the ratio of A260 to A280 was between 1.8 and 2.0.cDNA was synthesized via reverse transcription using total RNA as template strictly according to the instructions of TIANScript RT KIT.Real Time PCR reaction system was used for PCR amplification with cDNA as template.Primer sequence：upstream primer sequence of CK14 was 5′-GCAGCCGTCAGTTCCTCCT-3′， downstream primer sequence of CK14 was 5′-CCCCACCATAGCCACTTCCAA-3′； upstream primer sequence of CK19 was 5′-GGTGGAAGTTTTAGTGGGGC-3′，downstream primer sequence of CK19 was 5′-CAAGTAGGAGGCGAG GCGAT-3′； upstream primer sequence ofβ-actin was 5′-GGCACCA CACCTTCTAC-3′， downstream primer sequence of β-actin was 5′-CTGGGTCATCTTTTCAC-3′.Relative quantitative analysis was carried out with 2-△△CTmethod.

1.4 .Statistical analysis

SPSS22.0 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 multiple specimens， one-way ANOVA was used： LSD method for equal variances，and Games-Howell method for unequal variances.P ＜0.05 was considered statistically significant.

2.Results

2.1 .Analysis of Masson staining results

No significant difference was observed in the distribution of skin tissue cells at various time points in the blank group.On day 3 of treatment， obvious edema， extensive infiltration of inflammatory cells and a small number of new blood vessels were seen in the wound tissues of the acute wound group， chronic wound group， rb-bFGF group and MEBO group，and no significant difference was observed among the four groups.On day 7 of treatment，no significant difference was observed in the histological morphology of the wounds among the acute wound group， rb-bFGF group， and MEBO group， there were a large number of new blood vessels and some incomplete hair follicle structures，and significantly reduced inflammatory cells in all the three groups.In contrast， in the wounds of the chronic wound group， new blood vessels were relatively few，no marked improvement was seen on tissue edema and inflammatory cell infiltration，and skin appendages were not formed yet.On day 14 of treatment，neatly arranged new blood vessels， skin appendages including hair follicles and sebaceous glands，and basically normal tissue morphology were observable in the wound tissues of the acute wound group，rb-bFGF group and MEBOgroup.However， in the wound tissues of the chronic wound group， there were still a little inflammatory cell infiltration，and a large number of fibroblasts can be seen in some parts， gradually forming scar tissues （Fig.1）.

圖1 各時(shí)間點(diǎn)5組大鼠創(chuàng)面組織Masson染色圖 （×400）Fig.1 Masson staining results of wound tissues from the five groups at different time points（×400）

2.2 CK14、CK19蛋白表達(dá)及其mRNA轉(zhuǎn)錄水平對(duì)比

局部處理第3、7、14天，急創(chuàng)組、慢創(chuàng)組、rb-bFGF組及MEBO組大鼠創(chuàng)面組織中CK14蛋白表達(dá)及其mRNA轉(zhuǎn)錄水平均呈逐漸升高的趨勢(shì)，且局部處理第3天均低于空白組 （P均＜0.05），而4組間無(wú)明顯差異（P均＞0.05）；局部處理第7天4組仍低于空白組 （P均＜0.05），而急創(chuàng)組、rb-bFGF組及MEBO組均高于慢創(chuàng)組 （P均＜0.05），但3組間無(wú)明顯差異 （P均＞0.05）；局部處理第14天急創(chuàng)組、rb-bFGF組及MEBO組均升高至空白組水平 （P均＞0.05），而慢創(chuàng)組仍低于其它各組 （P均＜0.05），詳見(jiàn)表1、圖2。

2.2.Comparison of expression levels of CK14 and CK19 and their mRNA transcription levels

On the day 3， 7 and 14 of treatment， the expression level of CK14 and its mRNA transcription level in the wound tissues of the acute wound group， chronic wound group， rb-bFGF group and MEBO group showed a trend of gradual increase，and their levels in the above mentioned four groups on day 3 were lower than that in the blank group（allP＜0.05），while no significant difference was observed among the four groups（allP＞0.05）.On day 7 of treatment， the levels of CK14 expression and its mRNA transcription level in the four groups were still lower than that in the blank group（allP＜0.05） and the levels in the acute wound group，rb-bFGF group and MEBO group were all higher than that in the chronic wound group （allP＜0.05），but no significant difference was observed among the three groups （allP＞0.05）.On day 14 of treatment， the levels in the acute wound group，rb-bFGF group and MEBO group increased to the same level as that in the blank group（allP＞0.05）while the chronic wound group still had lower levels than that in the other groups（allP＜0.05）.See Table 1 and Fig.2.

On day 3， 7 and 14 of treatment， the expression level of CK19 and its mRNA transcription level in the wound tissues of the acute wound group， chronic wound group， rb-bFGF group and MEBO group showed a trend of increase followed by decrease； on day 3 and 7 of treatment， the levels of CK19 expression and its mRNA transcription in the four groups were all higher than that in the blank group（allP＜0.05），and the levels in the acute wound group， rb-bFGFgroup and MEBOgroup， were all higher than that in the chronic wound group（allP＜0.05），but no significant difference was observed among the three groups（allP＞0.05）.on day 14 of treatment， in the acute wound group， rb-bFGF group and MEBO group，the levels decreased to the same level as that in the blank group（allP＞0.05） while the chronic wound group still had higher levels than that in the other groups（allP＜0.05）.See Table 2 and Fig.3.

局部處理第3、7、14天，急創(chuàng)組、慢創(chuàng)組、rb-bFGF組及MEBO組大鼠創(chuàng)面組織中CK19蛋白表達(dá)及其mRNA轉(zhuǎn)錄水平均呈先升高后降低的趨勢(shì)，且局部處理第3、7天4組均高于空白組 （P均＜0.05），而急創(chuàng)組、rb-bFGF組及MEBO組又均高于慢創(chuàng)組 （P均＜0.05），但3組間無(wú)明顯差異 （P均 ＞0.05）；局部處理第14天急創(chuàng)組、rb-bFGF組及MEBO組均降低至空白組水平 （P均＞0.05），而慢創(chuàng)組仍高于其它各組 （P均＜0.05），詳見(jiàn)表2、圖3。

表1 5組大鼠皮膚或創(chuàng)面組織中CK14蛋白表達(dá)及其mRNA轉(zhuǎn)錄水平對(duì)比 （±s）Table 1 Comparison of expression level of CK14 and its mRNA transcription level in skin or wound tissues of the five groups（±s）

表1 5組大鼠皮膚或創(chuàng)面組織中CK14蛋白表達(dá)及其mRNA轉(zhuǎn)錄水平對(duì)比 （±s）Table 1 Comparison of expression level of CK14 and its mRNA transcription level in skin or wound tissues of the five groups（±s）

注：各時(shí)間點(diǎn)CK14蛋白表達(dá)及其mRNA轉(zhuǎn)錄水平組間兩兩對(duì)比，與空白組對(duì)比，a P＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與急創(chuàng)組對(duì)比，b P＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與慢創(chuàng)組對(duì)比，c P＜0.05，差異具有統(tǒng)計(jì)學(xué)意義Note：Pairwise comparisons of expression level of CK14 and its mRNA transcription level at different time points were conducted among the five groups.Comparison with the blank group showed statistically significant difference（a P＜0.05）；comparison with the acute wound group showed statistically significant difference（b P＜0.05）；comparison with the chronic wound group showed statistically significant difference（c P＜0.05）

組別Group鼠數(shù) （只）Number of rats（n）CK14蛋白CK14 protein CK14 mRNA第3天Day 3第7天Day 7第14天Day 14第3天Day 3第7天Day 7第14天Day 14空白組Blank group 6 1.30±0.08 1.24±0.13 1.22±0.04 1.12±0.12 1.10±0.06 1.16±0.08急創(chuàng)組Acute wound group 6 0.73±0.05a 1.01±0.05a 1.28±0.06 0.29±0.06a 0.69±0.05a 1.14±0.09慢創(chuàng)組Chronic wound group 6 0.68±0.10a 0.84±0.03ab 1.02±0.04ab 0.31±0.05a 0.46±0.03ab 0.68±0.07ab rb-bFGF組rb-bFGF group 6 0.75±0.03a 0.98±0.04ac 1.23±0.11c 0.30±0.06a 0.68±0.05ac 1.09±0.10c MEBO組MEBO group 6 0.76±0.07a 0.99±0.07ac 1.27±0.11c 0.29±0.06a 0.64±0.04ac 1.15±0.09c F值F value 80.080 23.250 10.870 146.600 149.100 33.700 P值P value 0.000 0.000 0.000 0.000 0.000 0.000

表2 5組大鼠皮膚或創(chuàng)面組織中CK19蛋白表達(dá)及其mRNA轉(zhuǎn)錄水平對(duì)比 （±s）Table 2 Comparison of expression level of CK19 and its mRNA transcription level in skin or wound tissues of the five groups（±s）

表2 5組大鼠皮膚或創(chuàng)面組織中CK19蛋白表達(dá)及其mRNA轉(zhuǎn)錄水平對(duì)比 （±s）Table 2 Comparison of expression level of CK19 and its mRNA transcription level in skin or wound tissues of the five groups（±s）

組別Group鼠數(shù) （只）Number of rats（n）CK19蛋白CK19 protein CK19 mRNA第3天Day 3第7天Day 7第14天Day 14第3天Day 3第7天Day 7第14天Day 14空白組Blank group 6 0.262±0.024 0.257±0.047 0.263±0.024 1 0.973±0.072 1.060±0.085急創(chuàng)組Acute wound group 6 0.536±0.019a 0.674±0.074a 0.254±0.041 2.434±0.171a 3.572±0.221a 1.195±0.138慢創(chuàng)組Chronic wound group 6 0.352±0.042ab 0.456±0.044ab 0.349±0.021ab 1.819±0.165ab 2.563±0.113ab 1.565±0.249ab rb-bFGF組rb-bFGF group 6 0.490±0.065ac 0.663±0.034ac 0.258±0.018c 2.347±0.200ac 3.527±0.281ac 1.133±0.098c MEBO組MEBO group 6 0.506±0.067ac 0.650±0.058ac 0.260±0.028c 2.363±0.170ac 3.600±0.277ac 1.124±0.083c F值F value 35.926 69.719 13.012 88.267 173.331 11.555 P值P value 0.000 0.000 0.000 0.000 0.000 0.000

注：各時(shí)間點(diǎn)CK19蛋白表達(dá)及其mRNA轉(zhuǎn)錄水平組間兩兩對(duì)比，與空白組對(duì)比，aP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與急創(chuàng)組對(duì)比，bP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義；與慢創(chuàng)組對(duì)比，cP＜0.05，差異具有統(tǒng)計(jì)學(xué)意義

Note：Pairwise comparisons of expression level of CK19 and its mRNA transcription level at different time points were conducted among the five groups.Comparison with the blank group showed statistically significant difference（aP＜0.05）；comparison with the acute wound group showed statistically significant difference（bP＜0.05）；comparison with the chronic wound group showed statistically significant difference（cP＜0.05）

圖2 5組大鼠皮膚或創(chuàng)面組織中CK14蛋白表達(dá)條帶圖；圖3 5組大鼠皮膚或創(chuàng)面組織中CK19蛋白表達(dá)條帶圖Fig.2 Histogram of expression level of CK14 in skin or wound tissues of the five groups； Fig.3 Histogram of expression level of CK19 in skin or wound tissues of the five groups

3 討論

慢性難愈合創(chuàng)面是機(jī)體正常微結(jié)構(gòu)被破壞后正常修復(fù)程序停滯而導(dǎo)致的創(chuàng)面經(jīng)久不愈［14］。近年來(lái)部分研究證實(shí)，表皮干細(xì)胞具有強(qiáng)大的增殖及多向分化能力［15-16］，能夠根據(jù)機(jī)體需要進(jìn)行增殖分化，以滿足表皮自我更新的需求，在創(chuàng)面修復(fù)過(guò)程中發(fā)揮著關(guān)鍵作用，且CK在表皮干細(xì)胞參與創(chuàng)面修復(fù)過(guò)程中的作用顯著［17］。 如舒清峰等［18］的研究顯示， MEBT/MEBO可通過(guò)調(diào)節(jié)慢性難愈合創(chuàng)面組織中CK10的表達(dá)水平而促進(jìn)創(chuàng)面的再生修復(fù)。鑒于此，筆者為進(jìn)一步探討MEBT/MEBO促進(jìn)慢性難愈合創(chuàng)面修復(fù)的部分分子生物學(xué)作用機(jī)制，于本研究中對(duì)比分析了MEBT/MEBO對(duì)大鼠慢性難愈合創(chuàng)面組織中CK14和CK19表達(dá)水平的影響。

3.Discussion

Chronic refractory wounds are wounds resulting from the stagnation of normal repair procedures after body’s normal microstructure is destroyed［14］.In recent years， some studies have confirmed that epidermal stem cells have strong ability to proliferate and differentiate in multi-directions［15-16］to achieve the self-renewal of epidermis based on the body’s needs， playing a key role in wound repair.Further，CK plays a significant role for epidermal stem cells to facilitate wound repair［17］.Studies by Shu Qingfeng， et al.［18］showed that MEBT/MEBO can promote the regenerative repair of wounds by regulating the expression level of CK10 in chronic refractory wound tissues.In order to further explore molecular biological mechanism of MEBT/MEBO in promoting healing of chronic refractory wounds，this study compared and analyzed the influence of MEBT/MEBO on the expression levels of CK14 and CK19 in chronic refractory wound tissues of rats.

相關(guān)研究顯示，CK14表達(dá)于基底層細(xì)胞并隨著基底層干細(xì)胞不斷增殖分化為基底上層細(xì)胞，且其表達(dá)水平也隨之不斷升高；CK19在創(chuàng)面修復(fù)過(guò)程中與瘢痕組織的形成密切相關(guān)［19-20］。本研究結(jié)果顯示，在正常皮膚組織中CK14及CK19的表達(dá)水平較為穩(wěn)定，而在急性創(chuàng)面組織中CK14的表達(dá)水平呈現(xiàn)出逐漸升高并趨于正常的趨勢(shì)，CK19的表達(dá)水平呈現(xiàn)出先升高后降低至正常的趨勢(shì)?？梢?jiàn)，CK14及CK19均參與了創(chuàng)面的修復(fù)。另外，本研究結(jié)果顯示，創(chuàng)面修復(fù)過(guò)程中急創(chuàng)組、rb-bFGF組及MEBO組大鼠創(chuàng)面組織中CK14表達(dá)水平始終高于慢創(chuàng)組，而CK19表達(dá)水平則是在創(chuàng)傷修復(fù)早期高于慢創(chuàng)組而晚期又低于慢創(chuàng)組。分析其原因可能與CK14參與干細(xì)胞的增殖分化而CK19參與瘢痕組織的形成，且慢創(chuàng)組創(chuàng)面更易形成瘢痕組織有關(guān)［21］。此外，CK14及 CK19在各組大鼠創(chuàng)面組織中的變化規(guī)律及創(chuàng)面組織的形態(tài)學(xué)改變也足以證明，MEBT/MEBO對(duì)慢性難愈合創(chuàng)面具有促愈合作用［22］，對(duì)創(chuàng)面組織中CK14、CK19具有調(diào)控作用，且其作用效果與已證實(shí)的對(duì)慢性難愈合創(chuàng)面具有促愈合作用的rb-bFGF凝膠相似。

綜上所述，MEBT/MEBO能夠促進(jìn)慢性難愈合創(chuàng)面愈合，且調(diào)控創(chuàng)面組織中CK14及CK19的表達(dá)水平可能是其部分作用機(jī)制。值得注意的是，已證實(shí)在創(chuàng)面修復(fù)過(guò)程中起到重要作用的PTEN/AKT信號(hào)通路是否能夠影響CK14及CK19的表達(dá)，以及角蛋白家族其他成員是否也參與了創(chuàng)面的修復(fù)過(guò)程仍需深入研究探討。

Related studies have shown that CK14 is expressed in cells at the basal layer.As stem cells at the basal layer differentiate into superbasal cells， the expression level of CK14 increases； CK19 is closely related to the formation of scar tissues during the wound repair［19-20］.The results of this study showed that the expression levels of CK14 and CK19 in normal skin tissues were relatively stable，whereas the expression level of CK14 in acute wound tissues showed a trend of gradual increase and then returned to normal level while the expression level of CK19 showed a trend of increase first followed by decrease and then gradually returned to normal，too.It can be concluded that CK14 and CK19 are both involved in wound repair.Further， the results of this study showed that during the wound repair，the expression levels of CK14 in wound tissues of the acute wound group，rb-bFGF group and MEBO group were always higher than that in the chronic wound group，whereas the expression levels of CK19 in the three groups were higher than that in the chronic wound group at the early stage of wound repair and lower than that in the chronic wound group at the later stage.The reason may lie in that CK14 is involved in the proliferation and differentiation of stem cells，while CK19 involves in the formation of scar tissue，and scar tissues are more likely to form on wounds in the chronic wound group［21］.In addition，the changes of CK14 and CK19 in the wound tissues and the morphological changes of the wound tissues in each group suffice to confirm that MEBT/MEBO can promote the healing of chronic refractory wounds［22］， and regulate the expression levels of CK14 and CK19 in wound tissue.Its effect is similar to that of rb-bFGF gel which has proven healing effect on chronic refractory wounds.

In summary，MEBT/MEBO can promote the healing of chronic refractory wounds，and its ability to regulate the expression levels of CK14 and CK19 in wound tissues may be part of its mechanism of action.It is worth noting that whether the PTEN/AKTsignaling pathway that has been proven playing an important role in wound repair can affect the expression levels of CK14 and CK19，and whether other members of the keratin family also participate in the wound repair require further research and discussion.

（收稿日期：2020-03-08）