• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

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

    2019-04-09 02:05:30舒清峰陳端凱唐乾利單云龍岑小寧卓臣義
    中國燒傷創(chuàng)瘍雜志 2019年2期
    關(guān)鍵詞:表皮創(chuàng)面皮膚

    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)

    猜你喜歡
    表皮創(chuàng)面皮膚
    第二層皮膚
    皮膚“出油”或許就能減肥
    建筑表皮中超薄基材的應用分析
    高頻超聲在皮膚惡性腫瘤中的應用
    rn-bFGH(蓋扶)對創(chuàng)面修復的影響
    人也會“蛻皮”,周期為一個月
    創(chuàng)面治療新技術(shù)的研發(fā)與轉(zhuǎn)化應用系列叢書
    表皮生長因子對HaCaT細胞miR-21/PCD4的表達研究
    我愛洗澡,皮膚好好
    城市綜合體表皮到表皮建筑的參數(shù)化設計
    国产色视频综合| 国产人伦9x9x在线观看| 高清欧美精品videossex| 晚上一个人看的免费电影| 久热这里只有精品99| 国产av精品麻豆| 免费人妻精品一区二区三区视频| 国产男女内射视频| 无限看片的www在线观看| 少妇粗大呻吟视频| 日韩,欧美,国产一区二区三区| 99精国产麻豆久久婷婷| 亚洲国产av新网站| 国产免费视频播放在线视频| 亚洲国产欧美日韩在线播放| 精品国产国语对白av| 日韩 亚洲 欧美在线| 亚洲av在线观看美女高潮| 人妻一区二区av| 国产精品 国内视频| 又粗又硬又长又爽又黄的视频| 午夜日韩欧美国产| 久久精品国产a三级三级三级| h视频一区二区三区| 人体艺术视频欧美日本| 十分钟在线观看高清视频www| 黑人欧美特级aaaaaa片| 国产视频一区二区在线看| 久久精品熟女亚洲av麻豆精品| 九草在线视频观看| 欧美亚洲 丝袜 人妻 在线| 熟女少妇亚洲综合色aaa.| 黄色毛片三级朝国网站| √禁漫天堂资源中文www| 亚洲人成网站在线观看播放| 91成人精品电影| 欧美日韩国产mv在线观看视频| 免费一级毛片在线播放高清视频 | 日本色播在线视频| 天天添夜夜摸| 国产熟女欧美一区二区| 色94色欧美一区二区| 欧美亚洲 丝袜 人妻 在线| 大片电影免费在线观看免费| 高清不卡的av网站| 国产精品国产三级国产专区5o| 涩涩av久久男人的天堂| 一本一本久久a久久精品综合妖精| 中文字幕人妻丝袜一区二区| 精品福利永久在线观看| 欧美日韩福利视频一区二区| 日韩大片免费观看网站| 欧美大码av| 老司机影院成人| 国产爽快片一区二区三区| a级片在线免费高清观看视频| 欧美xxⅹ黑人| 性少妇av在线| 亚洲欧洲精品一区二区精品久久久| 亚洲色图综合在线观看| 午夜福利视频精品| 国产无遮挡羞羞视频在线观看| 国产精品麻豆人妻色哟哟久久| 美女视频免费永久观看网站| 国产高清视频在线播放一区 | 中文字幕制服av| 亚洲国产精品999| 叶爱在线成人免费视频播放| 麻豆av在线久日| a级毛片在线看网站| 1024视频免费在线观看| 精品国产一区二区久久| 一边亲一边摸免费视频| 欧美另类一区| 国产成人a∨麻豆精品| 亚洲欧美激情在线| 一级黄片播放器| 999精品在线视频| 乱人伦中国视频| 无遮挡黄片免费观看| avwww免费| 另类亚洲欧美激情| 丁香六月天网| 热99久久久久精品小说推荐| 女人久久www免费人成看片| 最近最新中文字幕大全免费视频 | 日韩制服丝袜自拍偷拍| 亚洲精品国产av蜜桃| 日本欧美视频一区| 亚洲av电影在线进入| 免费人妻精品一区二区三区视频| 国产精品三级大全| av国产精品久久久久影院| 国产成人免费观看mmmm| 国产精品一二三区在线看| 老司机在亚洲福利影院| 免费在线观看视频国产中文字幕亚洲 | 成人亚洲精品一区在线观看| 国产高清不卡午夜福利| 香蕉丝袜av| 亚洲欧美精品自产自拍| 日韩一本色道免费dvd| 久久九九热精品免费| 久久久精品国产亚洲av高清涩受| 中文字幕色久视频| 亚洲国产精品一区三区| 999精品在线视频| 黄色一级大片看看| 日韩大片免费观看网站| 又紧又爽又黄一区二区| 大香蕉久久网| 国产成人欧美在线观看 | 亚洲国产欧美一区二区综合| 亚洲欧美日韩高清在线视频 | 少妇 在线观看| 久久久欧美国产精品| 又大又爽又粗| 国产免费视频播放在线视频| 观看av在线不卡| 国产片内射在线| 欧美亚洲日本最大视频资源| 色网站视频免费| 一本色道久久久久久精品综合| 一本综合久久免费| 久久精品aⅴ一区二区三区四区| 免费高清在线观看视频在线观看| 精品卡一卡二卡四卡免费| 国产亚洲精品第一综合不卡| 老鸭窝网址在线观看| 国产精品 欧美亚洲| 一二三四在线观看免费中文在| 久久毛片免费看一区二区三区| 国产一区二区三区av在线| 色视频在线一区二区三区| 每晚都被弄得嗷嗷叫到高潮| 夫妻性生交免费视频一级片| www.精华液| 黄网站色视频无遮挡免费观看| 人人妻人人爽人人添夜夜欢视频| 亚洲伊人久久精品综合| 免费日韩欧美在线观看| 欧美黄色片欧美黄色片| 日本vs欧美在线观看视频| 日韩免费高清中文字幕av| 在线观看一区二区三区激情| 嫁个100分男人电影在线观看 | 黑丝袜美女国产一区| 在线av久久热| 亚洲熟女毛片儿| www.999成人在线观看| 视频区图区小说| 亚洲国产最新在线播放| 80岁老熟妇乱子伦牲交| 国产成人一区二区三区免费视频网站 | 国产在线一区二区三区精| 久久中文字幕一级| 黑人猛操日本美女一级片| 婷婷成人精品国产| 男女免费视频国产| 色视频在线一区二区三区| av线在线观看网站| 亚洲av欧美aⅴ国产| 国产精品一区二区在线不卡| av线在线观看网站| 国产精品国产三级专区第一集| 我的亚洲天堂| 少妇精品久久久久久久| 嫁个100分男人电影在线观看 | 久久精品国产亚洲av高清一级| 午夜久久久在线观看| 午夜两性在线视频| 中文字幕另类日韩欧美亚洲嫩草| 黑人猛操日本美女一级片| 国产高清视频在线播放一区 | 狂野欧美激情性xxxx| 欧美精品亚洲一区二区| 成人免费观看视频高清| www.熟女人妻精品国产| 老熟女久久久| 嫁个100分男人电影在线观看 | 亚洲黑人精品在线| 丁香六月欧美| 欧美变态另类bdsm刘玥| 99热国产这里只有精品6| 欧美人与性动交α欧美软件| 国产欧美日韩精品亚洲av| 色婷婷av一区二区三区视频| 国产精品久久久久久精品古装| 最近中文字幕2019免费版| 欧美日韩亚洲国产一区二区在线观看 | avwww免费| 国产一区二区三区av在线| 欧美日韩国产mv在线观看视频| 午夜福利乱码中文字幕| 老司机午夜十八禁免费视频| 欧美成人午夜精品| 性色av乱码一区二区三区2| 男女高潮啪啪啪动态图| 国产亚洲精品第一综合不卡| 日本av免费视频播放| 热re99久久国产66热| avwww免费| 国产欧美日韩综合在线一区二区| 欧美国产精品va在线观看不卡| 国产欧美日韩一区二区三 | 成年女人毛片免费观看观看9 | 国产欧美日韩综合在线一区二区| 国产一区二区在线观看av| 一级毛片我不卡| av在线app专区| 精品视频人人做人人爽| netflix在线观看网站| 女性被躁到高潮视频| 亚洲精品中文字幕在线视频| 久久天堂一区二区三区四区| 色精品久久人妻99蜜桃| 日韩av免费高清视频| 一本久久精品| 悠悠久久av| 国产日韩欧美亚洲二区| 嫁个100分男人电影在线观看 | 99国产精品免费福利视频| 久久久久久亚洲精品国产蜜桃av| 性色av一级| 色网站视频免费| 国产人伦9x9x在线观看| 国产又爽黄色视频| 又大又黄又爽视频免费| 国产亚洲av高清不卡| 久久精品人人爽人人爽视色| 亚洲欧洲精品一区二区精品久久久| www.自偷自拍.com| 亚洲精品日韩在线中文字幕| 一区二区三区四区激情视频| 久久久久久人人人人人| 人人妻人人澡人人爽人人夜夜| 精品久久久久久电影网| 午夜福利影视在线免费观看| 日本91视频免费播放| 国产免费福利视频在线观看| 久久青草综合色| 亚洲欧美色中文字幕在线| 超色免费av| 欧美乱码精品一区二区三区| 亚洲国产日韩一区二区| 中文字幕最新亚洲高清| 国产日韩欧美在线精品| 国产极品粉嫩免费观看在线| 99热全是精品| 久久99一区二区三区| xxxhd国产人妻xxx| 亚洲精品一区蜜桃| 51午夜福利影视在线观看| 高清av免费在线| 好男人电影高清在线观看| 精品一区二区三卡| 波野结衣二区三区在线| 亚洲国产中文字幕在线视频| 91成人精品电影| 国产精品偷伦视频观看了| 王馨瑶露胸无遮挡在线观看| 国产精品二区激情视频| 久久久久精品国产欧美久久久 | 日韩 欧美 亚洲 中文字幕| 欧美日韩视频精品一区| 九色亚洲精品在线播放| 人人妻人人澡人人爽人人夜夜| 国产人伦9x9x在线观看| tube8黄色片| 成年人黄色毛片网站| 色婷婷久久久亚洲欧美| 美女脱内裤让男人舔精品视频| 亚洲国产精品一区三区| 午夜福利乱码中文字幕| 777米奇影视久久| 亚洲专区中文字幕在线| 欧美精品一区二区免费开放| 一级黄色大片毛片| 人成视频在线观看免费观看| 高清欧美精品videossex| 日本黄色日本黄色录像| 亚洲欧洲精品一区二区精品久久久| 婷婷成人精品国产| 美女脱内裤让男人舔精品视频| 天堂8中文在线网| 国产欧美日韩一区二区三区在线| 国产熟女欧美一区二区| 亚洲成人手机| 九草在线视频观看| 国产成人av激情在线播放| 日本欧美视频一区| 首页视频小说图片口味搜索 | 大码成人一级视频| av天堂在线播放| 国产精品偷伦视频观看了| 成年人午夜在线观看视频| 亚洲av日韩精品久久久久久密 | 在线观看一区二区三区激情| av片东京热男人的天堂| 黑人巨大精品欧美一区二区蜜桃| 男人操女人黄网站| 欧美成人午夜精品| 1024视频免费在线观看| 97精品久久久久久久久久精品| 人体艺术视频欧美日本| 王馨瑶露胸无遮挡在线观看| 成人免费观看视频高清| 日日夜夜操网爽| 亚洲精品一二三| 精品久久蜜臀av无| 波多野结衣av一区二区av| 操出白浆在线播放| 少妇粗大呻吟视频| 亚洲天堂av无毛| 狂野欧美激情性bbbbbb| 男的添女的下面高潮视频| 王馨瑶露胸无遮挡在线观看| 欧美激情极品国产一区二区三区| 久久精品久久精品一区二区三区| 日韩,欧美,国产一区二区三区| 国产人伦9x9x在线观看| 另类亚洲欧美激情| 丰满人妻熟妇乱又伦精品不卡| 国产亚洲av片在线观看秒播厂| 最黄视频免费看| 一级黄片播放器| 大型av网站在线播放| 女人爽到高潮嗷嗷叫在线视频| 捣出白浆h1v1| 亚洲情色 制服丝袜| 国产淫语在线视频| 精品久久蜜臀av无| 国产精品麻豆人妻色哟哟久久| 日本wwww免费看| 亚洲av综合色区一区| 日韩av不卡免费在线播放| 老司机影院成人| 国产精品av久久久久免费| 久久精品久久精品一区二区三区| 国产欧美日韩精品亚洲av| 久久午夜综合久久蜜桃| 91麻豆精品激情在线观看国产 | a 毛片基地| 欧美日韩亚洲综合一区二区三区_| 国产免费视频播放在线视频| 久久久久精品人妻al黑| 久久热在线av| 一本一本久久a久久精品综合妖精| 欧美另类一区| 国产av一区二区精品久久| 亚洲精品乱久久久久久| 国产亚洲精品第一综合不卡| 国产97色在线日韩免费| 国产在线观看jvid| 首页视频小说图片口味搜索 | 成人影院久久| 国产一卡二卡三卡精品| 久久久久精品国产欧美久久久 | 国产欧美亚洲国产| 午夜免费鲁丝| 看免费成人av毛片| 免费在线观看日本一区| 久久精品国产亚洲av涩爱| 丰满迷人的少妇在线观看| 人妻 亚洲 视频| 国产一区亚洲一区在线观看| av在线老鸭窝| 中文精品一卡2卡3卡4更新| 男女午夜视频在线观看| 两人在一起打扑克的视频| 成年av动漫网址| 亚洲色图 男人天堂 中文字幕| 亚洲欧美日韩另类电影网站| 十八禁人妻一区二区| 97人妻天天添夜夜摸| 永久免费av网站大全| 中文字幕精品免费在线观看视频| 日本a在线网址| 一本久久精品| 国产成人精品久久久久久| 高清欧美精品videossex| 丝袜美腿诱惑在线| 国产在视频线精品| 欧美xxⅹ黑人| 下体分泌物呈黄色| 亚洲精品自拍成人| 建设人人有责人人尽责人人享有的| 亚洲精品日韩在线中文字幕| 建设人人有责人人尽责人人享有的| 在线观看免费日韩欧美大片| 久久久精品区二区三区| 亚洲熟女精品中文字幕| 久久久久久久国产电影| 人人妻人人澡人人看| 三上悠亚av全集在线观看| 波多野结衣一区麻豆| 天堂俺去俺来也www色官网| 亚洲精品日韩在线中文字幕| 久久久精品区二区三区| 婷婷色综合大香蕉| 午夜精品国产一区二区电影| 精品国产一区二区久久| 亚洲成人免费av在线播放| 七月丁香在线播放| 波多野结衣av一区二区av| 国产成人欧美在线观看 | 久久影院123| 国产精品国产av在线观看| 成人影院久久| 久久精品国产亚洲av涩爱| 看免费av毛片| 国产成人精品在线电影| 精品卡一卡二卡四卡免费| 大陆偷拍与自拍| 91九色精品人成在线观看| 亚洲九九香蕉| 超碰成人久久| 欧美精品人与动牲交sv欧美| 欧美成人午夜精品| 波多野结衣一区麻豆| 欧美日韩亚洲高清精品| bbb黄色大片| 久久精品国产a三级三级三级| 性少妇av在线| 国产免费又黄又爽又色| 男女高潮啪啪啪动态图| 欧美人与善性xxx| 欧美久久黑人一区二区| 黄色一级大片看看| 欧美乱码精品一区二区三区| 最近最新中文字幕大全免费视频 | 欧美精品高潮呻吟av久久| 成人国语在线视频| 巨乳人妻的诱惑在线观看| 婷婷丁香在线五月| 热99久久久久精品小说推荐| 亚洲欧美日韩高清在线视频 | 亚洲成av片中文字幕在线观看| 亚洲精品久久午夜乱码| 女人久久www免费人成看片| 精品国产国语对白av| 中文字幕人妻丝袜制服| 欧美精品亚洲一区二区| 久久精品亚洲av国产电影网| 久久久久久久国产电影| 操美女的视频在线观看| 国产在线观看jvid| 男女国产视频网站| 一二三四社区在线视频社区8| 夜夜骑夜夜射夜夜干| 色网站视频免费| 热99国产精品久久久久久7| 久久久久久久久久久久大奶| 精品国产超薄肉色丝袜足j| 日本午夜av视频| 18禁观看日本| 少妇的丰满在线观看| 亚洲国产毛片av蜜桃av| 激情五月婷婷亚洲| 亚洲精品美女久久av网站| 久久精品国产综合久久久| 久久中文字幕一级| 欧美精品亚洲一区二区| 只有这里有精品99| 久久久久久人人人人人| 最新在线观看一区二区三区 | 菩萨蛮人人尽说江南好唐韦庄| av在线老鸭窝| 多毛熟女@视频| 国产成人一区二区在线| 丝袜美足系列| 香蕉丝袜av| a级毛片黄视频| 亚洲激情五月婷婷啪啪| 免费看av在线观看网站| 亚洲欧洲国产日韩| 国产精品麻豆人妻色哟哟久久| 一边亲一边摸免费视频| 欧美老熟妇乱子伦牲交| 中文字幕av电影在线播放| 丝袜美腿诱惑在线| a 毛片基地| 超色免费av| 男人舔女人的私密视频| 一本色道久久久久久精品综合| 国产主播在线观看一区二区 | 欧美激情极品国产一区二区三区| 日韩免费高清中文字幕av| 亚洲av欧美aⅴ国产| 久久久精品区二区三区| 极品人妻少妇av视频| 国产精品麻豆人妻色哟哟久久| 欧美少妇被猛烈插入视频| 十八禁网站网址无遮挡| 日韩免费高清中文字幕av| 亚洲国产欧美一区二区综合| 欧美日韩黄片免| 亚洲伊人久久精品综合| 国产主播在线观看一区二区 | 少妇裸体淫交视频免费看高清 | 男人舔女人的私密视频| xxx大片免费视频| 午夜免费观看性视频| 黑人欧美特级aaaaaa片| 成人亚洲欧美一区二区av| 下体分泌物呈黄色| 久9热在线精品视频| videos熟女内射| 亚洲国产中文字幕在线视频| 国产在线视频一区二区| 欧美 亚洲 国产 日韩一| 久久天躁狠狠躁夜夜2o2o | 国产97色在线日韩免费| 午夜免费男女啪啪视频观看| 亚洲,一卡二卡三卡| 免费女性裸体啪啪无遮挡网站| 侵犯人妻中文字幕一二三四区| 欧美日韩一级在线毛片| tube8黄色片| 国产精品一区二区在线不卡| 国产亚洲欧美在线一区二区| 美女国产高潮福利片在线看| 国产色视频综合| 国产亚洲精品第一综合不卡| 成人影院久久| 精品久久久久久久毛片微露脸 | 中文字幕亚洲精品专区| 国产成人欧美在线观看 | 午夜日韩欧美国产| 国产精品 国内视频| 中文字幕亚洲精品专区| 午夜激情久久久久久久| 亚洲,欧美,日韩| 国产在视频线精品| 只有这里有精品99| 精品亚洲成a人片在线观看| 亚洲精品国产色婷婷电影| 欧美激情极品国产一区二区三区| 超碰成人久久| 超色免费av| av在线app专区| 欧美精品av麻豆av| 黄色视频不卡| 欧美国产精品va在线观看不卡| 国产色视频综合| 午夜福利视频在线观看免费| 黄色a级毛片大全视频| 你懂的网址亚洲精品在线观看| 又紧又爽又黄一区二区| 国产精品久久久久久人妻精品电影 | 中国美女看黄片| 日本av手机在线免费观看| 久久久欧美国产精品| svipshipincom国产片| 亚洲第一青青草原| 色播在线永久视频| 日韩人妻精品一区2区三区| 久久久久久人人人人人| 国产精品免费大片| 看免费成人av毛片| 国产精品久久久久久精品电影小说| 超色免费av| 国产亚洲av片在线观看秒播厂| 制服人妻中文乱码| 精品国产乱码久久久久久男人| 一边摸一边做爽爽视频免费| 黄色片一级片一级黄色片| 美女中出高潮动态图| 高潮久久久久久久久久久不卡| 精品国产乱码久久久久久男人| 国产亚洲一区二区精品| 一级毛片 在线播放| 人妻一区二区av| 超碰成人久久| 亚洲一卡2卡3卡4卡5卡精品中文| 国产淫语在线视频| 午夜福利视频精品| 久久精品成人免费网站| 亚洲av在线观看美女高潮| 搡老乐熟女国产| 一级毛片黄色毛片免费观看视频| 亚洲成人免费电影在线观看 | 另类亚洲欧美激情| 午夜91福利影院| 又大又爽又粗| 在线观看人妻少妇| 人人妻人人添人人爽欧美一区卜| 狠狠婷婷综合久久久久久88av| 韩国高清视频一区二区三区| 成年女人毛片免费观看观看9 | 青春草亚洲视频在线观看| 欧美激情高清一区二区三区| 丰满饥渴人妻一区二区三| 久久久久国产一级毛片高清牌| 亚洲国产日韩一区二区| 777久久人妻少妇嫩草av网站| 久久99一区二区三区| 亚洲午夜精品一区,二区,三区| 天天躁夜夜躁狠狠躁躁| av福利片在线| 亚洲国产精品国产精品| 在线av久久热| 欧美精品高潮呻吟av久久| 亚洲精品国产色婷婷电影| 国产一区二区激情短视频 | 国产成人av激情在线播放| 亚洲中文av在线| 免费在线观看视频国产中文字幕亚洲 | 女警被强在线播放| 欧美老熟妇乱子伦牲交| 国产老妇伦熟女老妇高清| 极品人妻少妇av视频| 国产欧美日韩精品亚洲av| 97精品久久久久久久久久精品| 中文字幕色久视频|