維A酸涂膜劑對(duì)兔耳早期增生性瘢痕的影響

劉群英 段俊俊 邵家松

維A酸涂膜劑對(duì)兔耳早期增生性瘢痕的影響

劉群英 段俊俊 邵家松

目的研究維A酸涂膜劑對(duì)兔耳早期增生性瘢痕的影響,探討其防治瘢痕的可行性。方法選取新西蘭白兔24只,建立兔耳增生性瘢痕模型后,隨機(jī)分為4組,每組6只。A組:對(duì)照組,B組:涂膜劑組,C組:0.05%維A酸涂膜劑組,D組:0.1%維A酸涂膜劑組,連續(xù)用藥6周,期間觀察記錄瘢痕大小、厚度、顏色、硬度,6周后分別切取兔耳瘢痕組織,HE染色,膠原染色(VG法),行病理學(xué)觀察、檢測(cè)及分析。結(jié)果A組瘢痕顏色深、厚而硬,并明顯高于皮膚,表面凹凸不平;B組、C組、D組瘢痕顏色淺,質(zhì)地軟,厚度薄,皮下結(jié)節(jié)小,其中C、D組與周?chē)Ｆつw接近,D組瘢痕表面有脫皮現(xiàn)象。HE、VG染色中,A組膠原排列紊亂,有旋渦狀結(jié)構(gòu);C組和D組單位面積內(nèi)成纖維細(xì)胞、微血管數(shù)量、膠原沉積量較A組和B組少,且膠原排列整齊,與瘢痕長(zhǎng)軸平行。瘢痕增生指數(shù)(HI):A組3.17±0.26,B組2.46±0.19,C組1.91±0.21,D組1.90±0.23;成纖維細(xì)胞密度(NA):A組5836.70±527.03,B組4128.73±387.66,C組3207.59±439.17,D組3200.28±421.48;膠原纖維的面密度(AA):A組45.38±5.83,B組36.57±6.84,C組28.09±3.82,D組28.07±3.47。A組與B、C、D組比較,HI、NA、AA值差異均有統(tǒng)計(jì)學(xué)意義(P＜0.01);B組與C、D組比較,HI、NA、AA值差異均有統(tǒng)計(jì)學(xué)意義(P＜0.01);C組與D組比較,HI、NA、AA值差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。結(jié)論維A酸涂膜劑可以抑制兔耳早期瘢痕增生,可為防治瘢痕提供一種新的外用方法。

瘢痕,肥大性;維生素A酸類(lèi);疾病模型,動(dòng)物

瘢痕常發(fā)生于手術(shù)、外傷、燒傷和感染后,目前仍沒(méi)有有效的防治方法。硅膠膜防治瘢痕有一定療效,臨床使用已有幾十年的歷史。維A酸作為外用制劑,廣泛用于治療角化性及硬化性皮膚病,對(duì)瘢痕增生有一定效果[1-2]。本研究基于硅膠膜和維A酸對(duì)瘢痕的有效性,將維A酸加入有機(jī)成膜溶劑中,制成新型涂膜劑,探討其對(duì)兔耳早期增生性瘢痕的影響。

材料與方法

一、主要試劑與儀器

Van Gieson染色試劑盒(VG,上海源葉生物科技公司),維A酸涂膜劑為自制制劑。

二、動(dòng)物模型建立與分組

健康新西蘭純種系大耳白兔24只,體重(2.5±0.3)kg,雌雄各半,購(gòu)自桂林醫(yī)學(xué)院動(dòng)物實(shí)驗(yàn)中心,來(lái)源于北京科宇動(dòng)物飼養(yǎng)中心,清潔級(jí),編號(hào)為SCXK(京)2012-0003。兔耳增生性瘢痕模型的制作參考李薈元[3]和Morris等[4]的研究。24只大耳白兔適應(yīng)性喂養(yǎng)48 h,選取兔耳腹側(cè)面,切除全層皮膚并刮除軟骨膜,形成2 cm×2 cm的方形創(chuàng)面。每只兔耳2個(gè)創(chuàng)面,創(chuàng)面間隔2 cm以上,創(chuàng)面暴露直至愈合。28 d后,造模成功。將24只兔隨機(jī)分成4個(gè)組,A組:對(duì)照組,B組:涂膜劑組,C組:0.05%維A酸涂膜劑組,D組:0.1%維A酸涂膜劑組。

三、實(shí)驗(yàn)方法

術(shù)后第29天,A組不給予任何治療,B組采用涂膜劑外用2次/d,C組采用0.05%維A酸涂膜劑外用2次/d,D組采用0.1%維A酸涂膜劑外用2次/d,持續(xù)6周。藥物干預(yù)6周后,處死動(dòng)物,手術(shù)切取瘢痕組織及其周邊＜0.5 cm的正常組織,切片后做HE染色和VG染色。

四、觀察指標(biāo)與方法

1.大體觀察:觀察瘢痕顏色、大小、厚度、柔軟度的變化。

2.組織學(xué)觀察:觀察瘢痕增生情況,計(jì)算瘢痕增生指數(shù)(hypertrophic index,HI)[5]、成纖維細(xì)胞密度(numerical density on area,NA)[3]、膠原纖維的面密度(area density on area,AA)[3]。 ①HI:將切取的瘢痕及周?chē)＝M織制作成切片HE染色,低倍鏡下用測(cè)微尺測(cè)出耳腹側(cè)正常皮膚厚度a、耳軟骨厚度b、耳背皮膚厚度c、耳增生塊全厚度最大徑d,按以下公式計(jì)算HI,HI=(d-b-c)/a;②NA:于400倍視野下觀察VG染色切片,在瘢痕中央淺部和深部及其兩側(cè)各隨機(jī)選取10個(gè)矩形視野,目測(cè)計(jì)數(shù)并計(jì)算切片內(nèi)單位面積成纖維細(xì)胞的數(shù)量,結(jié)果取均數(shù),即為NA;③AA:方法同上,各隨機(jī)選取10個(gè)矩形視野,利用計(jì)算機(jī)輔助病理圖像分析系統(tǒng)計(jì)算紅染膠原的面密度,結(jié)果取均數(shù),即為AA。

3.統(tǒng)計(jì)分析及數(shù)據(jù)處理:采用SPSS19.0統(tǒng)計(jì)軟件,各組 HI、NA、AA 均用±s表示,各組比較采用方差分析,方差不齊時(shí)作秩和檢驗(yàn),以P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

一、大體標(biāo)本觀察結(jié)果

1.造模期:取預(yù)實(shí)驗(yàn)3只兔子的造模切片,術(shù)后第28天(圖1a),所有創(chuàng)面完全上皮化,創(chuàng)面增生性瘢痕形成,明顯高于周?chē)Ｆつw,質(zhì)硬,色紅。

2.藥物干預(yù)期:A組:瘢痕繼續(xù)增生,中心呈火山堆形隆起,并且明顯高于周?chē)Ｆつw,隆起的瘢痕體積較大,觸感硬(圖2a);B組:瘢痕顏色逐漸變淡,隆起的瘢痕體積漸變小變平,觸感稍軟(圖3a);C組:瘢痕顏色已與周?chē)Ｆつw接近,表面觸感軟(圖4a);D組:瘢痕顏色逐漸變淡,表面觸感軟,體積變小變平,與周?chē)Ｆつw接近,但有表皮剝脫現(xiàn)象(圖5a)。

二、組織病理學(xué)觀察

1.HE染色:28 d造模成功后(圖1b),可見(jiàn)成纖維細(xì)胞肥大,核濃染,膠原纖維排列紊亂。藥物干預(yù)6周后,A組(圖2b):炎性細(xì)胞浸潤(rùn),膠原紊亂排列,可見(jiàn)漩渦結(jié)構(gòu),核大深染,組織內(nèi)常見(jiàn)毛細(xì)血管增生;B組(圖3b):表皮細(xì)胞層數(shù)較A組明顯減少,炎性細(xì)胞浸潤(rùn),膠原排列較為整齊,組織內(nèi)可見(jiàn)毛細(xì)血管增生;C組(圖4b):成纖維細(xì)胞纖細(xì),膠原纖維排列疏松;D組(圖5b):瘢痕組織均較B組厚度薄,成纖維細(xì)胞較少,膠原纖維排列疏松,可見(jiàn)上皮不完整。

圖1 術(shù)后28 d,兔耳增生性瘢痕模型造模成功 1a:創(chuàng)面增生性瘢痕形成,明顯高于周?chē)Ｆつw,質(zhì)硬,色紅;1b:成纖維細(xì)胞肥大,核濃染,膠原排列紊亂(HE×400)

2.VG染色:A組(圖2c)膠原纖維染紅色,排列紊亂,可見(jiàn)旋渦狀結(jié)構(gòu),并有微血管形成。B組(圖3c)、C組(圖4c)、D組(圖5c)膠原排列均比A組整齊且疏松。與B組相比,C組和D組膠原纖維排列更為整齊,形態(tài)更為纖細(xì),微血管形成少。

3.各組 HI、NA、AA 比較:HI、NA、AA 值除 C 組與D組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)外,其余組間比較差異均有統(tǒng)計(jì)學(xué)意義(P＜0.01)。維A酸涂膜劑對(duì)增生性瘢痕的抑制作用比單純涂膜劑好,而0.05%維A酸涂膜劑和0.1%維A酸涂膜劑對(duì)增生性瘢痕的抑制差異無(wú)統(tǒng)計(jì)學(xué)意義。見(jiàn)表1。

表1 4組兔耳創(chuàng)面瘢痕增生指數(shù)、瘢痕成纖維細(xì)胞密度及膠原纖維的面密度比較(±s)

表1 4組兔耳創(chuàng)面瘢痕增生指數(shù)、瘢痕成纖維細(xì)胞密度及膠原纖維的面密度比較(±s)

注:A組:對(duì)照組;B組:涂膜劑組;C組:0.05%維A酸涂膜劑組;D組:0.1%維A酸涂膜劑組。a:與A組比較,P＜0.01;b:與B組比較,P＜0.01;c:與C組比較,P＞0.05

組別 n 瘢痕增生指數(shù) 成纖維細(xì)胞密度 膠原纖維面密度A組 24 3.17±0.26 5836.70±527.03 45.38±5.83 B組 24 2.46±0.19a 4128.73±387.66a 36.57±6.84a C組 24 1.91±0.21ab 3207.59±439.17ab 28.09±3.82ab D組 24 1.90±0.23abc 3200.28±421.48abc 28.07±3.47abc F值 173.21 185.24 61.14 P值 ＜0.01 ＜0.01 ＜0.01

圖2 兔耳增生性瘢痕對(duì)照組6周 2a:隆起的瘢痕體積較大;2b:成纖維細(xì)胞排列紊亂,核大深染,微血管形成,可見(jiàn)膠原漩渦狀結(jié)構(gòu)(HE×100);2c:膠原密度高,排列較整齊(VG染色×400)

圖3 兔耳增生性瘢痕涂膜劑治療6周 3a:瘢痕體積變小變平;3b:成纖維細(xì)胞較多,膠原致密,排列較整齊(HE×100);3c:膠原密度較低,排列紊亂(VG染色×400)

圖4 兔耳增生性瘢痕0.05%維A酸涂膜劑治療6周 4a:瘢痕表面平而軟,顏色已與周?chē)５钠つw顏色接近;4b:成纖維細(xì)胞纖細(xì),膠原纖維排列疏松(HE×100);4c:膠原密度低,排列整齊(VG染色×400)

圖5 兔耳增生性瘢痕0.1%維A酸涂膜劑治療6周 5a:瘢痕平而軟,表皮有剝脫現(xiàn)象;5b:成纖維細(xì)胞較少,膠原纖維排列疏松(HE×100);5c:膠原密度低,排列較整齊(VG染色×400)

討 論

維A酸外用制劑臨床上主要用于治療痤瘡、銀屑病、皮膚老化、色素性皮膚病、角化性皮膚病、硬化及萎縮性皮膚病、癌前病變以及加速創(chuàng)面的愈合等。Christophers和Lagner等[6]證實(shí),維A酸對(duì)上皮細(xì)胞的生長(zhǎng)有較強(qiáng)的促進(jìn)作用。Uchida等[7]研究發(fā)現(xiàn),維A酸通過(guò)影響人的成纖維細(xì)胞基質(zhì)金屬蛋白酶,減少膠原的沉積,達(dá)到抑制瘢痕的目的。宋維銘等[8]采用成纖維細(xì)胞體外培養(yǎng)技術(shù),將3H TdR(胸腺嘧啶核苷)摻入成纖維細(xì)胞DNA中,研究維A酸對(duì)體外培養(yǎng)成纖維細(xì)胞生長(zhǎng)增殖、DNA合成及超微結(jié)構(gòu)的影響。結(jié)果顯示,維A酸對(duì)成纖維細(xì)胞生長(zhǎng)增殖、DNA合成有不同程度的抑制作用,呈劑量效應(yīng)關(guān)系;掃描電鏡和透射電鏡觀察亦顯示,維A酸對(duì)成纖維細(xì)胞合成膠原有抑制作用。臨床研究[9]表明,維A酸對(duì)創(chuàng)傷后瘢痕抑制有一定作用。

硅膠膜敷貼在瘢痕防治中的效果得到肯定。Perkins等[10]在加壓療法中使用硅膠膜襯墊,發(fā)現(xiàn)在無(wú)壓力下硅膠膜也有松弛軟化瘢痕和使瘢痕平坦的作用。此后硅凝膠和硅彈力膜在增生性瘢痕的治療中得到較多應(yīng)用。Quinn等[11]對(duì)125例增生性瘢痕和瘢痕疙瘩患者用含硅酮的霜?jiǎng)┲委熢錾择：?按覆蓋方式分成紗布覆蓋組和塑料膜閉合覆蓋組,紗布覆蓋組僅有22%獲得輕微改善,而閉合覆蓋有82%獲得明顯改善,提示閉合和水化是其作用的主要方式。Sawada和Sone[12]采用不含硅酮的霜?jiǎng)╅]合覆蓋技術(shù)治療瘢痕,治療組的療效明顯優(yōu)于凡士林對(duì)照組,因此認(rèn)為閉合和水化是治療瘢痕的主要機(jī)制,而硅酮的存在是非必需的。Chang等[13]的工作也支持這一觀點(diǎn)。Ricketts等[14]在對(duì)未治療、用水膠體治療和用膠膜治療增生性瘢痕過(guò)程中,觀察到組織細(xì)胞分裂mRNA水平的一些差異,提出閉合可改變瘢痕細(xì)胞分裂水平,從而對(duì)瘢痕塑形產(chǎn)生作用。我們將維A酸加入有機(jī)成膜溶劑中,制成0.05%和0.1%維A酸凝膠后密閉待用。該涂膜劑涂抹于兔耳瘢痕后迅速成膜,具有硅凝膠膜的特性,同時(shí)又能發(fā)揮維A酸的作用。

實(shí)驗(yàn)結(jié)果表明:①維A酸涂膜劑對(duì)兔耳瘢痕組織中成纖維細(xì)胞及血管增生具有抑制作用;②維A酸涂膜劑抑制兔耳瘢痕增生,使瘢痕變平、變軟,顏色變淺;③維A酸涂膜劑對(duì)兔耳瘢痕的抑制效果顯著好于涂膜劑組;④0.1%維A酸與0.05%維A酸涂膜劑療效相當(dāng),但高濃度的維A酸有表皮剝脫現(xiàn)象。

雖然本試驗(yàn)證明了外用維A酸涂膜劑對(duì)兔耳早期增生性瘢痕有良好抑制效果,但仍有問(wèn)題需做進(jìn)一步研究,①本次試驗(yàn)只選用了0.05%和0.1%維A酸,并不清楚療效與藥物濃度的關(guān)系以及維A酸涂膜劑中維A酸的最佳有效濃度;②雖然目前已分別提出了維A酸和硅凝膠膜治療增生性瘢痕可能的機(jī)制,但這種新型的涂膜劑抑制瘢痕并沒(méi)有更進(jìn)一步理論依據(jù);③目前仍缺乏對(duì)于維A酸涂膜劑藥理毒理實(shí)驗(yàn)。

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2013-11-04)

(本文編輯:顏艷)

Effect of tretinoin gel sheeting on early-stage hyperplastic scars in rabbit ears

Liu Qunying*,Duan Junjun,Shao Jiasong.*Department of Dermatology,Affiliated Hospital,Guilin Medical College,Guilin 541001,China

Shao Jiasong,Email:shao1126@126.com

ObjectiveTo estimate the effect of tretinoin gel sheeting on early-stage hyperplastic scars in rabbit ears,and to evaluate the feasibility to prevent and treat hyperplastic scars with it.MethodsThe ears of 24 rabbits were used to establish a model of hyperplastic scar according to previously reported methods.Then,the rabbit ears were randomly divided into four groups:control group receiving no treatment,gel sheeting group treated with the vehicle of the tretinoin gel sheeting,0.05%tretinoin group treated with 0.05%tretinoin gel sheeting,0.1%tretinoin group treated with 0.1%tretinoin gel sheeting.All the gel sheetings were topically used twice daily for six consecutive weeks.During the treatment,the size,thickness,color and texture of scars were estimated.After six weeks of treatment,all the scar tissues were resected from the rabbit ears and subjected to hematoxylin and eosin(HE)staining and van Gieson(VG)staining.Statistical analysis was carried out by analysis of variance and rank sum test.ResultsThe scars were deeply colored,thickened,hard and elevated with an uneven surface in the control group,but lightly colored,thinned and soft with the presence of small subcutaneous nodules in the other three groups.The surface of scars in the two tretinoin groups was similar to that of adjacent normal skin,and scaling was observed on the scar surface in the 0.1%tretinoin group.HE and VG staining showed a disarrangement of collagen fibers with the formation of vortex-like structures in the control group.A significant decrease was noted in the number of fibroblasts and microvessels as well as amount of collagen deposition per unit cross-sectional area in the two tretinoin groups compared with the control group and gel sheeting group.Additionally,the collagen fibers were regularly arranged and parallel to the long axis of scars in the two tretinoin groups.The scar hyperplasia index(HI)was 3.173±0.26,2.465±0.19,1.906±0.21 and 1.903±0.23 in the control group,gel sheeting group,0.05%tretinoin group and 0.1%tretinoin group respectively,the fibroblast density(NA)was 5836.7±527.03,4128.73±387.66,3207.59±439.17 and 3200.28±421.48 respectively,and the area density of collagen fiber(AA)was 45.38±5.83,36.57±6.84,28.09±3.82 and 28.07±3.47 respectively.As far as HI,NA and AA were concerned,the control group differed significantly from the other three groups(allP＜0.01),and the gel sheeting group from the two tretinoin groups(allP＜0.01),but no significant difference was observed between the two tretinoin groups(allP＞0.05).ConclusionsTopical tretinoin gel sheeting can inhibit scar proliferation at early stage in rabbit ears,and may provide a new choice for the prevention and treatment of hyperplastic scars.

Cicatrix,hypertrophic;Retinoids;Disease models,animal

10.3760/cma.j.issn.0412-4030.2014.07.007

541001廣西,桂林醫(yī)學(xué)院附屬醫(yī)院皮膚科(劉群英),整形外科(段俊俊、邵家松)

邵家松,Email:shao1126@126.com