張 琪 劉李娜 鄧景成 曹衛(wèi)剛
瘢痕內(nèi)注射脂肪來源干細胞對兔耳增生性瘢痕的抑制作用研究
張 琪 劉李娜 鄧景成 曹衛(wèi)剛
目的研究瘢痕內(nèi)注射脂肪來源干細胞(ADSC)對兔耳增生性瘢痕的抑制作用及可能機制。方法選取12只新西蘭大白兔,制作兔耳增生性瘢痕模型,隨機平均分成3組,2周后各組右耳瘢痕內(nèi)注射DMEM作為自身對照,左耳分別注射ADSCs、脂肪來源干細胞條件培養(yǎng)基(ADSCs-CM)及不作處理。注射前及注射后1、2、3周檢測瘢痕增生情況,并于注射后3周取材,行組織化學(xué)及基因?qū)W檢測。結(jié)果造模2周后,傷口均完全上皮化;DMEM注射及未處理組的傷口逐漸出現(xiàn)增厚、變紅、變硬等增生表現(xiàn),注射后3周時最為明顯;ADSCs及ADSCs-CM注射組均未出現(xiàn)明顯增生反應(yīng)。取材后HE、Masson染色示,ADSCs及ADSCs-CM注射組瘢痕的膠原密度適中、排列整齊;對照及未處理組瘢痕可見大量致密、雜亂的膠原組織?;驒z測發(fā)現(xiàn),ADSCs及ADSCs-CM注射組瘢痕的α-SMA、CollagenⅠ表達顯著低于對照及未處理組。ADSCs注射組瘢痕冰凍切片Dil熒光染色可見大量存活脂肪來源干細胞。結(jié)論瘢痕內(nèi)注射脂肪來源干細胞可以降低α-SMA、CollagenⅠ基因表達,從而改善瘢痕內(nèi)膠原堆積,并最終改善瘢痕增生情況。
脂肪來源干細胞增生性瘢痕瘢痕內(nèi)注射
創(chuàng)面愈合過程包括炎癥期、增生期及重塑期。增生性瘢痕的發(fā)生是由于傷口愈合過程中某些重要因素的異常所致。T細胞及巨噬細胞免疫功能異常會導(dǎo)致炎癥期延長,從而產(chǎn)生過多的無功能的胞外基質(zhì)導(dǎo)致瘢痕增生[5-7]。中性粒細胞在傷口滅菌過程中產(chǎn)生的活性氧類物質(zhì)(ROS)亦是瘢痕增生的促進因素[8-9]。TGF-β1是目前已知的致纖維化因子之一,能強效刺激傷口組織中膠原纖維的增生堆積,并抑制基質(zhì)金屬蛋白酶對膠原等胞外基質(zhì)的分解,從而促進瘢痕增生[10-12]。傷口愈合過程中,肌成纖維細胞能有效縮小傷口,加速上皮化,但同時也合成分泌更多致密無序的膠原纖維,導(dǎo)致增生性瘢痕的攣縮[13-14]。傷口愈合過程中血供不足會導(dǎo)致愈合延遲從而加重瘢痕增生。因而及時、充足的微血管形成及向永久血管網(wǎng)絡(luò)轉(zhuǎn)化十分重要[15]。傷口愈合過程中的任何異常因素都可以影響組織再生從而產(chǎn)生增生性瘢痕。任何可以糾正或緩和傷口愈合過程中異常因素的藥物均可抑制或改善瘢痕增生。在許多纖維化疾病的研究中發(fā)現(xiàn),脂肪來源干細胞(ADSC)治療可以通過減輕炎癥反應(yīng)、抑制TGF-β1作用并促進組織再生從而抑制纖維化發(fā)生及進展[16-18]。本實驗通過建立兔耳瘢痕模型,研究瘢痕內(nèi)注射ADSCs對兔耳增生性瘢痕的抑制作用及其可能的機制。
1.1 主要材料與試劑
新西蘭大白兔,體質(zhì)量2.0~2.5 K g,雌雄不限,(我院實驗動物中心提供)。
Ⅰ型膠原酶(Sigma公司,美國);Dil(Invitrogen公司,美國);DAPI(Sigma公司,美國);TRIzol(Invitrogen公司,美國);SYBR Green試劑盒(Takara公司,日本);Nanodrop(Isogen公司,英國);小動物超聲測量儀(Esaote公司,意大利);倒置相差顯微鏡(Nikon公司,日本);電動高速勻漿器(新芝公司,中國);PCR儀(Applied Biosystems公司,美國)。
1.2 兔耳瘢痕模型的建立
12只新西蘭大白兔隨機平均分成3組。麻醉后,以直徑1 cm環(huán)轉(zhuǎn)于耳腹側(cè)制備6個相同的創(chuàng)面,去除皮膚全層及軟骨膜,按壓止血后均勻涂抹一層紅霉素眼膏,暴露創(chuàng)面,每日清除創(chuàng)面分泌物(圖1)。
1.3 ADSCs培養(yǎng)及ADSCs-CM準(zhǔn)備
取4周齡新西蘭大白兔腹股溝脂肪,0.075%Ⅰ型膠原酶37℃消化45 min;1 200 g離心10 min;沉淀物用常規(guī)細胞培養(yǎng)基(低糖DMEM加10%胎牛血清及1%雙抗)置于37℃細胞培養(yǎng)箱。取第三代ADSCs接種于六孔板中(5×104cells/cm2),培養(yǎng)16 h后棄去培養(yǎng)基,換無血清DMEM,孵育48 h,收集上清液,300 g離心5 min后過濾(0.22 μm濾器)備用。
1.4 瘢痕內(nèi)注射
取第三代ADSCs標(biāo)記Dil,細胞收集離心后PBS洗滌2次,重懸于5 μL/m L Dil稀釋液中(溶劑DMEM),避光,37℃孵育20 min,再次洗滌2遍,重懸于低糖DMEM中(2×107cells/mL)。1 mL注射器吸取細胞懸液或ADSCs-CM,用29 G針頭從瘢痕邊緣進針至中心處,緩慢推注0.2 mL,對照側(cè)同法推注0.2 mL低糖DMEM。
1.5 瘢痕大體觀察
術(shù)前及術(shù)后5周內(nèi)每周拍照,記錄瘢痕組織外觀變化,同時采用超聲測量儀記錄瘢痕內(nèi)部增生情況。
1.6 組織學(xué)檢測
術(shù)后5周取材,每塊瘢痕組織從中間最高處切成兩半,分別固定于4%多聚甲醛中,取其中1份行石蠟包埋,切片,HE染色,鏡下觀察拍照,計算瘢痕增生指數(shù)(SEI)。采用Masson三色染色法進一步觀察瘢痕組織中膠原的堆積及排列情況。
1.7 ADSCs熒光染色示蹤
瘢痕組織取材,4%多聚甲醛固定24 h后,換10%蔗糖溶液,4℃避光脫水12 h,30%蔗糖溶液4℃避光脫水24 h,OCT包埋,置于-80℃保存?zhèn)溆?。取OCT包埋組織,冰凍切片10 μm,貼片后PBS洗滌3次,置于空氣中干燥10 min,DAPI(1 μg/mL)襯染胞核,熒光顯微鏡觀察拍照。
1.8 實時熒光定量PCR檢測
瘢痕組織取材后去除表皮、軟骨及下層組織,置于液氮中速凍1 min,電動高速勻漿器勻漿,加入TRIzol(體積比1∶1),按操作說明提取組織總RNA,采用Nanodrop測量其濃度和純度。用反轉(zhuǎn)錄試劑盒生成cDNA,1∶25稀釋于去DNase水中備用。實時熒光定量PCR按SYBR Green試劑盒操作,結(jié)果采用β-actin為內(nèi)參,計算2-DDCt及倍數(shù)值。引物序列包括α-SMA:F 5’-CAGGGAGTAATGGTTGGAAT-3’,R 5’-TCTCAAACATAATCTGGGTCA-3’;Collagen type Ι:F 5’-CCCAACCAAGGATGCACTA-3’,R 5’-CTTGGCCTTGGAGCTCTTATAC-3’;β-actin:F 5’-GCTATTTGGCGCTGGACTT-3’,R 5’-GCG實驗GCTCGTAGCTCTTCTC-3’。
1.9 統(tǒng)計分析
數(shù)據(jù)統(tǒng)計分析采用GraphPad Prism 6軟件,Student’s-t檢驗比較實驗組及其自身對照的SEI值及基因表達。P<0.05為差異有顯著性。
2.1 ADSCs及ADSCs-CM注射均可抑制瘢痕增生
術(shù)后2周,大體觀察所有創(chuàng)面均已完全上皮化。DMEM注射及未處理組可見創(chuàng)面上皮化后逐漸增厚、變硬、變紅,并逐漸明顯突出于周圍正常組織,而ADSCs及ADSCs-CM注射組瘢痕未見明顯增生,并逐漸與正常組織相似(圖2)。
超聲檢查瘢痕組織可清晰觀察到表皮、真皮、軟骨、軟骨下結(jié)締組織,并可同時測量真皮組織的厚度,相比傳統(tǒng)測量瘢痕全層厚度的方法,該方法能更準(zhǔn)確地反應(yīng)真皮層的厚度。B超示,DMEM注射及未處理組真皮層逐漸增厚,明顯高于周圍正常組織;而ADSCs及ADSCs-CM注射組未見明顯增厚(圖3)。
術(shù)后5周,HE染色示DMEM注射及未處理組瘢痕顯著增厚,并伴輕度攣縮;而ADSCs及ADSCs-CM注射組平薄,類似正常組織(圖4)。
計算并比較SEI值,ADSCs及ADSCs-CM注射組的SEI值均顯著低于其自身對照(1.08±0.05 vs 1.93±0.09,**:P<0.01,n=24;1.33±0.10 vs 1.97±0.11,**:P<0.01,n=24)。而未處理組雙側(cè)SEI值無明顯差異(1.90±0.12 vs 1.94±0.06,P>0.05,n=24)。另外,ADSCs注射組瘢痕的SEI值低于ADSCs-CM注射組(△:P<0.01)(圖5)。
Masson染色觀察膠原增生及排列可見自身對照及未處理組膠原致密,排列雜亂,而ADSCs及ADSCs-CM注射組膠原較稀疏且排列整齊(圖6)。
2.2 ADSCs及ADSCs-CM抑制基因表達
實時熒光定量檢測示,ADSCs及ADSCs-CM注射可抑制α-SMA及Ι型膠原mRNA表達(**:P<0.01),而DMEM注射及未處理組基因表達無明顯差異。亦可見ADSCs注射組瘢痕α-SMA及Ι型膠原mRNA表達量顯著低于ADSCs-CM注射組(*:P<0.05)(圖7)。
2.3 熒光染色示蹤結(jié)果
Dil標(biāo)記ADSCs熒光染色示大量綠色熒光細胞,表明ADSCs注射后3周瘢痕內(nèi)存在大量存活的ADSCs(圖8)。
圖1 兔耳增生性瘢痕模型Fig.1 Hypertrophic scar model of rabbit ear
圖2 各組兔耳增生性瘢痕注射前后照片F(xiàn)ig.2 Photos of the hypertrophic scar of rabbit ear before and after injection in each group
圖3 各組兔耳增生性瘢痕注射前后B超檢測Fig.3 Utrasonography of the hypertrophic scar of rabbit ear before and after injection in each group
圖4 不同處理組術(shù)后5周HE染色結(jié)果Fig.4 H istological observation in each group 5 weeks after operation
圖5 各組瘢痕增生指數(shù)Fig.5 Calculation of scar elevated index in each group
圖6 不同處理組術(shù)后5周M asson染色結(jié)果Fig.6 Histological observation in each group by M asson staining 5 weeks after operation
圖7 各組α-SMA及Ι型膠原基因表達Fig.7 Gene expression of α-SMA and collagen Ι in each group
圖8 ADSCs的Dil熒光染色Fig.8 Dil staining of ADSCs
術(shù)后或創(chuàng)傷后瘢痕,輕者影響外觀,重者可導(dǎo)致器官功能障礙。臨床發(fā)現(xiàn),部分燒傷后瘢痕行脂肪填充后,瘢痕組織可能軟化,質(zhì)地向正常皮膚轉(zhuǎn)變,其組織學(xué)結(jié)構(gòu)亦趨于正常皮膚組織。該現(xiàn)象的機制目前并不清楚,可能是脂肪組織中ADSCs促進了瘢痕組織學(xué)及臨床表現(xiàn)的改變[19-21]。
對于創(chuàng)面愈合機制的研究理論可以用于解釋瘢痕增生的可能原因[1-2,22]。創(chuàng)面愈合過程較為復(fù)雜,可分為序貫并相互重疊的3個時期,即炎癥期、增生期及重塑期。此過程中的任何異常均可導(dǎo)致瘢痕增生。多項研究證實,間充質(zhì)干細胞(MSCs)可通過促進創(chuàng)面愈合,從而抑制增生性瘢痕產(chǎn)生。
2001年Zuk等發(fā)現(xiàn)了ADSCs,屬于MSCs的一種,但具有易獲取、易分離培養(yǎng)、量多的優(yōu)勢,被廣泛應(yīng)用于組織工程種子細胞,以及促進創(chuàng)面愈合、抗衰老、抗纖維化方面[23-27]。ADSCs應(yīng)用于各種纖維化疾病的治療已有廣泛研究。已在動物模型中證實,聲帶受損后注射ADSCs,可有效抑制萎縮性或增生性瘢痕形成[16];在急性心梗小鼠模型中注射ADSCs,可以縮小梗死面積、減輕心肌瘢痕形成,并促進心功能恢復(fù)。研究發(fā)現(xiàn),ADSCs可以遷移至心梗處,并表達內(nèi)皮細胞標(biāo)志,與血管重建密切相關(guān)[17]。
但是,ADSCs在皮膚瘢痕形成中的作用并未有相關(guān)研究。本實驗探索ADSCs在體內(nèi)瘢痕形成過程中的抗纖維化作用。實驗結(jié)果表明,ADSCs及其條件培養(yǎng)基對瘢痕增生均有不同程度的抑制作用,并可促進瘢痕向正常組織轉(zhuǎn)化。ADSCs-CM是ADSCs體外培養(yǎng)48小時內(nèi)的上清液,其中含有多種ADSCs分泌的細胞因子,如IL-10和HGF等。這些細胞因子可降低TGF-β1及膠原表達,促進MMPs表達,從而加速細胞外基質(zhì)更新,抑制纖維化[28-31]。其中,HGF還可抑制成纖維細胞向肌成纖維細胞分化,從而抑制其致纖維化作用[14]。在創(chuàng)面愈合的增生期,血管形成可以為成纖維細胞形成肉芽組織提供充足營養(yǎng)基質(zhì)[15]。ADSCs分泌的VEGF-A和b-FGF可以強效促進血管內(nèi)皮細胞遷移、增殖及分化,從而有利于血管生成及穩(wěn)定[32-35]。
本實驗同時發(fā)現(xiàn),ADSCs的抑制瘢痕增生作用顯著強于ADSCs-CM。ADSCs除通過分泌一些抗纖維化細胞因子抑制瘢痕增生外,還存在著其他抑制纖維化的作用機制。研究表明,MSCs可被創(chuàng)面的炎癥環(huán)境所激發(fā),啟動其免疫調(diào)節(jié)作用,上調(diào)前列腺素E2及環(huán)氧化酶-2的表達,減輕炎癥反應(yīng)并抑制炎癥反應(yīng)延長導(dǎo)致的T細胞及巨噬細胞免疫功能紊亂[6-7]。ROS是創(chuàng)面愈合過程中的一類致纖維化物質(zhì),可以誘導(dǎo)TGF-β1表達增強[8,36]。MSCs可以通過促進T細胞產(chǎn)生誘導(dǎo)性NO,從而改變ROS/RNS(反應(yīng)活性氮類物質(zhì))平衡,阻止纖維化形成[9]。另有研究表明,ADSCs能在傷口真表皮細胞的影響下通過表型轉(zhuǎn)換,分化成為成纖維細胞及角質(zhì)形成細胞,直接參與真皮和表皮的組織結(jié)構(gòu)再生[37]。本實驗發(fā)現(xiàn),ADSCs注射組在注射后3周,瘢痕組織內(nèi)存在大量存活的ADSCs,推測ADSCs積極參與了創(chuàng)面愈合及組織再生。由于Dil標(biāo)記熒光染色的時效性有限,未能追蹤到ADSCs的最終轉(zhuǎn)化,因而有待于進一步的研究,以證實ADSCs是否向創(chuàng)面愈合相關(guān)細胞轉(zhuǎn)化,如血管內(nèi)皮細胞、成纖維細胞及表皮細胞等。
本實驗采用穩(wěn)定的兔耳增生性瘢痕模型,研究ADSCs及其條件培養(yǎng)基的瘢痕增生抑制作用。我們證實,ADSCs及其條件培養(yǎng)基對瘢痕增生均有抑制作用,但ADSCs作用顯著強于ADSCs-CM,推測ADSCs不僅通過分泌抗纖維化因子抑制瘢痕增生,還可能通過ADSCs與創(chuàng)面內(nèi)細胞間相互作用,及ADSCs向其他細胞分化,參與創(chuàng)面愈合及組織再生。
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The Inhibition Effect of Intra-cicatrix Injection of ADSCs on Ear Hypertrophic Scar of Rabbit
ZHANG QI,LIU Lina,DENG Jingcheng,CAO Weigang.
Department of Plastic and Reconstructive Surgery,Shanghai Ninth peop le's Hospital, Shanghai Jiaotong University School of Medicine,Shanghai 200011,China.Corresponding author:CAO Weigang(E-mail: wgcao@sina.com).
Objective To investigate the inhibition effect and mechanism of intra-cicatrix injection of adipose derived stem cells on hypertrophic scar in rabbit ear.M ethods Twelve New Zealand white rabbits were used for establishing scar model and were random ly divided into 3 groups.Two weeks after the operation,all the right ears were injected DMEM as internal control while left ears of group 1,2 were injected ADSCs,ADSCs-CM respectively.Left ears in the third group were remained untouched.Photos and ultrasonography were taken before and 1,2,3 weeks after injection.Histochemical and genetic detection were used 3 weeks after the rabbits were sacrificed for offering scar tissue.Results All wounds were reepithelized 2 weeks after the injection.Wounds injected DMEM and untouched gradually grew thick,red and stiff which are the symptoms of hypertrophic scars,while ADSCs and ADSCs-CM injected ones showed no sign of growing hypertrophic.HE and Masson's staining showed collagen deposit and irregularly arrangement in the DMEM injection and untouched scars, while much less and better arranged collagen deposit were shown in both ADSCs and ADSCs-CM treated ones.Genetic detection showed lower expression of α-SMA,CollagenⅠin ADSCs and ADSCs-CM injection scars,compared with DMEM treated and untreated ones.Dil label staining showed a larger amount of ADSCs in the scar tissue of ADSCs treated group. Conclusion Intra-cicatrix injection of ADSCs can inhibit hypertrophic scar through ameliorating collagen deposit by down regulate the expression of α-SMA,CollagenⅠ.
Adipose derived stem cells;Hypertrophic scar;Intra-cicatrix injection增生性瘢痕的發(fā)生是由于真皮組織損傷導(dǎo)致細胞外基質(zhì),尤其是膠原組織的異常堆積及重塑[1]。其典型表現(xiàn)是增厚、變紅、變硬及伴癢感,影響美觀,甚至造成功能障礙[2]。目前,瘢痕的治療方法很多,包括切除、皮內(nèi)激素注射、加壓、激光等,但尚未有一種方法可單獨持久有效地抑制或治療瘢痕增生[3-4]。
R619+.6
A
1673-0364(2015)03-0139-05
10.3969/j.issn.1673-0364.2015.03.006
2015年3月20日;
2015年4月28日)
200011上海市上海交通大學(xué)醫(yī)學(xué)院附屬第九人民醫(yī)院整復(fù)外科。
曹衛(wèi)剛(E-mail:wgcao@sina.com)。