臭氧氣浴對(duì)深Ⅱ度燒傷大鼠創(chuàng)面病理變化及組織細(xì)胞因子表達(dá)的影響*

趙超莉， 葉子青， 阮瓊芳， 陳 斕， 張衛(wèi)東， 王 珊， 謝衛(wèi)國(guó)

(武漢大學(xué)同仁醫(yī)院暨武漢市第三醫(yī)院燒傷研究所， 湖北 武漢 430060)

臭氧氣浴對(duì)深Ⅱ度燒傷大鼠創(chuàng)面病理變化及組織細(xì)胞因子表達(dá)的影響*

趙超莉△， 葉子青， 阮瓊芳， 陳 斕， 張衛(wèi)東， 王 珊， 謝衛(wèi)國(guó)

(武漢大學(xué)同仁醫(yī)院暨武漢市第三醫(yī)院燒傷研究所， 湖北 武漢 430060)

目的探討臭氧氣浴干預(yù)對(duì)大鼠深Ⅱ度燒傷創(chuàng)面的病理變化及局部組織中細(xì)胞因子血小板源性生長(zhǎng)因子(PDGF)、轉(zhuǎn)化生長(zhǎng)因子β3(TGF-β3)和腫瘤壞死因子α(TNF-α)表達(dá)的影響。方法取80只雄性清潔級(jí)SD大鼠，隨機(jī)分為臭氧氣浴實(shí)驗(yàn)組和常規(guī)換藥對(duì)照組各40只。建立背部深Ⅱ度燒傷模型，傷后3 d、7 d、14 d和21 d，2組創(chuàng)面分別取材檢測(cè)。創(chuàng)面常規(guī)換藥,對(duì)照組的創(chuàng)面以生理鹽水清洗和碘伏油紗包扎，隔日換藥1次；臭氧氣浴實(shí)驗(yàn)組在換藥前將大鼠放入清潔泡沫盒內(nèi)，打開臭氧發(fā)生器開關(guān)，以輸出濃度為50 mg/L的臭氧熏蒸創(chuàng)面20 min，關(guān)閉開關(guān)，再行創(chuàng)面常規(guī)換藥，隔日1次，直至愈合。各時(shí)點(diǎn)每組大鼠創(chuàng)面打開時(shí)取1次創(chuàng)面中心組織標(biāo)本，然后實(shí)驗(yàn)組行生理鹽水棉球輕拭創(chuàng)面和臭氧氣熏蒸，對(duì)照組僅用生理鹽水棉球輕拭創(chuàng)面后再分別取材1次。標(biāo)本行HE染色組織學(xué)觀察和免疫組化染色半定量觀察，結(jié)合圖像數(shù)據(jù)分析及ELISA法檢測(cè)2組創(chuàng)面組織中細(xì)胞因子PDGF、TGF-β3和TNF-α含量。結(jié)果創(chuàng)面大體觀察可見，臭氧氣浴實(shí)驗(yàn)組的創(chuàng)面平整，邊緣清晰，炎性反應(yīng)輕，腫脹和滲出程度均較對(duì)照組弱，創(chuàng)面愈合率高于常規(guī)換藥對(duì)照組，2組比較差異有統(tǒng)計(jì)學(xué)意義。顯微鏡下觀察HE染色組織可見臭氧氣浴實(shí)驗(yàn)組的創(chuàng)面各時(shí)點(diǎn)炎性反應(yīng)程度比常規(guī)換藥對(duì)照組輕，而新生毛細(xì)血管、成纖維細(xì)胞和上皮細(xì)胞增生數(shù)量明顯優(yōu)于常規(guī)換藥對(duì)照組。與對(duì)照組比較，臭氧氣浴實(shí)驗(yàn)組各時(shí)點(diǎn)燒傷大鼠的創(chuàng)面組織勻漿上清液中PDGF和TGF-β3表達(dá)量均高于常規(guī)換藥對(duì)照組，而TNF-α表達(dá)量明顯低于對(duì)照組，2組比較差異均有統(tǒng)計(jì)學(xué)意義。結(jié)論臭氧氣浴療法干預(yù)大鼠深Ⅱ度燒傷創(chuàng)面，能改善局部病理變化，促進(jìn)與創(chuàng)面愈合相關(guān)的細(xì)胞因子PDGF和TGF-β3表達(dá)，同時(shí)減輕炎性介質(zhì)TNF-α的表達(dá)。

燒傷； 臭氧氣浴； 細(xì)胞因子

臭氧是一種強(qiáng)氧化劑，其消毒殺菌功能眾所周知。然而，臭氧對(duì)燒傷創(chuàng)面組織病理及生物學(xué)效應(yīng)影響卻罕見報(bào)道，但這些指標(biāo)的驗(yàn)證是燒傷臨床應(yīng)用臭氧所需依靠的理論依據(jù)和科學(xué)證明。本研究通過以臭氧氣浴干預(yù)大鼠深Ⅱ度燒傷創(chuàng)面，從HE染色觀察其對(duì)局部的炎性反應(yīng)、血管、肉芽等組織學(xué)變化的影響入手，結(jié)合ELISA法和免疫組織化學(xué)方法檢測(cè)與創(chuàng)面愈合相關(guān)的細(xì)胞因子[血小板源性生長(zhǎng)因子(platelet-derived growth factor，PDGF)、轉(zhuǎn)化生長(zhǎng)因子β3(transforming growth factor-β3，TGF-β3)和腫瘤壞死因子α(tumor necrosis factor-α， TNF-α)]的表達(dá)，探討臭氧氣浴對(duì)創(chuàng)面愈合影響的機(jī)制。

材 料 和 方 法

1材料

1.1材料及試劑 兔抗大鼠來源的PDGF、TGF-β3和TNF-α 多克隆抗體均購自Abcam；辣根過氧化物酶標(biāo)記的山羊抗兔IgG II 抗及大鼠PDGF、TGF-β3和TNF-α ELISA檢測(cè)試劑盒均購自湖北阿斯本生物有限公司。D-37520 型冷凍離心機(jī)購自Beckman。XMTE-6000數(shù)字式可控溫-電燙儀由上海復(fù)旦大學(xué)-長(zhǎng)海醫(yī)院聯(lián)合研制提供；YGO3-1型醫(yī)用臭氧治療儀(臭氧輸出濃度為50 mg/L)，由武漢華中科技大學(xué)陽光科技開發(fā)公司生產(chǎn)提供；Image-Pro Plus 6.0彩色病理圖像分析軟件購自Media Cybernetics。

1.2動(dòng)物 清潔級(jí)SD雄性大鼠80只，體重220～250 g，動(dòng)物批號(hào)為43004700019289，購自湖南斯萊克景達(dá)實(shí)驗(yàn)動(dòng)物有限公司。實(shí)驗(yàn)前12 h禁食，實(shí)驗(yàn)前2 h禁飲水。

2方法

2.1深Ⅱ度燒傷模型制備與分組 取80只經(jīng)1周適應(yīng)性飼養(yǎng)的SD大鼠，用1%戊巴比妥鈉腹腔注射(40 mg/kg)麻醉，背部剃毛，用電燙儀在背部制作一個(gè)深Ⅱ度燒傷創(chuàng)面(82 ℃，12 s)，創(chuàng)面為圓形，直徑30 mm，面積7.07 cm2(以上造模條件已經(jīng)病理切片證實(shí))。將大鼠隨機(jī)分為臭氧氣浴實(shí)驗(yàn)組和常規(guī)換藥對(duì)照組,每組40只，造模后單籠喂養(yǎng)進(jìn)行實(shí)驗(yàn)。每次打開創(chuàng)面后，常規(guī)換藥對(duì)照組在創(chuàng)面以生理鹽水棉球輕拭創(chuàng)面并碘伏油紗包扎，隔日換藥1次，直至愈合；臭氧氣浴實(shí)驗(yàn)組在打開創(chuàng)面后，將大鼠放入清潔泡沫盒內(nèi)，頭伸出盒孔外，打開臭氧氣發(fā)生器開關(guān)，先行臭氧氣浴(濃度為50 mg/L)20 min，關(guān)閉臭氧開關(guān)，再用常規(guī)換藥方法處理創(chuàng)面。

2.2標(biāo)本采集 2組大鼠分別于傷后3 d、7 d、14 d和21 d采集創(chuàng)面組織標(biāo)本，每組每時(shí)點(diǎn)選10只大鼠取材，取材前觀察創(chuàng)面大體情況，測(cè)量創(chuàng)面面積。取下組織，一部分以4%多聚甲醛溶液固定，行HE染色和免疫組化半定量檢測(cè)，一部分組織經(jīng)冷凝管入-80 ℃深低溫冰箱保存。

2.3HE染色 將已入蒸餾水后的切片放入蘇木精水溶液中染色；酸水及氨水中分色；流水沖洗1 h后入蒸餾水片刻;入70%和90%乙醇中脫水各10 min;入乙醇伊紅染色液染色2～3 min。染色后的切片經(jīng)純乙醇脫水，再經(jīng)二甲苯使切片透明。將已透明的切片滴上樹膠，蓋上蓋玻片封固。待樹膠略干后，貼上標(biāo)箋，光學(xué)顯微鏡下組織病理學(xué)觀察。

2.4免疫組化染色 石蠟切片免疫組化染色步驟按試劑盒說明書操作： 石蠟切片脫蠟至水；蒸餾水沖洗，PBS浸泡5 min，抗原修復(fù)；3% H2O2室溫孵育5～10 min，以消除內(nèi)源性過氧化物酶的活性，PBS沖洗，2 min、3次； 滴加適當(dāng)比例稀釋的Ⅰ抗工作液，37 ℃孵育1～2 h或4 ℃過夜； PBS沖洗，2 min×3次； 滴加試劑1，室溫或37 ℃孵育20 min，PBS或TBS沖洗，2 min×3次；滴加試劑2，室溫或37 ℃孵育20～30 min，PBS或TBS沖洗，2 min×3次；PBS沖洗，2 min×3次；DAB顯色劑顯色； 自來水充分沖洗，復(fù)染，脫水透明、封片。

2.5細(xì)胞因子水平的測(cè)定 免疫組化染色半定量觀察，并結(jié)合圖像數(shù)據(jù)分析，檢測(cè)創(chuàng)面組織中細(xì)胞因子PDGF、TGF-β3和TNF-α的表達(dá)量。取凍存的實(shí)驗(yàn)組和對(duì)照組燒傷大鼠各時(shí)點(diǎn)創(chuàng)面組織500 mg，在0.5 mL組織蛋白抽提試劑中加入5 μL苯甲基磺酰氟進(jìn)行勻漿，于4 ℃、10 000 r/min離心15 min，制備組織勻漿后取上清液蛋白定量，ELISA法檢測(cè)組織中PDGF、TGF-β3和TNF-α的含量，具體按照ELISA檢測(cè)試劑盒說明書操作。

3統(tǒng)計(jì)學(xué)處理

采用SPSS 18.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)處理。各項(xiàng)指標(biāo)數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，行兩因素析因設(shè)計(jì)的方差分析及LSD-t檢驗(yàn)。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1創(chuàng)面大體觀察

2組燒傷大鼠造模當(dāng)天，創(chuàng)面呈圓形，蒼白,創(chuàng)緣整齊，質(zhì)地稍硬。傷后第3天，臭氧氣浴實(shí)驗(yàn)組的創(chuàng)面略凹陷，炎性反應(yīng)不明顯，腫脹和滲出程度均較常規(guī)換藥對(duì)照組輕。傷后第7天，臭氧氣浴實(shí)驗(yàn)組的創(chuàng)面平整，呈淡紅色，邊緣清晰，肉芽組織生長(zhǎng)迅速，質(zhì)地較軟，炎性反應(yīng)程度明顯比常規(guī)換藥對(duì)照組輕，壞死組織呈黃褐色；傷后第10～14天，臭氧氣浴實(shí)驗(yàn)組的創(chuàng)面明顯縮小，壞死組織逐漸變軟、脫落，在創(chuàng)緣處可見痂殼與創(chuàng)面分離，基底紅潤(rùn)；傷后第16～18天，臭氧氣浴實(shí)驗(yàn)組大鼠造模創(chuàng)面已完全愈合，愈合的面積與造模的面積基本吻合，實(shí)驗(yàn)組所有動(dòng)物創(chuàng)面未見明顯滲出和感染現(xiàn)象。常規(guī)換藥對(duì)照組的早期創(chuàng)面腫脹凸起，色澤稍暗，炎性滲出明顯；傷后第7～14天，創(chuàng)面變硬變厚，壞死組織附著緊，肉芽生長(zhǎng)相對(duì)臭氧氣浴實(shí)驗(yàn)組遲緩；傷后第18天，創(chuàng)面壞死組織脫落，肉芽組織生長(zhǎng)，一部分大鼠創(chuàng)面愈合，一部創(chuàng)面未愈合；傷后約21 d左右全部愈合，見圖1。

2創(chuàng)面愈合率

臭氧氣浴實(shí)驗(yàn)組傷后3、7、14和21 d的創(chuàng)面愈合率明顯高于常規(guī)換藥對(duì)照組，2組比較差異有統(tǒng)計(jì)學(xué)意義(P<0.01)，見表1。

3組織學(xué)觀察

傷后當(dāng)天各組的組織切片HE染色顯示，表皮脫落壞死，真皮組織明顯損害，皮膚附件結(jié)構(gòu)大部分受損，炎性細(xì)胞浸潤(rùn)，呈現(xiàn)出深Ⅱ度燒傷創(chuàng)面病理改變。傷后第3天，臭氧氣浴實(shí)驗(yàn)組的皮下組織內(nèi)無明顯炎性細(xì)胞浸潤(rùn)，有壞死表皮脫落；傷后7天，臭氧氣浴實(shí)驗(yàn)組的創(chuàng)面可見表皮細(xì)胞、成纖維細(xì)胞(fibroblast，F(xiàn)b)和毛細(xì)血管增生，炎性細(xì)胞數(shù)較少；傷后第14天，臭氧氣浴實(shí)驗(yàn)組可見新生表皮組織向上、向創(chuàng)面方向生長(zhǎng)，肉芽組織增生活躍，F(xiàn)b及毛細(xì)血管含量更豐富，炎性細(xì)胞數(shù)更稀少；傷后第16～21天，臭氧氣浴實(shí)驗(yàn)組創(chuàng)緣上皮增生活躍，大量Fb和毛細(xì)血管朝著創(chuàng)緣方向生長(zhǎng)，鏡下可見“釘突樣”上皮島結(jié)構(gòu)，未見炎性細(xì)胞，新生表皮細(xì)胞已基本覆蓋創(chuàng)面愈合。臭氧氣浴實(shí)驗(yàn)組的創(chuàng)面各時(shí)點(diǎn)炎性反應(yīng)程度較常規(guī)換藥對(duì)照組輕，而新生毛細(xì)血管、Fb和上皮細(xì)胞增生狀況明顯優(yōu)于常規(guī)換藥對(duì)照組，見圖2。

Figure 1. Comparison of deep second-degree burn wounds of the rats in the 2 groups 10 d after injury. A: ozone gas bath group; B: routine dressing group.

圖12組大鼠深I(lǐng)I度燒傷10d創(chuàng)面的比較

表1不同時(shí)點(diǎn)2組大鼠燒傷創(chuàng)面愈合率的比較

Table 1. Comparison of wound healing rate between the 2 groups at different time points (%. Mean±SD.n=40)

Group3d7d14d21dOzonebath17．3±0．438．7±0．683．5±1．899．1±0．7Routinedressing13．1±0．3??27．6±0．2??68．0±0．6??89．5±1．6??

**P<0.01vsozone bath group.

4創(chuàng)面組織中PDGF、TGF-β3和TNF-α表達(dá)的變化

臭氧氣浴實(shí)驗(yàn)組各時(shí)點(diǎn)燒傷大鼠創(chuàng)面組織中細(xì)胞因子PDGF和TGF-β3的表達(dá)量均高于常規(guī)換藥對(duì)照組(P<0.01)；同組內(nèi)各時(shí)點(diǎn)PDGF、TGF-β3和TNF-α總體比較差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。燒傷后第3、7天，TNF-α在2組皮下組織及真皮層下部均有陽性表達(dá)；傷后第14天，常規(guī)換藥對(duì)照組的TNF-α陽性表達(dá)有所上調(diào)，臭氧氣浴組表達(dá)較弱。第21天，臭氧氣浴實(shí)驗(yàn)組的TNF-α表達(dá)下調(diào)回落，常規(guī)換藥對(duì)照組表達(dá)較強(qiáng)(P<0.01)，見圖3～5及表2、3。

Figure 2. Pathological observation of wound tissue samples in the 2 groups of scalded rats (HE staining, ×200). In ozone bath group, after the injury for 3 d, epidermal shedding, inflammatory cells in the dermis and subcutaneous tissue (↑) infiltration were observed; after the injury for 7 d, blood capillary hyperplasia and fibroblast (←) dense distribution were observed; after the injury for 14 d, a large number of epithelial migration to the surface, and visible spikes-like skin islands (←) were observed; after the injury for 21 d, the epithelial tissue completely covered the wound (←). In routine dressing group, after the injury for 3 d, inflammatory cell infiltration (↑) in epithelial tissue was observed, accompanied by necrosis of skin appendage residual contour; after the injury for 7 d, the skin deep tissue edema (→), disarrangement, inflammatory cell infiltration (↑) were observed; after the injury for 14 d, the arrangement of collagen tissue was obviously disordered; after the injury for 21 d, most of the new epithelium covered, with partial defect (↑).

圖22組燙傷大鼠創(chuàng)面組織樣本病理學(xué)觀察

Figure 3. The expression of PDGF in wound tissues in the 2 groups of scalded rats (×200). The rabbit anti-rat PDGF polyclonal antibody was used.

圖3燙傷大鼠創(chuàng)面組織樣本PDGF表達(dá)的觀察和比較

討 論

創(chuàng)面愈合是一個(gè)非常復(fù)雜的病理生理過程，大致分為炎癥期、增殖期和重塑期[1]。在這一過程中有眾多免疫細(xì)胞和細(xì)胞因子參與，啟動(dòng)機(jī)體局部和全身的免疫防御、細(xì)胞再生和組織重建過程[2]。

臭氧是一種強(qiáng)氧化劑，有強(qiáng)大的殺菌作用，使微生物細(xì)胞中的多種成分產(chǎn)生氧化，從而產(chǎn)生不可逆的變化導(dǎo)致死亡[3]。依據(jù)臭氧所公認(rèn)的殺菌消毒原理，本人前期已將臭氧氣浴體外干預(yù)燒傷創(chuàng)面分泌物中分離的致病及耐藥菌株，取得顯著殺菌效果[4]。在進(jìn)一步開展臭氧氣干預(yù)治療的動(dòng)物實(shí)驗(yàn)中發(fā)現(xiàn)，臭氧對(duì)創(chuàng)面組織的病理變化及生物學(xué)效應(yīng)會(huì)產(chǎn)生一定影響，為此，本人深入進(jìn)行了這方面的研究，并取得滿意的成果。實(shí)驗(yàn)結(jié)果顯示，臭氧能改善燒傷創(chuàng)面組織病理學(xué)變化，調(diào)控與創(chuàng)面愈合相關(guān)的細(xì)胞因子表達(dá)。

通過免疫組化半定量檢測(cè)實(shí)驗(yàn)結(jié)果顯示，臭氧氣浴干預(yù)大鼠深Ⅱ度燒傷創(chuàng)面，能促進(jìn)與創(chuàng)面愈合相關(guān)的細(xì)胞因子PDGF和TGF-β3表達(dá)，表現(xiàn)為各時(shí)點(diǎn)創(chuàng)面內(nèi)PDGF和TGF-β3陽性細(xì)胞表達(dá)量均高于常規(guī)換藥對(duì)照組。PDGF是由2個(gè)糖肽亞基構(gòu)成的復(fù)合體，對(duì)間充質(zhì)細(xì)胞和膠質(zhì)細(xì)胞增殖具有顯著的調(diào)節(jié)作用[5]。TGF-β3在創(chuàng)傷處的高表達(dá)能促進(jìn)表皮細(xì)胞的遷移運(yùn)動(dòng)，加速創(chuàng)面封閉，促進(jìn)創(chuàng)面愈合。TGF-β3能刺激Fb合成膠原、整合素等細(xì)胞外基質(zhì)及加快血管化進(jìn)程，促進(jìn)創(chuàng)傷愈合，但并不增加瘢痕的形成[6]。也有文獻(xiàn)報(bào)道，臭氧治療改善糖尿病足潰瘍的愈合，臭氧組創(chuàng)面縮小幅度明顯高于對(duì)照組,且臭氧組血清VEGF、TGF-β和PDGF在11 d中明顯高于對(duì)照組[7]。

Figure 4. The expression of TNF-α in wound tissues in the 2 groups of scalded rats (×200). The rabbit anti-rat TNF-α polyclonal antibody was used.

圖4燙傷大鼠創(chuàng)面組織樣本TNF-α表達(dá)的觀察和比較

Figure 5. The expression of TGF-β3in wound tissues in the 2 groups of scalded rats (×200). The rabbit anti-rat TGF-β3polyclonal antibody was used.

圖5燙傷大鼠創(chuàng)面組織樣本TGF-β3表達(dá)的觀察和比較

表2免疫組化半定量法檢測(cè)2組燒傷大鼠創(chuàng)面組織PDGF、TGF-β3和TNF-α表達(dá)

Table 2. The expression of PDGF, TGF-β3and TNF-α in wound tissues in the 2 groups of burned rats detected by semiquantitative immunohistochemical method (Mean±SD.n=10)

**P<0.01vsozone bath group.

表3ELISA法檢測(cè)2組燒傷大鼠創(chuàng)面組織標(biāo)本中細(xì)胞因子含量

Table 3. ELISA method was used to detect the contents of cytokines in wound tissues in the 2 groups of burned rats (ng/g protein. Mean±SD.n=10)

TimepointsafterinjuryOzonebathRoutinedressingPDGFTGF?β3TNF?αPDGFTGF?β3TNF?α3d85．5±3．472．4±3．0105．2±12．062．0±4．0??46．5±2．7??127．5±9．0??7d152．0±11．7120．9±12．3142．4±7．5100．7±12．1??74．3±4．1??190．4±13．2??14d195．8±10．0170．0±10．083．6±3．5141．6±8．2??135．6±8．4??158．3±9．5??21d173．1±7．6162．7±11．011．2±3．7112．2±11．5??149．2±7．4??65．0±7．1??

**P<0.01vsozone bath group.

TNF-α是一種促炎細(xì)胞因子，可直接損傷血管內(nèi)皮，誘導(dǎo)血管內(nèi)皮細(xì)胞產(chǎn)生血小板活化因子，促進(jìn)血栓形成[8]。TNF-α和IL-β參與機(jī)體免疫反應(yīng)和炎癥反應(yīng)的調(diào)節(jié)，若產(chǎn)生過多，或與其它細(xì)胞因子的關(guān)系失調(diào)，會(huì)引起一系列的炎性損害，阻礙創(chuàng)面愈合[9]。研究表明，臭氧氣浴實(shí)驗(yàn)組各時(shí)點(diǎn)創(chuàng)面組織中TNF-α含量均低于常規(guī)換藥對(duì)照組，說明臭氧可通過調(diào)控和降低TNF-α表達(dá)而減輕炎性反應(yīng)。

本研究通過組織病理學(xué)觀察、ELISA法和免疫組化檢測(cè)PDGF、TGF-β3、TNF-α表達(dá)等實(shí)驗(yàn)表明，臭氧氣浴干預(yù)可促進(jìn)與創(chuàng)面愈合相關(guān)的生長(zhǎng)因子PDGF和TGF-β3表達(dá)，可減輕深Ⅱ度燒傷大鼠創(chuàng)面炎性反應(yīng)，促進(jìn)血管形成、肉芽組織增生及再上皮化，縮短創(chuàng)面愈合時(shí)間，創(chuàng)面愈合率明顯高于常規(guī)換藥對(duì)照組。局部高濃度的臭氧對(duì)機(jī)體免疫調(diào)理具有一定的促進(jìn)作用，可加速患者創(chuàng)面的愈合，此外其在治療結(jié)束后會(huì)自行分解為O2，不會(huì)產(chǎn)生任何殘留和二次污染，因?yàn)橐脖环Q為“綠色環(huán)保元素”[10]。

綜上所述，臭氧可影響燒傷創(chuàng)面炎癥反應(yīng)期和增殖修復(fù)期的某些因子的表達(dá)，改善局部病理變化，促進(jìn)創(chuàng)面消炎愈合。為進(jìn)一步臨床應(yīng)用打下實(shí)驗(yàn)基礎(chǔ)。

[1] Maxson S, Lopez EA, Yoo D, et al. Concise review: role of mesenchymal stem cell in wound repair[J]. Stem Cells Transl Med, 2012, 1(2):142-149.

[2] 何曉峰. 臭氧治療的臨床應(yīng)用[M]. 北京： 科學(xué)出版社， 2009：9-11.

[3] 張 威， 王雪山， 徐業(yè)凱， 等. 臭氧對(duì)感染創(chuàng)面治療的探討[J]. 醫(yī)學(xué)信息， 2011, 24(5):2013.

[4] 趙超莉， 葉子青， 謝瓊慧， 等. 醫(yī)用臭氧氣浴干預(yù)對(duì)燒傷常見致病菌體外殺菌作用的實(shí)驗(yàn)研究[J]. 中華損傷與修復(fù)雜志：電子版，2015, 10(6):480-484.

[5] 周 俊， 羅高興， 吳 軍. 生長(zhǎng)因子促進(jìn)創(chuàng)面愈合研究進(jìn)展[J]. 中華燒傷雜志, 2010, 26(2):164-166.

[6] 李明勇， 邱 林. 轉(zhuǎn)化生長(zhǎng)因子β3與創(chuàng)面愈合及瘢痕形成關(guān)系研究進(jìn)展[J].中華燒傷雜志, 2010, 26(1):59-61.

[7] Zhang J, Guan M, Xie C, et al. Increased growth factors play a role in wound healing promoted by noninvasive oxygen-ozone therapy in diabetic patients with foot ulcers[J]. Oxid Med Cell Longev, 2014, 2014:273475.

[8] Swadzba J, Iwaniec T, Musial J. Increased level of tumor necrosis factor-α in patients with antiphospholipid syndrome: markernot only of inflammation but also of the prothrombotic state[J]. Rheumatol Int, 2011, 31(3):307-313.

[9] 王姝月， 樊 磊， 白曉智， 等. 重度燒傷大鼠血清對(duì)巨噬細(xì)胞Notch1的激活分泌功能的影響[J]. 中華損傷與修復(fù)雜志： 電子版， 2015, 10(2):120-125.

[10] 李 琳， 杜智勇， 徐 暉， 等. 濕潤(rùn)燒傷膏聯(lián)合臭氧在治療闌尾切除術(shù)切口脂肪液化的臨床研究[J]. 中國(guó)生化藥物雜志， 2015, 35(5):137-139.

(責(zé)任編輯： 陳妙玲， 羅 森)

Effect of ozone bath on pathological changes and expression of cytokines in rats with deep second-degree burns

ZHAO Chao-li, YE Zi-qing, RUAN Qiong-fang, CHEN Lan, ZHANG Wei-dong, WANG Shan, XIE Wei-guo

(InstituteofBurns,TheThirdHospitalofWuhanCity&TongrenHospitalofWuhanUniversity,Wuhan430060,China.E-mail:zhaochaoli17@sina.cn)

AIM: To investigate the effect of ozone bath on the pathological changes and the expression of cytokines, platelet-derived growth factor (PDGF), transforming growth factor-β3(TGF-β3), and tumor necrosis factor-α (TNF-α), in the wounds of deep second-degree burns in rats.METHODSMale clean-grade SD rats (n=80) were randomly divided into 2 groups, ozone bath group and routine dressing group (control group), with 40 rats in each group. Deep second-degree burn wound was established on the back of the rats, and then the examinations were conducted at 3 d, 7 d, 14 d and 21 d after burn. For the routine dressing group, the wound was cleaned by normal saline and covered with iodophor vaseline gauze every 2 d. For the ozone bath group, before the dressing, the rats were put into the clean foam box to accept ozone fumigation for 20 min (50 mg/L), and then accepted dressing change as the same as that in control group every 2 d. At each time point, the tissue specimens from these rat wounds (at wound center) were taken. The rats in ozone bath group

cleaning by saline cotton and then the ozone bath fumigation, while the rats in control group only received cleaning by saline. After that, the tissue specimens were taken again for HE staining, immunohistochemical staining and semiquantitative observation combined with image data analysis. The concentrations of the cytokines PDGF, TGF-β3and TNF-α in the wound were measured by double-antibody sandwich ELISA.RESULTSIn ozone bath group, the wounds were smooth with clear edge and slight inflammatory reaction, swelling and exudation were weaker, and the wound healing rate was higher than that in control group with significant difference. Under microscopic observation with HE staining, slighter inflammatory reaction in ozone bath group was observed than that in control group at each time point, and the numbers of fresh capillaries, fibroblasts and epithelial cells were significantly larger than those in control group. The expression levels of PDGF and TGF-β3in the wound tissue homogenate in ozone bath group were higher, and the expression level of TNF-α was significantly lower than those in control group at each time point with significant difference.CONCLUSIONThe ozone bath therapy improves the local pathological changes and promotes the expression of cytokines PDGF and TGF-β3, which are associated with wound healing, as well as reduces the expression of inflammatory mediator TNF-α in the rats with deep second-degree burns, thus promoting the wound healing and anti-inflammatory responses.

Burns; Ozone bath; Cytokines

1000- 4718(2017)11- 2067- 06

2016- 11- 23

2017- 06- 05

武漢市衛(wèi)計(jì)委臨床醫(yī)學(xué)科研項(xiàng)目[武衛(wèi)2014(92)號(hào)WX14C21]

△通訊作者 Tel： 027-68894836； E-mail: zhaochaoli17@sina.cn

R644; R363.1+23

A

10.3969/j.issn.1000- 4718.2017.11.023