目的 研究大鼠全层皮肤缺损创面愈合过程中巨噬细胞的浸润及表型变化. 方法 取30只健康SD大鼠,按随机数字表法分为损伤组24只、对照组6只.损伤组大鼠于脊柱两侧用自制环钻及手术剪制成2个直径为11 mm的全层皮肤缺损创面,致伤后即刻测量创面面积,每日碘伏消毒;对照组大鼠仅行麻醉脱毛处理.伤后1、3、7、13 d,分别取损伤组6只大鼠测量创面面积(计算创面愈合率)后处死,沿创缘切取创面组织达健康筋膜层,HE染色观察组织学表现,免疫组织化学染色观察组织中巨噬细胞表面标志物CD68表达,免疫荧光染色分别观察组织中CD68与诱导型一氧化氮合酶(iNOS)双阳性(Ⅰ型巨噬细胞)、CD68与精氨酸酶1(Arg-1)双阳性(Ⅱ型巨噬细胞)表达情况,双抗体夹心ELISA法检测创面组织中γ干扰素、TNF-α、IL-4、IL-13、IL-10和IL-12的水平并计算IL-10/IL-12比值.对照组大鼠在与损伤组相同部位切取直径为11 mm的全层正常皮肤组织,同前行组织学及细胞因子检测.对数据行单因素方差分析或LSD-t检验. 结果 损伤组大鼠伤后创面逐渐缩小,伤后各时相点创面愈合率总体比较差异有统计学意义(F=358.55,P＜0.01).对照组大鼠皮肤组织形态未见异常.损伤组大鼠伤后1、3d,创面组织中炎性细胞明显浸润;伤后7、13d,可见明显血管腔结构、新生胶原.对照组大鼠正常组织和损伤组大鼠伤后1、3、7、13d创面组织每200倍视野下的CD68阳性细胞数分别为(2.7±1.5)、(31.8±3.5)、(40.8±4.7)、(20.8±2.8)、(3.2±2.4)个(F=180.55,P＜0.01).损伤组大鼠伤后1、3、7 d CD68阳性细胞数明显高于对照组(t值分别为18.81、18.79、14.05,P值均小于0.01).对照组大鼠正常组织中未见CD68与iNOS双阳性或者CD68与Arg-1双阳性细胞.损伤组大鼠伤后1、3、7、13 d CD68与iNOS双阳性细胞百分比分别为(12.2±2.8)％、(16.5±2.9)％、(4.2±2.3)％、(0.7±0.8)％(F=72.50,P＜0.01),CD68与Arg-1双阳性细胞百分比分别为0、(8.2±1.9)％、(21.5±3.4)％、(4.7±2.0)％(F=120.93,P＜0.01).损伤组大鼠伤后3 d CD68与iNOS双阳性细胞百分比显著高于组内其他时相点(t值分别为2.65、8.17、12.95,P值均小于0.05),伤后7 d CD68与Arg-1双阳性细胞百分比显著高于组内其他时相点(t值分别为15.27、8.25、10.38,P值均小于0.01).CD68与iNOS双阳性细胞百分比于伤后1、3d显著高于CD68与Arg-1双阳性细胞百分比(t值分别为10.71、5.88,P值均小于0.01),伤后7、13d则显著低于CD68与Arg-1双阳性细胞百分比(t值分别为10.24、4.60,P值均小于0.01).对照组大鼠正常组织及损伤组大鼠伤后各时相点创面组织中γ干扰素、TNF-α、IL-4、IL-13水平及IL-10/IL-12比值总体比较,差异均有统计学意义(F值为14.08 ～631.03,P值均小于0.01).与对照组比较,损伤组大鼠伤后各时相点创面组织中γ干扰素、TNF-α、IL-4和IL-13水平均显著增高(t值为4.58～ 9.17,P值均小于0.05),伤后1、3、7 d IL-10/IL-12比值明显增高(t值分别为27.70、30.51、9.49,P值均小于0.05).损伤组大鼠伤后1dγ干扰素水平[(61±5) pg/mL]及伤后3 d IL-10/IL-12比值(1.647 ±0.098),显著高于对照组及损伤组其余时相点[γ干扰素水平依次为(32±4)、(54±6)、(46±7)、(47 ±4) pg/mL,IL-10/IL-12比值依次为0.328±0.045、0.960±0.034、0.530±0.028、0.289±0.040,t值分别为3.19 ～ 8.20、16.59 ～31.84,P值均小于0.05]. 结论 大鼠全层皮肤缺损创面愈合过程中巨噬细胞浸润明显增加并呈现不同表型,其中Ⅰ型巨噬细胞出现在炎症期,而增殖期以Ⅱ型巨噬细胞为主.
目的 研究大鼠全層皮膚缺損創麵愈閤過程中巨噬細胞的浸潤及錶型變化. 方法 取30隻健康SD大鼠,按隨機數字錶法分為損傷組24隻、對照組6隻.損傷組大鼠于脊柱兩側用自製環鑽及手術剪製成2箇直徑為11 mm的全層皮膚缺損創麵,緻傷後即刻測量創麵麵積,每日碘伏消毒;對照組大鼠僅行痳醉脫毛處理.傷後1、3、7、13 d,分彆取損傷組6隻大鼠測量創麵麵積(計算創麵愈閤率)後處死,沿創緣切取創麵組織達健康觔膜層,HE染色觀察組織學錶現,免疫組織化學染色觀察組織中巨噬細胞錶麵標誌物CD68錶達,免疫熒光染色分彆觀察組織中CD68與誘導型一氧化氮閤酶(iNOS)雙暘性(Ⅰ型巨噬細胞)、CD68與精氨痠酶1(Arg-1)雙暘性(Ⅱ型巨噬細胞)錶達情況,雙抗體夾心ELISA法檢測創麵組織中γ榦擾素、TNF-α、IL-4、IL-13、IL-10和IL-12的水平併計算IL-10/IL-12比值.對照組大鼠在與損傷組相同部位切取直徑為11 mm的全層正常皮膚組織,同前行組織學及細胞因子檢測.對數據行單因素方差分析或LSD-t檢驗. 結果 損傷組大鼠傷後創麵逐漸縮小,傷後各時相點創麵愈閤率總體比較差異有統計學意義(F=358.55,P＜0.01).對照組大鼠皮膚組織形態未見異常.損傷組大鼠傷後1、3d,創麵組織中炎性細胞明顯浸潤;傷後7、13d,可見明顯血管腔結構、新生膠原.對照組大鼠正常組織和損傷組大鼠傷後1、3、7、13d創麵組織每200倍視野下的CD68暘性細胞數分彆為(2.7±1.5)、(31.8±3.5)、(40.8±4.7)、(20.8±2.8)、(3.2±2.4)箇(F=180.55,P＜0.01).損傷組大鼠傷後1、3、7 d CD68暘性細胞數明顯高于對照組(t值分彆為18.81、18.79、14.05,P值均小于0.01).對照組大鼠正常組織中未見CD68與iNOS雙暘性或者CD68與Arg-1雙暘性細胞.損傷組大鼠傷後1、3、7、13 d CD68與iNOS雙暘性細胞百分比分彆為(12.2±2.8)％、(16.5±2.9)％、(4.2±2.3)％、(0.7±0.8)％(F=72.50,P＜0.01),CD68與Arg-1雙暘性細胞百分比分彆為0、(8.2±1.9)％、(21.5±3.4)％、(4.7±2.0)％(F=120.93,P＜0.01).損傷組大鼠傷後3 d CD68與iNOS雙暘性細胞百分比顯著高于組內其他時相點(t值分彆為2.65、8.17、12.95,P值均小于0.05),傷後7 d CD68與Arg-1雙暘性細胞百分比顯著高于組內其他時相點(t值分彆為15.27、8.25、10.38,P值均小于0.01).CD68與iNOS雙暘性細胞百分比于傷後1、3d顯著高于CD68與Arg-1雙暘性細胞百分比(t值分彆為10.71、5.88,P值均小于0.01),傷後7、13d則顯著低于CD68與Arg-1雙暘性細胞百分比(t值分彆為10.24、4.60,P值均小于0.01).對照組大鼠正常組織及損傷組大鼠傷後各時相點創麵組織中γ榦擾素、TNF-α、IL-4、IL-13水平及IL-10/IL-12比值總體比較,差異均有統計學意義(F值為14.08 ～631.03,P值均小于0.01).與對照組比較,損傷組大鼠傷後各時相點創麵組織中γ榦擾素、TNF-α、IL-4和IL-13水平均顯著增高(t值為4.58～ 9.17,P值均小于0.05),傷後1、3、7 d IL-10/IL-12比值明顯增高(t值分彆為27.70、30.51、9.49,P值均小于0.05).損傷組大鼠傷後1dγ榦擾素水平[(61±5) pg/mL]及傷後3 d IL-10/IL-12比值(1.647 ±0.098),顯著高于對照組及損傷組其餘時相點[γ榦擾素水平依次為(32±4)、(54±6)、(46±7)、(47 ±4) pg/mL,IL-10/IL-12比值依次為0.328±0.045、0.960±0.034、0.530±0.028、0.289±0.040,t值分彆為3.19 ～ 8.20、16.59 ～31.84,P值均小于0.05]. 結論 大鼠全層皮膚缺損創麵愈閤過程中巨噬細胞浸潤明顯增加併呈現不同錶型,其中Ⅰ型巨噬細胞齣現在炎癥期,而增殖期以Ⅱ型巨噬細胞為主.
목적 연구대서전층피부결손창면유합과정중거서세포적침윤급표형변화. 방법 취30지건강SD대서,안수궤수자표법분위손상조24지、대조조6지.손상조대서우척주량측용자제배찬급수술전제성2개직경위11 mm적전층피부결손창면,치상후즉각측량창면면적,매일전복소독;대조조대서부행마취탈모처리.상후1、3、7、13 d,분별취손상조6지대서측량창면면적(계산창면유합솔)후처사,연창연절취창면조직체건강근막층,HE염색관찰조직학표현,면역조직화학염색관찰조직중거서세포표면표지물CD68표체,면역형광염색분별관찰조직중CD68여유도형일양화담합매(iNOS)쌍양성(Ⅰ형거서세포)、CD68여정안산매1(Arg-1)쌍양성(Ⅱ형거서세포)표체정황,쌍항체협심ELISA법검측창면조직중γ간우소、TNF-α、IL-4、IL-13、IL-10화IL-12적수평병계산IL-10/IL-12비치.대조조대서재여손상조상동부위절취직경위11 mm적전층정상피부조직,동전행조직학급세포인자검측.대수거행단인소방차분석혹LSD-t검험. 결과 손상조대서상후창면축점축소,상후각시상점창면유합솔총체비교차이유통계학의의(F=358.55,P＜0.01).대조조대서피부조직형태미견이상.손상조대서상후1、3d,창면조직중염성세포명현침윤;상후7、13d,가견명현혈관강결구、신생효원.대조조대서정상조직화손상조대서상후1、3、7、13d창면조직매200배시야하적CD68양성세포수분별위(2.7±1.5)、(31.8±3.5)、(40.8±4.7)、(20.8±2.8)、(3.2±2.4)개(F=180.55,P＜0.01).손상조대서상후1、3、7 d CD68양성세포수명현고우대조조(t치분별위18.81、18.79、14.05,P치균소우0.01).대조조대서정상조직중미견CD68여iNOS쌍양성혹자CD68여Arg-1쌍양성세포.손상조대서상후1、3、7、13 d CD68여iNOS쌍양성세포백분비분별위(12.2±2.8)％、(16.5±2.9)％、(4.2±2.3)％、(0.7±0.8)％(F=72.50,P＜0.01),CD68여Arg-1쌍양성세포백분비분별위0、(8.2±1.9)％、(21.5±3.4)％、(4.7±2.0)％(F=120.93,P＜0.01).손상조대서상후3 d CD68여iNOS쌍양성세포백분비현저고우조내기타시상점(t치분별위2.65、8.17、12.95,P치균소우0.05),상후7 d CD68여Arg-1쌍양성세포백분비현저고우조내기타시상점(t치분별위15.27、8.25、10.38,P치균소우0.01).CD68여iNOS쌍양성세포백분비우상후1、3d현저고우CD68여Arg-1쌍양성세포백분비(t치분별위10.71、5.88,P치균소우0.01),상후7、13d칙현저저우CD68여Arg-1쌍양성세포백분비(t치분별위10.24、4.60,P치균소우0.01).대조조대서정상조직급손상조대서상후각시상점창면조직중γ간우소、TNF-α、IL-4、IL-13수평급IL-10/IL-12비치총체비교,차이균유통계학의의(F치위14.08 ～631.03,P치균소우0.01).여대조조비교,손상조대서상후각시상점창면조직중γ간우소、TNF-α、IL-4화IL-13수평균현저증고(t치위4.58～ 9.17,P치균소우0.05),상후1、3、7 d IL-10/IL-12비치명현증고(t치분별위27.70、30.51、9.49,P치균소우0.05).손상조대서상후1dγ간우소수평[(61±5) pg/mL]급상후3 d IL-10/IL-12비치(1.647 ±0.098),현저고우대조조급손상조기여시상점[γ간우소수평의차위(32±4)、(54±6)、(46±7)、(47 ±4) pg/mL,IL-10/IL-12비치의차위0.328±0.045、0.960±0.034、0.530±0.028、0.289±0.040,t치분별위3.19 ～ 8.20、16.59 ～31.84,P치균소우0.05]. 결론 대서전층피부결손창면유합과정중거서세포침윤명현증가병정현불동표형,기중Ⅰ형거서세포출현재염증기,이증식기이Ⅱ형거서세포위주.
Objective To study the infiltration of macrophages and their phenotype in the healing process of full-thickness wound in rat.Methods Thirty healthy SD rats were divided into control group (n =6) and injury group (n =24) according to the random number table.Two round full-thickness skin defects (11 mm diameter) were created on both sides of dorsal spine of rats in injury group with surgical scissors and homemade trephine.After injury,wound area was measured immediately.The wounds were disinfected with iodophor every day.Rats in control group received anesthesia and hair removal only.On post injury day (PID) 1,3,7,and 13,respectively,6 rats of injury group were sacrificed after the measurement of wound area (wound healing rate was calculated).Wound samples were obtained by excision down to healthy fascia along wound edge.Histological study was done with HE staining.The expression of CD68 (the surface marker of macrophage) in the wound tissue was observed with immunohistochemical staining.The double positive expressions of induced nitric oxide synthase (iNOS) plus CD68 (type Ⅰ macrophage) and arginase 1 (Arg-1)plus CD68 (type Ⅱ macrophage)were observed with immunofluorescence staining.The levels of interferon-γ (IFN-γ),TNF-o,IL-4,IL-13,IL-10,and IL-12 in wound tissue were assayed by double-antibody sandwich ELISA,and the ratio of IL-10/IL-12 was calculated.Full-thickness skin tissues (11 mm diameter) in rats of control group were excised at the same site as rats in injury group,and the histological observation and cytokines assay were performed as well.Data were processed with one-way analysis of variance or LSD-t test.Results Wound area of rats in injury group was gradually reduced after injury,and the overall difference of the wound healing rate on each PID was statistically significant (F =358.55,P ＜0.01).No abnormal appearance of skin tissue was observed in rats of control group.In injury group,inflammatory cell infiltration was obvious in wound tissue on PID 1 and 3; vascular structure and fresh collagen were observed in wound tissue on PID 7 and 13.Numbers of CD68 positive cells in skin tissue of rats in control group and wound tissue of rats in injury group on PID 1,3,7,and 13 were respectively (2.7±1.5),(31.8 ±3.5),(40.8 ±4.7),(20.8 ±2.8),(3.2 ±2.4) per 200 times visual field (F =180.55,P ＜0.01).Compared with that in control group,the number of CD68 positive cells of rats in injury group was increased on PID 1,3,and 7 (with t values respectively 18.81,18.79,14.05,P values below 0.01).No double positive expression of iNOS plus CD68 or Arg-1 plus CD68 was observed in normal tissue of rats in control group.In injury group,proportions of iNOS plus CD68 double positive cells on PID 1,3,7,and 13 were respectively (12.2±2.8)％,(16.5 ±2.9)％,(4.2 ±2.3)％,(0.7 ±0.8)％ (F =72.50,P ＜0.01); proportions of Arg-1 plus CD68 double positive cells on PID 1,3,7,and 13 were respectively 0,(8.2±1.9)％,(21.5±3.4)％,(4.7±2.0)％ (F =120.93,P ＜0.01).In injury group,proportion of iNOS plus CD68 double positive cells on PID 3 was significantly higher than that on other PID (with t values respectively 2.65,8.17,12.95,P values below 0.05) ; proportion of Arg1 plus CD68 double positive cells on PID 7 was higher than that on other PID (with t values respectively 15.27,8.25,10.38,P values below 0.01).Compared with that of Arg-1 plus CD68 double positive cells,proportion of iNOS plus CD68 double positive cells was higher on PID 1 and 3 (with t values respectively 10.71 and 5.88,P values below 0.01) and lower on PID 7 and 13 (with t values respectively 10.24 and 4.60,P values below 0.01).The overall differences of IFN-γ,TNF-α,IL-4,IL-13,and IL-10/IL-12 ratio in skin tissue of rats in control group and wound tissue of rats in injury group on every PID were statistically significant (with F values from 14.08 to 631.03,P values below 0.01).Compared with those in control group,levels of IFN-γ,TNF-α,IL-4,and IL-13 in wound tissue of rats in injury group were significantly higher on every PID (with t values from 4.58 to 9.17,P values below 0.05),while IL-10/IL-12 ratio was significantly higher on PID 1,3,and 7 (with t values respectively 27.70,30.51,9.49,P values below 0.05).In injury group,IFN-γ level on PID 1 [(61 ± 5) pg/mL] and IL-10/IL-12 ratio on PID 3 (1.647 ± 0.098)were significantly higher than those of control group and those on other PID in injury group [with IFN-γ level respectively (32 ±4),(54 ±6),(46 ±7),(47 ±4) pg/mL and IL-10/IL-12 ratio respectively0.328 ± 0.045,0.960 ±0.034,0.530 ±0.028,0.289 ±0.040,with t values respectively from 3.19 to 8.20 and from 16.59 to 31.84,P values below 0.05].Conclusions Macrophage infiltration increases in the healing process of full-thickness wound in rat with different phenotypes,among which type Ⅰ macrophage appears in the inflammatory stage,and type Ⅱ macrophage predominates in the proliferative stage.
