油酸对人正常和瘢痕成纤维细胞增殖和分泌炎症介质的影响
유산대인정상화반흔성섬유세포증식화분비염증개질적영향
Effect of oleic acid on the proliferation and secretion of pro-inflammatory mediators of human normal fibroblasts and scar fibroblasts
  • 万方数据
    中华烧伤杂志 중화소상잡지
    16
    2012年 6期 444-450 ,共7页

    江琼%吴伯瑜%陈晓东

    강경%오백유%진효동

    油酸%成纤维细胞%细胞增殖%一氧化氮%炎症介质 油痠%成纖維細胞%細胞增殖%一氧化氮%炎癥介質
    유산%성섬유세포%세포증식%일양화담%염증개질
    Oleic acid%Fibroblasts%Cell proliferation%Nitric oxide%Inflammatory mediator
    目的 探讨油酸对人正常Fb和瘢痕Fb增殖和分泌炎症介质的影响. 方法 体外培养人正常Fb和瘢痕Fb,分别按照随机数字表法分为7组(各组样本数为8):空白对照组,除常规成分外培养液中不再添加其他物质；乙醇对照组,培养液中加入终浓度为体积分数2％无水乙醇；不同浓度油酸组,即0.25、0.50、1.00、2.00以及4.00 mmol/L油酸组,培养液中分别加入相应终浓度的油酸(以含体积分数2％无水乙醇培养液配制).采用锥虫蓝染色法观察各组细胞培养1～5d的生长情况.于倒置相差显微镜和透射电镜下观察2种细胞2个对照组、1.00 mmol/L油酸组细胞培养2d的细胞结构,采用流式细胞仪检测2种细胞2个对照组及1.00 mmol/L油酸组细胞培养2d的细胞周期.噻唑蓝法检测各组细胞培养2d的增殖情况.取各组细胞培养2d时的培养上清液,采用改良Griess法测定NO含量,ELISA法检测TNF-α、IL-6、IL-1β、IL-8含量.对数据进行多因素方差分析及重复测量设计的方差分析. 结果 (1)正常Fb和瘢痕Fb的空白对照组与乙醇对照组比较,各指标均无明显差别.(2)培养2～5d,正常Fb和瘢痕Fb 2.00、4.00 mmol/L油酸组细胞数量均低于对应的2个对照组(F值分别为13.773、11.344,P值均小于0.01).(3)培养2d,正常Fb和瘢痕Fb1.00 mmol/L油酸组细胞数量明显减少,部分细胞开始堆积、变圆易脱落；细胞膜不完整,线粒体空泡变性,核固缩,胞内可见脂滴.(4)正常Fb 1.00 mmol/L油酸组的G0/G1期和G2/M期细胞百分比[(93.56±9.98)％、(2.01士0.75)％]显著高于空白对照组[(84.23±10.96)％、(0.37±0.16)％],F值分别为3.026、34.751,P＜0.05或P＜0.01；S期细胞百分比为(4.42 ±0.87)％,明显低于空白对照组的(16.06±1.74)％,F=136.120,P＜0.01.瘢痕Fb 1.00 mmol/L油酸组G0/G1期和G2/M期细胞百分比分别为(93.86±13.90)％、(1.89±0.66)％,显著高于空白对照组[(83.88±10.42)％、(0.41±0.17)％],F值分别为3.529、32.710,P＜0.05或P＜0.01；S期细胞百分比为(3.87±0.63)％,明显低于空白对照组的(15.89±2.02)％,F=116.508,P＜0.01.(5)正常Fb和瘢痕Fb 0.50 ～4.00 mmol/L油酸组细胞增殖率显著低于对应的2个对照组(F值分别为215.945、194.555,P＜0.05或P＜0.01).(6)正常Fb各浓度油酸组细胞分泌NO水平明显高于2个对照组(F=30.240,P＜0.05或P＜0.01)；瘢痕Fb 1.00～ 4.00 mmol/L油酸组细胞分泌NO水平明显高于2个对照组(F=12.495,P＜0.01).正常Fb和瘢痕Fb 2.00、4.00 mmol/L油酸组细胞分泌TNF-α、IL-6水平明显高于对应的2个对照组(F TNF-α值分别为6.911、3.818,FIL-6值分别为16.939、11.600,P ＜0.05或P＜0.01).正常Fb和瘢痕Fb各浓度油酸组细胞分泌IL-1β水平显著高于对应的2个对照组(F值分别为25.117、9.137,P值均小于0.01).正常Fb 1.00～4.00 mmol/L油酸组细胞分泌IL-8水平明显高于2个对照组(F=2.717,P＜0.05或P＜0.01),瘢痕Fb 2.00、4.00 mmol/L油酸组细胞分泌的IL-8水平明显高于2个对照组(F=3.338,P＜0.05).正常Fb和瘢痕Fb相同浓度油酸组各炎症因子水平比较无明显差异(F值为0.120 ～3.766,P值均大于0.05). 结论 高浓度油酸虽能抑制瘢痕Fb增殖,但同时也抑制正常Fb增殖,且高浓度油酸能同时促进正常Fb和瘢痕Fb分泌炎症介质,从而导致过度、持续的炎症反应,不利于创面愈合.
    목적 탐토유산대인정상Fb화반흔Fb증식화분비염증개질적영향. 방법 체외배양인정상Fb화반흔Fb,분별안조수궤수자표법분위7조(각조양본수위8):공백대조조,제상규성분외배양액중불재첨가기타물질；을순대조조,배양액중가입종농도위체적분수2％무수을순；불동농도유산조,즉0.25、0.50、1.00、2.00이급4.00 mmol/L유산조,배양액중분별가입상응종농도적유산(이함체적분수2％무수을순배양액배제).채용추충람염색법관찰각조세포배양1～5d적생장정황.우도치상차현미경화투사전경하관찰2충세포2개대조조、1.00 mmol/L유산조세포배양2d적세포결구,채용류식세포의검측2충세포2개대조조급1.00 mmol/L유산조세포배양2d적세포주기.새서람법검측각조세포배양2d적증식정황.취각조세포배양2d시적배양상청액,채용개량Griess법측정NO함량,ELISA법검측TNF-α、IL-6、IL-1β、IL-8함량.대수거진행다인소방차분석급중복측량설계적방차분석. 결과 (1)정상Fb화반흔Fb적공백대조조여을순대조조비교,각지표균무명현차별.(2)배양2～5d,정상Fb화반흔Fb 2.00、4.00 mmol/L유산조세포수량균저우대응적2개대조조(F치분별위13.773、11.344,P치균소우0.01).(3)배양2d,정상Fb화반흔Fb1.00 mmol/L유산조세포수량명현감소,부분세포개시퇴적、변원역탈락；세포막불완정,선립체공포변성,핵고축,포내가견지적.(4)정상Fb 1.00 mmol/L유산조적G0/G1기화G2/M기세포백분비[(93.56±9.98)％、(2.01사0.75)％]현저고우공백대조조[(84.23±10.96)％、(0.37±0.16)％],F치분별위3.026、34.751,P＜0.05혹P＜0.01；S기세포백분비위(4.42 ±0.87)％,명현저우공백대조조적(16.06±1.74)％,F=136.120,P＜0.01.반흔Fb 1.00 mmol/L유산조G0/G1기화G2/M기세포백분비분별위(93.86±13.90)％、(1.89±0.66)％,현저고우공백대조조[(83.88±10.42)％、(0.41±0.17)％],F치분별위3.529、32.710,P＜0.05혹P＜0.01；S기세포백분비위(3.87±0.63)％,명현저우공백대조조적(15.89±2.02)％,F=116.508,P＜0.01.(5)정상Fb화반흔Fb 0.50 ～4.00 mmol/L유산조세포증식솔현저저우대응적2개대조조(F치분별위215.945、194.555,P＜0.05혹P＜0.01).(6)정상Fb각농도유산조세포분비NO수평명현고우2개대조조(F=30.240,P＜0.05혹P＜0.01)；반흔Fb 1.00～ 4.00 mmol/L유산조세포분비NO수평명현고우2개대조조(F=12.495,P＜0.01).정상Fb화반흔Fb 2.00、4.00 mmol/L유산조세포분비TNF-α、IL-6수평명현고우대응적2개대조조(F TNF-α치분별위6.911、3.818,FIL-6치분별위16.939、11.600,P ＜0.05혹P＜0.01).정상Fb화반흔Fb각농도유산조세포분비IL-1β수평현저고우대응적2개대조조(F치분별위25.117、9.137,P치균소우0.01).정상Fb 1.00～4.00 mmol/L유산조세포분비IL-8수평명현고우2개대조조(F=2.717,P＜0.05혹P＜0.01),반흔Fb 2.00、4.00 mmol/L유산조세포분비적IL-8수평명현고우2개대조조(F=3.338,P＜0.05).정상Fb화반흔Fb상동농도유산조각염증인자수평비교무명현차이(F치위0.120 ～3.766,P치균대우0.05). 결론 고농도유산수능억제반흔Fb증식,단동시야억제정상Fb증식,차고농도유산능동시촉진정상Fb화반흔Fb분비염증개질,종이도치과도、지속적염증반응,불리우창면유합.
    Objective To investigate the effect of oleic acid on the proliferation and secretion of pro-inflammatory mediators of human normal fibroblasts and scar fibroblasts.Methods Human normal fibroblasts and scar fibroblasts were cultured in vitro and respectively divided into seven groups according to the random number table,with 8 samples in each group.Cells in blank control (BC) group were routinely cultured without addition of other agents.Cells in ethanol-control (EC) group were cultured with aedium with the addition of 2％ absolute ethanol.Cells in oleic acid groups were cultured with the addition of oleic acid in concentration of 0.25,0.50,1.00,2.00,or 4.00 mmol/L in 2％ absolute ethanol.The growth of cells in each group was observed with trypan blue staining on post culture day (PCD) 1-5.On PCD 2,structure of cells in BC,EC,and 1.00 mmol/L oleic acid groups was observed under inverted phase contrast microscope and transmission electron microscope ; cell cycle of BC,EC,and 1.00 mmol/L oleic acid groups was measured by flow cytometer; cell proliferation activity in each group was measured by MTT assay; the level of NO in supernatant was assayed by Griess assay; the levels of TNF-α,IL-1β,IL-6,and IL-8 in supernatants in each group were determined by enzyme-linked immunosorbent assay.Data were processed with multifactor and repeated measurement design analysis of variance.Results (1) There was no significant difference in each index of normal fibroblasts and scar fibroblasts between BC group and EC group.(2) The numbers of normal fibroblasts and scar fibroblasts in 2.00 and 4.00 mmol/L oleic acid groups were significantly lower than those in corresponding BC and EC groups on PCD 2-5 (with F values respectively 13.773and 11.344,P values all below 0.01).(3) On PCD 2,the numbers of normal fibroblasts and scar fibroblasts in 1.00 mmol/L oleic acid groups decreased,and the cells were aggregating,rounding,and easy to drop off.Cellular membrane disruption,vacuolar degeneration of mitochondrion,pyknosis,and lipid droplets were observed.(4) The percentages of G0/G1 and G2/M phases of normal fibroblasts in 1.00 mmol/L oleic acid group [(93.56 ± 9.98) ％,(2.01 ± 0.75) ％] were significantly higher than those in BC group [(84.23 ±10.96)％,(0.37 ±0.16)％,with F values respectively 3.026,34.751,P ＜0.05 or P ＜0.01],while the percentage of normal fibroblasts in S phase [(4.42 ± 0.87) ％] was markedly lower than that in BC group [(16.06 ±1.74)％,F =136.120,P ＜0.01].The percentages of scar fibroblasts of G0/G1 and G2/M phases in 1.00 mmol/L oleic acid group [(93.86 ± 13.90) ％,(1.89 ±0.66) ％] were significantly higher than those in BC group [(83.88 ± 10.42)％,(0.41 ±0.17)％,with F values respectively 3.529,32.710,P ＜ 0.05 or P ＜ 0.01],and the percentage of scar fibroblasts in S phase [(3.87 ±0.63)％] was markedly lower than that in BC group [(15.89 ±2.02)％,F =116.508,P ＜0.01].(5)The proliferation rates of normal fibroblasts and scar fibroblasts in 0.50-4.00 mmol/L oleic acid groups were significantly lower than those in corresponding BC and EC groups (with F values respectively 215.945 and 194.555,P ＜ 0.05 or P ＜ 0.01).(6) The content of NO in supernatant of normal fibroblasts in all oleic acid groups was obviously higher than that in BC and EC groups (F =30.240,P ＜ 0.05 or P ＜ 0.01).The contents of NO in supernatants of scar fibroblasts in 1.00-4.00 mmol/L oleic acid groups were significantly higher than that in BC and EC groups (F =12.495,P ＜0.01).The contents of TNF-α and IL-6 in supernatants of normal fibroblasts and scar fibroblasts in 2.00 and 4.00 mmol/L oleic acid groups were obviously higher than those in corresponding BC and EC groups (with F TNF-α values respectively 6-911,3.818,F IL-6 values respectively 16.939,11.600,P ＜ 0.05 or P ＜ 0.01).The contents of IL-1β in supematants of normal fibroblasts and scar fibroblasts in groups of every concentration of oleic acid were significantly higher than those in corresponding BC and EC groups (with F values respectively 25.117,9.137,P values all below 0.01).The contents of IL-8 in supernatants of normal fibroblasts in 1.00-4.00 mmol/L oleic acid groups were markedly higher than those in BC and EC groups (F =2.717,P ＜0.05 or P ＜0.01).The contents of IL-8 in supernatants of scar fibrob]asts in 2.00 and 4.00 mmol/L oleic acid groups were significantly higher than those in BC and EC groups (F =3.338,P ＜ 0.05).There was no statistically significant difference in above indexes between normal fibroblasts and scar fibroblasts in the same concentration of oleic acid group (with F values from 0.120 to 3.766,P values all above 0.05).Conclusions Although oleic acid in high concentration inhibits the proliferation of scar fibroblasts,it also inhibits the proliferation of normal fibroblasts.Oleic acid in high concentration can cause excessive and continued inflammatory reaction by promoting the secretion of pro-inflammatory mediators of normal fibroblasts and scar fibroblasts,and they are detrimental to wound healing.
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