维生素A棕榈酸酯对兔机械性角膜上皮损伤愈合及结膜杯状细胞的作用研究
유생소A종려산지대토궤계성각막상피손상유합급결막배상세포적작용연구
Research on effects of vitamin A palmitate on repair of mechanical corneal epithelial defects and conjunctival goblet cells in rabbits
  • 万方数据
    中华眼科杂志 중화안과잡지
    Chinese Journal of Ophthalmology
    2010年 2期 151-160 ,共10页

    邱晓頔%龚岚%陈敏洁

    구효적%공람%진민길

    上皮%角膜%伤口愈合%杯状细胞%维生素A%成纤维细胞生长因子2 上皮%角膜%傷口愈閤%杯狀細胞%維生素A%成纖維細胞生長因子2
    상피%각막%상구유합%배상세포%유생소A%성섬유세포생장인자2
    Epithelium,corneal%Wound healing%Goblet cells%Vitamin A%Fibroblast growth factor 2
    目的 探讨维生素A棕榈酸酯与重组牛碱性成纤维细胞生长因子对兔机械性角膜上皮损伤愈合及结膜上皮细胞、杯状细胞的作用.方法 实验研究.选取雄性新西兰大白兔120只,建立机械性角膜上皮损伤模型(角膜中央直径8 mm上皮刮除),随机数字表法分为4组,每组30只.A组使用盐酸林可霉素滴眼液,B组合用维生素A棕榈酸酯凝胶与盐酸林可霉素滴眼液,C组合用重组牛碱性成纤维细胞生长因子凝胶与盐酸林可霉素滴眼液,D组合用重组牛碱性成纤维细胞生长因子凝胶与维生素A棕榈酸酯凝胶及盐酸林可霉素滴眼液.各组均于机械性角膜上皮损伤建模后当日开始用药.用法均为3次/d,1滴/次,观察时间为7 d.在模型建立第0、1、4、7天共4个时间点采用前节裂隙灯显微镜照相系统进行眼表照相,并计算角膜上皮损伤及其修复面积;在模型建立前及建立后第1、4、7天共4个时间点进行角结膜组织透射电镜检查、角膜光镜组织学检查及结膜印迹细胞学检查,对角膜上皮、结膜上皮细胞修复情况及结膜杯状细胞形态数量进行分析.采用方差分析、Tukey显著性检验等对数据进行统计学分析.结果 造模后第1天各组角膜上皮已愈合面积比较差异有统计学意义(F=17.663,P=0.000),角膜上皮已愈合面积A组(53.512±18.850)mm~2,B组(92.194±14.367)mm~2,C组(89.779±20.535)mm~2,D组(127.816±16.379)mm~2,B组与C组之间差异无统计学意义(P=0.995);结膜印迹细胞学检查显示,各组结膜杯状细胞数量(每740μm×550μm 面积)明显下降,A组(10.083±4.441)个,B组(10.667±3.551)个,C组(9.583±4.502)个,D组(9.167±5.606)个,但各组之间的差异无统计学意义(F=0.239,P=0.868).造模后第4天各组角膜上皮已愈合面积比较差异有统计学意义(F=37.665,P=0.000),角膜上皮已愈合面积A组(120.369±11.839)mm~2,B组(156.606±8.087)mm~2,C组(154.216±9.990)mm~2,D组(175.181±5.168)mm~2,B组与C组之间差异无统计学意义(P=0.968);结膜印迹细胞学检查显示,各组结膜杯状细胞数量(每740 μm ×550 μm 面积)开始恢复,A组(41.250±4.575)个,B组(56.083±6.374)个,C组(48.417±4.562)个,D组(61.917±5.017)个,各组之间比较差异有统计学意义(F=36.210,P=0.000).造模后第7天各组角膜上皮已愈合面积A组(177.472±3.585)mm~2,B组(186.715±3.022)mm~2,C组(182.293±3.158)mm~2,D组(194.106±2.176)mm~2,D组角膜上皮已愈合面积大于其他3组(P<0.05);结膜印迹细胞学检查显示,各组结膜杯状细胞数量(每 740 μm×550 μm面积)明显恢复,A组(63.167±11.488)个,B组(99.501±15.877)个,C组(82.015±9.175)个,D组(104.750±9.659)个,各组之间比较差异有统计学意义(F=30.312,P=0.000),B组与D组之间比较差异无统计学意义(P=0.700).透射电镜下观察角膜组织,维生素A棕榈酸酯和重组牛碱性成纤维细胞生长因子均能促进角膜上皮细胞之间连接的建立,维生素A棕榈酸酯具有保护角膜上皮细胞、防止上皮角化、促进角膜上皮增殖分化的作用.电镜检查结膜组织,可见B、D组新生的结膜杯状细胞数量较多,胞内分泌颗粒密集,与A、C组有明显差异,B、C、D组新生的结膜上皮细胞连接紧密.结论 维生素A棕榈酸酯和重组牛碱性成纤维细胞生长因子均能够有效促进机械性角膜上皮损伤的角膜上皮修复,合并用药时作用最明显.维生素A棕榈酸酯可促进结膜杯状细胞的再生,促进结膜上皮细胞间连接的建立,明显优于重组牛碱性成纤维细胞生长因子.
    목적 탐토유생소A종려산지여중조우감성성섬유세포생장인자대토궤계성각막상피손상유합급결막상피세포、배상세포적작용.방법 실험연구.선취웅성신서란대백토120지,건립궤계성각막상피손상모형(각막중앙직경8 mm상피괄제),수궤수자표법분위4조,매조30지.A조사용염산림가매소적안액,B조합용유생소A종려산지응효여염산림가매소적안액,C조합용중조우감성성섬유세포생장인자응효여염산림가매소적안액,D조합용중조우감성성섬유세포생장인자응효여유생소A종려산지응효급염산림가매소적안액.각조균우궤계성각막상피손상건모후당일개시용약.용법균위3차/d,1적/차,관찰시간위7 d.재모형건립제0、1、4、7천공4개시간점채용전절렬극등현미경조상계통진행안표조상,병계산각막상피손상급기수복면적;재모형건립전급건립후제1、4、7천공4개시간점진행각결막조직투사전경검사、각막광경조직학검사급결막인적세포학검사,대각막상피、결막상피세포수복정황급결막배상세포형태수량진행분석.채용방차분석、Tukey현저성검험등대수거진행통계학분석.결과 조모후제1천각조각막상피이유합면적비교차이유통계학의의(F=17.663,P=0.000),각막상피이유합면적A조(53.512±18.850)mm~2,B조(92.194±14.367)mm~2,C조(89.779±20.535)mm~2,D조(127.816±16.379)mm~2,B조여C조지간차이무통계학의의(P=0.995);결막인적세포학검사현시,각조결막배상세포수량(매740μm×550μm 면적)명현하강,A조(10.083±4.441)개,B조(10.667±3.551)개,C조(9.583±4.502)개,D조(9.167±5.606)개,단각조지간적차이무통계학의의(F=0.239,P=0.868).조모후제4천각조각막상피이유합면적비교차이유통계학의의(F=37.665,P=0.000),각막상피이유합면적A조(120.369±11.839)mm~2,B조(156.606±8.087)mm~2,C조(154.216±9.990)mm~2,D조(175.181±5.168)mm~2,B조여C조지간차이무통계학의의(P=0.968);결막인적세포학검사현시,각조결막배상세포수량(매740 μm ×550 μm 면적)개시회복,A조(41.250±4.575)개,B조(56.083±6.374)개,C조(48.417±4.562)개,D조(61.917±5.017)개,각조지간비교차이유통계학의의(F=36.210,P=0.000).조모후제7천각조각막상피이유합면적A조(177.472±3.585)mm~2,B조(186.715±3.022)mm~2,C조(182.293±3.158)mm~2,D조(194.106±2.176)mm~2,D조각막상피이유합면적대우기타3조(P<0.05);결막인적세포학검사현시,각조결막배상세포수량(매 740 μm×550 μm면적)명현회복,A조(63.167±11.488)개,B조(99.501±15.877)개,C조(82.015±9.175)개,D조(104.750±9.659)개,각조지간비교차이유통계학의의(F=30.312,P=0.000),B조여D조지간비교차이무통계학의의(P=0.700).투사전경하관찰각막조직,유생소A종려산지화중조우감성성섬유세포생장인자균능촉진각막상피세포지간련접적건립,유생소A종려산지구유보호각막상피세포、방지상피각화、촉진각막상피증식분화적작용.전경검사결막조직,가견B、D조신생적결막배상세포수량교다,포내분비과립밀집,여A、C조유명현차이,B、C、D조신생적결막상피세포련접긴밀.결론 유생소A종려산지화중조우감성성섬유세포생장인자균능구유효촉진궤계성각막상피손상적각막상피수복,합병용약시작용최명현.유생소A종려산지가촉진결막배상세포적재생,촉진결막상피세포간련접적건립,명현우우중조우감성성섬유세포생장인자.
    Objective Randomized controlled experimental study to investigate the influence of vitamin A palmitate and bovine recombinant basic fiboblast growth factor(bFGF)on repair of mechanical corneal epithelial defects,conjunctival epithelial cells and goblet cells in rabbits.Methods One hundred and twenty New Zealand rabbits (all males) were selected to establish the mechanical corneal epithelial defects models (scratching out a round area with the diameter of 8 mm in the centre of cornea).Forty eight New Zealand rabbits were randomly divided into 4 groups:group A used lincomycin hydrochloride eye drops (LED)after the model had been established;group B used vitamin A palmitate eye gel and LED;group C used recombinant bFGF eye gel and LED;group D used vitamin A palmitate eye gel,bFGF eye gel and LED.Photo slit lamp examination and measurement of repaired area were performed on day O,day 1,day 4 and day 7;transmission electron microscopy,histological microscope examination and impression cytology were performed on day O,day 1,day 4 and day 7 to analysis the morphology and repairment of corneal epithelium,conjunctival epithelial cells and the goblet cells.The variants were tested using analysis of variance and Tukey's test.Results Statistic analysis showed that on day 1,the size of areas of repaired corneal epithelium was:group A(53.512±18.850)mm~2,group B(92.194±14.367)mm~2,group C (89.779±20.535)mm~2,group D(127.816±16.379)mm~2.The difference in size of repaired areas between different groups was statistically significant(F=17.663,P=0.000),with exception of the difference between groups B and C(P=0.995).Conjunctival impression cytology showed that,the average number of conjunctival goblet cells per 740 μm×550 μm at day 1 was decreased,group A(10.083±4.441),group B(10.667±3.551),group C(9.583±4.502),group D(9.167±5.606).The difference between these four groups was not significant(F=0.239,P=0.868).At day 4,the size of areas of repaired corneal epithelium was;group A(120.369±11.839)mm~2,group B(156.606±8.087)mm~2,group C (154.216±9.990)mm~2,group D(175.181±5.168)mm~2,which showed a significant difference between each two groups (F=37.665,P=0.000),with exception between groups B and C(P=0.968).The average number of goblet cells at day 4 was recovered,which was:group A(41.250±4.575),group B (56.083 ±6.374),group C(48.417±4.562),group D(61.917±5.017),with a significant difference between these four groups(F=36.210,P=0.000).At day 7,the size of areas of repaired corneal epithelium had a statistical significance(F=32.656,P=0.000)between these four groups,which was group A(177.472±3.585)mm~2,group B(186.715±3.022)mm~2,group C(182.293±3.158)mm~2,group D (194.106±2.176)mm~2.The area of repaired corneal epithelium in group D was larger than that of other groups(P<0.05).The average number of goblet cells was recovered significantly,which was:group A (63.167 ±11.488),group B(99.501±15.877),group C(82.015±9.175),group D(104.750±9.659).There was a significant difference in goblet cell number between these groups(F=30.312,P=0.000)with exception between groups B and D(P=0.700).In transmission electron microscope examination of the cornea,we found that vitamin A palmitate and bFGF could both promote the development of intracellular conjunction.Vitamin A palmitate protected corneal epithelial cells,prevented cell keratinization,promoted proliferation and differentiation of corneal epithelial cells.In transmission electron microscopy examination of the conjunctiva,conjunctival goblet cells in groups B and D recovered well with rich secretary granules,which were quite different from groups A and C.Conjunctival epithelium of groups B,C and D were well-differentiated with tight intracellular conjunction.Conclusions Vitamin A palmitate and bFGF could promote the repair of mechanical corneal epithelial defects and the development of intracellular conjunction.The effect is more significant when vitamin A palmitate is combined with bFGF.Vitamin A palmitate promotes regeneration of conjunctival goblet cells and can re-establish intracellular conjunction of conjunctival epithelium.The protective effect of vitamin A palmitate on conjunctival goblet cells is better than that of bFGF.
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