CAJ | 학술논문

背景角膜的创伤或手术可导致角膜基质细胞纤维化,进而形成瘢痕.研究表明姜黄素可明显减轻组织的纤维化程度,但姜黄素是否会影响角膜基质细胞纤维化的研究尚少.目的观察不同质量浓度的姜黄素对小鼠角膜基质细胞向成纤维细胞转化过程的影响,探讨姜黄素抗角膜基质纤维化的作用.方法 150只6～8周龄BALB/c小鼠,分离角膜基质细胞并在含质量分数10%胎牛血清(FBS)的DMEM中进行培养,以原代角膜基质细胞重悬于DMEM中并分为5组:(1)空白对照组(DMEM+10%FBS,含质量分数1‰DMSO,CG组).(2)低剂量组(CG+7.5 mg/L姜黄素组).(3)中剂量组(CG+10 mg/L姜黄素组).(4)高剂量组(CG+12.5 mg/L姜黄素组).(5)无诱导剂组(DMEM,含1‰DMSO).上述因素干预7 d后,用逆转录聚合酶链反应(RT-PCR)法检测各组中细胞表型keratocan、醛脱氢酶(ALDH)、CD90、decorin、fibrenectin-1的表达.用MTS法检测姜黄素对角膜基质细胞增生的影响.制备小鼠角膜冰冻切片,采用免疫荧光技术检测角膜基质细胞内fibrenectin-1的表达.结果原代培养的角膜基质细胞呈梭形,为细胞质丰富且核大的角膜基质成纤维细胞.随着姜黄素质量浓度的增加,角膜基质细胞中keratocan mRNA、ALDH mRNA的表达量增加,CD90 mRNA和decorin mRNA的表达量减少,差异均有统计学意义(P<0.05),fibronectin-1 mRNA的表达变化差异无统计学意义(P>0.05).MTS法检测发现,随着姜黄素质量浓度的增加,对角膜基质细胞增生的抑制率逐渐增加(F=956.00,P<0.05).免疫荧光技术检测发现角膜基质细胞中fibronectin-1的表达呈红色荧光.结论姜黄素对离体小鼠角膜基质细胞纤维化有明显的抑制作用,可减轻角膜基质创伤修复过程中的过度纤维化.
배경 각막적창상혹수술가도치각막기질세포섬유화,진이형성반흔.연구표명강황소가명현감경조직적섬유화정도,단강황소시부회영향각막기질세포섬유화적연구상소.목적 관찰불동질량농도적강황소대소서각막기질세포향성섬유세포전화과정적영향,탐토강황소항각막기질섬유화적작용.방법 150지6～8주령BALB/c소서,분리각막기질세포병재함질량분수10%태우혈청(FBS)적DMEM중진행배양,이원대각막기질세포중현우DMEM중병분위5조:(1)공백대조조(DMEM+10%FBS,함질량분수1‰DMSO,CG조).(2)저제량조(CG+7.5 mg/L강황소조).(3)중제량조(CG+10 mg/L강황소조).(4)고제량조(CG+12.5 mg/L강황소조).(5)무유도제조(DMEM,함1‰DMSO).상술인소간예7 d후,용역전록취합매련반응(RT-PCR)법검측각조중세포표형keratocan、철탈경매(ALDH)、CD90、decorin、fibrenectin-1적표체.용MTS법검측강황소대각막기질세포증생적영향.제비소서각막빙동절편,채용면역형광기술검측각막기질세포내fibrenectin-1적표체.결과 원대배양적각막기질세포정사형,위세포질봉부차핵대적각막기질성섬유세포.수착강황소질량농도적증가,각막기질세포중keratocan mRNA、ALDH mRNA적표체량증가,CD90 mRNA화decorin mRNA적표체량감소,차이균유통계학의의(P<0.05),fibronectin-1 mRNA적표체변화차이무통계학의의(P>0.05).MTS법검측발현,수착강황소질량농도적증가,대각막기질세포증생적억제솔축점증가(F=956.00,P<0.05).면역형광기술검측발현각막기질세포중fibronectin-1적표체정홍색형광.결론강황소대리체소서각막기질세포섬유화유명현적억제작용,가감경각막기질창상수복과정중적과도섬유화.
Background The injury or surgery of cornea cause the proliferation of corneal stromal cells and scar formation.Recent research showed that cureumin can obviously reduce the degree of fibrosis of tissue.But if curcumm play inhibitory effect on corneal keratocytes fibrosis is rarely reported.Objecttve This studv was to investigate the effect of curcumin on the transformation of corneal keratocytes into fibroblasts in vitro and further explore the antifibrotic effect of curcumin on corneal keratocytes.Methods The murine corneal keratocytes from 150 BALB/c mice were isolated and primary culture in DMEM culture medium containing 10% fetal bovine serum and then divided into blank control group(inducer group,CG),low-dose group(CG+7.5 mg/L curcumin),mediumdose group(CG+10.0 mg/L curcumin),high-dose group(CG+12.5 mg/L curcumin),non-inducer group.Seven days following intervention,the expression of cell markers such as keratocan,aldehyde dehydrogenase(ALDH),decorin and fibronectin-1 in keratocytes were analyzed by RT-PCR.The effect of curcumin on cultured murine corneal keratocytes proliferation was evaluated by MTS technique.The expression of fibronectin-1 in murine cornea was investigated by immunofluorescence assay.Results The primarily cultured keratocytes showed tlIe fusiform-like shape with the abundant cytoplasm and big nuclei.In the presence of curcumin,the mRNA levels of keratocan and ALDH were down-regulated and those of CD90 and decorin were up-regulated,showing the significantly differences with the increase of dose(P<0.05),but the expression pf fibronectin-i was not obviously changed with the alteration of dose of curcumin. MTS showed that the inhibitory rates of curcumin on keratocytes in 10.0 mg/L and 2. 5 mg/L groups were enhanced in comparison with 7.5 mg/L group, showing statistically significant difference among three groups( F = 956.00, P<0.05). The expression of fibronectin-1 was found in the corneal keratocytes with the red fluorescence in stroma. Conclusion Curcumin can inhibit the fibrosis of corneal keratoeytes in a dose-dependent manner. These results offer a preliminary theoretical basis for the application of curcumin in controlling corneal scar formation during wound healing.