中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
10期
873-876
,共4页
炎性因子%晶状体%上皮细胞%细胞移行
炎性因子%晶狀體%上皮細胞%細胞移行
염성인자%정상체%상피세포%세포이행
Inflammatory cytokine%Lens%Epithelial cell%Cell migration
背景 晶状体上皮细胞(LECs)在保持晶状体的透明性方面发挥重要的作用.研究表明炎症反应参与了一些类型白内障的形成过程,且炎性因子对LECs的生物学行为产生影响,但其对LECs移行的影响尚不明确. 目的 探讨白细胞介素1α(IL-1α)、IL-1β、IL-6、肿瘤坏死因子α(TNF-α)及4种炎性因子混合物对人LECs细胞系HLEB-3迁移的影响. 方法 HLEB-3细胞用含质量分数0.5％胎牛血清的DMEM培养液进行饥饿培养,将10 μg/L的IL-1α、IL-1β、IL-6、TNF-α及这4种炎性因子的混合物分别加入培养液中作用24 h后进行细胞划痕实验和Transwell实验.于上述因子处理后即刻和24 h在倒置显微镜下观察并用数码相机记录划痕区,在200倍显微镜下计数划痕线内移行的细胞数量.Transwell实验24 h后取出小室用质量分数4％多聚甲醛溶液固定15 min,草酸铵结晶紫溶液染色2 min,于倒置显微镜下观察,选取12:00、3:00、6:00和9:00位以及中心部位5个观察区在200倍显微镜下进行细胞计数,以评价炎性因子对LECs移行的作用.用仅含0.5％胎牛血清的DMEM培养液进行培养的LECs作为空白对照组. 结果 划痕实验结果表明,各种炎性因子作用后24 h,对照组划痕区LECs数量明显低于IL-1β作用组及TNF-α作用组,差异均有统计学意义(P=0.000、0.000),混合因子组划痕区细胞数量均明显高于IL-1β作用组及TNF-α作用组,差异均有统计学意义(P=0.000、0.000)；IL-1α作用组、IL-6作用组与对照组比较,划痕区细胞数量差异均无统计学意义(P=0.600、0.098).Transwell实验显示,混合因子组作用24 h小室膜下层LECs密度最大,与IL-1β作用组、TNF-α作用组相比差异均有统计学意义(P=0.000、0.000),且IL-1 β作用组、TNF-α作用组小室膜下层LECs数量均多于对照组,差异均有统计学意义(P=0.000、0.000).IL-1α作用组和IL-6作用组可见少量穿膜细胞的生长和迁移,与对照组相比差异均无统计学意义(P=0.056、0.327). 结论 IL-1α和IL-6对LECs的移行无明显影响,IL-1β和TNF-α能够促进LECs移行,当多种炎性因子共同作用时促进LECs移行的作用强于单个细胞因子.
揹景 晶狀體上皮細胞(LECs)在保持晶狀體的透明性方麵髮揮重要的作用.研究錶明炎癥反應參與瞭一些類型白內障的形成過程,且炎性因子對LECs的生物學行為產生影響,但其對LECs移行的影響尚不明確. 目的 探討白細胞介素1α(IL-1α)、IL-1β、IL-6、腫瘤壞死因子α(TNF-α)及4種炎性因子混閤物對人LECs細胞繫HLEB-3遷移的影響. 方法 HLEB-3細胞用含質量分數0.5％胎牛血清的DMEM培養液進行饑餓培養,將10 μg/L的IL-1α、IL-1β、IL-6、TNF-α及這4種炎性因子的混閤物分彆加入培養液中作用24 h後進行細胞劃痕實驗和Transwell實驗.于上述因子處理後即刻和24 h在倒置顯微鏡下觀察併用數碼相機記錄劃痕區,在200倍顯微鏡下計數劃痕線內移行的細胞數量.Transwell實驗24 h後取齣小室用質量分數4％多聚甲醛溶液固定15 min,草痠銨結晶紫溶液染色2 min,于倒置顯微鏡下觀察,選取12:00、3:00、6:00和9:00位以及中心部位5箇觀察區在200倍顯微鏡下進行細胞計數,以評價炎性因子對LECs移行的作用.用僅含0.5％胎牛血清的DMEM培養液進行培養的LECs作為空白對照組. 結果 劃痕實驗結果錶明,各種炎性因子作用後24 h,對照組劃痕區LECs數量明顯低于IL-1β作用組及TNF-α作用組,差異均有統計學意義(P=0.000、0.000),混閤因子組劃痕區細胞數量均明顯高于IL-1β作用組及TNF-α作用組,差異均有統計學意義(P=0.000、0.000)；IL-1α作用組、IL-6作用組與對照組比較,劃痕區細胞數量差異均無統計學意義(P=0.600、0.098).Transwell實驗顯示,混閤因子組作用24 h小室膜下層LECs密度最大,與IL-1β作用組、TNF-α作用組相比差異均有統計學意義(P=0.000、0.000),且IL-1 β作用組、TNF-α作用組小室膜下層LECs數量均多于對照組,差異均有統計學意義(P=0.000、0.000).IL-1α作用組和IL-6作用組可見少量穿膜細胞的生長和遷移,與對照組相比差異均無統計學意義(P=0.056、0.327). 結論 IL-1α和IL-6對LECs的移行無明顯影響,IL-1β和TNF-α能夠促進LECs移行,噹多種炎性因子共同作用時促進LECs移行的作用彊于單箇細胞因子.
배경 정상체상피세포(LECs)재보지정상체적투명성방면발휘중요적작용.연구표명염증반응삼여료일사류형백내장적형성과정,차염성인자대LECs적생물학행위산생영향,단기대LECs이행적영향상불명학. 목적 탐토백세포개소1α(IL-1α)、IL-1β、IL-6、종류배사인자α(TNF-α)급4충염성인자혼합물대인LECs세포계HLEB-3천이적영향. 방법 HLEB-3세포용함질량분수0.5％태우혈청적DMEM배양액진행기아배양,장10 μg/L적IL-1α、IL-1β、IL-6、TNF-α급저4충염성인자적혼합물분별가입배양액중작용24 h후진행세포화흔실험화Transwell실험.우상술인자처리후즉각화24 h재도치현미경하관찰병용수마상궤기록화흔구,재200배현미경하계수화흔선내이행적세포수량.Transwell실험24 h후취출소실용질량분수4％다취갑철용액고정15 min,초산안결정자용액염색2 min,우도치현미경하관찰,선취12:00、3:00、6:00화9:00위이급중심부위5개관찰구재200배현미경하진행세포계수,이평개염성인자대LECs이행적작용.용부함0.5％태우혈청적DMEM배양액진행배양적LECs작위공백대조조. 결과 화흔실험결과표명,각충염성인자작용후24 h,대조조화흔구LECs수량명현저우IL-1β작용조급TNF-α작용조,차이균유통계학의의(P=0.000、0.000),혼합인자조화흔구세포수량균명현고우IL-1β작용조급TNF-α작용조,차이균유통계학의의(P=0.000、0.000)；IL-1α작용조、IL-6작용조여대조조비교,화흔구세포수량차이균무통계학의의(P=0.600、0.098).Transwell실험현시,혼합인자조작용24 h소실막하층LECs밀도최대,여IL-1β작용조、TNF-α작용조상비차이균유통계학의의(P=0.000、0.000),차IL-1 β작용조、TNF-α작용조소실막하층LECs수량균다우대조조,차이균유통계학의의(P=0.000、0.000).IL-1α작용조화IL-6작용조가견소량천막세포적생장화천이,여대조조상비차이균무통계학의의(P=0.056、0.327). 결론 IL-1α화IL-6대LECs적이행무명현영향,IL-1β화TNF-α능구촉진LECs이행,당다충염성인자공동작용시촉진LECs이행적작용강우단개세포인자.
Background Lens epithelial cells (LECs)play an important role in maintaining the lens transparency.Inflammatory cytokines have been clarified to participate in the formation of traumatic and after cataracts.However,the influence of inflammatory cytokines on the migration of LECs is still studying.Objective This study was to investigate the effects of interleukin-1 alpha (IL-1α),interleukin-1 beta (IL-lβ),IL-6,tumor necrosis factor alpha(TNF-α) and their mixture on the migration of human LECs in vitro.Methods HLE-B3 cells were cultured in DMEM containing 0.5％ fetal bovine serum.10 μg/L of IL-1α,IL-1β,IL-6,TNF-α or the mixture was added in the medium for 24 hours,respectively.Wound-scratch assay and transwell monolayer permeability assay were used to evaluate the effects of different cytokines on LECs migration.The migrating cell numbers at the scratch zone and the transmembrane cell numbers at 12:00,3:00,6:00,9:00 and the center areas on the transwell chamber were calculated under the inverted microscope.In the control group,LECs were cultured in DMEM with 0.5％ fetal bovine serum only.Results Wound-scratch assay revealed that 24 hours after cytokine incubation,the LECs numbers in the scratch zone were significantly less in the control group compared with the IL-1β group and TNF-αgroup (P =0.000,0.000),and those in the mixture group were higher than the IL-1β group and TNF-α group (P=O.000,0.000).However,no significant change was found in migrating LECs number between IL-1o group or IL-6 group and the control group (P =0.600,0.098).Transwell assay displayed that after 24-hour culture with cytokines,the largest density of transmembrane LECs was obtained in the mixture group,with a significant difference in comparison with the IL-1β group or TNF-α group (P=0.000,0.000).However,the transmembrane LECs were much more in the IL-1 β group or TNF-α group compared with the control group (P =0.000,0.000).Only a few transmembrane LECs were seen in the IL-1α group and IL-6 group in comparison with the control group(P =0.056,0.327).Conclusions IL-lα and IL-6 can not stimulate the migration of human LECs,but TNF-α and IL-1β can enhance their migration markedly.Mixture of these cytokines may play a much powerful influence on LECs migration.
