中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
8期
695-698
,共4页
人%成纤维细胞%Tenon囊%结缔组织生长因子%转分化%青光眼/手术%滤过泡%瘢痕化
人%成纖維細胞%Tenon囊%結締組織生長因子%轉分化%青光眼/手術%濾過泡%瘢痕化
인%성섬유세포%Tenon낭%결체조직생장인자%전분화%청광안/수술%려과포%반흔화
Humans%Fibroblasts%Tenon capsule%Connective tissue growth factor%Transdifferentiation%Glaucoma/surgery%Bleb%Scarring
背景 青光眼滤过手术后滤过通道的瘢痕化是手术失败的主要原因,其主要病理机制是成纤维细胞的异常增生、间质-上皮转分化及细胞外基质的重塑.结缔组织生长因子(CTGF)是促进瘢痕形成的关键因子,而CTGF是否能促进人Tenon囊成纤维细胞(HTFs)的间质-上皮转分化尚不清楚.目的 观察CTGF对HTFs间质-上皮转分化的影响.方法 用体积分数10％胎牛血清的DMEM高糖型培养液进行HTFs体外常规培养和传代,取3～6代细胞用于实验.将培养的HTFs分为空白对照组和CTGF处理组,空白对照组用DMEM完全培养液培养细胞,CTGF处理组在培养液中加入CTGF,使终质量浓度为50 ng/ml.细胞培养后48 h,采用细胞免疫荧光染色技术鉴定HTFs中上皮钙黏素(E-cadherin)蛋白的表达,采用Western blot法对HTFs中E-cadherin蛋白的表达量进行检测.结果 空白对照组及CTGF处理组HTFs均生长良好,呈长梭形,漩涡状排列.细胞免疫荧光染色显示,CTGF处理组HTFs细胞质呈红色荧光,细胞核呈蓝色荧光;空白对照组HTFs仅见DAPI蓝染的细胞核,无E-cadherin表达的红色荧光.Western blot法检测结果显示,空白对照组HTFs中E-cadherin蛋白无表达,而CTGF处理组HTFs中E-cadherin蛋白的相对表达量为0.63±0.08.结论 间叶组织来源的成纤维细胞本身不表达E-cadherin,在CTGF的刺激下成纤维细胞能够表达E-cadherin,CTGF促进HTFs的间质-上皮转分化.
揹景 青光眼濾過手術後濾過通道的瘢痕化是手術失敗的主要原因,其主要病理機製是成纖維細胞的異常增生、間質-上皮轉分化及細胞外基質的重塑.結締組織生長因子(CTGF)是促進瘢痕形成的關鍵因子,而CTGF是否能促進人Tenon囊成纖維細胞(HTFs)的間質-上皮轉分化尚不清楚.目的 觀察CTGF對HTFs間質-上皮轉分化的影響.方法 用體積分數10％胎牛血清的DMEM高糖型培養液進行HTFs體外常規培養和傳代,取3～6代細胞用于實驗.將培養的HTFs分為空白對照組和CTGF處理組,空白對照組用DMEM完全培養液培養細胞,CTGF處理組在培養液中加入CTGF,使終質量濃度為50 ng/ml.細胞培養後48 h,採用細胞免疫熒光染色技術鑒定HTFs中上皮鈣黏素(E-cadherin)蛋白的錶達,採用Western blot法對HTFs中E-cadherin蛋白的錶達量進行檢測.結果 空白對照組及CTGF處理組HTFs均生長良好,呈長梭形,漩渦狀排列.細胞免疫熒光染色顯示,CTGF處理組HTFs細胞質呈紅色熒光,細胞覈呈藍色熒光;空白對照組HTFs僅見DAPI藍染的細胞覈,無E-cadherin錶達的紅色熒光.Western blot法檢測結果顯示,空白對照組HTFs中E-cadherin蛋白無錶達,而CTGF處理組HTFs中E-cadherin蛋白的相對錶達量為0.63±0.08.結論 間葉組織來源的成纖維細胞本身不錶達E-cadherin,在CTGF的刺激下成纖維細胞能夠錶達E-cadherin,CTGF促進HTFs的間質-上皮轉分化.
배경 청광안려과수술후려과통도적반흔화시수술실패적주요원인,기주요병리궤제시성섬유세포적이상증생、간질-상피전분화급세포외기질적중소.결체조직생장인자(CTGF)시촉진반흔형성적관건인자,이CTGF시부능촉진인Tenon낭성섬유세포(HTFs)적간질-상피전분화상불청초.목적 관찰CTGF대HTFs간질-상피전분화적영향.방법 용체적분수10％태우혈청적DMEM고당형배양액진행HTFs체외상규배양화전대,취3～6대세포용우실험.장배양적HTFs분위공백대조조화CTGF처리조,공백대조조용DMEM완전배양액배양세포,CTGF처리조재배양액중가입CTGF,사종질량농도위50 ng/ml.세포배양후48 h,채용세포면역형광염색기술감정HTFs중상피개점소(E-cadherin)단백적표체,채용Western blot법대HTFs중E-cadherin단백적표체량진행검측.결과 공백대조조급CTGF처리조HTFs균생장량호,정장사형,선와상배렬.세포면역형광염색현시,CTGF처리조HTFs세포질정홍색형광,세포핵정람색형광;공백대조조HTFs부견DAPI람염적세포핵,무E-cadherin표체적홍색형광.Western blot법검측결과현시,공백대조조HTFs중E-cadherin단백무표체,이CTGF처리조HTFs중E-cadherin단백적상대표체량위0.63±0.08.결론 간협조직래원적성섬유세포본신불표체E-cadherin,재CTGF적자격하성섬유세포능구표체E-cadherin,CTGF촉진HTFs적간질-상피전분화.
Background Scarring of filtration channel following glaucoma filtering surgery is a main cause of the failure of the surgery.The proliferation,epithelial-mesenchymal transition and extracellular matrix remodeling of fibroblasts are thought to be the primary pathological mechanism of scarring.Connective tissue growth factor (CTGF) plays a promoting role in the formation of scar.Whether CTGF participates in mesenchymal-epithelial transition of human Tenon capsule fibroblasts (HTFs) is not clear yet.Objective This study attempted to investigate the effect of CTGF on the mesenchymal-epithelial transition of HTFs in vitro.Methods HTFs were cultured and passaged in high glucose DMEM medium with 10％ fetal bovine serum,and the cells of generation 3-6 were used in this experiment.The cells were divided into the blank control group and CTGF-treated group and were routinely cultured in the blank control group.CTGF was added in the medium in the CTGF-treated group,with the final concentration 50 ng/ml CTGF.Immunofluorescence staining was used to identify and locate the expression of E-cadherin protein in the cells,and Western blot assay was employed to quantitatively analyze the expression level of E-cadherin protein in 48 hours after culture.Results The HTFs grew well with the spindle-like shape and vortex-like arrangement.The red fluorescence (E-cadherin protein) in the cytoplasm and blue fluorescence in the cellular nucleus were seen in the CTGF-treated group,but only nucleus with blue fluorescence were obtained in the blank control group.Western blot assay showed that the E-cadherin protein expression was absent in the blank control group,however,the relative expression level of E-cadherin protein in the cells was 0.63± 0.08.Conclusions E-cadherin protein is not expressed in fibroblast derived from mesenchymal tissue.However,CTGF can induce the expression of E-cadherin in HTFs.This study suggests that CTGF promotes the mesenchymal-epithelial transition of HTFs in vitro.
