中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
5期
406-411
,共6页
靳荷%罗世男%范梓晰%李杰%周卫为%李霞
靳荷%囉世男%範梓晰%李傑%週衛為%李霞
근하%라세남%범재석%리걸%주위위%리하
角膜基质细胞%创伤修复%细胞培养技术/方法%转化生长因子-β1%细胞外基质%纤维化%Ⅰ型胶原%Ⅲ型胶原
角膜基質細胞%創傷脩複%細胞培養技術/方法%轉化生長因子-β1%細胞外基質%纖維化%Ⅰ型膠原%Ⅲ型膠原
각막기질세포%창상수복%세포배양기술/방법%전화생장인자-β1%세포외기질%섬유화%Ⅰ형효원%Ⅲ형효원
Corneal keratocyte%Wound healing%Cell culture techniques/methods%Transforming growth factor-β1%Extracellular matrix%Fibrosis%Collagen type Ⅰ%Collagen type Ⅲ
背景 角膜损伤修复过程中细胞外基质(ECM)纤维化是角膜瘢痕形成的基础,转化生长因子-β1(TGF-β1)可引起角膜基质过度产生ECM.我们在前期的研究工作中构建了角膜基质三维培养模型,而将TGF-β1添加于该三维培养体系中是否可达到构建角膜基质ECM纤维化的体外三维培养模型有待研究.目的 评估TGF-β1对该三维培养模型中ECM纤维化相关基因表达的影响,确定该三维培养体系是否可以作为角膜基质ECM纤维化的体外三维培养候选模型.方法分离新鲜成年牛角膜,并在基础培养液(DMEM/F12+体积分数10％胎牛血清)中进行角膜基质细胞的培养,将5×105个牛角膜基质细胞收集于15 ml离心管中构建体外三维培养模型(Pellet),根据培养液中添加的TGF-β1质量浓度的不同分为基础培养液组(无TGF-β1)、基础培养液+0.5 ng/ml TGF-β1组和基础培养液+1.0 ng/ml TGF-β1组,于培养2周时用钙黄绿素-AM/碘化丙啶法(Calcein-AM/PI)法进行细胞活性测定;分别于培养后48 h、1周和2周应用实时荧光定量PCR(real-time PCR)和Western blot法检测各组Pellet中α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(Col Ⅰ)、ColⅢmRNA及其蛋白的表达. 结果 Pellet模型培养后48 h,角膜基质细胞开始抱团,培养后2周经Calcein AM/PI染色证实绝大多数细胞存活.培养后48 h、1周和2周,基础培养液+0.5 ng/ml TGF-β1组和基础培养液+1.0 ng/ml TGF-β1组角膜基质细胞中α-SMA、Col Ⅰ和ColⅢmRNA的相对表达量均明显高于基础培养液组,3个组间的总体差异均有统计学意义(F分组=696.745,P＜0.001;F分组=35.166,P＜0.001;F分组=33.677,P＜0.001),且随着培养时间的延长,α-SMA、Col Ⅰ和ColⅢmRNA的相对表达量逐渐升高,差异均有统计学意义(F时间=5.863,P＜0.05;F时间=298.614,P＜0.001;F时间=607.472,P＜0.001);ColⅢmRNA合成速率均大于Col Ⅰ mRNA的合成速率;Western blot检测发现,培养后48 h和1周α-SMA、Col Ⅰ和ColⅢ蛋白的表达量为微量,培养后2周基础培养液+0.5 ng/ml TGF-β1组Pellet模型中α-SMA、Col Ⅰ和ColⅢ蛋白的相对灰度表达量分别为0.395±0.208、1.060±0.175和0.629±0.382,基础培养液+1.0 ng/ml TGF-β1组o-SMA、Col Ⅰ和ColⅢ蛋白的灰度表达量分别为0.758±0.228、1.201±0.187和0.753 ±0.468,组间总体比较差异均有统计学意义(α-SMA:F=10.691,P＜0.05;Col Ⅰ:F=14.094,P＜0.05;ColⅢ:F=10.995,P＜0.05). 结论 培养液中添加TGF-β1和血清促进角膜ECM的纤维化,表现为Pellet三维培养模型中牛角膜基质细胞高表达纤维化特异性标志物.该培养体系可作为角膜基质ECM纤维化研究的体外三维培养模型.
揹景 角膜損傷脩複過程中細胞外基質(ECM)纖維化是角膜瘢痕形成的基礎,轉化生長因子-β1(TGF-β1)可引起角膜基質過度產生ECM.我們在前期的研究工作中構建瞭角膜基質三維培養模型,而將TGF-β1添加于該三維培養體繫中是否可達到構建角膜基質ECM纖維化的體外三維培養模型有待研究.目的 評估TGF-β1對該三維培養模型中ECM纖維化相關基因錶達的影響,確定該三維培養體繫是否可以作為角膜基質ECM纖維化的體外三維培養候選模型.方法分離新鮮成年牛角膜,併在基礎培養液(DMEM/F12+體積分數10％胎牛血清)中進行角膜基質細胞的培養,將5×105箇牛角膜基質細胞收集于15 ml離心管中構建體外三維培養模型(Pellet),根據培養液中添加的TGF-β1質量濃度的不同分為基礎培養液組(無TGF-β1)、基礎培養液+0.5 ng/ml TGF-β1組和基礎培養液+1.0 ng/ml TGF-β1組,于培養2週時用鈣黃綠素-AM/碘化丙啶法(Calcein-AM/PI)法進行細胞活性測定;分彆于培養後48 h、1週和2週應用實時熒光定量PCR(real-time PCR)和Western blot法檢測各組Pellet中α-平滑肌肌動蛋白(α-SMA)、Ⅰ型膠原(Col Ⅰ)、ColⅢmRNA及其蛋白的錶達. 結果 Pellet模型培養後48 h,角膜基質細胞開始抱糰,培養後2週經Calcein AM/PI染色證實絕大多數細胞存活.培養後48 h、1週和2週,基礎培養液+0.5 ng/ml TGF-β1組和基礎培養液+1.0 ng/ml TGF-β1組角膜基質細胞中α-SMA、Col Ⅰ和ColⅢmRNA的相對錶達量均明顯高于基礎培養液組,3箇組間的總體差異均有統計學意義(F分組=696.745,P＜0.001;F分組=35.166,P＜0.001;F分組=33.677,P＜0.001),且隨著培養時間的延長,α-SMA、Col Ⅰ和ColⅢmRNA的相對錶達量逐漸升高,差異均有統計學意義(F時間=5.863,P＜0.05;F時間=298.614,P＜0.001;F時間=607.472,P＜0.001);ColⅢmRNA閤成速率均大于Col Ⅰ mRNA的閤成速率;Western blot檢測髮現,培養後48 h和1週α-SMA、Col Ⅰ和ColⅢ蛋白的錶達量為微量,培養後2週基礎培養液+0.5 ng/ml TGF-β1組Pellet模型中α-SMA、Col Ⅰ和ColⅢ蛋白的相對灰度錶達量分彆為0.395±0.208、1.060±0.175和0.629±0.382,基礎培養液+1.0 ng/ml TGF-β1組o-SMA、Col Ⅰ和ColⅢ蛋白的灰度錶達量分彆為0.758±0.228、1.201±0.187和0.753 ±0.468,組間總體比較差異均有統計學意義(α-SMA:F=10.691,P＜0.05;Col Ⅰ:F=14.094,P＜0.05;ColⅢ:F=10.995,P＜0.05). 結論 培養液中添加TGF-β1和血清促進角膜ECM的纖維化,錶現為Pellet三維培養模型中牛角膜基質細胞高錶達纖維化特異性標誌物.該培養體繫可作為角膜基質ECM纖維化研究的體外三維培養模型.
배경 각막손상수복과정중세포외기질(ECM)섬유화시각막반흔형성적기출,전화생장인자-β1(TGF-β1)가인기각막기질과도산생ECM.아문재전기적연구공작중구건료각막기질삼유배양모형,이장TGF-β1첨가우해삼유배양체계중시부가체도구건각막기질ECM섬유화적체외삼유배양모형유대연구.목적 평고TGF-β1대해삼유배양모형중ECM섬유화상관기인표체적영향,학정해삼유배양체계시부가이작위각막기질ECM섬유화적체외삼유배양후선모형.방법분리신선성년우각막,병재기출배양액(DMEM/F12+체적분수10％태우혈청)중진행각막기질세포적배양,장5×105개우각막기질세포수집우15 ml리심관중구건체외삼유배양모형(Pellet),근거배양액중첨가적TGF-β1질량농도적불동분위기출배양액조(무TGF-β1)、기출배양액+0.5 ng/ml TGF-β1조화기출배양액+1.0 ng/ml TGF-β1조,우배양2주시용개황록소-AM/전화병정법(Calcein-AM/PI)법진행세포활성측정;분별우배양후48 h、1주화2주응용실시형광정량PCR(real-time PCR)화Western blot법검측각조Pellet중α-평활기기동단백(α-SMA)、Ⅰ형효원(Col Ⅰ)、ColⅢmRNA급기단백적표체. 결과 Pellet모형배양후48 h,각막기질세포개시포단,배양후2주경Calcein AM/PI염색증실절대다수세포존활.배양후48 h、1주화2주,기출배양액+0.5 ng/ml TGF-β1조화기출배양액+1.0 ng/ml TGF-β1조각막기질세포중α-SMA、Col Ⅰ화ColⅢmRNA적상대표체량균명현고우기출배양액조,3개조간적총체차이균유통계학의의(F분조=696.745,P＜0.001;F분조=35.166,P＜0.001;F분조=33.677,P＜0.001),차수착배양시간적연장,α-SMA、Col Ⅰ화ColⅢmRNA적상대표체량축점승고,차이균유통계학의의(F시간=5.863,P＜0.05;F시간=298.614,P＜0.001;F시간=607.472,P＜0.001);ColⅢmRNA합성속솔균대우Col Ⅰ mRNA적합성속솔;Western blot검측발현,배양후48 h화1주α-SMA、Col Ⅰ화ColⅢ단백적표체량위미량,배양후2주기출배양액+0.5 ng/ml TGF-β1조Pellet모형중α-SMA、Col Ⅰ화ColⅢ단백적상대회도표체량분별위0.395±0.208、1.060±0.175화0.629±0.382,기출배양액+1.0 ng/ml TGF-β1조o-SMA、Col Ⅰ화ColⅢ단백적회도표체량분별위0.758±0.228、1.201±0.187화0.753 ±0.468,조간총체비교차이균유통계학의의(α-SMA:F=10.691,P＜0.05;Col Ⅰ:F=14.094,P＜0.05;ColⅢ:F=10.995,P＜0.05). 결론 배양액중첨가TGF-β1화혈청촉진각막ECM적섬유화,표현위Pellet삼유배양모형중우각막기질세포고표체섬유화특이성표지물.해배양체계가작위각막기질ECM섬유화연구적체외삼유배양모형.
Background Extracellular matrix (ECM) fibrosis leads to corneal scaring during the process of cornea wound healing.Transforming growth factor-β1 (TGF-β1) is known to mediate overproduce of ECM components.Our previous study developed a three-dimensional model for corneal stromal cells culture in vitro.Objective The hypothesis of this study was to apply TGF-β1 in the three-dimensional culture system to establish a corneal stroma ECM fibrosis model.Methods Fresh bovine corneas were extracted for the culture of bovine keratocytes in constructed three-dimension culture system.The Pellets were cultured in the DMEM/F12+ 10％ fetal bovine serum (FBS) medium with 0.5 ng/ml or 1.0 ng/ml TGF-β1 or without TGF-β1,respectively.Calcein AM/(propidium iodide) PI staining was employed to assay the cell viability 2 weeks after culture.The expressions of α-smooth muscle actin (α-SMA),type Ⅰ collagen (Col Ⅰ) and Col Ⅲ mRNA and protein in the cells were detected by real-time PCR and Western blot respectively 48 hours,1 week and 2 weeks after cultured.The results were statistically analyzed.Results Cultured for 48 hours in the Pellet system,corneal stromal cells clustered and was identified alive by Calcein-AM/PI staining in 2 weeks.The relative expression levels of α-SMA,Col Ⅰ and Col m mRNA were elevated in both the 0.5 ng/ml and 1.0 ng/ml TGF-β1 supplement groups in comparison with the only DMEM/F12+10％ FBS group,with marked difference among the three groups (Fgroup =696.745,P＜0.001;Fgroup =35.166,P＜0.001;Fgroup =33.677,P＜0.001),and the expression levels increased with the lapse of culture time (Ftime =5.863,P＜0.05;Ftime =298.614,P＜0.001;Ftime =607.472,P＜0.001).The synthetic rate of Col Ⅲ mRNA was obviously faster than that of Col Ⅰ mRNA.Western blot showed that only a trace of α-SMA,Col Ⅰ and Col Ⅲ were detected 48 hours and 1 week after culture.The expression levels of α-SMA,Col Ⅰ and Col Ⅲ in Pellet system in 0.5 ng/ml TGF-β1 medium were 0.395±0.208,1.060±0.175 and 0.629±0.382,and in 1.0 ng/ml TGF-β1 medium were 0.758±0.228,1.201 ±0.187 and 0.753±0.468,respectively 2 weeks after culture,significant differences were shown among the three groups (α-SMA:F=10.691,P＜0.05;Col Ⅰ:F=14.094,P＜0.05;Col Ⅲ:F=10.995,P＜0.05).Conclusions Addition of TGF-β1 and serum enhance the assembly and fibrosis of ECM,showing the higher expressions of specific fibrotic markers in bovine keratocytes Pellet.This culture systerm can be used as a candidate three-dimensional model for corneal stroma ECM fibrosis.
