中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
10期
930-934
,共5页
崔琨明%张凤妍%祈颖%王蒙蒙%高航
崔琨明%張鳳妍%祈穎%王矇矇%高航
최곤명%장봉연%기영%왕몽몽%고항
灯盏花素%晶状体上皮细胞%上皮-间质转化%后发性白内障
燈盞花素%晶狀體上皮細胞%上皮-間質轉化%後髮性白內障
등잔화소%정상체상피세포%상피-간질전화%후발성백내장
Breviscapine%Lens epithelial cell%Epithelial-mesenchymal transition%Posterior capsular opacification
背景 晶状体上皮细胞(LECs)的上皮-间质转化(EMT)是后发性白内障的主要病理改变,而EMT的主要标志是α-平滑肌肌动蛋白(α-SMA)的表达.研究发现,灯盏花素具有抑制组织纤维化病变进展及EMT的作用,但对LECs的EMT及其增生是否有抑制作用尚不清楚. 目的 探讨灯盏花素对转化生长因子-β2(TGF-β2)诱导的人LECs表达α-SMA和纤维连接蛋白(FN)的影响. 方法 将人LECs HLE-B3细胞株在含质量分数10％胎牛血清的DMEM培养基中进行培养和传代,在培养基中分别加入质量浓度为6.75、12.75、25.00、50.00和100.00 mg/L的灯盏花素作用24、48和72 h,采用细胞计数试剂盒-8(CCK-8)法检测不同质量浓度灯盏花素作用不同时间对HLE-B3细胞增生的抑制率.此外取生长状态良好的HLE-B3细胞以1×106个/孔的密度接种于培养板中,将其分为4个组,无血清培养基培养的HLE-B3为正常对照组；加入10 μg/L TGF-β2处理的HLE-B3为TGF-β2组；10 mg/L灯盏花素处理的HLE-B3为灯盏花素组；用10 mg/L灯盏花素与10 μg/L TGF-β2共同处理的HLE-B3为联合用药组,药物干预72 h后分别用实时荧光定量PCR法及Western blot法检测α-SMA、FN的mRNA及蛋白在HLE-B3中的表达. 结果 随着灯盏花素质量浓度的逐渐增加,对HLE-B3增生的抑制率逐渐增加,差异有统计学意义(F=292.851,P=0.000)；随着灯盏花素作用时间的延长,对HLE-B3增生的抑制率逐渐增加,差异有统计学意义(F=65.037,P=0.000)；HLE-B3培养72 h的半抑制率(IC50)为22 mg/L,故本实验用10 mg/L(≈1/2 IC50)药物质量浓度作用72 h行后续实验.正常对照组HLE-B3可表达一定量的α-SMA及FN,正常对照组、TGF-β2组、灯盏花素组、联合用药组间HLE-B3中α-SMA mRNA及FN mRNA的表达差异均有统计学意义(F=105.490,P=0.000;F=1041.414,P=0.000),HLE-B3中α-SMA及FN蛋白的表达差异均有统计学意义(F=136.872,P=0.000;F=119.820,P=0.000).与正常对照组相比,TGF-β2组HLE-B3中α-SMA、FN的mRNA及蛋白表达量均明显增加,差异均有统计学意义(均P=0.000).与TGF-β2组比较,联合用药组HLE-B3中α-SMA和FN mRNA及其蛋白的表达量均明显减少,差异均有统计学意义(P=0.001、0.001、0.001、0.010)；但灯盏花素组与正常对照组相比,HLE-B3细胞中α-SMA和FN mRNA及其蛋白的表达量差异均无统计学意义(P=0.551、0.292、0.551、0.360). 结论 10 mg/L灯盏花素能够抑制LECs的增生及EMT,从而发挥防治后发性白内障的作用.
揹景 晶狀體上皮細胞(LECs)的上皮-間質轉化(EMT)是後髮性白內障的主要病理改變,而EMT的主要標誌是α-平滑肌肌動蛋白(α-SMA)的錶達.研究髮現,燈盞花素具有抑製組織纖維化病變進展及EMT的作用,但對LECs的EMT及其增生是否有抑製作用尚不清楚. 目的 探討燈盞花素對轉化生長因子-β2(TGF-β2)誘導的人LECs錶達α-SMA和纖維連接蛋白(FN)的影響. 方法 將人LECs HLE-B3細胞株在含質量分數10％胎牛血清的DMEM培養基中進行培養和傳代,在培養基中分彆加入質量濃度為6.75、12.75、25.00、50.00和100.00 mg/L的燈盞花素作用24、48和72 h,採用細胞計數試劑盒-8(CCK-8)法檢測不同質量濃度燈盞花素作用不同時間對HLE-B3細胞增生的抑製率.此外取生長狀態良好的HLE-B3細胞以1×106箇/孔的密度接種于培養闆中,將其分為4箇組,無血清培養基培養的HLE-B3為正常對照組；加入10 μg/L TGF-β2處理的HLE-B3為TGF-β2組；10 mg/L燈盞花素處理的HLE-B3為燈盞花素組；用10 mg/L燈盞花素與10 μg/L TGF-β2共同處理的HLE-B3為聯閤用藥組,藥物榦預72 h後分彆用實時熒光定量PCR法及Western blot法檢測α-SMA、FN的mRNA及蛋白在HLE-B3中的錶達. 結果 隨著燈盞花素質量濃度的逐漸增加,對HLE-B3增生的抑製率逐漸增加,差異有統計學意義(F=292.851,P=0.000)；隨著燈盞花素作用時間的延長,對HLE-B3增生的抑製率逐漸增加,差異有統計學意義(F=65.037,P=0.000)；HLE-B3培養72 h的半抑製率(IC50)為22 mg/L,故本實驗用10 mg/L(≈1/2 IC50)藥物質量濃度作用72 h行後續實驗.正常對照組HLE-B3可錶達一定量的α-SMA及FN,正常對照組、TGF-β2組、燈盞花素組、聯閤用藥組間HLE-B3中α-SMA mRNA及FN mRNA的錶達差異均有統計學意義(F=105.490,P=0.000;F=1041.414,P=0.000),HLE-B3中α-SMA及FN蛋白的錶達差異均有統計學意義(F=136.872,P=0.000;F=119.820,P=0.000).與正常對照組相比,TGF-β2組HLE-B3中α-SMA、FN的mRNA及蛋白錶達量均明顯增加,差異均有統計學意義(均P=0.000).與TGF-β2組比較,聯閤用藥組HLE-B3中α-SMA和FN mRNA及其蛋白的錶達量均明顯減少,差異均有統計學意義(P=0.001、0.001、0.001、0.010)；但燈盞花素組與正常對照組相比,HLE-B3細胞中α-SMA和FN mRNA及其蛋白的錶達量差異均無統計學意義(P=0.551、0.292、0.551、0.360). 結論 10 mg/L燈盞花素能夠抑製LECs的增生及EMT,從而髮揮防治後髮性白內障的作用.
배경 정상체상피세포(LECs)적상피-간질전화(EMT)시후발성백내장적주요병리개변,이EMT적주요표지시α-평활기기동단백(α-SMA)적표체.연구발현,등잔화소구유억제조직섬유화병변진전급EMT적작용,단대LECs적EMT급기증생시부유억제작용상불청초. 목적 탐토등잔화소대전화생장인자-β2(TGF-β2)유도적인LECs표체α-SMA화섬유련접단백(FN)적영향. 방법 장인LECs HLE-B3세포주재함질량분수10％태우혈청적DMEM배양기중진행배양화전대,재배양기중분별가입질량농도위6.75、12.75、25.00、50.00화100.00 mg/L적등잔화소작용24、48화72 h,채용세포계수시제합-8(CCK-8)법검측불동질량농도등잔화소작용불동시간대HLE-B3세포증생적억제솔.차외취생장상태량호적HLE-B3세포이1×106개/공적밀도접충우배양판중,장기분위4개조,무혈청배양기배양적HLE-B3위정상대조조；가입10 μg/L TGF-β2처리적HLE-B3위TGF-β2조；10 mg/L등잔화소처리적HLE-B3위등잔화소조；용10 mg/L등잔화소여10 μg/L TGF-β2공동처리적HLE-B3위연합용약조,약물간예72 h후분별용실시형광정량PCR법급Western blot법검측α-SMA、FN적mRNA급단백재HLE-B3중적표체. 결과 수착등잔화소질량농도적축점증가,대HLE-B3증생적억제솔축점증가,차이유통계학의의(F=292.851,P=0.000)；수착등잔화소작용시간적연장,대HLE-B3증생적억제솔축점증가,차이유통계학의의(F=65.037,P=0.000)；HLE-B3배양72 h적반억제솔(IC50)위22 mg/L,고본실험용10 mg/L(≈1/2 IC50)약물질량농도작용72 h행후속실험.정상대조조HLE-B3가표체일정량적α-SMA급FN,정상대조조、TGF-β2조、등잔화소조、연합용약조간HLE-B3중α-SMA mRNA급FN mRNA적표체차이균유통계학의의(F=105.490,P=0.000;F=1041.414,P=0.000),HLE-B3중α-SMA급FN단백적표체차이균유통계학의의(F=136.872,P=0.000;F=119.820,P=0.000).여정상대조조상비,TGF-β2조HLE-B3중α-SMA、FN적mRNA급단백표체량균명현증가,차이균유통계학의의(균P=0.000).여TGF-β2조비교,연합용약조HLE-B3중α-SMA화FN mRNA급기단백적표체량균명현감소,차이균유통계학의의(P=0.001、0.001、0.001、0.010)；단등잔화소조여정상대조조상비,HLE-B3세포중α-SMA화FN mRNA급기단백적표체량차이균무통계학의의(P=0.551、0.292、0.551、0.360). 결론 10 mg/L등잔화소능구억제LECs적증생급EMT,종이발휘방치후발성백내장적작용.
Background Epithelial-mesenchymal transition (EMT) is a major event in the pathogenesis of posterior capsular opacification (PCO),and the expressions of α-smooth muscle actin (α-SMA) is the marker of EMT.Previous studies showed that breviscapine plays an important role in anti-fibrosis and suppression EMT,however,the mechanism of its effect on EMT in LECs is unclear.Objective Present study was to investigate the effect of breviscapine on the expression of α-SMA and fibronectin (FN) in human LECs induced by transforming growth factor-β2 (TGF-β2).Methods Human LECs strain,HLE-B3,was cultured and passaged in DMEM containing 10％ fetal bovine serum.Different concentrations of breviscapine (6.75,12.75,25.00,50.00 and 100.00 mg/L)were added into the medium for 24,48 and 72 hours respectively,and then cell cunting kit-8 (CCK-8) was used to evaluate the half maximal inhibitory concentration (IC50) of breviscapine to HLE-B3.In addition,HLE-B3 was subcultured at the density of 1 × 106/hole and divided into 4 groups.The cells were in free-serum medium as the normal control;the cells were exposed in 10 μg/L TGF-β2 as TGF-β2 group;while in the breviscapine group,10 mg/L of breviscapine was added into the culture medium and another group was the combination of 10 mg/L breviscapine and 10 μg/L of TGF-β2 treatment.Real-time PCR and Western blot were used to detect the mRNA expressions of α-SMA and FN as well as their protein in HLE-B3 72 hours after cultured.Results The inhibitory rate of breviscapine to HLE-B3 proliferation was gradually elevated with the increase of concentration of breviscapine,showing a significant inhibition of the cell proliferation among the different groups and at various time points (F =292.851,P=0.000;F =65.037,P=0.000).IC50 of HLE-B3 at 72 hours was 22 mg/L,and therefore,in rest of experiment 10 mg/L of breviscapine was used which was 1/2 only half of the IC50.α-SMA and FN were expressed in cultured normal HLE-B3.The expressing level of α-SMA mRNA and FN mRNA in the HLE-B3 was significantly different among the normal group,TGF-β22 group,breviscapine treatment group and combination group as well as at various time points (F =105.490,P =0.000 ; F =1041.414,P =0.000).Similarly,the protein expressions of α-SM A and FN in the HLE-B3 was significantly different among the four groups and different time points (F=136.872,P=0.000;F=119.820,P=0.000).The expression levels of α-SMA and FN mRNA and their proteins in HLE-B3 were remarkably increased in the 10 μg/L TGF-β2 group compared with the normal control group (at all P=0.000),and those in the combination group were obviously declined in comparison to the TGF-β2 group (P =0.001,0.001,0.001,0.010).No significant difference was found in the expressions of α-SMA and FN in the HLE-B3 between the breviscapine group and normal control group in both transcriptional level and protein level (P =0.551,0.292,0.551,0.360).Conclusions 10 mg/L breviscapine can arrest the proliferation and EMT of human LECs.This result suggests that using breviscapine may be a potential prophylactic approach in the prevention of PCO.
