CAJ | 학술논문

背景氯离子通道阻滞剂5-硝基-2-(3-苯丙胺)苯甲酸(NPPB)对家兔心肌细胞缺血-再灌注损伤时的细胞凋亡有促进作用,而小梁细胞上也存在ClC型氯离子通道及容积敏感性氯离子通道,但NPPB究竟会对小梁细胞的形态和功能产生什么影响尚不清楚. 目的探讨氯离子通道阻滞剂NPPB对人眼小梁细胞增生及细胞周期、细胞凋亡的影响.方法将处于对数生长期的永生株人眼小梁细胞进行培养并以1×106个/ml的密度接种于96孔培养板,将不同浓度(10、50、100 μmol/L)的NPPB分别加入小梁细胞培养基中,通过四甲基偶氮唑蓝(MTT)法检测各组小梁细胞的增生情况以吸光度(A)值表示；采用流式细胞术测定小梁细胞的细胞周期.将100 mg/L 5-氟尿嘧啶(5-FU)加入培养的细胞中,而另一组培养的细胞中加入100 mg/L 5-FU+100μmol/L NPPB,用Annexin V-PI法检测各组小梁细胞的凋亡情况；罗丹明123法检测细胞线粒体膜电位(△ψm),分别评估各浓度NPPB对培养的人小梁细胞生长和存活的影响.结果 3种不同浓度的NPPB与小梁细胞共同孵育48 h后4个组小梁细胞的增生率差异有统计学意义(F=7.230,P=0.006),50 μmol/L、100μmoL/L NPPB作用后小梁细胞的A值明显低于10 μmol/L NPPB组,50 μmol/L NPPB组、100μmol/LNPPB组与空白对照组比较差异均有统计学意义(t=1.610,P=0.025;t=12.270,P=0.001).小梁细胞培养基中加入NPPB 48 h后,G0/G1期的细胞比例增多,S期细胞比例减少,各细胞周期小梁细胞的比例与未加NPPB的组比较,差异均有统计学意义(P＜0.05).用5-FU作用24 h及48 h后,100 mg/L 5-FU组和100 mg/L5-FU+100μmol/L NPPB组的细胞凋亡率均较其各自空白对照组增加(t24h=2.130,P=0.023;t48h=4.810,P=0.011)；100 mg/L 5-FU+100 μmol/L NPPB组细胞凋亡率明显高于100 mg/L 5-FU组,差异均有统计学意义(t24h=1.980,P=0.037;t48h=1.290,P=0.028),小梁细胞线粒体膜电位降低,差异均有统计学意义(t24h=1.580,P=0.029；F48h=6.200,P=0.015).结论氯离子通道阻滞剂NPPB可抑制小梁细胞增生,抑制小梁细胞通过G1/S期限制点进入S期；NPPB对5-FU诱导的小梁细胞凋亡有促进作用,与内源性线粒体凋亡通路相关. 揹景氯離子通道阻滯劑5-硝基-2-(3-苯丙胺)苯甲痠(NPPB)對傢兔心肌細胞缺血-再灌註損傷時的細胞凋亡有促進作用,而小樑細胞上也存在ClC型氯離子通道及容積敏感性氯離子通道,但NPPB究竟會對小樑細胞的形態和功能產生什麽影響尚不清楚. 目的探討氯離子通道阻滯劑NPPB對人眼小樑細胞增生及細胞週期、細胞凋亡的影響.方法將處于對數生長期的永生株人眼小樑細胞進行培養併以1×106箇/ml的密度接種于96孔培養闆,將不同濃度(10、50、100 μmol/L)的NPPB分彆加入小樑細胞培養基中,通過四甲基偶氮唑藍(MTT)法檢測各組小樑細胞的增生情況以吸光度(A)值錶示；採用流式細胞術測定小樑細胞的細胞週期.將100 mg/L 5-氟尿嘧啶(5-FU)加入培養的細胞中,而另一組培養的細胞中加入100 mg/L 5-FU+100μmol/L NPPB,用Annexin V-PI法檢測各組小樑細胞的凋亡情況；囉丹明123法檢測細胞線粒體膜電位(△ψm),分彆評估各濃度NPPB對培養的人小樑細胞生長和存活的影響.結果 3種不同濃度的NPPB與小樑細胞共同孵育48 h後4箇組小樑細胞的增生率差異有統計學意義(F=7.230,P=0.006),50 μmol/L、100μmoL/L NPPB作用後小樑細胞的A值明顯低于10 μmol/L NPPB組,50 μmol/L NPPB組、100μmol/LNPPB組與空白對照組比較差異均有統計學意義(t=1.610,P=0.025;t=12.270,P=0.001).小樑細胞培養基中加入NPPB 48 h後,G0/G1期的細胞比例增多,S期細胞比例減少,各細胞週期小樑細胞的比例與未加NPPB的組比較,差異均有統計學意義(P＜0.05).用5-FU作用24 h及48 h後,100 mg/L 5-FU組和100 mg/L5-FU+100μmol/L NPPB組的細胞凋亡率均較其各自空白對照組增加(t24h=2.130,P=0.023;t48h=4.810,P=0.011)；100 mg/L 5-FU+100 μmol/L NPPB組細胞凋亡率明顯高于100 mg/L 5-FU組,差異均有統計學意義(t24h=1.980,P=0.037;t48h=1.290,P=0.028),小樑細胞線粒體膜電位降低,差異均有統計學意義(t24h=1.580,P=0.029；F48h=6.200,P=0.015).結論氯離子通道阻滯劑NPPB可抑製小樑細胞增生,抑製小樑細胞通過G1/S期限製點進入S期；NPPB對5-FU誘導的小樑細胞凋亡有促進作用,與內源性線粒體凋亡通路相關.
배경 록리자통도조체제5-초기-2-(3-분병알)분갑산(NPPB)대가토심기세포결혈-재관주손상시적세포조망유촉진작용,이소량세포상야존재ClC형록리자통도급용적민감성록리자통도,단NPPB구경회대소량세포적형태화공능산생십요영향상불청초. 목적 탐토록리자통도조체제NPPB대인안소량세포증생급세포주기、세포조망적영향.방법 장처우대수생장기적영생주인안소량세포진행배양병이1×106개/ml적밀도접충우96공배양판,장불동농도(10、50、100 μmol/L)적NPPB분별가입소량세포배양기중,통과사갑기우담서람(MTT)법검측각조소량세포적증생정황이흡광도(A)치표시；채용류식세포술측정소량세포적세포주기.장100 mg/L 5-불뇨밀정(5-FU)가입배양적세포중,이령일조배양적세포중가입100 mg/L 5-FU+100μmol/L NPPB,용Annexin V-PI법검측각조소량세포적조망정황；라단명123법검측세포선립체막전위(△ψm),분별평고각농도NPPB대배양적인소량세포생장화존활적영향.결과 3충불동농도적NPPB여소량세포공동부육48 h후4개조소량세포적증생솔차이유통계학의의(F=7.230,P=0.006),50 μmol/L、100μmoL/L NPPB작용후소량세포적A치명현저우10 μmol/L NPPB조,50 μmol/L NPPB조、100μmol/LNPPB조여공백대조조비교차이균유통계학의의(t=1.610,P=0.025;t=12.270,P=0.001).소량세포배양기중가입NPPB 48 h후,G0/G1기적세포비례증다,S기세포비례감소,각세포주기소량세포적비례여미가NPPB적조비교,차이균유통계학의의(P＜0.05).용5-FU작용24 h급48 h후,100 mg/L 5-FU조화100 mg/L5-FU+100μmol/L NPPB조적세포조망솔균교기각자공백대조조증가(t24h=2.130,P=0.023;t48h=4.810,P=0.011)；100 mg/L 5-FU+100 μmol/L NPPB조세포조망솔명현고우100 mg/L 5-FU조,차이균유통계학의의(t24h=1.980,P=0.037;t48h=1.290,P=0.028),소량세포선립체막전위강저,차이균유통계학의의(t24h=1.580,P=0.029；F48h=6.200,P=0.015).결론 록리자통도조체제NPPB가억제소량세포증생,억제소량세포통과G1/S기한제점진입S기；NPPB대5-FU유도적소량세포조망유촉진작용,여내원성선립체조망통로상관.
Background 5-Nitro-2-(3-styrene-acrylic amine) benzoic acid ( NPPB),a chloride channel inhibitor,has a promoting effect on cell apoptosis in myocardial ischemia and reperfusion of domestic rabbit.The CIC chloride channel has been found in the ocular trabecular cells.However,the effect of NPPB on the shape and function of trabecular cells is unclear. Objective This study was performed to investigate the effect of NPPB on the proliferation,cell cycle progression and apoptosis of human trabecular meshwork cells. Methods The immortalized human trabcular cell strain was cultured,and logarithmic-phase cells were incubated in 96-well plates at a density of 1 ×106/ml.Different concentrations of NPPB (10,50,100 μ mol/L) were added to the medium,and the MTT assay was used to assess the growth and proliferation of the cells.Flow cytometry was used to evaluate the cell cycle.Then,100 mg/L 5-FU or 100 mg/L 5-FU + 100 μmol/L NPPB was used to induce cell apoptosis,which was assessed by Annexin V-PI.The membrane potential of mitochondria was examined using rhodamine 123 (△ψm). Results After 48 hours of treatment with NPPB,the abosorbency (A value) of the cells was gradually lowered with the increasing dose of NPPB,with significant differences among the 4 groups (F =7.230,P =0.006).Compared with the 10 μmol/L NPPB group,the A values were significantly declined in the 50 and 100 μmol/L NPPB groups (t =1.610,P =0.025 ;t =12.270,P =0.001 ).Forty-eight hours after exposure to NPPB,the percentage of cells in G0/G1 phase was increased and that in the S phase was decreased.The percentages of cells in different phases of cell cycle were significantly different in comparison with their control groups (without NPPB)( P＜0.05 ).Twenty-four and 48 hours after the treatment with 100 mg/L 5-FU,the apoptosis rates of the cells were raised in the 100 mg/L 5-FU group and 100 mg/L 5-FU + 100 μmol/L NPPB group compared to the without NPPB group (t24h =2.130,P =0.023;t48h =4.810,P=0.011 ) ;while that in the 100 mg/L 5-FU+100 μmol/L NPPB group was higher than the 100 mg/L 5-FU group ( t24 h =1.980,P =0.037 ; t48 h =1.290,P =0.028 ),and the mitochondrial membrane potential was lowered ( t24h =1.580,P =0.029 ; F48 h =6.200,P =0.015 ). Conclusions NPPB suppresses the proliferation of human trabecular cells and promotes the cells to enter S phase via the G1/S check point.In addition,ClC might be involved in an anti-apoptosis mechanism through the internal mitochondrial pathway.