中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2013年
11期
1014-1019
,共6页
晶体%上皮细胞%细胞增殖%氯化物通道%4,4'-二异硫氰基苯-2,2'-二磺酸
晶體%上皮細胞%細胞增殖%氯化物通道%4,4'-二異硫氰基苯-2,2'-二磺痠
정체%상피세포%세포증식%록화물통도%4,4'-이이류청기분-2,2'-이광산
Lens,crystalline%Epithelial cells%Cell proliferation%Chloride channels%4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid
目的 观察氯通道阻断剂5-硝基-2(3-苯丙胺)苯甲酸(NPPB)和二异硫氰基芪-2,2'-二磺酸(DIDS)对碱性成纤维细胞生长因子(bFGF)诱导的人永生系晶状体上皮细胞(HLE) B-3增殖的影响.方法 实验研究.取对数生长期的HLE B-3细胞为研究对象,用10 μg/L bFGF作用的细胞作为bFGF组,常规培养的细胞作为空白对照组,NPPB(50、100、150 μmol/L)及DIDS(10、50、100 μmol/L)分别加入含bFGF的HLE B-3细胞的培养液中共同作用24 h、48 h及72 h,采用CCK-8法、免疫细胞化学法及PI单染流式细胞术检测细胞增殖率、细胞增殖核抗原Ki-67蛋白的表达率及细胞周期的变化,进而观察氯通道阻断剂对其影响.不同浓度的NPPB和DIDS在三个时间段作用后A值差异比较采用双因素方差分析,组内各因素的多重比较采用LSD-t检验.各组细胞Ki-67表达情况及细胞周期构成比均采用单因素方差分析,组间多重比较采用LSD-t检验,空白对照组与bFGF处理组的两样本比较采用t检验.结果 CCK-8检测表明10 μg/L bFGF对HLE B-3细胞具有促增殖作用,24 h、48 h及72 h的增殖率分别为(18.77±6.53)％、(92.15±8.38)％和(37.09 ±2.89)％.不同浓度NPPB和DIDS在3个时间段内作用细胞后A值的变化表现出剂量及时间依赖性(bFGF+NPPB组:F时间=305.28,F浓度=18.76,P=0.000; bFGF+ DIDS组:F时间=94.43,F浓度=24.41,P=0.000),其中在作用48 h及72 h后,与bFGF处理组比较,各浓度的NPPB和DIDS均使得细胞A值有着明显的下降(P＜0.05).不同浓度的NPPB和DIDS作用于细胞48 h后,细胞内Ki-67蛋白表达细胞数有不同程度下降,差异存在统计学意义(bFGF+ NPPB组:F=580.32,P=0.000;bFGF+ DIDS组:F =507.43,P=0.000),其中150 μmol/L NPPB组和100 μmol/L DIDS组Ki-67的阳性细胞率分别下降至(18.32±1.23)％和(11.21±1.02)％,与bFGF处理组相比较,差异均有统计学意义(P＜0.01,P＜0.01).药物作用48 h后PI染色流式细胞周期检测结果,bFGF组G0/G1期及S期细胞比例分别为(45.35±1.00)％和(31.51±1.04)％,150 μmol/L NPPB组G0/G1期细胞比例升高至(66.53±1.28)％,S期减少至(15.49±1.31)％,100 μmol/L DIDS组G0/G1期细胞比例分别升高至(67.37±0.63)％,S期减少至(13.88±0.53)％,与bFGF组相比差异均存在统计学意义(F=390.75,P=0.000;F=166.24,P=0.000).结论 NPPB和DIDS能够抑制bFGF诱导的HLE B-3细胞增殖,并在G1/S期限制点抑制其进入S期.
目的 觀察氯通道阻斷劑5-硝基-2(3-苯丙胺)苯甲痠(NPPB)和二異硫氰基芪-2,2'-二磺痠(DIDS)對堿性成纖維細胞生長因子(bFGF)誘導的人永生繫晶狀體上皮細胞(HLE) B-3增殖的影響.方法 實驗研究.取對數生長期的HLE B-3細胞為研究對象,用10 μg/L bFGF作用的細胞作為bFGF組,常規培養的細胞作為空白對照組,NPPB(50、100、150 μmol/L)及DIDS(10、50、100 μmol/L)分彆加入含bFGF的HLE B-3細胞的培養液中共同作用24 h、48 h及72 h,採用CCK-8法、免疫細胞化學法及PI單染流式細胞術檢測細胞增殖率、細胞增殖覈抗原Ki-67蛋白的錶達率及細胞週期的變化,進而觀察氯通道阻斷劑對其影響.不同濃度的NPPB和DIDS在三箇時間段作用後A值差異比較採用雙因素方差分析,組內各因素的多重比較採用LSD-t檢驗.各組細胞Ki-67錶達情況及細胞週期構成比均採用單因素方差分析,組間多重比較採用LSD-t檢驗,空白對照組與bFGF處理組的兩樣本比較採用t檢驗.結果 CCK-8檢測錶明10 μg/L bFGF對HLE B-3細胞具有促增殖作用,24 h、48 h及72 h的增殖率分彆為(18.77±6.53)％、(92.15±8.38)％和(37.09 ±2.89)％.不同濃度NPPB和DIDS在3箇時間段內作用細胞後A值的變化錶現齣劑量及時間依賴性(bFGF+NPPB組:F時間=305.28,F濃度=18.76,P=0.000; bFGF+ DIDS組:F時間=94.43,F濃度=24.41,P=0.000),其中在作用48 h及72 h後,與bFGF處理組比較,各濃度的NPPB和DIDS均使得細胞A值有著明顯的下降(P＜0.05).不同濃度的NPPB和DIDS作用于細胞48 h後,細胞內Ki-67蛋白錶達細胞數有不同程度下降,差異存在統計學意義(bFGF+ NPPB組:F=580.32,P=0.000;bFGF+ DIDS組:F =507.43,P=0.000),其中150 μmol/L NPPB組和100 μmol/L DIDS組Ki-67的暘性細胞率分彆下降至(18.32±1.23)％和(11.21±1.02)％,與bFGF處理組相比較,差異均有統計學意義(P＜0.01,P＜0.01).藥物作用48 h後PI染色流式細胞週期檢測結果,bFGF組G0/G1期及S期細胞比例分彆為(45.35±1.00)％和(31.51±1.04)％,150 μmol/L NPPB組G0/G1期細胞比例升高至(66.53±1.28)％,S期減少至(15.49±1.31)％,100 μmol/L DIDS組G0/G1期細胞比例分彆升高至(67.37±0.63)％,S期減少至(13.88±0.53)％,與bFGF組相比差異均存在統計學意義(F=390.75,P=0.000;F=166.24,P=0.000).結論 NPPB和DIDS能夠抑製bFGF誘導的HLE B-3細胞增殖,併在G1/S期限製點抑製其進入S期.
목적 관찰록통도조단제5-초기-2(3-분병알)분갑산(NPPB)화이이류청기기-2,2'-이광산(DIDS)대감성성섬유세포생장인자(bFGF)유도적인영생계정상체상피세포(HLE) B-3증식적영향.방법 실험연구.취대수생장기적HLE B-3세포위연구대상,용10 μg/L bFGF작용적세포작위bFGF조,상규배양적세포작위공백대조조,NPPB(50、100、150 μmol/L)급DIDS(10、50、100 μmol/L)분별가입함bFGF적HLE B-3세포적배양액중공동작용24 h、48 h급72 h,채용CCK-8법、면역세포화학법급PI단염류식세포술검측세포증식솔、세포증식핵항원Ki-67단백적표체솔급세포주기적변화,진이관찰록통도조단제대기영향.불동농도적NPPB화DIDS재삼개시간단작용후A치차이비교채용쌍인소방차분석,조내각인소적다중비교채용LSD-t검험.각조세포Ki-67표체정황급세포주기구성비균채용단인소방차분석,조간다중비교채용LSD-t검험,공백대조조여bFGF처리조적량양본비교채용t검험.결과 CCK-8검측표명10 μg/L bFGF대HLE B-3세포구유촉증식작용,24 h、48 h급72 h적증식솔분별위(18.77±6.53)％、(92.15±8.38)％화(37.09 ±2.89)％.불동농도NPPB화DIDS재3개시간단내작용세포후A치적변화표현출제량급시간의뢰성(bFGF+NPPB조:F시간=305.28,F농도=18.76,P=0.000; bFGF+ DIDS조:F시간=94.43,F농도=24.41,P=0.000),기중재작용48 h급72 h후,여bFGF처리조비교,각농도적NPPB화DIDS균사득세포A치유착명현적하강(P＜0.05).불동농도적NPPB화DIDS작용우세포48 h후,세포내Ki-67단백표체세포수유불동정도하강,차이존재통계학의의(bFGF+ NPPB조:F=580.32,P=0.000;bFGF+ DIDS조:F =507.43,P=0.000),기중150 μmol/L NPPB조화100 μmol/L DIDS조Ki-67적양성세포솔분별하강지(18.32±1.23)％화(11.21±1.02)％,여bFGF처리조상비교,차이균유통계학의의(P＜0.01,P＜0.01).약물작용48 h후PI염색류식세포주기검측결과,bFGF조G0/G1기급S기세포비례분별위(45.35±1.00)％화(31.51±1.04)％,150 μmol/L NPPB조G0/G1기세포비례승고지(66.53±1.28)％,S기감소지(15.49±1.31)％,100 μmol/L DIDS조G0/G1기세포비례분별승고지(67.37±0.63)％,S기감소지(13.88±0.53)％,여bFGF조상비차이균존재통계학의의(F=390.75,P=0.000;F=166.24,P=0.000).결론 NPPB화DIDS능구억제bFGF유도적HLE B-3세포증식,병재G1/S기한제점억제기진입S기.
Objective To study the effect of chloride channel on proliferation induced by basicfibroblast growth factor (bFGF) in human lens epithelial cells HLE B-3 (LEC).Methods HLE B-3 cells at Logarithmic growth phase were incubated in the 6 or 96 well plate overnight and the proliferation was induced by 10 μg/L bFGF.The cells were divided into bFGF group treated with bFGF and the blank control group with the regular cells culture.LEC were treated with different concentrations of chloride channel inhibitors:5-nitro-2-[3-henylpro-pylamino] benzoic acid (NPPB) at 50,100,150 μmol/L and 4,4'-2 diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) at 10,50,100 μmol/L with bFGF or without bFGF in 10％ serum for 24,48,72 hours.The cell viability and the drug toxicity were detected by CCK-8 colorimetric assay.The expression of proliferating cell nuclear antigen (Ki-67) of LEC treated with NPPB or DIDS were observed by immunocytochemistry staining assay.The phase change of cell cycle was examined by flow cytometry.Each experiment group and control group were compared using two-way ANOVA,further pairwise comparisons using LSD-t test or by the Independent-Samples t test.Results 10 μg/L bFGF induced LEC proliferation and the values of A stage were gradually declined with the increase of chloride channel inhibitos' concentrations and time at 24 hours,48 hours and 72 hours (bFGF + NPPB group:Ftime =305.28,F trations =18.76,P =0.000; bFGF + DIDS group:Ftme =94.44,Fconcentrations =24.42,P =0.000).Different concentrations of chloride channel inhibitor reduced the values of a stage with 10 μg/L bFGF in a dose-and time-dependent manner.The expression of Ki-67 was significantly lowered compared with the bFGF group (bFGF + NPPB group:18.32％ ± 1.23 ％,F =580.3,P =0.000 ;bFGF + DIDS group:11.21％ ± 1.02％,F =507.4,P =0.000),when 150 μmol/L NPPB and 100 μmol/L DIDS with 10 μg/L bFGF were added at 48 hours.After 150 μmol/L NPPB and 100 μmol/L DIDS treatment at the 48th hour,the rate of G1 stage was significantly increased (F =390.754,P =0.000),where as that of S stage decreased significantly (F =166.240,P =0.000).Conclusions These results indicate that chloride channel inhibitors play an important role in modulating the proliferation cycle of LEC treated with bFGF by limiting the cell at cycle S/G1 stage from dividing into S stage.
