中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
3期
216-219
,共4页
血管内皮细胞%视网膜%血管生成%雷帕霉素%细胞周期蛋白D1
血管內皮細胞%視網膜%血管生成%雷帕黴素%細胞週期蛋白D1
혈관내피세포%시망막%혈관생성%뢰파매소%세포주기단백D1
Vascular endothelial cell%Retina%Angiogenesis%Rapamycin%Cyclin D1
背景 哺乳动物雷帕霉素靶蛋白(mTOR)信号通路在细胞周期调控机制中扮演着重要角色,其抑制剂雷帕霉素(RAPA)可通过调控细胞周期抑制细胞增生. 目的 观察RAPA对体外培养的猴视网膜微血管内皮细胞RF/6A细胞系增生的影响. 方法 体外培养RF/6A细胞系,分别在培养液中加入PBS、10 μg/L RAPA和5μg/L RAPA.采用Western blot法检测cyclin D1蛋白在各组RF/6A细胞中的表达；采用流式细胞仪检测不同质量浓度RAPA作用后G0/G1期RF/6A细胞的比例；行血管内皮细胞体外成管实验观察不同质量浓度RAPA对RF/6A细胞成管的抑制作用. 结果 Western blot检测表明,PBS组、10μg/LRAPA组和5μg/L RAPA组RF/6A细胞中cyclin D1蛋白的相对表达量分别为0.92±0.04、0.58±0.02和0.73±0.02,3个组间的差异有统计学意义(F=246.320,P=0.000),其中10 μg/L RAPA组和5μg/L RAPA组cyclin D1蛋白在RF/6A细胞中的相对表达量均明显低于PBS组,差异均有统计学意义(P＜0.05).细胞周期检测发现,PBS组、10 μg/L RAPA组和5μg/L RAPA组G0/G1期细胞比例分别为(42.13±0.57)％、(65.15±0.64)％和(54.09±0.78)％,3个组间差异有统计学意义(F=887.815,P=0.000),其中10 μg/L RAPA组和5 μg/L RAPA组RF/6A细胞G0/G1期比例明显高于PBS组,差异有统计学意义(P＜0.05).体外成管实验显示,PBS组、10 μg/L RAPA组及5μg/L RAPA组每个视野下血管内皮细胞成管数分别为(9.67±1.53)、(4.33±0.58)和(6.33±0.58)个/视野,3个组间比较差异有统计学意义(F=21.778,P=0.002),10 μg/LRAPA组内皮细胞管数最少,其次为5μg/L RAPA组,均明显少于PBS组,差异均有统计学意义(P＜0.05).结论 RAPA可通过下调cyclin D1蛋白的表达抑制RF/6A细胞的增生,较高质量浓度的RAPA作用更强.
揹景 哺乳動物雷帕黴素靶蛋白(mTOR)信號通路在細胞週期調控機製中扮縯著重要角色,其抑製劑雷帕黴素(RAPA)可通過調控細胞週期抑製細胞增生. 目的 觀察RAPA對體外培養的猴視網膜微血管內皮細胞RF/6A細胞繫增生的影響. 方法 體外培養RF/6A細胞繫,分彆在培養液中加入PBS、10 μg/L RAPA和5μg/L RAPA.採用Western blot法檢測cyclin D1蛋白在各組RF/6A細胞中的錶達；採用流式細胞儀檢測不同質量濃度RAPA作用後G0/G1期RF/6A細胞的比例；行血管內皮細胞體外成管實驗觀察不同質量濃度RAPA對RF/6A細胞成管的抑製作用. 結果 Western blot檢測錶明,PBS組、10μg/LRAPA組和5μg/L RAPA組RF/6A細胞中cyclin D1蛋白的相對錶達量分彆為0.92±0.04、0.58±0.02和0.73±0.02,3箇組間的差異有統計學意義(F=246.320,P=0.000),其中10 μg/L RAPA組和5μg/L RAPA組cyclin D1蛋白在RF/6A細胞中的相對錶達量均明顯低于PBS組,差異均有統計學意義(P＜0.05).細胞週期檢測髮現,PBS組、10 μg/L RAPA組和5μg/L RAPA組G0/G1期細胞比例分彆為(42.13±0.57)％、(65.15±0.64)％和(54.09±0.78)％,3箇組間差異有統計學意義(F=887.815,P=0.000),其中10 μg/L RAPA組和5 μg/L RAPA組RF/6A細胞G0/G1期比例明顯高于PBS組,差異有統計學意義(P＜0.05).體外成管實驗顯示,PBS組、10 μg/L RAPA組及5μg/L RAPA組每箇視野下血管內皮細胞成管數分彆為(9.67±1.53)、(4.33±0.58)和(6.33±0.58)箇/視野,3箇組間比較差異有統計學意義(F=21.778,P=0.002),10 μg/LRAPA組內皮細胞管數最少,其次為5μg/L RAPA組,均明顯少于PBS組,差異均有統計學意義(P＜0.05).結論 RAPA可通過下調cyclin D1蛋白的錶達抑製RF/6A細胞的增生,較高質量濃度的RAPA作用更彊.
배경 포유동물뢰파매소파단백(mTOR)신호통로재세포주기조공궤제중분연착중요각색,기억제제뢰파매소(RAPA)가통과조공세포주기억제세포증생. 목적 관찰RAPA대체외배양적후시망막미혈관내피세포RF/6A세포계증생적영향. 방법 체외배양RF/6A세포계,분별재배양액중가입PBS、10 μg/L RAPA화5μg/L RAPA.채용Western blot법검측cyclin D1단백재각조RF/6A세포중적표체；채용류식세포의검측불동질량농도RAPA작용후G0/G1기RF/6A세포적비례；행혈관내피세포체외성관실험관찰불동질량농도RAPA대RF/6A세포성관적억제작용. 결과 Western blot검측표명,PBS조、10μg/LRAPA조화5μg/L RAPA조RF/6A세포중cyclin D1단백적상대표체량분별위0.92±0.04、0.58±0.02화0.73±0.02,3개조간적차이유통계학의의(F=246.320,P=0.000),기중10 μg/L RAPA조화5μg/L RAPA조cyclin D1단백재RF/6A세포중적상대표체량균명현저우PBS조,차이균유통계학의의(P＜0.05).세포주기검측발현,PBS조、10 μg/L RAPA조화5μg/L RAPA조G0/G1기세포비례분별위(42.13±0.57)％、(65.15±0.64)％화(54.09±0.78)％,3개조간차이유통계학의의(F=887.815,P=0.000),기중10 μg/L RAPA조화5 μg/L RAPA조RF/6A세포G0/G1기비례명현고우PBS조,차이유통계학의의(P＜0.05).체외성관실험현시,PBS조、10 μg/L RAPA조급5μg/L RAPA조매개시야하혈관내피세포성관수분별위(9.67±1.53)、(4.33±0.58)화(6.33±0.58)개/시야,3개조간비교차이유통계학의의(F=21.778,P=0.002),10 μg/LRAPA조내피세포관수최소,기차위5μg/L RAPA조,균명현소우PBS조,차이균유통계학의의(P＜0.05).결론 RAPA가통과하조cyclin D1단백적표체억제RF/6A세포적증생,교고질량농도적RAPA작용경강.
Background The signal pathway of mammalian target of rapamycin (mTOR) plays an important role in the regulation of cell cycle.Rapamycin(RAPA) can inhibit the proliferation of cells by regulating of cell cycle.Objective This study was to investigate the effect of RAPA on the proliferation of Rhesus retinal vascular endothelial cells (RF/6A).Methods RF/6A cell strains were cultured in vitro,and PBS,10 μg/L RAPA or 5 μg/L RAPA was added into the medium respectively.The expression of cyclin D1 protein in the RF/6A cells (absorbancy) were detected by Western blot.The cell cycle distribution after RAPA action was analyzed by flow cytometry.Matrigel was used in endothelial-cell tube formation to evaluate the effect of RAPA on angiogenesis.Results Western blot assay showed that the expressions of cyclin D z protein were 0.92±0.04,0.58±0.02 and 0.73±0.02 in the PBS group,10 μg/L RAPA group and 5 μg/L RAPA group,showing a significant difference among the three groups (F =246.320,P =0.000),and the relative expressing level of cyclin D1 protein in the l0 μg/L RAPA group and 5 μg/L RAPA group was significantly lower than that of the PBS group (both at P＜0.05).The proportion of G0/G1 cells were (42.13±0.57)％,(65.15±0.64)％ and (54.09± 0.78)％ respectively in the PBS group,10 μg/L RAPA group and 5 μg/L RAPA group,which was significantly different among the three groups (F=887.815,P=0.000).The number of endothelial-cell tubes were (9.67 ± 1.53)/field,(4.33 ± 0.58)/field and (6.33 ±0.58)/field respectively in the PBS group,10 μg/L RAPA group and 5 μg/L RAPA group,and the number of endothelial-cell tubes in 10 μg/L RAPA group and 5 μg/L RAPA group was less than that in the PBS group,with a significant difference among the three groups (F =21.778,P =0.002).Conclusions RAPA can inhibit the proliferation of RF/6A cells in vitro by down-regulating the expression of cyclin D1.
