中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2009年
1期
43-47
,共5页
陈美%江警予%张瑜%王奕%郝燕%周建华
陳美%江警予%張瑜%王奕%郝燕%週建華
진미%강경여%장유%왕혁%학연%주건화
细胞增殖%细胞凋亡%细胞周期蛋白类%系膜细胞%寡核苷酸序列分析%FRY720
細胞增殖%細胞凋亡%細胞週期蛋白類%繫膜細胞%寡覈苷痠序列分析%FRY720
세포증식%세포조망%세포주기단백류%계막세포%과핵감산서렬분석%FRY720
Cell proloferation%Apoptosis%Cyclins%Mesangial cells%Oligonucleotide Array Sequence Analysis%FTY720
目的 观察新型免疫抑制剂FTY720对体外培养的正常大鼠肾小球系膜细胞(GMC)的促凋亡作用及其对细胞周期调节蛋白基因表达谱的影响,以探讨其作用机制.方法 体外培养大鼠GMC,加入20 μmol/L FTY720,分别作用6 h、12 h、24 h、48 h后,以MTT法检测GMC增殖情况;流式细胞术检测GMC凋亡;Hoechst33258和PI染色观察凋亡细胞形态变化;琼脂糖DNA凝胶电泳法观察凋亡细胞核小体DNA的断裂现象.并通过SuperArray实时定量PCR细胞周期基因芯片测定FTY720对细胞周期调节蛋白基因表达谱的影响.结果 加入FYY720培养6 h后,流式细胞术检测发现,GMC出现典型细胞凋亡的亚二倍体峰;12 h后Hoechst33258和PI荧光染色观察发现亮蓝色的凋亡小体,且开始出现典型细胞凋亡形态改变;24 h后DNA琼脂糖凝胶电泳可见凋亡梯度的出现.随FTY720作用时间延长,细胞凋亡明显增加,不同时间组间细胞凋亡率差异有统计学意义(P<0.01).SuperArray实时定量PCR细胞周期基因芯片发现FTY720分别显著上调系膜细胞Dnajc2、LOC688900、RGD1562436_predicted基因表达,分别达41.6、38和16倍.结论 FTY720在体外可呈时间依赖诱导正常大鼠GMC凋亡,其机制与影响细胞周期调节蛋白及凋亡相关基因的表达有关.
目的 觀察新型免疫抑製劑FTY720對體外培養的正常大鼠腎小毬繫膜細胞(GMC)的促凋亡作用及其對細胞週期調節蛋白基因錶達譜的影響,以探討其作用機製.方法 體外培養大鼠GMC,加入20 μmol/L FTY720,分彆作用6 h、12 h、24 h、48 h後,以MTT法檢測GMC增殖情況;流式細胞術檢測GMC凋亡;Hoechst33258和PI染色觀察凋亡細胞形態變化;瓊脂糖DNA凝膠電泳法觀察凋亡細胞覈小體DNA的斷裂現象.併通過SuperArray實時定量PCR細胞週期基因芯片測定FTY720對細胞週期調節蛋白基因錶達譜的影響.結果 加入FYY720培養6 h後,流式細胞術檢測髮現,GMC齣現典型細胞凋亡的亞二倍體峰;12 h後Hoechst33258和PI熒光染色觀察髮現亮藍色的凋亡小體,且開始齣現典型細胞凋亡形態改變;24 h後DNA瓊脂糖凝膠電泳可見凋亡梯度的齣現.隨FTY720作用時間延長,細胞凋亡明顯增加,不同時間組間細胞凋亡率差異有統計學意義(P<0.01).SuperArray實時定量PCR細胞週期基因芯片髮現FTY720分彆顯著上調繫膜細胞Dnajc2、LOC688900、RGD1562436_predicted基因錶達,分彆達41.6、38和16倍.結論 FTY720在體外可呈時間依賴誘導正常大鼠GMC凋亡,其機製與影響細胞週期調節蛋白及凋亡相關基因的錶達有關.
목적 관찰신형면역억제제FTY720대체외배양적정상대서신소구계막세포(GMC)적촉조망작용급기대세포주기조절단백기인표체보적영향,이탐토기작용궤제.방법 체외배양대서GMC,가입20 μmol/L FTY720,분별작용6 h、12 h、24 h、48 h후,이MTT법검측GMC증식정황;류식세포술검측GMC조망;Hoechst33258화PI염색관찰조망세포형태변화;경지당DNA응효전영법관찰조망세포핵소체DNA적단렬현상.병통과SuperArray실시정량PCR세포주기기인심편측정FTY720대세포주기조절단백기인표체보적영향.결과 가입FYY720배양6 h후,류식세포술검측발현,GMC출현전형세포조망적아이배체봉;12 h후Hoechst33258화PI형광염색관찰발현량람색적조망소체,차개시출현전형세포조망형태개변;24 h후DNA경지당응효전영가견조망제도적출현.수FTY720작용시간연장,세포조망명현증가,불동시간조간세포조망솔차이유통계학의의(P<0.01).SuperArray실시정량PCR세포주기기인심편발현FTY720분별현저상조계막세포Dnajc2、LOC688900、RGD1562436_predicted기인표체,분별체41.6、38화16배.결론 FTY720재체외가정시간의뢰유도정상대서GMC조망,기궤제여영향세포주기조절단백급조망상관기인적표체유관.
Objective To observe the effects of a new immunosuppressive agent, FFY720, on rat glomerular mesangial cell (GMC) apoptosis and on gene expression profiles of cell cycle regulatory proteins. Methods Rat GMCs were cultured with 20 μmol/L FTY720 for 6 h, 12 h, 24 h and 48 h, and then were evaluated for proliferation through MTT method, and for apoptosis by flow cytometry and fluorescence stainig with Hoechst33258 and PI, and DNA fragmentation analysis. The gene expression profile of cell cycle regulatory proteins was characterized in rat GMCs before and after FTY720 treatment by SuperArray real-time PCR microarray analysis. Results After incubation with FTY720 for 6 h, apoptotic sub-G1 peak was identified in GMC through flow cytometry. After incubation with FTY720 for 12 h, not only apoptosis bodies of GMC were observed by fluorescence staining with Hoechst33258 and PI, but also typical morphological changes of apoptosis were found in GMC. After incubation with FRY720 for 24 h, typical DNA ladder pattern was identified. The percentage of FTY720-iuduced GMC apoptosis gradually increased with the extension of incubation time. SuperArray real-time PCRmicroarray analysis revealed that FTY720 could respectively up-regulate the expression of Dnajc2, LOC688900 and RGDi562436_predicted genes to 41.6, 38 and 16 folds. Conclusion FTY720 can induce GMC apoptosis in a time-dependent manner, probably through influencing gene expression of cell cycle regulatory proteins.
