国际移植与血液净化杂志
國際移植與血液淨化雜誌
국제이식여혈액정화잡지
INTERNATIONAL JOURNAL OF TRANSPLANTATION AND HEMOPURIFICATION
2014年
4期
42-45
,共4页
雷帕霉素%足细胞%p-P70S6K%Nephrin
雷帕黴素%足細胞%p-P70S6K%Nephrin
뢰파매소%족세포%p-P70S6K%Nephrin
Rapamycin%Podocytes%p-p70S6K%Nephrin
目的 通过观察不同嘌呤霉素氨基核苷浓度下不同剂量雷帕霉素对足细胞Nephrin及p-P70S6K表达的影响,探讨雷帕霉素对嘌呤霉素氨基核苷损伤足细胞的保护作用及可能的机制.方法 (1)将同步化的足细胞分成对照组及20、50、100 mg/L嘌呤霉素氨基核苷组,对照组加入无血清RPMI 1640培养基培养,其余三组分别加入含有嘌呤霉素氨基核苷(终浓度为20、50、100 mg/L)的无血清RPMI 1640培养基培养.24h后收集细胞,采用Western blot法检测细胞Nephrin蛋白相对表达量.(2)将同步化的足细胞分成模型对照组及10、100、1000 μg/L雷帕霉素组.所有细胞加入含嘌呤霉素氨基核苷(终浓度为50mg/L)的无血清RPMI 1640培养液,培养24 h后弃去嘌呤霉素氨基核苷培养液,模型对照组加入等量雷帕霉素溶解液,其余三组分别加入终浓度为10、100、1 000 μg/L的雷帕霉素培养,24及48 h后采用Western blot法检测细胞Nephrin蛋白相对表达量.(3)将同步化的足细胞分成正常对照组(以无血清的RPMI 1640培养液培养)、模型对照组(以终浓度为50 mg/L的嘌呤霉素氨基核苷+无血清的RPMI 1640培养液培养)、雷帕霉素组(以终浓度为100 μg/L的雷帕霉素+终浓度为50 mg/L的嘌呤霉素氨基核苷+无血清RPMI 1640培养液培养).3组细胞分别培养24及48h,收集细胞,观察细胞形态,采用Western blot检测p-P70S6K蛋白相对表达量.结果 (1)嘌呤霉素氨基核苷50、100 mg/L组与空白对照组相比,Nephrin蛋白表达显著减少(P＜0.05);嘌呤霉素氨基核苷50 mg/L与嘌呤霉素氨基核苷100 mg/L组相比,Nephrin蛋白表达差异无统计学意义,与20 mg/L组相比,Nephrin蛋白表达显著减少.(2)雷帕霉素100 mg/L组与10、1 000 mg/L组相比,Nephrin蛋白表达明显增加(P＜0.05).(3)与正常对照组、嘌呤霉素氨基核苷模型组相比,雷帕霉素(100 μg/L)组作用嘌呤霉素氨基核苷损伤模型24h后nephrin及p-P70S6K蛋白表达无明显变化;作用48h后,雷帕霉素(100 μg/L)组与嘌呤霉素氨基核苷模型组相比,Nephrin蛋白表达显著增加,p-P70S6K蛋白表达较对照组、嘌呤霉素氨基核苷模型组显著减少.结论 嘌呤霉素氨基核苷50 mg/L的剂量即为体外嘌呤霉素氨基核苷损伤足细胞模型的最佳浓度.100 mg/L雷帕霉素对嘌呤霉素氨基核苷导致的足细胞损伤有很好的保护作用.雷帕霉素的这一保护作用可能是通过抑制mTOR实现的,但其具体机制仍需进一步研究.
目的 通過觀察不同嘌呤黴素氨基覈苷濃度下不同劑量雷帕黴素對足細胞Nephrin及p-P70S6K錶達的影響,探討雷帕黴素對嘌呤黴素氨基覈苷損傷足細胞的保護作用及可能的機製.方法 (1)將同步化的足細胞分成對照組及20、50、100 mg/L嘌呤黴素氨基覈苷組,對照組加入無血清RPMI 1640培養基培養,其餘三組分彆加入含有嘌呤黴素氨基覈苷(終濃度為20、50、100 mg/L)的無血清RPMI 1640培養基培養.24h後收集細胞,採用Western blot法檢測細胞Nephrin蛋白相對錶達量.(2)將同步化的足細胞分成模型對照組及10、100、1000 μg/L雷帕黴素組.所有細胞加入含嘌呤黴素氨基覈苷(終濃度為50mg/L)的無血清RPMI 1640培養液,培養24 h後棄去嘌呤黴素氨基覈苷培養液,模型對照組加入等量雷帕黴素溶解液,其餘三組分彆加入終濃度為10、100、1 000 μg/L的雷帕黴素培養,24及48 h後採用Western blot法檢測細胞Nephrin蛋白相對錶達量.(3)將同步化的足細胞分成正常對照組(以無血清的RPMI 1640培養液培養)、模型對照組(以終濃度為50 mg/L的嘌呤黴素氨基覈苷+無血清的RPMI 1640培養液培養)、雷帕黴素組(以終濃度為100 μg/L的雷帕黴素+終濃度為50 mg/L的嘌呤黴素氨基覈苷+無血清RPMI 1640培養液培養).3組細胞分彆培養24及48h,收集細胞,觀察細胞形態,採用Western blot檢測p-P70S6K蛋白相對錶達量.結果 (1)嘌呤黴素氨基覈苷50、100 mg/L組與空白對照組相比,Nephrin蛋白錶達顯著減少(P＜0.05);嘌呤黴素氨基覈苷50 mg/L與嘌呤黴素氨基覈苷100 mg/L組相比,Nephrin蛋白錶達差異無統計學意義,與20 mg/L組相比,Nephrin蛋白錶達顯著減少.(2)雷帕黴素100 mg/L組與10、1 000 mg/L組相比,Nephrin蛋白錶達明顯增加(P＜0.05).(3)與正常對照組、嘌呤黴素氨基覈苷模型組相比,雷帕黴素(100 μg/L)組作用嘌呤黴素氨基覈苷損傷模型24h後nephrin及p-P70S6K蛋白錶達無明顯變化;作用48h後,雷帕黴素(100 μg/L)組與嘌呤黴素氨基覈苷模型組相比,Nephrin蛋白錶達顯著增加,p-P70S6K蛋白錶達較對照組、嘌呤黴素氨基覈苷模型組顯著減少.結論 嘌呤黴素氨基覈苷50 mg/L的劑量即為體外嘌呤黴素氨基覈苷損傷足細胞模型的最佳濃度.100 mg/L雷帕黴素對嘌呤黴素氨基覈苷導緻的足細胞損傷有很好的保護作用.雷帕黴素的這一保護作用可能是通過抑製mTOR實現的,但其具體機製仍需進一步研究.
목적 통과관찰불동표령매소안기핵감농도하불동제량뢰파매소대족세포Nephrin급p-P70S6K표체적영향,탐토뢰파매소대표령매소안기핵감손상족세포적보호작용급가능적궤제.방법 (1)장동보화적족세포분성대조조급20、50、100 mg/L표령매소안기핵감조,대조조가입무혈청RPMI 1640배양기배양,기여삼조분별가입함유표령매소안기핵감(종농도위20、50、100 mg/L)적무혈청RPMI 1640배양기배양.24h후수집세포,채용Western blot법검측세포Nephrin단백상대표체량.(2)장동보화적족세포분성모형대조조급10、100、1000 μg/L뢰파매소조.소유세포가입함표령매소안기핵감(종농도위50mg/L)적무혈청RPMI 1640배양액,배양24 h후기거표령매소안기핵감배양액,모형대조조가입등량뢰파매소용해액,기여삼조분별가입종농도위10、100、1 000 μg/L적뢰파매소배양,24급48 h후채용Western blot법검측세포Nephrin단백상대표체량.(3)장동보화적족세포분성정상대조조(이무혈청적RPMI 1640배양액배양)、모형대조조(이종농도위50 mg/L적표령매소안기핵감+무혈청적RPMI 1640배양액배양)、뢰파매소조(이종농도위100 μg/L적뢰파매소+종농도위50 mg/L적표령매소안기핵감+무혈청RPMI 1640배양액배양).3조세포분별배양24급48h,수집세포,관찰세포형태,채용Western blot검측p-P70S6K단백상대표체량.결과 (1)표령매소안기핵감50、100 mg/L조여공백대조조상비,Nephrin단백표체현저감소(P＜0.05);표령매소안기핵감50 mg/L여표령매소안기핵감100 mg/L조상비,Nephrin단백표체차이무통계학의의,여20 mg/L조상비,Nephrin단백표체현저감소.(2)뢰파매소100 mg/L조여10、1 000 mg/L조상비,Nephrin단백표체명현증가(P＜0.05).(3)여정상대조조、표령매소안기핵감모형조상비,뢰파매소(100 μg/L)조작용표령매소안기핵감손상모형24h후nephrin급p-P70S6K단백표체무명현변화;작용48h후,뢰파매소(100 μg/L)조여표령매소안기핵감모형조상비,Nephrin단백표체현저증가,p-P70S6K단백표체교대조조、표령매소안기핵감모형조현저감소.결론 표령매소안기핵감50 mg/L적제량즉위체외표령매소안기핵감손상족세포모형적최가농도.100 mg/L뢰파매소대표령매소안기핵감도치적족세포손상유흔호적보호작용.뢰파매소적저일보호작용가능시통과억제mTOR실현적,단기구체궤제잉수진일보연구.
Objective To study the protective effect of mpamycin on puromycin aminonucleoside (PAN) induced podocyte injury and its possible mechanisms.Methods (1) The synchronized podocytes were divided into four groups:the control group (RPMI 1640 culture medium without serum) and the 20,50,100 mg/L PAN group (a final concentration of 20,50,100 mg/L PAN RPMI 1640 culture medium without serum).The cells were collected after 24 h before Western blot assay was performed to assess relative expression of Nephrin protein.(2) The synchronization podocytes were assigned to the model group and 10,100,1 000μg/L mpamycin group.All the cells were added to a final concentration of 50 mg/L PAN RPMI 1640 serum-free culture medium,incubated for 24 h.The model control group was added to dissolve the same amount of mpamycin solution,and the remaining three groups were added to a final concentration of 10,100,1 000μg/L rapamycin training.After 24 and 48 h,Western blot assay was used to test the relative expression of Nephrin protein.(3) The synchronization podocytes were assigned to thee normal control group (serum-free RPMI 1640 culture solution),model control group (the final concentration of 50 mg/L PAN + serum-free RPMI 1640 culture hquid) and mpamycin group (final concentration of 100 μg/L rapamycin + 50 mg/L PAN + serum free RPMI 1640 medium).The cells were cultured for 24 and 48 h,and their morphology was observed.Western blot was used to detect p-P70S6K relative expression.Results (1) Compared with the control group,Nephrin protein expression was significantly decreased in 50 or 100 mg/L PAN group (P ＜ 0.05).No significant difference in nephrin protein expression was found between 50 or 100 mg/L PAN group.(2) Compared with 10 or 1 000μg/L group,Nephrin protein expression was significantly increased in 100 μg/L mpamycin group (P ＜ 0.05).(3) Compared with the normal control group,Nephrin protein expression did not significantly change in the PAN or mpamycin group.Conclusions 50 mg/L PAN could damage the optimal concentration of PAN in vitro cell model.100 μg/L mpamycinon may protect podocytes from PAN induced injury,which may contribute to the inhibition of mTOR.
