中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2013年
5期
358-363
,共6页
苏晓乐%乔晞%李荣山%王艳红%刘新艳
囌曉樂%喬晞%李榮山%王豔紅%劉新豔
소효악%교희%리영산%왕염홍%류신염
肾上腺髓质素%再灌注损伤%细胞凋亡%半胱氨酸天冬氨酸蛋白酶
腎上腺髓質素%再灌註損傷%細胞凋亡%半胱氨痠天鼕氨痠蛋白酶
신상선수질소%재관주손상%세포조망%반광안산천동안산단백매
Adrenomedullin%Reperfusion injury%Apoptosis%Caspase
目的 探讨肾上腺髓质素(ADM)对缺氧复氧(HR)诱导的大鼠近端肾小管上皮细胞(NRK-52E)凋亡的影响及其机制.方法 体外培养的NRK-52E细胞,随机分为4组:正常对照组、HR组、空质粒+HR组、ADM质粒+HR组.采用Fugene HD转染试剂,将pcDNA3.1-myc-his B空质粒和ADM真核表达质粒分别转染至NRK-52E细胞内,应用免疫细胞化学的方法检测转染效率,转染成功48 h后制作HR模型.锥虫蓝摄取法进行细胞计数并计算细胞存活率,分光光度法检测上清液中乳酸脱氢酶(LDH)含量评价细胞活力；Annexin V和PI染色结合流式细胞仪技术检测细胞凋亡率；RT-PCR法检测ADM、Bax、Bcl-2、Fas的mRNA表达；Western印迹法检测活性半胱氨酸天冬氨酸蛋白酶3、8、9(Caspase-3、8、9)的蛋白表达.结果 HR组ADM的表达显著高于正常对照组(P＜0.05).ADM质粒+HR组的ADM表达显著高于空质粒+ HR组(P＜0.05).与正常对照组相比,HR组活细胞计数和细胞存活率显著降低,LDH含量及细胞凋亡率显著升高,Bax、Bcl-2、Fas、活性Caspase-3、8、9表达显著上调,Bax上调更明显,故Bax/Bcl-2升高(均P＜0.05).与HR组相比,ADM质粒+HR组活细胞计数和细胞存活率显著升高,LDH含量及细胞凋亡率显著降低,Bax、Fas、活性Caspase-3、8、9表达显著下调,Bcl-2表达进一步上调,Bax/Bcl-2降低(均P＜0.05).空质粒+HR组和HR组比较,上述各指标差异均无统计学意义(P＞0.05).结论 ADM在NRK-52E细胞中的高表达可以减轻HR诱导的细胞凋亡,其机制至少部分是通过抑制线粒体通路和死亡受体通路实现的.
目的 探討腎上腺髓質素(ADM)對缺氧複氧(HR)誘導的大鼠近耑腎小管上皮細胞(NRK-52E)凋亡的影響及其機製.方法 體外培養的NRK-52E細胞,隨機分為4組:正常對照組、HR組、空質粒+HR組、ADM質粒+HR組.採用Fugene HD轉染試劑,將pcDNA3.1-myc-his B空質粒和ADM真覈錶達質粒分彆轉染至NRK-52E細胞內,應用免疫細胞化學的方法檢測轉染效率,轉染成功48 h後製作HR模型.錐蟲藍攝取法進行細胞計數併計算細胞存活率,分光光度法檢測上清液中乳痠脫氫酶(LDH)含量評價細胞活力；Annexin V和PI染色結閤流式細胞儀技術檢測細胞凋亡率；RT-PCR法檢測ADM、Bax、Bcl-2、Fas的mRNA錶達；Western印跡法檢測活性半胱氨痠天鼕氨痠蛋白酶3、8、9(Caspase-3、8、9)的蛋白錶達.結果 HR組ADM的錶達顯著高于正常對照組(P＜0.05).ADM質粒+HR組的ADM錶達顯著高于空質粒+ HR組(P＜0.05).與正常對照組相比,HR組活細胞計數和細胞存活率顯著降低,LDH含量及細胞凋亡率顯著升高,Bax、Bcl-2、Fas、活性Caspase-3、8、9錶達顯著上調,Bax上調更明顯,故Bax/Bcl-2升高(均P＜0.05).與HR組相比,ADM質粒+HR組活細胞計數和細胞存活率顯著升高,LDH含量及細胞凋亡率顯著降低,Bax、Fas、活性Caspase-3、8、9錶達顯著下調,Bcl-2錶達進一步上調,Bax/Bcl-2降低(均P＜0.05).空質粒+HR組和HR組比較,上述各指標差異均無統計學意義(P＞0.05).結論 ADM在NRK-52E細胞中的高錶達可以減輕HR誘導的細胞凋亡,其機製至少部分是通過抑製線粒體通路和死亡受體通路實現的.
목적 탐토신상선수질소(ADM)대결양복양(HR)유도적대서근단신소관상피세포(NRK-52E)조망적영향급기궤제.방법 체외배양적NRK-52E세포,수궤분위4조:정상대조조、HR조、공질립+HR조、ADM질립+HR조.채용Fugene HD전염시제,장pcDNA3.1-myc-his B공질립화ADM진핵표체질립분별전염지NRK-52E세포내,응용면역세포화학적방법검측전염효솔,전염성공48 h후제작HR모형.추충람섭취법진행세포계수병계산세포존활솔,분광광도법검측상청액중유산탈경매(LDH)함량평개세포활력；Annexin V화PI염색결합류식세포의기술검측세포조망솔；RT-PCR법검측ADM、Bax、Bcl-2、Fas적mRNA표체；Western인적법검측활성반광안산천동안산단백매3、8、9(Caspase-3、8、9)적단백표체.결과 HR조ADM적표체현저고우정상대조조(P＜0.05).ADM질립+HR조적ADM표체현저고우공질립+ HR조(P＜0.05).여정상대조조상비,HR조활세포계수화세포존활솔현저강저,LDH함량급세포조망솔현저승고,Bax、Bcl-2、Fas、활성Caspase-3、8、9표체현저상조,Bax상조경명현,고Bax/Bcl-2승고(균P＜0.05).여HR조상비,ADM질립+HR조활세포계수화세포존활솔현저승고,LDH함량급세포조망솔현저강저,Bax、Fas、활성Caspase-3、8、9표체현저하조,Bcl-2표체진일보상조,Bax/Bcl-2강저(균P＜0.05).공질립+HR조화HR조비교,상술각지표차이균무통계학의의(P＞0.05).결론 ADM재NRK-52E세포중적고표체가이감경HR유도적세포조망,기궤제지소부분시통과억제선립체통로화사망수체통로실현적.
Objective To investigate the effect of adrenomedullin on rat renal tubular epithelial cell line (NRK-52E) apoptosis induced by hypoxia-reoxygenation (HR) injury and its mechanism.Methods NRK-52E cells were cultured and randomly allotted to the following 4 groups:control group,HR group,empty plasmid + HR group,ADM + HR group.NRK-52E cells were transfected with pcDNA3.1-myc-his B empty vector or pcDNA3.1-ADM by transfection complex comprising optimal proportion of plasmid and Fugene HD reagents.Cells were counted by trypanblau and cell survival rate was computed.The concentration of lactate dehydrogenase (LDH) was detected by spectrophotometric method to evaluate cell vitality.The apoptotic rate of NRK-52E cells was measured by flow cytometry.The transfer efficiency was detected by immunocytochemistry.The mRNA expressions of ADM,Bax,Bcl-2 and Fas were determined by Semi-quantitative RT-PCR,and active caspase-3,8,9 protein expression were examined by Western blotting.Results Compared with control group,the expression of ADM significantly increased in HR group (P ＜ 0.05).The expression of ADM significantly increased in ADM + HR group than that in empty plasmid + HR group.Compared with control group,in HR group,the living cell counts and cell survival rate significantly decreased; the LDH concentration in media,apoptotic rate and the levels of Bax,Bcl-2,Bax/Bcl-2,Fas,active Caspase -3,8,9 significantly increased (all P ＜ 0.05).Compared with HR group,in ADM + HR group,the living cell counts and cell survival rate significantly increased; the LDH concentration in media,the cell apoptotic rate and the levels of Bax,Bax/Bcl-2,Fas,active Caspase-3,8,9 significantly decreased,while Bcl-2 was promoted (all P ＜ 0.05).The above indexes had no differences between empty plasmid + HR group and HR group (all P ＞ 0.05).Conclusion The increased expression of ADM can inhibit NRK-52E apoptosis induced by HR,and the mechanism might be achieved by inhibiting mitochondrial and death receptor-mediated apoptotic pathways.
