中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2009年
4期
299-304
,共6页
陈刚%沈小燕%林旭%游婷婷%乔玉芳%姚瑾%林苗%朱香清%牟伦盼%方晓文%邹欣%林丽香
陳剛%瀋小燕%林旭%遊婷婷%喬玉芳%姚瑾%林苗%硃香清%牟倫盼%方曉文%鄒訢%林麗香
진강%침소연%림욱%유정정%교옥방%요근%림묘%주향청%모륜반%방효문%추흔%림려향
RNA干扰%NF-κB%细胞凋亡%高糖%人脐静脉内皮细胞
RNA榦擾%NF-κB%細胞凋亡%高糖%人臍靜脈內皮細胞
RNA간우%NF-κB%세포조망%고당%인제정맥내피세포
RNA interference%NF-kappa B%Apoptosis%High glucose%Human umbilical vein endothelial cells
目的 研究高糖尤其是波动性高糖对人脐静脉内皮细胞(HUVEC)凋亡的影响,以及NF-κB信号通路在高糖诱导HUVEC凋亡中的作用.方法 构建针对NF-κBp65 mRNA序列1566位点的重组RNAi腺病毒表达载体,并利用RNAi腺病毒抑制HUVEC p65表达.应用流式细胞术及脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)研究NF-κB信号通路在高糖诱导HUVEC凋亡的作用.结果 高糖(20.5 mmol/L或30.5 mmol/L)可以促进HUVEC凋亡.NF-κBp65特异腺病毒感染HUVEC后,可以明显抑制高糖刺激的NF-κBp65核蛋白转录,使核内p65蛋白表达处于基础水平.TUNEL结果示高糖作用后5 d,高糖组细胞凋亡率显著高于正常葡萄糖组(25.81%±1.77%比8.20%±0.63%,P<0.05),Ad-DEST+高糖组(26.10%±0.98%)与单独高糖组的细胞凋亡率差异无统计学意义(P>0.05).Ad-1566+高糖组的细胞凋亡率(11.49%±0.92%)比Ad-DEST+高糖组显著下降(P<0.01).TUNEL法及流式细胞术检测结果均显示NF-κBp65特异腺病毒可以降低高糖诱导的HUVEC凋亡.结论 高糖可以促进HUVEC凋亡.腺病毒感染HUVEC可以明显抑制高糖刺激的NF-κBp65核转录,从而保护高糖作用的HUVEC凋亡.
目的 研究高糖尤其是波動性高糖對人臍靜脈內皮細胞(HUVEC)凋亡的影響,以及NF-κB信號通路在高糖誘導HUVEC凋亡中的作用.方法 構建針對NF-κBp65 mRNA序列1566位點的重組RNAi腺病毒錶達載體,併利用RNAi腺病毒抑製HUVEC p65錶達.應用流式細胞術及脫氧覈糖覈苷痠末耑轉移酶介導的缺口末耑標記法(TUNEL)研究NF-κB信號通路在高糖誘導HUVEC凋亡的作用.結果 高糖(20.5 mmol/L或30.5 mmol/L)可以促進HUVEC凋亡.NF-κBp65特異腺病毒感染HUVEC後,可以明顯抑製高糖刺激的NF-κBp65覈蛋白轉錄,使覈內p65蛋白錶達處于基礎水平.TUNEL結果示高糖作用後5 d,高糖組細胞凋亡率顯著高于正常葡萄糖組(25.81%±1.77%比8.20%±0.63%,P<0.05),Ad-DEST+高糖組(26.10%±0.98%)與單獨高糖組的細胞凋亡率差異無統計學意義(P>0.05).Ad-1566+高糖組的細胞凋亡率(11.49%±0.92%)比Ad-DEST+高糖組顯著下降(P<0.01).TUNEL法及流式細胞術檢測結果均顯示NF-κBp65特異腺病毒可以降低高糖誘導的HUVEC凋亡.結論 高糖可以促進HUVEC凋亡.腺病毒感染HUVEC可以明顯抑製高糖刺激的NF-κBp65覈轉錄,從而保護高糖作用的HUVEC凋亡.
목적 연구고당우기시파동성고당대인제정맥내피세포(HUVEC)조망적영향,이급NF-κB신호통로재고당유도HUVEC조망중적작용.방법 구건침대NF-κBp65 mRNA서렬1566위점적중조RNAi선병독표체재체,병이용RNAi선병독억제HUVEC p65표체.응용류식세포술급탈양핵당핵감산말단전이매개도적결구말단표기법(TUNEL)연구NF-κB신호통로재고당유도HUVEC조망적작용.결과 고당(20.5 mmol/L혹30.5 mmol/L)가이촉진HUVEC조망.NF-κBp65특이선병독감염HUVEC후,가이명현억제고당자격적NF-κBp65핵단백전록,사핵내p65단백표체처우기출수평.TUNEL결과시고당작용후5 d,고당조세포조망솔현저고우정상포도당조(25.81%±1.77%비8.20%±0.63%,P<0.05),Ad-DEST+고당조(26.10%±0.98%)여단독고당조적세포조망솔차이무통계학의의(P>0.05).Ad-1566+고당조적세포조망솔(11.49%±0.92%)비Ad-DEST+고당조현저하강(P<0.01).TUNEL법급류식세포술검측결과균현시NF-κBp65특이선병독가이강저고당유도적HUVEC조망.결론 고당가이촉진HUVEC조망.선병독감염HUVEC가이명현억제고당자격적NF-κBp65핵전록,종이보호고당작용적HUVEC조망.
Objective To verify whether the periodic or continuous exposure to high glucose may have different effects on human umbilical vein endothelial cell (HUVEC)apoptosis, and to explore the effect of NF-κB pathway on apoptosis of HUVEC induced by high glucose using the RNAi adenovirus vector. Methods RNAi combinant adenovirus vector which targeted 1566 site of NF-κB p65 mRNA was constructed and the effect of p65 gene knockdown in HUVEC was detected by Western blot analysis. Then, the RNAi adenovirus was transducted to explore the role of NF-κB pathway on the regulation of apoptosis in HUVEC induced by high glucose. The apoptosis of HUVEC was tested by flow cytometry and TUNEL assay. Results High glucose could induce apoptosis of HUVEC. p65 protein expression of nuclear extracts was significantly increased in high glucose culture as compared to control group, but only slightly increased in NF-κB-specific knockdown group, which maintained at basal state. Compared with normal glucose group, the number of TUNEL-positive cells in high glucose group was significantly increased (25.81%±1.77% vs 8.20%±0.63%, P<0.05). The number of TUNEL-positive cells was decreased in 30.5 rmnol/L glucose plus Ad-1566 than that in 30.5 mmol/L glucose plus Ad-DEST (11.49%±0.92% vs 26.10%±0.98%, P<0.01). Flow cytometry and TUNEL assay showed that the apoptosis of human umbilical vein endothelial cells induced by high glucose was inhibited by the RNAi adenovirus. Conclusion High glucose induces apoptosis of HUVEC. Knockdown of NF-κB p65 may protect HUVEC from apoptosis by preventing high glucose-induced NF-κB nuclear translocation.
