国际内分泌代谢杂志
國際內分泌代謝雜誌
국제내분비대사잡지
INTERNATIONAL JOURNAL OF ENDOCRINOLOGY AND METABOLISM
2013年
4期
217-220,225
,共5页
陈丽琴%李梦辰%孙承军%周均%李双%杨菊红
陳麗琴%李夢辰%孫承軍%週均%李雙%楊菊紅
진려금%리몽신%손승군%주균%리쌍%양국홍
胰岛微血管内皮细胞%SIRT1%炎症%细胞因子%2型糖尿病
胰島微血管內皮細胞%SIRT1%炎癥%細胞因子%2型糖尿病
이도미혈관내피세포%SIRT1%염증%세포인자%2형당뇨병
Islet microvascular endothelial cells%SIRT1%Inflammation%Cytokines%Type 2 diabetes mellitus
目的 分析高糖、高脂环境下胰岛微血管内皮细胞(IMVC)分泌E-选择素、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β的变化,研究沉默信息调节蛋白1(SIRT1)/核因子-κB信号途径异常在内皮细胞炎性反应激活中的作用.方法 合成含绿色荧光蛋白报告基因的小鼠SIRT1基因重组质粒,以Lipofectamine 2000转染IMVC.之后将IMVC分为4组:正常对照组、高糖高脂组、高糖高脂+ SIRT1基因转染组、高糖高脂+基因转染对照组.高糖高脂组以33.3 mmol/L葡萄糖+0.5mmol/L棕榈酸处理48 h.高糖高脂+SIRT1基因转染组在高糖高脂处理前以SIRT1基因重组质粒预处理48 h；高糖高脂+基因转染对照组在高糖高脂处理前以空质粒进行预处理48 h.应用实时荧光定量PCR法检测SIRT1基因转染效果及各组核因子-κB的mRNA水平,应用酶联免疫吸附法测定细胞培养上清中TNF-α、IL-1β、E-选择素的水平.结果 与正常对照组相比,高糖高脂组SIRT1 mRNA表达明显下降(0.58 ±0.12,P＜0.01)；而高糖高脂+SIRT1基因转染组的SIRT1基因表达明显升高,与高糖高脂组相比差异显著[(1.55±0.43)比(0.65±0.37),P＜0.01].与正常对照组相比,高糖高脂组核因子-κB mRNA(1.59 ±0.32,P＜0.01)、E-选择素[(48.46±1.04)比(67.12±0.57) ng/L,P＜0.01]、TNF-α[(467.46±8.98)比(621.14±11.26) ng/L,P＜0.01]、IL-1β[(63.32±1.48)比(118.43±1.40) ng/L,P＜0.01]的水平明显升高(P＜0.01).与高糖高脂组相比,高糖高脂+SIRT1基因转染组核因子-κB mRNA (0.95±0.31,P＜0.01)及TNF-α[(451.38±15.91)比(618.89±7.23) ng/L,P＜0.01]明显下降(P＜0.01),E-选择素、IL-1β无明显变化(P＞0.05).结论 高糖高脂环境下,IMVC存在炎性反应激活,可释放大量炎性反应细胞因子.SIRT1-核因子-κB-TNF-α通路异常与IMVC的炎性反应激活密切相关,早期干预该信号通路可能在预防及治疗2型糖尿病的胰岛炎性反应中具有重要意义.
目的 分析高糖、高脂環境下胰島微血管內皮細胞(IMVC)分泌E-選擇素、腫瘤壞死因子(TNF)-α、白細胞介素(IL)-1β的變化,研究沉默信息調節蛋白1(SIRT1)/覈因子-κB信號途徑異常在內皮細胞炎性反應激活中的作用.方法 閤成含綠色熒光蛋白報告基因的小鼠SIRT1基因重組質粒,以Lipofectamine 2000轉染IMVC.之後將IMVC分為4組:正常對照組、高糖高脂組、高糖高脂+ SIRT1基因轉染組、高糖高脂+基因轉染對照組.高糖高脂組以33.3 mmol/L葡萄糖+0.5mmol/L棕櫚痠處理48 h.高糖高脂+SIRT1基因轉染組在高糖高脂處理前以SIRT1基因重組質粒預處理48 h；高糖高脂+基因轉染對照組在高糖高脂處理前以空質粒進行預處理48 h.應用實時熒光定量PCR法檢測SIRT1基因轉染效果及各組覈因子-κB的mRNA水平,應用酶聯免疫吸附法測定細胞培養上清中TNF-α、IL-1β、E-選擇素的水平.結果 與正常對照組相比,高糖高脂組SIRT1 mRNA錶達明顯下降(0.58 ±0.12,P＜0.01)；而高糖高脂+SIRT1基因轉染組的SIRT1基因錶達明顯升高,與高糖高脂組相比差異顯著[(1.55±0.43)比(0.65±0.37),P＜0.01].與正常對照組相比,高糖高脂組覈因子-κB mRNA(1.59 ±0.32,P＜0.01)、E-選擇素[(48.46±1.04)比(67.12±0.57) ng/L,P＜0.01]、TNF-α[(467.46±8.98)比(621.14±11.26) ng/L,P＜0.01]、IL-1β[(63.32±1.48)比(118.43±1.40) ng/L,P＜0.01]的水平明顯升高(P＜0.01).與高糖高脂組相比,高糖高脂+SIRT1基因轉染組覈因子-κB mRNA (0.95±0.31,P＜0.01)及TNF-α[(451.38±15.91)比(618.89±7.23) ng/L,P＜0.01]明顯下降(P＜0.01),E-選擇素、IL-1β無明顯變化(P＞0.05).結論 高糖高脂環境下,IMVC存在炎性反應激活,可釋放大量炎性反應細胞因子.SIRT1-覈因子-κB-TNF-α通路異常與IMVC的炎性反應激活密切相關,早期榦預該信號通路可能在預防及治療2型糖尿病的胰島炎性反應中具有重要意義.
목적 분석고당、고지배경하이도미혈관내피세포(IMVC)분비E-선택소、종류배사인자(TNF)-α、백세포개소(IL)-1β적변화,연구침묵신식조절단백1(SIRT1)/핵인자-κB신호도경이상재내피세포염성반응격활중적작용.방법 합성함록색형광단백보고기인적소서SIRT1기인중조질립,이Lipofectamine 2000전염IMVC.지후장IMVC분위4조:정상대조조、고당고지조、고당고지+ SIRT1기인전염조、고당고지+기인전염대조조.고당고지조이33.3 mmol/L포도당+0.5mmol/L종려산처리48 h.고당고지+SIRT1기인전염조재고당고지처리전이SIRT1기인중조질립예처리48 h；고당고지+기인전염대조조재고당고지처리전이공질립진행예처리48 h.응용실시형광정량PCR법검측SIRT1기인전염효과급각조핵인자-κB적mRNA수평,응용매련면역흡부법측정세포배양상청중TNF-α、IL-1β、E-선택소적수평.결과 여정상대조조상비,고당고지조SIRT1 mRNA표체명현하강(0.58 ±0.12,P＜0.01)；이고당고지+SIRT1기인전염조적SIRT1기인표체명현승고,여고당고지조상비차이현저[(1.55±0.43)비(0.65±0.37),P＜0.01].여정상대조조상비,고당고지조핵인자-κB mRNA(1.59 ±0.32,P＜0.01)、E-선택소[(48.46±1.04)비(67.12±0.57) ng/L,P＜0.01]、TNF-α[(467.46±8.98)비(621.14±11.26) ng/L,P＜0.01]、IL-1β[(63.32±1.48)비(118.43±1.40) ng/L,P＜0.01]적수평명현승고(P＜0.01).여고당고지조상비,고당고지+SIRT1기인전염조핵인자-κB mRNA (0.95±0.31,P＜0.01)급TNF-α[(451.38±15.91)비(618.89±7.23) ng/L,P＜0.01]명현하강(P＜0.01),E-선택소、IL-1β무명현변화(P＞0.05).결론 고당고지배경하,IMVC존재염성반응격활,가석방대량염성반응세포인자.SIRT1-핵인자-κB-TNF-α통로이상여IMVC적염성반응격활밀절상관,조기간예해신호통로가능재예방급치료2형당뇨병적이도염성반응중구유중요의의.
Objective To explore the alteration of E-selectin,tumor necrosis factor (TNF)-α,interleukin (IL)-1β in high-glucose and lipid cultured islet microvascular endothelial cells (IMVC),and analysis the effects of sirtuin 1 (SIRT) 1/nuclear factor-κB (NF-κB) signaling pathway on the inflammatory activation of IMVC.Methods Recombinant mouse SIRT1 plasmid including green fluorescent protein report gene was synthesized and transfected to IMVC by using Lipofectamine 2000.IMVC were divided into 4 groups:normal control group,high glucose/lipid group,SIRT1 plasmid plus high glucose/lipid group,and empty plasmid plus high glucose/lipid group.Cells in high glucose/lipid group were cultured in 33.3 mmol/L glucose and 0.5 mmol/L palmic acid for 48 hours.Cells in SIRT1 plasmid plus high glucose/lipid group were pretreated with SIRT1 recombinant plasmid for 48 hours before the treatment with high glucose and lipid.Cells in empty plasmid plus high glucose/lipid treatment group were pretreated with empty plasmid before the treatment with high glucose and lipid.Real time quantitive-PCR was used to test the transfecting efficacy of SIRT1 recombinant plasmid and NF-κB mRNA expression in each group.ELISA was used to exam the levels of TNF-α,IL-1 β,E-selectin in cell culture supernatant.Results Compared with normal control group,the expression of SIRT1 mRNA in high glucose/lipid group was decreased significantly (0.58 ± 0.12,P ＜ 0.01).However,in SIRT1 plasmid plus high glucose/lipid group,the expression of SIRT1 mRNA was increased greatly compared with that in high glucose/lipid treatment group [(1.55 ± 0.43) vs.(0.65 ± 0.37),P ＜0.01].Compared with normal control group,the expressions of NF-κB mRNA (1.59 ± 0.32,P ＜ 0.01),E-selectin [(48.46 ± 1.04)vs.(67.12 ±0.57) ng/L,P ＜ 0.01],TNF-α [(467.46 ± 8.98) vs.(621.14 ±11.26)ng/L,P ＜0.01],IL-1 β[(63.32 ± 1.48) vs.(118.43 ± 1.40) ng/L,P ＜0.01] were increased in high glucose/lipid group.However,in SIRT1 plasmid plus high glucose/lipid group,the expression of NF-κB mRNA (0.95 ± 0.31,P ＜ 0.01) and TNF-α level [(451.38 ± 15.91)vs.(618.89 ± 7.23) ng/L,P ＜ 0.01]were decreased compared with high glucose/lipid group,but E-selectin and IL-1 β level were not changed significantly(P ＞0.05).Conclusions In hyperglycemic and hyperlipid situation,IMVC is activated and can release many inflammatory factors.Abnormal SIRT1-NF-κB-TNF-α pathway plays roles in the inflammatory activation of IMVC,and early intervention may have great promise in the prevention and treatment of islet inflammation in type 2 diabetes.
