中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
1期
57-60
,共4页
主父中印%林樾%刘恿铂%蒋亚楠%谭谦
主父中印%林樾%劉恿鉑%蔣亞楠%譚謙
주부중인%림월%류용박%장아남%담겸
葡萄糖%游离脂肪酸%β-连环蛋白%糖尿病溃疡
葡萄糖%遊離脂肪痠%β-連環蛋白%糖尿病潰瘍
포도당%유리지방산%β-련배단백%당뇨병궤양
High-glucose%Free fatty acid%β-catenin%Diabetic ulcer
目的 观察高浓度葡萄糖、高浓度游离脂肪酸对人皮肤成纤维细胞β-连环蛋白(β-catenin)表达的影响,探讨糖尿病皮肤溃疡难愈合的机制.方法 复苏正常成人皮肤成纤维细胞株,高糖改良伊格尔培养基(DMEM,25 mmol/L)培养基进行培养.首先,噻唑蓝(MTT)法筛选葡萄糖和游离脂肪酸对成纤维细胞最佳作用浓度:将细胞分为葡萄糖处理组和游离脂肪酸处理组,葡萄糖处理组含不同浓度葡萄糖(30、35、40、50、60、70 mmol/L),对照组不加葡萄糖；游离脂肪酸处理组含不同浓度软脂酸(100、200、400、600、800、1000 μmol/L),对照组仅为高糖DMEM培养基.以MTT法检测不同培养条件下成纤维细胞吸光度(A)值,结合细胞形态学观察,选择一个对细胞会造成一定损伤,但又不会使细胞大量死亡的浓度作为处理成纤维细胞的最佳浓度.其次,以最佳浓度培养成纤维细胞不同时间,Western blot法检测胞质内β-catenin蛋白的表达.取对数生长期的细胞分为3组:高浓度葡萄糖组(35 mmol/L)、高浓度游离脂肪酸组(200 μmol/L)、高浓度葡萄糖±高浓度游离脂肪酸(35 mmol/L ±200 μmol/L)联合组.于加入葡萄糖和软脂酸培养前(0 h)以及培养后4、8、12、16、20、24、48 h,收集各组细胞,采用Western blot法检测各组细胞3-catenin蛋白的表达.结果 (1)培养24 h后,成纤维细胞A值于葡萄糖浓度为35 mmol/L和游离脂肪酸为200 μmol/L时分别为(0.741 ±0.012、0.676±0.043),与对照组(0.934 ±0.007)比较开始下降(t值分别为1.70和2.48,P＜0.05),后随着葡萄糖和软脂酸浓度的增加,各组A值均减小(P＜0.05).(2)35 mmol/L葡萄糖组、200 μmol/L游离脂肪酸组及两者联合培养组细胞内3-catenin蛋白的表达分别于12、4、8h开始降低(t值分别为7.45、5.12、3.33,P＜0.05),48 h降至最低(P＜0.05).结论 高葡萄糖短期内能延缓高游离脂肪酸诱导的细胞β-catenin低表达,但在持续高糖存在的情况下,成纤维细胞β-catenin低表达程度比两者单独作用时更显著,高葡萄糖对高游离脂肪酸诱导的细胞β-catenin低表达呈现出先抑制后促进的作用.
目的 觀察高濃度葡萄糖、高濃度遊離脂肪痠對人皮膚成纖維細胞β-連環蛋白(β-catenin)錶達的影響,探討糖尿病皮膚潰瘍難愈閤的機製.方法 複囌正常成人皮膚成纖維細胞株,高糖改良伊格爾培養基(DMEM,25 mmol/L)培養基進行培養.首先,噻唑藍(MTT)法篩選葡萄糖和遊離脂肪痠對成纖維細胞最佳作用濃度:將細胞分為葡萄糖處理組和遊離脂肪痠處理組,葡萄糖處理組含不同濃度葡萄糖(30、35、40、50、60、70 mmol/L),對照組不加葡萄糖；遊離脂肪痠處理組含不同濃度軟脂痠(100、200、400、600、800、1000 μmol/L),對照組僅為高糖DMEM培養基.以MTT法檢測不同培養條件下成纖維細胞吸光度(A)值,結閤細胞形態學觀察,選擇一箇對細胞會造成一定損傷,但又不會使細胞大量死亡的濃度作為處理成纖維細胞的最佳濃度.其次,以最佳濃度培養成纖維細胞不同時間,Western blot法檢測胞質內β-catenin蛋白的錶達.取對數生長期的細胞分為3組:高濃度葡萄糖組(35 mmol/L)、高濃度遊離脂肪痠組(200 μmol/L)、高濃度葡萄糖±高濃度遊離脂肪痠(35 mmol/L ±200 μmol/L)聯閤組.于加入葡萄糖和軟脂痠培養前(0 h)以及培養後4、8、12、16、20、24、48 h,收集各組細胞,採用Western blot法檢測各組細胞3-catenin蛋白的錶達.結果 (1)培養24 h後,成纖維細胞A值于葡萄糖濃度為35 mmol/L和遊離脂肪痠為200 μmol/L時分彆為(0.741 ±0.012、0.676±0.043),與對照組(0.934 ±0.007)比較開始下降(t值分彆為1.70和2.48,P＜0.05),後隨著葡萄糖和軟脂痠濃度的增加,各組A值均減小(P＜0.05).(2)35 mmol/L葡萄糖組、200 μmol/L遊離脂肪痠組及兩者聯閤培養組細胞內3-catenin蛋白的錶達分彆于12、4、8h開始降低(t值分彆為7.45、5.12、3.33,P＜0.05),48 h降至最低(P＜0.05).結論 高葡萄糖短期內能延緩高遊離脂肪痠誘導的細胞β-catenin低錶達,但在持續高糖存在的情況下,成纖維細胞β-catenin低錶達程度比兩者單獨作用時更顯著,高葡萄糖對高遊離脂肪痠誘導的細胞β-catenin低錶達呈現齣先抑製後促進的作用.
목적 관찰고농도포도당、고농도유리지방산대인피부성섬유세포β-련배단백(β-catenin)표체적영향,탐토당뇨병피부궤양난유합적궤제.방법 복소정상성인피부성섬유세포주,고당개량이격이배양기(DMEM,25 mmol/L)배양기진행배양.수선,새서람(MTT)법사선포도당화유리지방산대성섬유세포최가작용농도:장세포분위포도당처리조화유리지방산처리조,포도당처리조함불동농도포도당(30、35、40、50、60、70 mmol/L),대조조불가포도당；유리지방산처리조함불동농도연지산(100、200、400、600、800、1000 μmol/L),대조조부위고당DMEM배양기.이MTT법검측불동배양조건하성섬유세포흡광도(A)치,결합세포형태학관찰,선택일개대세포회조성일정손상,단우불회사세포대량사망적농도작위처리성섬유세포적최가농도.기차,이최가농도배양성섬유세포불동시간,Western blot법검측포질내β-catenin단백적표체.취대수생장기적세포분위3조:고농도포도당조(35 mmol/L)、고농도유리지방산조(200 μmol/L)、고농도포도당±고농도유리지방산(35 mmol/L ±200 μmol/L)연합조.우가입포도당화연지산배양전(0 h)이급배양후4、8、12、16、20、24、48 h,수집각조세포,채용Western blot법검측각조세포3-catenin단백적표체.결과 (1)배양24 h후,성섬유세포A치우포도당농도위35 mmol/L화유리지방산위200 μmol/L시분별위(0.741 ±0.012、0.676±0.043),여대조조(0.934 ±0.007)비교개시하강(t치분별위1.70화2.48,P＜0.05),후수착포도당화연지산농도적증가,각조A치균감소(P＜0.05).(2)35 mmol/L포도당조、200 μmol/L유리지방산조급량자연합배양조세포내3-catenin단백적표체분별우12、4、8h개시강저(t치분별위7.45、5.12、3.33,P＜0.05),48 h강지최저(P＜0.05).결론 고포도당단기내능연완고유리지방산유도적세포β-catenin저표체,단재지속고당존재적정황하,성섬유세포β-catenin저표체정도비량자단독작용시경현저,고포도당대고유리지방산유도적세포β-catenin저표체정현출선억제후촉진적작용.
Objective To study the effects of high glucose,high free fatty acids (FFA) and combination on the expression of β-catenin in skin fibroblasts in vivo.Methods Normal human skin firbroblasts (HSFs) were pelleted and grown in high-glucose Dulbecco' s modified Eagle' s medium (DMEM,glucose concentration:25 mmol/L) supplemented with 10％ fetal bovine serum (FBS).Cells in passage three were used for the following experiments.They were seeded in 96-well plates and cultured in nutrient solution with different concentrations of glucose and FFA respectively.First,the optimized concentration was selected by methyl thiazol tetrazolium (MTT) assay.Following two groups were set up:high-glucose medium groups (with different glucose concentrations of 30,35,40,50,60 and 70 mmol/L) and highFFA groups (with different concentrations of 100,200,400,600,800 amd 1000 μmol/L).Neither glucose nor FFA was given in control group.80％ confluent fibroblasts were incubated for 24 h and then proliferation was measured by using MTT assay.Second,in another experiment,the expression of β-catenin was detected by using Western blotting.HSFs were harvested before (0 h) or 4,8,12,16,20,24,and 48 h after stimulation with glucose (35 mmol/L),FFA (200 μmol/L) and both (35 mmol/L ± 200 μmol/L).Total protein was extracted from those cells.Protein levels of β-catenin were determined using Western blotting.Results (1) As compared with control group,the absorbance values of HSFs in glucose 35 mmol/L group and FFA 200 μmol/L group after culture for 24 h began to reduce (t =1.70 and 2.48respectively,P ＜ 0.05).With the increases of glucose and FFA,the absorbance values were significantly decreased (P ＜ 0.05).(2) HSFs exposed to high-FFA showed a low expression of β-catenin protein at 4.h (P ＜0.05),and the level of β-catenin protein began to reduce at 12 h after treatment with high-glucose medium (P＜0.05).However,the β-catenin protein expression in HSFs stimulated with glucose combined with FFA was decreased at 8 h,reaching the minimum at 48 h (P ＜ 0.05).Conclusion Glucose and FFA can both inhibit the expression of β-catenin.In a short time,high FFA exhibit inhibition of the expression on β-catenin,but this suppressive effect of high glucose was not obvious.When combined intervention,high glucose could prevent the low expression of β-catenin induced by FFA.In the case of the presence of sustained high glucose level,the inhibition of β-catenin was more significant than their respective role.
