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短暂高糖环境暴露对人真皮微血管内皮细胞生物学行为的影响

Effects of transient exposure to high glucose on biological behaviors of human dermal microvascular endothelial cells

摘要:

目的 观察短暂高糖环境暴露对体外培养人真皮微血管内皮细胞生物学行为的影响.方法 本研究所有检测指标对应的细胞分组及处理如下:将第4代人真皮微血管内皮细胞按随机数字表法分成3组,每组12孔.对照组细胞采用含5 mmol/L D-葡萄糖的完全培养液培养7d;短暂高糖组细胞在含30 mmol/L D-葡萄糖的完全培养液中培养2d,再改用含5 mmol/L D-葡萄糖的完全培养液继续培养5d;长时高糖组细胞采用含30 mmol/L D-葡萄糖的完全培养液培养7d.(1)采用倒置光学显微镜观察对照组和长时高糖组培养7d以及短暂高糖组培养2、7d细胞形态学变化.(2)培养0、2、4、7d,采用细胞存活率分析计数器检测细胞增殖率.(3)培养2d后进行划痕实验,划痕后继续培养,于划痕后0、24、48、72、96、120 h测量划痕面积,计算后5个时相点细胞迁移率.(4)培养0、2、4、7d,采用细胞分析仪检测细胞凋亡率.(5)培养7d后再在基质胶上培养24 h,采用倒置光学显微镜观察血管样结构形成情况,并计算血管样结构长度、分叉点数量.(6)培养2、4、7d,采用实时荧光定量RT-PCR法检测血管化相关基因基质金属蛋白酶组织抑制剂3(TIMP-3)mRNA表达.对数据行析因设计方差分析、重复测量方差分析、单因素方差分析、LSD检验. 结果 (1)对照组细胞培养7d,细胞呈卵圆形,铺路石样排列.短暂高糖组细胞培养2d,形态变细长,排列丧失铺路石样;培养7d仍保持细长,丧失铺路石样排列.长时高糖组细胞培养7d,形态变细长,排列丧失铺路石样.(2)培养0d,3组细胞增殖率差异无统计学意义(F=0.23,P>0.05).培养2d,短暂高糖组和长时高糖组细胞增殖率相近(P>0.05),且均低于对照组(P值均小于0.01);培养4、7d,短暂高糖组和对照组细胞增殖率相近(P值均大于0.05),且均高于长时高糖组(P值均小于0.01).(3)划痕后24~ 120 h,短暂高糖组和长时高糖组细胞迁移率均相近(P值均大于0.05),且均低于对照组(P值均小于0.01).(4)培养0d,3组细胞凋亡率差异无统计学意义(F=0.78,P>0.05).培养2d,短暂高糖组和长时高糖组细胞凋亡率相近(P>0.05),且均高于对照组(P值均小于0.01);培养4、7d,短暂高糖组和对照组细胞凋亡率相近(P值均大于0.05),且均低于长时高糖组(P值均小于0.01).(5)短暂高糖组和长时高糖组细胞形成血管样结构长度分别为(1.84±0.10) ×105、(1.82±0.11) ×105 μm(P>0.05),均短于对照组的(2.75±0.23) ×105 μm(P值均小于0.01);短暂高糖组和长时高糖组细胞形成血管样结构分叉点数量分别为(43±5)、(46 ±8)个(P>0.05),均少于对照组的(103 ±21)个(P值均小于0.01).(6)培养2、4、7d,短暂高糖组和长时高糖组细胞TIMP-3的mRNA表达量相近(P值均大于0.05),且均低于对照组(P值均小于0.05). 结论 短暂高糖环境暴露能诱导体外培养的人真皮微血管内皮细胞形态、迁移和血管形成发生“代谢记忆”从而导致其产生持久的生物学行为变化,其机制可能与调控血管化相关基因发生改变有关.

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abstracts:

Objective To observe the effects of transient exposure to high glucose on biological behaviors of human dermal microvascular endothelial cells cultured in vitro.Methods The dividing method and treatment of cells for the detection of all indexes in this study were as follows.Human dermal microvascular endothelial cells of the 4th passage were divided into 3 groups according to the random number table,with 12 wells in each group.Cells in control group (C) were cultured with complete culture solution containing 5 mmol/L D-glucose for 7 d.Cells in transient high glucose group (THG) were cultured with complete culture solution containing 30 mmol/L D-glucose for 2 d and complete culture solution containing 5 mmol/L D-glucose for 5 d.Cells in prolonged high glucose group (PHG) were cultured with complete culture solution containing 30 mmol/L D-glucose for 7 d.(1) The cell morphology in groups C and PHG on culture day 7 and that in group THG on culture day 2 and 7 was observed by inverted optical microscope.(2) On culture day 0,2,4,and 7,cell proliferation rate was determined by cell viability analyzing counter.(3) After culture day 2,the scratch experiment was performed,and the cells were further cultured.At post scratch hour (PSH) 0,24,48,72,96,and 120,the scratch area was measured,and the cell migration rates of the latter 5 time points were calculated.(4) On culture day 0,2,4,and 7,the cell apoptosis rate was determined by cell analyzer.(5) Cells were seeded into Matrigel to culture for 24 h after culture day 7.The formation of vessel-like structure was observed by inverted optical microscope.The length and number of branch point of vessel-like structure were calculated.(6) On culture day 2,4,and 7,mRNA expression of vascularization-related gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) was determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction.Data were processed with analysis of variance of factorial design,analysis of variance for repeated measurement,one-way analysis of variance,and LSD test.Results (1) Cells in group C exhibited ovary shape in cobble stone order on culture day 7.Cells in group THG exhibited long ovary shape and lost cobble stone order on culture day 2 and kept the same changes on culture day 7.Cells in group PHG exhibited long ovary shape and lost cobble stone order on culture day 7.(2) On culture day 0,there was no significant difference in cell proliferation rate among the 3 groups (F =0.23,P > 0.05).On culture day 2,cell proliferation rates in groups THG and PHG were similar (P > 0.05),which were significantly lower than the cell proliferation rate in group C (with P values below 0.01).On culture day 4 and 7,the cell proliferation rates in groups THG and C were similar (with P values above 0.05),which were significantly higher than those in group PHG (with P values below 0.01).(3) At PSH 24-120,the cell migration rates in groups THG and PHG were similar (with P values above 0.05),which were significantly lower than those in group C (with P values below 0.01).(4) On culture day 0,there was no statistically significant difference in cell apoptosis rate among the 3 groups (F =0.78,P > 0.05).On culture day 2,cell apoptosis rates in groups THG and PHG were similar (P > 0.05),which were significantly higher than the cell apoptosis rate in group C (with P values below 0.01).On culture day 4 and 7,the cell apoptosis rates in groups THG and C were similar (with P values above 0.05),which were significantly lower than those in group PHG (with P values below 0.01).(5) The length of vessel-like structure of cells in group THG was (1.84 ± 0.10) × 105 μm,close to (1.82 ± 0.11) ×105 μmin group PHG (P >0.05),both significantly shorter than (2.75±0.23) ×105 μm in group C (with P values below 0.01).The numbers of branch point of vessel-like structure of cells in groups THG and PHG were 43 ±5 and 46 ± 8 respectively,which were close to each other (P >0.05) and both significantly less than 103 ± 21 in group C (with P values below 0.01).(6) On culture day 2,4,and 7,mRNA expressions of TIMP-3 of cells in groups THG and PHG were similar (with P values above 0.05),which were significantly lower than those in group C (with P values below 0.05).Conclusions Transient exposure to high glucose can cause metabolic memory of morphology,migration,and angiogenesis in human dermal microvascular endothelial cells cultured in vitro,resulting in sustained changes in biological behaviors.The mechanism may be related to the changes of vascularization-related genes.

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作者: 乔亮 [1] 杨惠忠 [1] 李学川 [1] 黄晓琴 [1] 原博 [1] 周增丁 [1]
期刊: 《中华烧伤杂志》2017年33卷2期 77-82页 MEDLINEISTICPKUCSCD
栏目名称: 慢性难愈性创面
DOI: 10.3760/cma.j.issn.1009-2587.2017.02.004
发布时间: 2017-03-14
基金项目:
国家自然科学基金(81272112)National Natural Science Foundation of China
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