探讨不同培养条件对糖尿病细胞模型的内皮细胞(VEC)和平滑肌细胞(VSMC)增殖、凋亡的影响,检测糖尿病模型中的VEC和VSMC的代谢产物, 为糖尿病心血管并发症早期诊断的代谢性生物标记前体物的筛选提供实验依据.通过模拟糖尿病血液环境,分别建立糖尿病VEC和VSMC的细胞模型,采用MTT法和Hoechst 33258荧光染色法观察不同培养条件下细胞的形态和生长变化,并检测乳酸脱氢酶(LDH)、活性氧簇(ROS)的活性.结果显示:高糖培养短期内诱导VEC和VSMC增殖,且VEC 24 h达到最大增殖速率,VSMC在48 h达到最大增殖速率,但高糖长时程作用导致细胞凋亡,细胞形态变化显著;30 mM高糖培养48 h后,VEC细胞的LDH、ROS较对照组显著升高,凋亡率明显;而白藜芦醇亚甲胺(RMI)可翻转ROS及LDH的升高,减少细胞在模拟糖尿病内环境中受到的损伤,提示培养VEC 24 h及VSMC在48 h时间点收集细胞代谢气体可作为质谱分析数据.糖尿病细胞模型中ROS、LDH显著升高,诱导细胞凋亡,暗示糖尿病严重患者血液的血糖、血酮严重失常,酸中毒明显,推测其代谢性前体物质可能为丙酮酸、LDH等,呼吸气体中丙酮及CO2可作为糖尿病心血管并发症早期诊断的候选物质.
Abstract
To explore the effect of different culture conditions on endothelial cells of diabetic cell model (VEC) and smooth muscle cells (VSMC) proliferation, apoptosis; to test metabolites in the diabetic model of VEC and VSMC, to provide experimental evidence for the selection of metabolic biomarkers precursor in the early diagnosis of diabetic cardiovascular complications. By simulating the diabetes blood environment, establishing diabetes VEC and VSMC cell model separately, using MTT method and Hoechst 33258 fluorescent staining method to observe the morphology and growth of cells under different culture conditions, the lactate dehydrogenase (LDH), reactive oxygen species (ROS) activity will be tested. Result showed that high sugar culture induced the proliferation of VEC and VSMC in a short term, VEC reached its maximum growth rate within 24 h while VSMC reached its maximum growth rate in 48 h. However, high sugar led to the cell death and morphology changes in long range interactions significantly. After culturing 30 mM for 48 h, the LDH, ROS in VEC cells grew significantly, the apoptosis rate can be noticed easily. As to the flipped ROS and LDH in reseratrol methylene amine (RMI) increased, the suffered damage in simulated environment diabetes decreased. Tipped cultured metabolic gas point in VSMC VEC cells was collected to use as mass spectrometry data within 48 h. ROS, LDH in cell model of diabetes increased significantly. The induced cell death by activation suggested severe diabetes blood glucose, blood ketone serious disorders, acidosis significantly of patients. Metabolic precursors can be presumed as pyruvate, LDH, etc. Acetone and CO2 in the breathing gas are substance candidate for early diagnosis of cardiovascular complications of diabetes.
关键词
糖尿病心血管并发症 /
内皮细胞 /
平滑肌细胞 /
LDH /
丙酮 /
CO2
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Key words
cardiovascular complications of diabetes /
endothelial cells /
smooth muscle cells /
LDH /
Acetone /
CO2
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参考文献
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脚注
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基金
国家自然科学基金资助项目(31171281,31301116);广州市教育局科技计划资助项目(10A044);广东省大学生创新训练资助项目(1107812068)
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