【作者】 刘文；

【导师】 管庆波；

【作者基本信息】 山东大学 ， 内分泌及代谢病， 2011， 硕士

【摘要】 [目的]硫化氢(hydrogen sulphide, H2S)是继NO和CO之后发现的第三种内源性气体信号分子,具有保护血管内皮,减轻内皮损伤的作用。糖尿病血管病变的基础是血管内皮损伤,高浓度葡萄糖是血管内皮损伤的重要危险因素之一。糖尿病高糖毒性引起内皮细胞损伤,是糖尿病血管性并发症发生的重要原因。外源性的H2S是否可以改善高浓度葡萄糖对内皮细胞的损害,至今少有报道。本研究的目的是通过原代培养人脐静脉内皮细胞(HUVECs)并进行细胞鉴定,观察硫氢化钠(NaHS,外源性的H2S供体)对高浓度葡萄糖环境下HUVECs的细胞存活率及H2S生成酶胱硫醚一γ-裂解酶(cystathionine-y-Iyase, CSE)的影响。[方法]0.125%胰酶和0.01%EDTA灌注法以获得HUVECs,将细胞置于含10%胎牛血清的M199培养基中；放于37℃、5%C2O的细胞培养箱中培养。第2、3代细胞用于实验。(1) HUVECs密度达到70%左右时在倒置显微镜下摄片观察细胞形态,用兔抗人八因子相关抗原(Ⅷ因子-R Ag)多克隆抗体进行细胞鉴定。(2)当细胞70%-80%融合时,换成含2%FBS的培养基继续培养,分别用5.5mmol/L葡萄糖、25 mmol/L葡萄糖、25 mmol/L葡萄糖+50μmol/L NaHS及50μmol/LNaHS处理72 h,采用MTT方法检测各组细胞的存活率,用Western blot方法检测CSE蛋白水平的变化。[结果](1)倒置显微镜下可见,HUVECs单层贴壁生长,第1天呈小团状聚集,3-5 d后呈铺路石样生长,细胞鉴定显示Ⅷ因子阳性,胞浆呈棕黄色,阴性对照组胞浆未见着色。(2)MTT检测表明,25 mmol/L葡萄糖组的HUVECs存活率比5.5 mmol/L葡萄糖组下降28.37%(P<0.05),而50μmol/L NaHS和25 mmol/L葡萄糖共同处理组的细胞存活率比25 mmol/L葡萄糖组上升30.3%(P<0.05),50μmol/L NaHS组与5.5 mmol/L葡萄糖组无明显差异(P>0.05)。(3)与5.5 mmol/L葡萄糖组相比,以25 mmol/L葡萄糖处理72 h后能使CSE蛋白的表达下降,而25 mmol/L葡萄糖+50μmol/L NaHS则能改善这种损害作用,CSE蛋白的表达比25 mmol/L葡萄糖组增强,CSE蛋白相对定量分析显示25 mmol/L葡萄糖处理HUVECs 72 h后,能使CSE蛋白的表达下降53.7%(P<0.05),50μmol/L NaHS和25 mmol/L葡萄糖共处理组使CSE的表达较25mmol/L葡萄糖组上升19%(P<0.05)。[结论](1)0.125%胰酶+0.01%EDTA消化法可获得数量可观的脐静脉内皮细胞,为体外实验提供可靠的人脐静脉内皮细胞模型。(2)高浓度葡萄糖降低人原代脐静脉内皮细胞的生长和存活率,减少CSE蛋白的表达；而NaHS能够减轻高浓度葡萄糖对CSE的作用。更多还原

【Abstract】 [Objective] Hydrogen sulfide (H2S) is the third endogenous gaseous signal molecule besides nitric oxide and carbon monoxide, H2S has protective effect on endothelial cells and can reduce endothelial injury. Endothelial injury is the basis of diabetic vascular disease. Among many impairment endothelial factors, over high level of glucose is one of the main factors. High glucose toxicity can cause endothelial injury, which is the important reason of diabetic vascular diseases. Up to date, there is little research on whether exogenous H2S can ameliorate the damage by high glucose on endothelial cells. In this study, primary human umbilical vein endothelial cells (HUVECs) were cultured and identified by the presence of von Willebrand factor, and then we aimed to observe the effect of sodium hydrosulfide (exogenous doner of H2S) on HUVECs viability and the expression of cystathionine-γ-Iyase (CSE) when the cells cultured with high glucose.【Methods】0.125% trypsin and 0.01% EDTA perfusion method was used to isolate HUVEC, and then the gained cells were cultured in M199 medium containing 10% fetal bovine serum (FBS) in the 37℃、5% carbon dioxide incubator. All experiments were performed using cells bwteen passages 2 to 3.(1)When HUVECs reached 70% fusion, morphological observation were obtained by phase contrast microscope, and HUVECs were identified by detection ofⅧfactor.(2) When the cells reached 70%～80% fusion, medium containing 2% FBS was used instead of 10% FBS. The cells were treated for 72 h with 5.5 mmol/L glucose,25 mmol/L glucose,25 mmol/L glucose with 50μmol/L NaHS and 50μmol/L NaHS. MTT method was used for cells viability. Protein level of CSE was measured by western blot.[Results] (1)HUVECs could adhere to the plate and were monolayer, and they were small group distributed on the frist day, after 3 to 5 days, HUVECs were shaped like paving stone. And the cytoplasm of HUVECs were yellowish-brown identified by theⅧfactor, while the negative group were not.(2) MTT showed that compared to 5.5 mmol/L glucose group, cells viability of 25 mmol/L glcouse decreased 28.37%(P<0.05), while 50μmol/L NaHS with 25 mmol/L glucose group rose 30.3%(P<0.05) compared to 25 mmol/L glucose group, and there was no significant difference between 50μmol/L NaHS group with 5.5 mmol/L glucose group.(3) Western blot showed that 25 mmol/L glcouse could reduce the protein expression of CSE compared to 5.5 mmol/L glucose and 50μmol/L NaHS with 25 mmol/L glucose group could ameliorate this change that the expression of CSE protein was enhanced. Relative quantitative analysis of CSE showed that the expression of CSE protein in 25 mmol/L glucose group decreased 53.7%(P<0.05) compared to 5.5 mmol/L glucose group, while 50μmol/L NaHS with 25 mmol/L glucose group rose 19%(P<0.05) compared to 25 mmol/L glucose group.[Conclusion] (1) 0.125% trypsin and 0.01% EDTA perfusion method was feasible to obtain HUVECs and provided a reliable cell model.(2) High glucose could lower cells viability and decrease the expression of CSE in HUVECs, and NaHS could ameliorate the effect caused by high glucose.更多还原