α-硫辛酸协同角化上皮生长因子KGF对胎鼠肺泡Ⅱ型上皮细胞高氧损伤的保护作用及机制研究

发布时间：2018-01-24 15:43

本文关键词：高氧肺损伤氧化应激角化上皮生长因子肺泡Ⅱ型上皮细胞氧化应激角化上皮生长因子 p53 组蛋白去乙酰化酶1 高氧肺损伤肺泡Ⅱ型上皮细胞角化上皮细胞生长因子 α-硫辛酸高氧肺损伤氧化应激　出处：《华中科技大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分氧化应激抑制KGF对胎鼠肺泡Ⅱ型上皮细胞的保护作用 [目的]建立早产新生大鼠高氧暴露肺损伤细胞模型,探讨常氧及高氧环境下,角化上皮生长因子KGF对胎鼠肺泡Ⅱ型上皮细胞生存凋亡的影响。 [方法]建立原代培养的胎鼠肺泡Ⅱ型上皮细胞高氧损伤模型,随机分为KGF干预组和对照组,采用流式细胞仪检测细胞内活性氧ROS水平,采用MTT、LDH法检测细胞存活、死亡情况,Western Blot检测活化caspase-3蛋白的表达。 [结果] (1)常氧环境下加入KGF,0-25ng/ml范围内,与空白对照组相比,各组MTT依次升高、LDH依次下降,呈剂量依赖性；50ng/ml时,MTT、LDH变化无显著差异。 (2)高氧环境下培养细胞,随通氧时间的延长,与空气对照组相比,细胞内ROS水平显著升高、MTT明显降低、LDH显著升高；高氧4h、8h时,活化的Caspase-3表达依次升高。 (3)高氧环境下培养细胞0.5h～12h,分别给予15-100ng/ml KGF。0.5h、1h时,KGF各浓度组MTT值高于对照组,LDH低于空气对照组,50ng/ml范围内呈现剂量依赖性；高氧4h,KGF50ng/ml时LDH低于对照组,15-25ng/ml范围内MTT高于对照组；8h时,需提高KGF浓度到50ng/ml、75ng/ml,对应的MTT值、LDH值与对照组相比具有统计学意义；12h时,KGF各浓度组与对照组相比均无统计学意义。高氧4h、8h时,分别给予25、75ng/ml KGF,激活的Caspase-3表达明显低于对照组。 [结论]KGF能够在常氧、短时间高氧情况下促进肺泡Ⅱ型上皮细胞的增殖、抑制凋亡,但延长的高氧暴露会降低KGF敏感性,抑制KGF保护效应第二部分氧化应激诱导胎鼠肺泡Ⅱ型上皮细胞发生KGF抵抗的机制探究 [目的]通过早产新生大鼠肺泡Ⅱ型上皮细胞高氧损伤的模型,探讨高氧环境下,长时间高浓度氧暴露诱导ATIICs发生KGF抵抗的分子机制。 [方法]无菌、氧浓度95%的高氧环境中培养原代SD大鼠胎鼠肺泡Ⅱ型上皮细胞。随机将细胞分为单纯高氧暴露组和角化上皮因子(Keratinocyte growth factor, KGF)处理组。高氧暴露0-36h,MTT法检测细胞存活增殖情况；LDH法检测细胞死亡情况；实时荧光定量PCR测定KGFR mRNA的表达; Western Blot测定KGFR蛋白、磷酸化p53、HDAC1、乙酰化H3、乙酰化H4的表达；免疫共沉淀技术检测磷酸化p53与HDAC1之间的联系；抗氧化酶系的活性由相应的商品化试剂盒测定。 [结果]与单纯高氧组细胞相比： 1)高氧暴露4h,KFGR mRNA的表达开始降低,8h、12h时,KFGR mRNA的表达明显低于对照组；相应的,高氧暴露24-36h,KFGR蛋白的表达明显降低； 2)高氧暴露4h、8h、12h后,p53392位丝氨酸位点的磷酸化(Phospho-p53(ser392))水平显著升高,总p53表达量不变； 3)免疫共沉淀实验证明磷酸化p53(ser392)的活化进一步招募下游分子HDAC1的表达增加,p53抑制齐pifithrin-a可以抑制磷酸化p53(ser392)的活化,同时抑制p53对HDAC1的招募。 4)乙酰化组蛋白H4在高氧暴露4h、8h后表达水平明显降低,而乙酰化组蛋白H3的表达未见明显变化；HDAC抑制剂TSA可以抑制高氧诱导的乙酰化组蛋白H4的表达下调 5)高氧环境下,KGF对抗氧化酶系的表达无显著影响 [结论]高氧环境,KGFR表达水平下调是诱导ATIICs引起KGF抵抗的主要原因,其中Phospho-p53(ser392)-HDAC1-乙酰化组蛋白H4信号通路的活化是导致KGFR表达水平下调的关键第三部分 α-硫辛酸协同KGF对对胎鼠肺泡Ⅱ型上皮细胞高氧损伤的保护机制 [目的]通过早产新生大鼠肺泡Ⅱ型上皮细胞高氧损伤的模型,探讨高氧环境下,α-硫辛酸协同KGF联合应用对肺泡Ⅱ型上皮细胞高氧损伤的的保护作用及机制 [方法]无菌、氧浓度95%的高氧环境中培养原代SD大鼠胎鼠肺泡Ⅱ型上皮细胞。随机将细胞分为单纯高氧暴露组、KGF处理组、α-硫辛酸(α-Lipoic acid, α-LA)处理组及KGF-αt-硫辛酸共处理组。高氧暴露下,采用流式细胞仪检测细胞内活性氧族(reactive oxygen species, ROS)水平,MTT法检测细胞存活增殖情况；LDH法检测细胞死亡情况；实时荧光定量PCR测定KGFR mRNA的表达；Western Blot测定KGFR蛋白、磷酸化p53的表达。抗氧化酶系(超氧化物歧化酶、谷胱甘肽还原酶、过氧化氢酶、总抗氧化能力)的活性由相应的商品化试剂盒测定。 [结果] 1)与单纯高氧组相比,随着高氧暴露时间的延长,α-硫辛酸组细胞内活性氧ROS水平显著降低； 2)与单纯高氧组相比,高氧暴露4h、8h,α-硫辛酸组磷酸化p53(ser392)的表达明显降低；高氧暴露24h、48h时,KGFR的表达明显高于前两组； 3)与单纯高氧组、KGF处理组相比,高氧暴露8h、12h, KGF-α-硫辛酸共处理组ATⅡCs MTT水平明显高于前两组；LDH释放水平显著低于前两组,细胞保护作用显著； 4)与单纯高氧组相比,aα-硫辛酸组可以显著提高细胞内谷胱甘肽还原酶的活性,提高总抗氧化能力； [结论]高氧暴露环境下,α-硫辛酸能明显降低细胞内氧化应激水平,抑制磷酸化p53(ser392)的活化、抑制高氧诱导的KGFR表达下调,从根本上解决KGFR抵抗现象。因此,KGF与α-硫辛酸联合应用可以最大程度的发挥KGF对肺泡Ⅱ型上皮细胞高氧损伤的的保护作用。
[Abstract]:Part 1 the protective effect of oxidative stress inhibition of KGF on alveolar type II epithelial cells in fetal rats
[Objective] to establish a rat model of hyperoxia induced lung injury in premature rats, and to explore the effect of keratinocyte growth factor KGF on the survival and apoptosis of fetal alveolar type II epithelial cells in normoxic and hyperoxic environment.
[Methods] established in alveolar epithelial type high oxygen injury model of primary cultured KGF, were randomly divided into intervention group and control group. Flow cytometry was used to detect the intracellular ROS level, using MTT LDH method to detect cell survival and death, Western Blot to detect the expression of activated caspase-3 protein.
[results]
(1) in the range of KGF and 0-25ng/ml added to normoxic environment, MTT increased sequentially and LDH decreased in a dose-dependent manner compared with blank control group, while MTT and LDH did not change significantly at 50ng/ml.
(2) when cultured in hyperoxia environment, the level of ROS increased significantly, the MTT decreased and LDH increased significantly compared with the air control group. When the time of hyperoxia 4H and 8h increased, the expression of activated Caspase-3 increased.
(3) 12h cells 0.5h to high oxygen environment, were treated with 15-100ng/ml KGF.0.5h, 1H, KGF, MTT of each group was higher than those of the control group, LDH lower than those of the control group, the range of 50ng/ml in a dose-dependent manner; high oxygen 4h, KGF50ng/ml LDH lower than the control group, the range of 15-25ng/ml MTT is higher than that of control group; 8h, to increase the concentration of KGF to 50ng/ml, 75ng/ml, MTT values, LDH values compared with the control group with statistical significance; 12h, different concentration of KGF group compared with the control group had no statistical significance. The high oxygen 4h, 8h, 25,75ng/ml were given KGF, activated the expression of Caspase-3 was significantly lower than the control group.
[conclusion]KGF can promote proliferation and inhibit apoptosis of alveolar type II epithelial cells under normal oxygen and short time hyperoxia, but prolonged hyperoxia exposure can reduce KGF sensitivity and inhibit KGF protection.
The mechanism of KGF resistance induced by second parts of oxidative stress in fetal rat alveolar type II epithelial cells
[Objective] to explore the molecular mechanism of KGF resistance induced by prolonged exposure to hyperoxia in high oxygen environment through the model of hyperoxia induced by alveolar type II epithelial cells in preterm newborn rats.
[method] aseptic culture, high oxygen environment with 95% oxygen concentration in primary cultured SD rat alveolar type II cells were randomly divided into simple cells exposed to hyperoxia group and keratinized epithelial (Keratinocyte growth factor, KGF factor) treatment group. Hyperoxia exposure 0-36h, MTT method to detect cell survival and proliferation; the cell death was detected by LDH; the expression was detected by real-time PCR KGFR mRNA Western Blot; KGFR protein, phosphorylation of p53, HDAC1, H3 acetylation, expression of acetylated H4; CO immunoprecipitation between detection of phosphorylated p53 and HDAC1; antioxidant enzyme activity by the corresponding commercial reagent the box was determined.
[results] compared with the simple hyperoxic group cells:
1) after high oxygen exposure, the expression of KFGR mRNA began to decrease, while the expression of KFGR mRNA in 8h and 12h was significantly lower than that in the control group. Correspondingly, the expression of KFGR protein decreased significantly in 24-36h exposed to high oxygen.
2) after hyperoxia exposure 4h, 8h, 12h, the level of phosphorylation (Phospho-p53 (ser392)) of p53392 serine site increased significantly, and the total p53 expression was unchanged.
3) Co immunoprecipitation assay showed that the activation of phosphorylated p53 (ser392) further increased the expression of HDAC1 downstream of the downstream molecule. P53 inhibited pifithrin-a activation and inhibited p53 recruitment to HDAC1.
4) acetylated histone H4 decreased significantly after hyperoxia exposure to 4h and 8h, while the expression of acetylated histone H3 did not change significantly. HDAC inhibitor TSA could inhibit the expression of histone H4 in hyperoxia induced acetylation.
5) there was no significant effect of KGF on the expression of antioxidant enzymes in high oxygen environment
[Conclusion] the reduction of KGFR expression in hyperoxia environment is the main reason to induce KGF resistance induced by ATIICs. The activation of Phospho-p53 (ser392) -HDAC1- acetylation histone H4 signal pathway is the key to decrease KGFR expression level.
The third part
Protective mechanism of alpha lipoic acid synergistic KGF on hyperoxia injury of fetal rat alveolar type II epithelial cells
[Objective] to explore the protective effect and mechanism of alpha lipoic acid combined with KGF on alveolar type II epithelial cells hyperoxia induced by hyperoxia in preterm newborn rats.
[method] aseptic culture, high oxygen environment with 95% oxygen concentration in primary cultured SD rat alveolar type II cells were randomly divided into simple cells exposed to hyperoxia group, KGF treatment group, alpha lipoic acid (-Lipoic, alpha acid, alpha -LA) treatment group and KGF- t- alpha lipoic acid treatment group. Hyperoxia exposure, flow cytometry was used to detect intracellular reactive oxygen species (reactive oxygen, species, ROS) level, MTT method to detect cell survival and proliferation of cells by LDH assay; death; expression was detected by real-time PCR KGFR mRNA Western Blot; KGFR protein expression and phosphorylation of p53. Antioxidant enzymes (superoxide dismutase, glutathione reductase, catalase activity, total antioxidant capacity) by the commercial kit.
[results]
1) compared with the hyperoxia group, the ROS level in the cells of the alpha lipoic acid group decreased significantly with the prolongation of high oxygen exposure time.
2) compared with the hyperoxia group, the expression of phosphorylated p53 (ser392) in hyperoxia exposed 4h, 8h and alpha lipoic acid group was significantly lower than that in the hyperoxia group. The expression of KGFR in hyperoxia exposed 24h and 48h was significantly higher than that in the first two groups.
3) compared with the hyperoxia group and KGF treatment group, the level of AT II Cs MTT in hyperoxia exposed 8h, 12h, KGF- alpha lipoic acid co treatment group was significantly higher than that in the first two groups, LDH release level was significantly lower than that in the first two groups, and the cytoprotection effect was significant.
4) compared with the simple hyperoxia group, the a alpha lipoic acid group could significantly increase the activity of the Uchiya Ka reductase and improve the total antioxidant capacity.
[Conclusion] exposure to high oxygen environment, alpha lipoic acid can significantly reduce the level of oxidative stress in cells, inhibit the phosphorylation of p53 (ser392) activation, inhibition of hyperoxia induced the expression of KGFR and KGFR to solve the resistance phenomenon fundamentally. Therefore, most can play a protective role of KGF on alveolar type II epithelial cell injury and high oxygen KGF alpha lipoic acid combined with application.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

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