本文關(guān)鍵詞： 高氧肺損傷 氧化應(yīng)激 角化上皮生長(zhǎng)因子 肺泡Ⅱ型上皮細(xì)胞 氧化應(yīng)激 角化上皮生長(zhǎng)因子 p53 組蛋白去乙酰化酶1 高氧肺損傷 肺泡Ⅱ型上皮細(xì)胞 角化上皮細(xì)胞生長(zhǎng)因子 α-硫辛酸 高氧肺損傷 氧化應(yīng)激　出處：《華中科技大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分氧化應(yīng)激抑制KGF對(duì)胎鼠肺泡Ⅱ型上皮細(xì)胞的保護(hù)作用 [目的]建立早產(chǎn)新生大鼠高氧暴露肺損傷細(xì)胞模型,探討常氧及高氧環(huán)境下,角化上皮生長(zhǎng)因子KGF對(duì)胎鼠肺泡Ⅱ型上皮細(xì)胞生存凋亡的影響。 [方法]建立原代培養(yǎng)的胎鼠肺泡Ⅱ型上皮細(xì)胞高氧損傷模型,隨機(jī)分為KGF干預(yù)組和對(duì)照組,采用流式細(xì)胞儀檢測(cè)細(xì)胞內(nèi)活性氧ROS水平,采用MTT、LDH法檢測(cè)細(xì)胞存活、死亡情況,Western Blot檢測(cè)活化caspase-3蛋白的表達(dá)。 [結(jié)果] (1)常氧環(huán)境下加入KGF,0-25ng/ml范圍內(nèi),與空白對(duì)照組相比,各組MTT依次升高、LDH依次下降,呈劑量依賴性；50ng/ml時(shí),MTT、LDH變化無(wú)顯著差異。 (2)高氧環(huán)境下培養(yǎng)細(xì)胞,隨通氧時(shí)間的延長(zhǎng),與空氣對(duì)照組相比,細(xì)胞內(nèi)ROS水平顯著升高、MTT明顯降低、LDH顯著升高；高氧4h、8h時(shí),活化的Caspase-3表達(dá)依次升高。 (3)高氧環(huán)境下培養(yǎng)細(xì)胞0.5h～12h,分別給予15-100ng/ml KGF。0.5h、1h時(shí),KGF各濃度組MTT值高于對(duì)照組,LDH低于空氣對(duì)照組,50ng/ml范圍內(nèi)呈現(xiàn)劑量依賴性；高氧4h,KGF50ng/ml時(shí)LDH低于對(duì)照組,15-25ng/ml范圍內(nèi)MTT高于對(duì)照組；8h時(shí),需提高KGF濃度到50ng/ml、75ng/ml,對(duì)應(yīng)的MTT值、LDH值與對(duì)照組相比具有統(tǒng)計(jì)學(xué)意義；12h時(shí),KGF各濃度組與對(duì)照組相比均無(wú)統(tǒng)計(jì)學(xué)意義。高氧4h、8h時(shí),分別給予25、75ng/ml KGF,激活的Caspase-3表達(dá)明顯低于對(duì)照組。 [結(jié)論]KGF能夠在常氧、短時(shí)間高氧情況下促進(jìn)肺泡Ⅱ型上皮細(xì)胞的增殖、抑制凋亡,但延長(zhǎng)的高氧暴露會(huì)降低KGF敏感性,抑制KGF保護(hù)效應(yīng) 第二部分氧化應(yīng)激誘導(dǎo)胎鼠肺泡Ⅱ型上皮細(xì)胞發(fā)生KGF抵抗的機(jī)制探究 [目的]通過早產(chǎn)新生大鼠肺泡Ⅱ型上皮細(xì)胞高氧損傷的模型,探討高氧環(huán)境下,長(zhǎng)時(shí)間高濃度氧暴露誘導(dǎo)ATIICs發(fā)生KGF抵抗的分子機(jī)制。 [方法]無(wú)菌、氧濃度95%的高氧環(huán)境中培養(yǎng)原代SD大鼠胎鼠肺泡Ⅱ型上皮細(xì)胞。隨機(jī)將細(xì)胞分為單純高氧暴露組和角化上皮因子(Keratinocyte growth factor, KGF)處理組。高氧暴露0-36h,MTT法檢測(cè)細(xì)胞存活增殖情況；LDH法檢測(cè)細(xì)胞死亡情況；實(shí)時(shí)熒光定量PCR測(cè)定KGFR mRNA的表達(dá); Western Blot測(cè)定KGFR蛋白、磷酸化p53、HDAC1、乙酰化H3、乙�；疕4的表達(dá)；免疫共沉淀技術(shù)檢測(cè)磷酸化p53與HDAC1之間的聯(lián)系；抗氧化酶系的活性由相應(yīng)的商品化試劑盒測(cè)定。 [結(jié)果]與單純高氧組細(xì)胞相比： 1)高氧暴露4h,KFGR mRNA的表達(dá)開始降低,8h、12h時(shí),KFGR mRNA的表達(dá)明顯低于對(duì)照組；相應(yīng)的,高氧暴露24-36h,KFGR蛋白的表達(dá)明顯降低； 2)高氧暴露4h、8h、12h后,p53392位絲氨酸位點(diǎn)的磷酸化(Phospho-p53(ser392))水平顯著升高,總p53表達(dá)量不變； 3)免疫共沉淀實(shí)驗(yàn)證明磷酸化p53(ser392)的活化進(jìn)一步招募下游分子HDAC1的表達(dá)增加,p53抑制齊pifithrin-a可以抑制磷酸化p53(ser392)的活化,同時(shí)抑制p53對(duì)HDAC1的招募。 4)乙�；M蛋白H4在高氧暴露4h、8h后表達(dá)水平明顯降低,而乙酰化組蛋白H3的表達(dá)未見明顯變化；HDAC抑制劑TSA可以抑制高氧誘導(dǎo)的乙酰化組蛋白H4的表達(dá)下調(diào) 5)高氧環(huán)境下,KGF對(duì)抗氧化酶系的表達(dá)無(wú)顯著影響 [結(jié)論]高氧環(huán)境,KGFR表達(dá)水平下調(diào)是誘導(dǎo)ATIICs引起KGF抵抗的主要原因,其中Phospho-p53(ser392)-HDAC1-乙�；M蛋白H4信號(hào)通路的活化是導(dǎo)致KGFR表達(dá)水平下調(diào)的關(guān)鍵 第三部分 α-硫辛酸協(xié)同KGF對(duì)對(duì)胎鼠肺泡Ⅱ型上皮細(xì)胞高氧損傷的保護(hù)機(jī)制 [目的]通過早產(chǎn)新生大鼠肺泡Ⅱ型上皮細(xì)胞高氧損傷的模型,探討高氧環(huán)境下,α-硫辛酸協(xié)同KGF聯(lián)合應(yīng)用對(duì)肺泡Ⅱ型上皮細(xì)胞高氧損傷的的保護(hù)作用及機(jī)制 [方法]無(wú)菌、氧濃度95%的高氧環(huán)境中培養(yǎng)原代SD大鼠胎鼠肺泡Ⅱ型上皮細(xì)胞。隨機(jī)將細(xì)胞分為單純高氧暴露組、KGF處理組、α-硫辛酸(α-Lipoic acid, α-LA)處理組及KGF-αt-硫辛酸共處理組。高氧暴露下,采用流式細(xì)胞儀檢測(cè)細(xì)胞內(nèi)活性氧族(reactive oxygen species, ROS)水平,MTT法檢測(cè)細(xì)胞存活增殖情況；LDH法檢測(cè)細(xì)胞死亡情況；實(shí)時(shí)熒光定量PCR測(cè)定KGFR mRNA的表達(dá)；Western Blot測(cè)定KGFR蛋白、磷酸化p53的表達(dá)。抗氧化酶系(超氧化物歧化酶、谷胱甘肽還原酶、過氧化氫酶、總抗氧化能力)的活性由相應(yīng)的商品化試劑盒測(cè)定。 [結(jié)果] 1)與單純高氧組相比,隨著高氧暴露時(shí)間的延長(zhǎng),α-硫辛酸組細(xì)胞內(nèi)活性氧ROS水平顯著降低； 2)與單純高氧組相比,高氧暴露4h、8h,α-硫辛酸組磷酸化p53(ser392)的表達(dá)明顯降低；高氧暴露24h、48h時(shí),KGFR的表達(dá)明顯高于前兩組； 3)與單純高氧組、KGF處理組相比,高氧暴露8h、12h, KGF-α-硫辛酸共處理組ATⅡCs MTT水平明顯高于前兩組；LDH釋放水平顯著低于前兩組,細(xì)胞保護(hù)作用顯著； 4)與單純高氧組相比,aα-硫辛酸組可以顯著提高細(xì)胞內(nèi)谷胱甘肽還原酶的活性,提高總抗氧化能力； [結(jié)論]高氧暴露環(huán)境下,α-硫辛酸能明顯降低細(xì)胞內(nèi)氧化應(yīng)激水平,抑制磷酸化p53(ser392)的活化、抑制高氧誘導(dǎo)的KGFR表達(dá)下調(diào),從根本上解決KGFR抵抗現(xiàn)象。因此,KGF與α-硫辛酸聯(lián)合應(yīng)用可以最大程度的發(fā)揮KGF對(duì)肺泡Ⅱ型上皮細(xì)胞高氧損傷的的保護(hù)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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 邵寒娟,陳亮,林濤;角質(zhì)細(xì)胞生長(zhǎng)因子研究進(jìn)展[J];生命科學(xué);2004年01期

，

本文編號(hào)：1460393