發(fā)布時間：2018-04-22 20:34

  本文選題：高氧 + 肺損傷　； 參考：《重慶醫(yī)科大學》2012年碩士論文

【摘要】：背景 氧療是臨床上治療呼吸系統(tǒng)疾病的常用方法，但長時間高濃度氧，可產(chǎn)生大量的氧自由基，超過細胞的防御能力，從而引起炎癥，細胞損傷，壞死和凋亡相關(guān)基因的過表達，從而致各種急性或慢性肺損傷。肺泡上皮細胞是高氧暴露后氧化損傷的主要靶點，其損傷后的修復(fù)主要依靠肺泡Ⅱ型上皮細胞（AECⅡ）的增殖、分化為肺泡I型上皮細胞（AECⅠ）。AECⅡ細胞的功能狀態(tài)是決定肺損傷病理轉(zhuǎn)歸的主要因素。 神經(jīng)肽P物質(zhì)（SP）廣泛分布于氣道上皮細胞層內(nèi)，可啟動早期的神經(jīng)源性炎性反應(yīng)，參與對損傷細胞的修復(fù)、增殖、遷移、分化調(diào)控。本課題組前期研究發(fā)現(xiàn)神經(jīng)肽P物質(zhì)能夠降低高氧誘導的氧化損傷，但調(diào)節(jié)的分子機制尚不完全清楚。 目的 （1）早產(chǎn)鼠AECⅡ的體外分離、培養(yǎng)、純化及鑒定，以及氧化損 傷細胞模型的建立。 （2）高氧暴露對早產(chǎn)鼠肺泡Ⅱ型上皮細胞(AECⅡ)的氧化損傷以及神經(jīng)肽P物質(zhì)(SP)對AECⅡ的保護作用。 方法 （1）原代分離培養(yǎng)孕19d早產(chǎn)鼠AECⅡ，隨機分為4組：空氣組、高氧組、高氧+SP組、高氧+SP+SP拮抗劑組�？諝饨M和高氧組分別置于氧體積分數(shù)為0.21的空氣和氧體積分數(shù)為0.95的氧氣中暴露24h；高氧+SP組在置于空氣或高氧環(huán)境前加入SP；高氧+SP+SP拮抗劑組同時加入SP和SP受體拮抗劑L703.606后，再置于氧體積分數(shù)為0.95的氧氣中培養(yǎng)24h。 （2）倒置相差顯微鏡及電鏡下觀察高氧暴露及神經(jīng)肽P物質(zhì)干預(yù)對AECⅡ細胞的形態(tài)變化。 （3）DCFH-DA分子探針法檢查細胞內(nèi)ROS水平。 （4）MTT法和Annexin-V和PI雙染色法分別檢測細胞存活率和凋亡率。 結(jié)果 與空氣組比較，高氧組暴露24h，光鏡和電鏡觀察AECⅡ出現(xiàn)明顯的氧化損傷，活性氧水平明顯增加，進一步檢測可見細胞凋亡率明顯增加，存活率明顯降低。神經(jīng)肽SP預(yù)處理后形態(tài)學觀察可見細胞損傷明顯減輕，其活性氧水平明顯降低，凋亡率明顯降低，存活率明顯升高。然而，神經(jīng)肽SP受體拮抗劑L703.606干預(yù)后上述的保護作用明顯減弱，細胞氧化損傷較神經(jīng)肽SP預(yù)處理組明顯增加。 結(jié)論 高氧暴露24小時，可導致早產(chǎn)鼠AECⅡ發(fā)生氧化性損傷，誘導細胞凋亡。而神經(jīng)肽SP可部分減輕AECⅡ的氧化損傷，減少凋亡，促進細胞存活，對高氧損傷的AECⅡ起保護作用。 背景 SHH信號通路是一種重要的信號轉(zhuǎn)導通路，在細胞損傷修復(fù)中有著重要的作用。SHH產(chǎn)生有生物學活性的N末端片段(Shh-N),與其受體Patched1(Ptch1)結(jié)合。通過Hedgehog信號復(fù)合物(Hedgehogsignaling complex)介導,使信號轉(zhuǎn)錄因子Gli進入細胞核內(nèi),進一步激活其下游信號分子，起到基因和蛋白調(diào)控作用。大量研究表明SHH信號通路在胚胎形成，組織修復(fù)，創(chuàng)傷愈合，腫瘤形成方面有重要作用。在鼠肺組織，研究發(fā)現(xiàn)Shh信號通路在肺的形態(tài)發(fā)生和器官形成中起著至關(guān)重要的作用。 前一部分已經(jīng)證實了神經(jīng)肽SP可部分減輕AECⅡ的氧化損傷，，減少凋亡，促進細胞存活，對高氧損傷的AECⅡ起保護作用。然而SP降低氧化損傷的機制尚不明確，是否與調(diào)控Shh信號通路有關(guān)，目前研究甚少。本部分進一步研究SP干預(yù)對高氧暴露下AECⅡ的損傷的作用，及是否有SHH信號通路的參與及SHH信號通路的調(diào)控機制。 目的 （1）早產(chǎn)鼠AECⅡ的原代培養(yǎng)及氧化損傷細胞模型的建立。 （2）研究高氧暴露及SP干預(yù)對早產(chǎn)鼠肺泡Ⅱ型上皮細胞(AECⅡ)中的SHH信號通路分子的活化情況，從而研究是否有SHH信號通路的參與及神經(jīng)肽P物質(zhì)(SP)干預(yù)對SHH信號通路的調(diào)控作用。 方法 （1）早產(chǎn)鼠AECⅡ的體外分離、培養(yǎng)、純化及鑒定，以及氧化損傷細胞模型的建立。 （2）熒光定量PCR檢測高氧暴露及SP干預(yù)對AECⅡ中SHH信號通路分子Shh和Patched1（Ptch1）基因表達的影響。 （3）Western-Blot檢測高氧暴露及SP干預(yù)對AECⅡ中SHH信號通路分子Shh和Patched1（Ptch1）基因表達的影響。 結(jié)果 與空氣組比較，高氧暴露24h后，AECⅡ內(nèi)SHH信號通路分子shhmRNA和蛋白水平明顯升高（P＜0.05），而Patched1mRNA和蛋白表達水平明顯降低。SP干預(yù)后，可促進高氧組AECⅡ內(nèi)Shh信號通路激活，高氧+SP組Shh和Patched1mRNA水平較單純高氧組明顯升高（P＜0.05）。而L703.606干預(yù)后可明顯逆轉(zhuǎn)上述SP的作用。 結(jié)論 SHH信號通路參與了高氧誘導細胞凋亡的信號轉(zhuǎn)導系統(tǒng)，介導了神經(jīng)肽SP對細胞的信號轉(zhuǎn)導過程，參與了對高氧誘導AECⅡ損傷的保護作用，其通路的各分子，執(zhí)行了SHH信號途徑的保護作用。
[Abstract]:background
Oxygen therapy is a commonly used method for clinical treatment of respiratory diseases, but a long time of high concentration of oxygen can produce a large number of oxygen free radicals, which exceed the defense ability of cells, thus causing inflammation, cell damage, necrosis and apoptosis related genes, and causing various acute or slow lung injury. Alveolar epithelial cells are oxidative damage after hyperoxia exposure. The main target of injury is that the repair of the injury mainly depends on the proliferation of the alveolar type II epithelial cells (AEC II), and the functional state of the I cell (AEC I) cells of the alveolar type (AEC I) is the main factor determining the pathological changes of the lung injury.
Neuropeptide P (SP) is widely distributed in the epithelial cell layer of the airway. It can initiate early neurogenic inflammatory response and participate in the repair, proliferation, migration and differentiation of damaged cells. Earlier studies have found that neuropeptide P can reduce oxidative damage induced by hyperoxia, but the molecular mechanism of regulation is not completely clear.
objective
(1) isolation, culture, purification and identification of AEC II from premature rats and oxidative damage.
The establishment of the injury cell model.
(2) oxidative damage of alveolar type II epithelial cells (AEC II) induced by hyperoxia exposure in premature rats and neuropeptide substance P (SP) protection against AEC II.
Method
(1) AEC II of preconceived 19d preterm rats were divided into 4 groups randomly: air group, hyperoxia group, hyperoxic +SP group, hyperoxic +SP+SP antagonist group. Air group and hyperoxic group exposed 24h in oxygen volume fraction of air and oxygen volume fraction 0.95 respectively; high oxygen +SP group was added to SP before air or high oxygen environment; high oxygen +SP+ SP antagonist group was added SP and SP receptor antagonist L703.606 at the same time, and then cultured in oxygen volume fraction of 0.95 oxygen to culture 24h..
(2) phase contrast microscope and electron microscope were used to observe the morphological changes of AEC II cells after hyperoxia exposure and neuropeptide P substance intervention.
(3) DCFH-DA molecular probe was used to examine the level of ROS in cells.
(4) cell viability and apoptosis rate were detected by MTT and Annexin-V and PI double staining respectively.
Result
Compared with the air group, the hyperoxic group exposed 24h, and the AEC II showed obvious oxidative damage, and the active oxygen level was obviously increased. The apoptosis rate was obviously increased and the survival rate was obviously decreased. The morphological observation of neuropeptide SP showed that the damage of cell damage was obviously reduced and the active oxygen level was obviously reduced and apoptosis was decreased. However, the protective effect of neuropeptide SP receptor antagonist L703.606 was obviously weakened, and the oxidative damage of cells was significantly increased than that of the neuropeptide SP preconditioning group.
conclusion
Hyperoxia exposure for 24 hours can induce oxidative damage and induce apoptosis of AEC II in premature rats. Neuropeptide SP can partially alleviate the oxidative damage of AEC II, reduce apoptosis, promote cell survival, and protect AEC II damaged by hyperoxia.
background
SHH signaling pathway is an important signal transduction pathway, which plays an important role in the repair of cell damage..SHH produces biologically active N terminal fragment (Shh-N) and its receptor Patched1 (Ptch1). Through the Hedgehog signal complex (Hedgehogsignaling complex), the signal transcription factor Gli enters the nucleus and further enters the nucleus. Activation of its downstream signal molecules plays a role in gene and protein regulation. A large number of studies have shown that SHH signaling pathway plays an important role in embryonic formation, tissue repair, wound healing, and tumor formation. In rat lung tissue, the Shh signaling pathway plays a vital role in lung morphogenesis and organ formation.
The previous part has confirmed that neuropeptide SP can partially alleviate oxidative damage in AEC II, reduce apoptosis, promote cell survival, and protect AEC II from hyperoxia damage. However, the mechanism of SP to reduce oxidative damage is not clear, and is not related to the regulation of the Shh signaling pathway. This part further studies the SP intervention for hyperoxia exposure. The role of AEC II injury and whether there are SHH signaling pathways and the regulation mechanism of SHH signaling pathway.
objective
(1) primary culture of AEC II and establishment of oxidative damage cell model in preterm rats.
(2) to study the activation of SHH signaling molecules in the alveolar type II epithelial cells (AEC II) of preterm rats by hyperoxia exposure and SP intervention, and to investigate whether there is the participation of SHH signaling pathway and the regulation of neuropeptide P substance (SP) intervention on SHH signaling pathway.
Method
(1) isolation, culture, purification and identification of AEC II from premature rats and establishment of an oxidative damage cell model.
(2) fluorescent quantitative PCR was used to detect the effects of hyperoxia exposure and SP intervention on the expression of Shh and Patched1 (Ptch1) genes in SHH signaling pathway in AEC II.
(3) Western-Blot was used to detect the effects of hyperoxia exposure and SP intervention on the expression of Shh and Patched1 (Ptch1) genes in SHH signaling pathway in AEC II.
Result
Compared with the air group, the level of shhmRNA and protein in the SHH signaling pathway in AEC II was significantly increased (P < 0.05) after high oxygen exposure (P < 0.05). The Patched1mRNA and protein expression level obviously decreased the prognosis of.SP stem, which could promote the activation of Shh signaling pathway in the hyperoxia group AEC II, and the Shh and Patched1mRNA levels in the hyperoxic +SP group were significantly higher than those in the simple hyperoxia group (0.05). L703.606 can significantly reverse the effect of SP after intervention.
conclusion
SHH signaling pathway participates in the signal transduction system of hyperoxia induced cell apoptosis, mediates the signal transduction of neuropeptide SP to cells, and participates in the protection of AEC II injury induced by hyperoxia, and the various molecules of the pathway have performed the protection of the SHH signal pathway.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R722.6

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