神经肽P物质在高氧诱导肺泡Ⅱ型上皮细胞损伤中的作用及其对SHH信号通路影响的研究

发布时间：2018-04-22 20:34

本文选题：高氧 + 肺损伤　；参考：《重庆医科大学》2012年硕士论文

【摘要】：背景氧疗是临床上治疗呼吸系统疾病的常用方法，但长时间高浓度氧，可产生大量的氧自由基，超过细胞的防御能力，从而引起炎症，细胞损伤，坏死和凋亡相关基因的过表达，从而致各种急性或慢性肺损伤。肺泡上皮细胞是高氧暴露后氧化损伤的主要靶点，其损伤后的修复主要依靠肺泡Ⅱ型上皮细胞（AECⅡ）的增殖、分化为肺泡I型上皮细胞（AECⅠ）。AECⅡ细胞的功能状态是决定肺损伤病理转归的主要因素。神经肽P物质（SP）广泛分布于气道上皮细胞层内，可启动早期的神经源性炎性反应，参与对损伤细胞的修复、增殖、迁移、分化调控。本课题组前期研究发现神经肽P物质能够降低高氧诱导的氧化损伤，但调节的分子机制尚不完全清楚。目的（1）早产鼠AECⅡ的体外分离、培养、纯化及鉴定，以及氧化损伤细胞模型的建立。（2）高氧暴露对早产鼠肺泡Ⅱ型上皮细胞(AECⅡ)的氧化损伤以及神经肽P物质(SP)对AECⅡ的保护作用。方法（1）原代分离培养孕19d早产鼠AECⅡ，随机分为4组：空气组、高氧组、高氧+SP组、高氧+SP+SP拮抗剂组。空气组和高氧组分别置于氧体积分数为0.21的空气和氧体积分数为0.95的氧气中暴露24h；高氧+SP组在置于空气或高氧环境前加入SP；高氧+SP+SP拮抗剂组同时加入SP和SP受体拮抗剂L703.606后，再置于氧体积分数为0.95的氧气中培养24h。（2）倒置相差显微镜及电镜下观察高氧暴露及神经肽P物质干预对AECⅡ细胞的形态变化。（3）DCFH-DA分子探针法检查细胞内ROS水平。（4）MTT法和Annexin-V和PI双染色法分别检测细胞存活率和凋亡率。结果与空气组比较，高氧组暴露24h，光镜和电镜观察AECⅡ出现明显的氧化损伤，活性氧水平明显增加，进一步检测可见细胞凋亡率明显增加，存活率明显降低。神经肽SP预处理后形态学观察可见细胞损伤明显减轻，其活性氧水平明显降低，凋亡率明显降低，存活率明显升高。然而，神经肽SP受体拮抗剂L703.606干预后上述的保护作用明显减弱，细胞氧化损伤较神经肽SP预处理组明显增加。结论高氧暴露24小时，可导致早产鼠AECⅡ发生氧化性损伤，诱导细胞凋亡。而神经肽SP可部分减轻AECⅡ的氧化损伤，减少凋亡，促进细胞存活，对高氧损伤的AECⅡ起保护作用。背景 SHH信号通路是一种重要的信号转导通路，在细胞损伤修复中有着重要的作用。SHH产生有生物学活性的N末端片段(Shh-N),与其受体Patched1(Ptch1)结合。通过Hedgehog信号复合物(Hedgehogsignaling complex)介导,使信号转录因子Gli进入细胞核内,进一步激活其下游信号分子，起到基因和蛋白调控作用。大量研究表明SHH信号通路在胚胎形成，组织修复，创伤愈合，肿瘤形成方面有重要作用。在鼠肺组织，研究发现Shh信号通路在肺的形态发生和器官形成中起着至关重要的作用。前一部分已经证实了神经肽SP可部分减轻AECⅡ的氧化损伤，，减少凋亡，促进细胞存活，对高氧损伤的AECⅡ起保护作用。然而SP降低氧化损伤的机制尚不明确，是否与调控Shh信号通路有关，目前研究甚少。本部分进一步研究SP干预对高氧暴露下AECⅡ的损伤的作用，及是否有SHH信号通路的参与及SHH信号通路的调控机制。目的（1）早产鼠AECⅡ的原代培养及氧化损伤细胞模型的建立。（2）研究高氧暴露及SP干预对早产鼠肺泡Ⅱ型上皮细胞(AECⅡ)中的SHH信号通路分子的活化情况，从而研究是否有SHH信号通路的参与及神经肽P物质(SP)干预对SHH信号通路的调控作用。方法（1）早产鼠AECⅡ的体外分离、培养、纯化及鉴定，以及氧化损伤细胞模型的建立。（2）荧光定量PCR检测高氧暴露及SP干预对AECⅡ中SHH信号通路分子Shh和Patched1（Ptch1）基因表达的影响。（3）Western-Blot检测高氧暴露及SP干预对AECⅡ中SHH信号通路分子Shh和Patched1（Ptch1）基因表达的影响。结果与空气组比较，高氧暴露24h后，AECⅡ内SHH信号通路分子shhmRNA和蛋白水平明显升高（P＜0.05），而Patched1mRNA和蛋白表达水平明显降低。SP干预后，可促进高氧组AECⅡ内Shh信号通路激活，高氧+SP组Shh和Patched1mRNA水平较单纯高氧组明显升高（P＜0.05）。而L703.606干预后可明显逆转上述SP的作用。结论 SHH信号通路参与了高氧诱导细胞凋亡的信号转导系统，介导了神经肽SP对细胞的信号转导过程，参与了对高氧诱导AECⅡ损伤的保护作用，其通路的各分子，执行了SHH信号途径的保护作用。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.6

【参考文献】