CGRP在高氧诱导肺泡Ⅱ型上皮细胞损伤中的作用及其信号转导途径探讨

发布时间：2018-06-03 13:12

本文选题：胎鼠 + 肺泡Ⅱ型上皮细胞　；参考：《重庆医科大学》2009年博士论文

【摘要】： 第一部分早产胎鼠肺泡II型上皮细胞分离、纯化、培养及鉴定目的探讨胎鼠肺泡Ⅱ型上皮细胞(AECⅡ)的分离、纯化及原代培养方法,建立胎鼠AECⅡ细胞模型,为有关胎儿肺发育及新生儿肺部疾病的研究奠定基础。方法采用胰酶结合胶原酶的消化方法,分离肺组织细胞成份,然后经差速离心和差速贴壁的方法纯化AECⅡ,进行原代培养;通过台盼蓝染色检测细胞活力,倒置相差显微镜观察细胞生长特点及形态特征,透射电镜鉴定,改良巴氏染色检测细胞纯度以及免疫荧光技术检测表面蛋白C(SP-C)的表达。结果每3～5只胎鼠可获得AECⅡ(36±5)×106,活力(98±2)%。镜下观察原代AECⅡ呈岛状生长,外观呈多边形或立方形。透射电镜可见特征性的板层小体,改良巴氏染色见胞质内有较多颗粒,纯度为96±3%,呈现SP-C绿色免疫荧光的细胞占96%以上。结论利用胰酶和胶原酶消化,以及差速离心和差速贴壁的方法可成功分离出高产量、高纯度的胎鼠AECⅡ,能满足体外进一步实验的需要。第二部分高氧暴露对早产鼠肺泡Ⅱ型上皮细胞的氧化损伤及CGRP的保护作用目的观察高氧对早产鼠肺泡Ⅱ型上皮细胞(AECⅡ)的影响以及降钙素基因相关肽(CGRP)对AECⅡ的保护作用。方法将原代分离培养的孕19d早产鼠AECⅡ接种至6孔培养板,实验随机分为空气组、高氧组、高氧CGRP组、高氧CGRP受体拮抗剂组。空气组和高氧组分别置于体积分数为21%的空气和60%的氧气中暴露24h;高氧CGRP组在暴露前加入CGRP;高氧CGRP拮抗剂组在高氧CGRP组基础上加入CGRP受体拮抗剂(CGRP8-37)。培养24h后,用分光光度计测定各组丙二醛(MDA)、总抗氧化能力(TAOC)、超氧化物歧化酶(SOD)水平;用流式细胞仪检测活性氧(ROS)和细胞凋亡率;用逆转录聚合酶链反应(RT-PCR)测定表面活性蛋白C (SP-C)的mRNA表达。结果与空气组比较,高氧组MDA、ROS及细胞调亡率均显著增高,TAOC、SOD水平及SP-C mRNA表达均显著降低(P均0.01)。与高氧组比较,高氧CGRP组细胞MDA、ROS水平及细胞调亡率均显著下降;而TAOC、SOD水平及SP-C mRNA表达均明显增高(P0.01)。高氧CGRP拮抗剂组与高氧组各指标比较差异均无统计学意义。结论 60%氧暴露24h可导致早产鼠AECⅡ发生氧化损伤,诱导细胞凋亡及SP-C mRNA表达下降;而CGRP可部分减轻AECⅡ的氧化损伤,减少凋亡,促进SP-CmRNA表达,对高氧损伤的AECⅡ起保护作用。第三部分CGRP对60%氧暴露早产鼠AECⅡ生长增殖的影响目的探讨降钙素基因相关肽(CGRP)对60%氧暴露早产鼠肺泡Ⅱ型上皮细胞(AECⅡ)生长增殖的影响。方法原代分离培养孕19d早产鼠AECⅡ,随机分为6组:空气组、空气CGRP组、空气CGRP拮抗剂组、高氧组、高氧CGRP组、高氧CGRP拮抗剂组。空气组和高氧组分别在氧体积分数为21%的空气和60%的氧气中暴露24h;空气或高氧CGRP组在置于空气或高氧环境前加入CGRP;空气或高氧CGRP拮抗剂组同时加入CGRP和CGRP受体拮抗剂后,再置于空气或60%的氧气中培养24h。先用MTT比色法测定不同浓度CGRP(10-10～10-7M)对正常AECⅡ生长的影响,以确定CGRP的实验最佳浓度;分别采用MTT法和流式细胞术测定各组细胞增殖能力和细胞周期,逆转录聚合酶链反应和免疫荧光技术测定表面活性蛋白C (SP-C)的mRNA及蛋白表达。结果 MTT法结果显示,CGRP从10-10～10-8M,呈剂量依赖方式促进正常培养的早产鼠AECⅡ生长,因此选择10-8M CGRP来干预细胞。加入10-8M CGRP还可使正常培养的AECⅡ进入G2/M及S期的比例增多,SP-C mRNA及SP-C蛋白表达增高(与空气组相比,P均 0.01)。暴露于60%氧24h后,细胞存活率下降,G0/G1期细胞比例增高,G2/M及S期细胞相应降低,SP-C mRNA及SP-C蛋白表达低下(与空气组比较,P均0.01)。而预先加入10-8M CGRP后,促进了高氧暴露AECⅡ的增殖能力,使S及G2/M期细胞增多,并可提高AECⅡ的SP-C mRNA及SP-C蛋白表达水平(与高氧组及高氧CGRP拮抗剂组比较,P0.01)。结论 60%氧暴露24h可抑制早产鼠AECⅡ增殖分化,而CGRP可促进AECⅡ生长,部分解除高氧对AECⅡ的抑制作用。第四部分PKCα信号转导途径介导了CGRP对高氧肺泡Ⅱ型上皮细胞损伤的保护作用目的通过测定PKCα和NF-κB的活化情况,从细胞内信号转导通路这一角度探讨CGRP对高氧肺泡上皮细胞损伤的保护作用机制。方法原代分离培养孕19d早产鼠AECⅡ,随机分为6组:空气组、空气CGRP组、空气CGRP拮抗剂组、高氧组、高氧CGRP组、高氧CGRP拮抗剂组。空气组和高氧组分别在氧体积分数为21%的空气和60%的氧气中暴露24h;空气或高氧CGRP组在置于空气或高氧环境前加入CGRP,空气或高氧CGRP拮抗剂组同时加入CGRP和CGRP受体拮抗剂,再置于空气或60%的氧气中培养24h。用Western blot检测胞膜和胞浆PKCα的表达变化,激光共聚焦检测NF-κB的核表达情况。结果在正常培养的AECⅡ细胞中加入CGRP后,胞膜与胞浆PKCα的比值显著增高,NFκB的荧光较强,与空气组及空气CGRP拮抗剂组相比有显著性差异(P0.01)。细胞暴露于高氧后,胞膜与胞浆PKC比值显著低于空气组,而NFκB的荧光强度高于空气组(p0.01)。高氧CGRP组二者比值及核内NFκB荧光强度高于高氧组及高氧CGRP拮抗剂组,差异有显著性意义(p0.01)。结论 PKCα介导了CGRP对细胞的信号传递过程,参与了CGRP对高氧AECⅡ损伤的保护作用,而NF-κB是PKCα的下游信号,部分执行了PKCα传递的保护功能
[Abstract]:Part 1 isolation, purification, culture and identification of alveolar type II epithelial cells from preterm fetal rats
objective
To explore the isolation, purification and primary culture of type II epithelial cells (AEC II) of fetal rat alveolar type II epithelial cells (AEC II), and to establish a model of AEC II cells in fetal mice, which lay the foundation for the study of fetal lung development and neonatal pulmonary disease.
Method
The cell components of lung tissue were separated by trypsin and collagenase digestion. Then AEC II was purified by differential centrifugation and differential adherence to primary culture. Cell viability was detected by trypan blue staining, and cell growth characteristics and morphological characteristics were observed by inverted phase contrast microscope. Transmission electron microscopy was used to identify cells and improve the detection of cells by PAP staining. Purity and immunofluorescence technique were used to detect the expression of surface protein C (SP-C).
Result
AEC II (36 + 5) * 106 and vitality (98 + 2) were obtained in every 3~5 fetal mice. The original AEC II was observed under the microscope. The appearance was polygonal or cubic. The transmission electron microscope showed the characteristic lamellar body. The improved PAP staining showed that there were more granules in the cytoplasm, the purity was 96 + 3%, and the cells showing SP-C green immunofluorescence were more than 96%.
conclusion
The use of trypsin and collagenase digestion, differential centrifugation and differential adherence can successfully isolate AEC II of high yield and high purity fetal mice, which can meet the needs of further experiments in vitro.
The second part is the oxidative damage of alveolar type II epithelial cells induced by hyperoxia exposure in premature rats and the protective effect of CGRP.
objective
Objective To observe the effect of hyperoxia on alveolar type II epithelial cells (AEC II) in premature rats and the protective effect of calcitonin gene-related peptide (CGRP) on AEC II.
Method
The pregnant 19d preterm rat AEC II was inoculated to 6 Hole culture plate. The experiment was randomly divided into air group, hyperoxia group, hyperoxic CGRP group and hyperoxic CGRP receptor antagonist group. Air group and hyperoxic group were exposed to 24h in the air of volume fraction of 21% and 60% of oxygen in 60% oxygen; high oxygen CGRP group was added to CGRP before exposure; the group of high oxygen CGRP antagonist was in the group The CGRP receptor antagonist (CGRP8-37) was added to the hyperoxic CGRP group. After the culture of 24h, a spectrophotometer was used to determine all groups of malondialdehyde (MDA), the total antioxidant capacity (TAOC), the level of superoxide dismutase (SOD), the detection of reactive oxygen species (ROS) and the apoptosis rate by flow cytometry, and the determination of the surface active protein C (SP-) by reverse transcription polymerase chain reaction (RT-PCR). MRNA expression of C).
Result
Compared with the air group, the MDA, ROS and cell apoptosis rate of hyperoxia group were significantly increased, TAOC, SOD level and SP-C mRNA expression were significantly decreased (P 0.01). Compared with the hyperoxia group, the MDA, ROS level and cell apoptosis rate of the hyperoxic CGRP group decreased significantly, while TAOC, SOD level and higher expression were higher. There was no significant difference in each index of oxygen group.
conclusion
60% oxygen exposure to 24h can lead to oxidative damage of AEC II in premature rats, induce apoptosis and decrease the expression of SP-C mRNA, and CGRP can partially alleviate the oxidative damage of AEC II, decrease apoptosis, promote the expression of SP-CmRNA, and protect the AEC II injured by hyperoxia.
The third part is the effect of CGRP on the growth and proliferation of AEC II in premature rats exposed to 60% oxygen.
objective
Objective to investigate the effects of calcitonin gene-related peptide (CGRP) on the growth and proliferation of alveolar type II epithelial cells (AEC II) in 60% oxygen exposed premature rats.
Method
AEC II of preterm 19d preterm rats was randomly divided into 6 groups: air group, air CGRP group, air CGRP antagonist group, hyperoxia group, hyperoxic CGRP group, hyperoxic CGRP antagonist group. Air group and hyperoxic group exposed 24h in oxygen volume fraction of 21% air and 60% oxygen respectively; air or hyperoxic CGRP group was added before air or high oxygen environment. After entering CGRP, air or hyperoxic CGRP antagonist group was added to CGRP and CGRP receptor antagonists, and then cultured in air or 60% of oxygen, 24h. was first used to determine the effect of CGRP (10-10 ~ 10-7M) on normal AEC II growth by MTT colorimetry, in order to determine the optimal concentration of CGRP, and the cells were determined by MTT method and flow cytometry respectively. Proliferative capacity and cell cycle, reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence technique were used to detect the expression of mRNA and protein of surface active protein C (SP-C).
Result
The results of MTT showed that CGRP from 10-10 to 10-8M promoted the growth of AEC II in normal cultured preterm rats, so 10-8M CGRP was selected to interfere with the cells. The proportion of AEC II into G2/M and S phase in normal culture was increased by adding 10-8M CGRP, and increased (0.01) from the air group (compared with the air group). 60% After oxygen 24h, the cell survival rate decreased, the proportion of G0/G1 cells increased, G2/M and S cells decreased correspondingly, SP-C mRNA and SP-C protein were low (P was 0.01). The proliferation of AEC II was promoted after the addition of 10-8M CGRP. Reached the level (compared with hyperoxia group and hyperoxia CGRP antagonist group, P0.01).
conclusion
60% oxygen exposure 24h can inhibit the proliferation and differentiation of AEC II in premature rats, while CGRP can promote AEC II growth, partially relieving the inhibitory effect of hyperoxia on AEC II.
In the fourth part, PKC alpha signal transduction mediates the protective effect of CGRP on hyperoxia alveolar type II epithelial cells.
objective
By measuring the activation of PKC and NF- kappa B, we explored the protective mechanism of CGRP on the injury of hyperoxia alveolar epithelial cells from the signal transduction pathway.
Method
AEC II of preterm 19d preterm rats was randomly divided into 6 groups: air group, air CGRP group, air CGRP antagonist group, hyperoxia group, hyperoxic CGRP group, hyperoxic CGRP antagonist group. Air group and hyperoxic group exposed 24h in oxygen volume fraction of 21% air and 60% oxygen respectively; air or hyperoxic CGRP group was added before air or high oxygen environment. In CGRP, air or hyperoxic CGRP antagonist group was added to CGRP and CGRP receptor antagonists, and then in air or 60% oxygen, 24h. was cultured with Western blot to detect the expression of PKC alpha in cell membrane and cytoplasm, and the nuclear expression of NF- kappa B was detected by confocal laser.
Result
After adding CGRP to normal AEC II cells, the ratio of cell membrane to cytoplasmic PKC a was significantly higher, and the fluorescence of NF kappa B was stronger than that in air group and air CGRP antagonist group (P0.01). After exposure to hyperoxia, the ratio of cell membrane to cytoplasmic PKC was significantly lower than that in air group, and the fluorescence intensity of NF kappa B was higher than that of air group (P0.01). The ratio of the two groups and the fluorescence intensity of NF kappa B in the oxygen CGRP group were higher than those in the hyperoxia group and the hyperoxia CGRP antagonist group (P0.01).
conclusion
PKC - alpha mediates the signaling process of CGRP to cells, and participates in the protective effect of CGRP on hyperoxia AEC II damage, and NF- - kappa B is a downstream signal of PKC alpha, partially performing the protection function of PKC alpha transmission
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363

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