【作者】 付红敏；

【导师】 匡凤梧； 许峰；

【作者基本信息】 重庆医科大学 ， 儿科学， 2009， 博士

【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(P<0.01)。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,P<0.01)。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(P<0.01)。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(p<0.01)。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(p<0.01)。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传递的保护功能更多还原

【Abstract】 Part one An enhanced method for isolation,primary culture,and identification of typeⅡalveolar Epithelial cells from fetal ratsObjectiveTo develop the isolation and purification technology for typeⅡalveolar epithelial cells (AECⅡ) of fetal rat, thus providing experimental means for the study of lung development or neonatal lung disease.MethodsLung tissues of 19-day fetal rats were digested with trypsin and collagenase, then purified for AECⅡwith different centrifugal force and repeated attachment. Cell viability was evaluated by typran inclusion dying before plating into six-well flask. Growth status and shape of attached cells were observed with inverted phase contrast microscope. AECⅡwere identified by electron microscope and its percentage was assessed by modified Papanicolaou dying and immunofluorescence ,the latter aiming to detect expression of surfactant protein C(SP-C) in AECⅡ.Results The total amount of cells we harvested from 3 to5 fetal rat was (36±5)×106 with a viability of (98±2)%. Cells were like polygonal and connected like island under microscope.AECⅡwas ascertained by lamellar bodies found in cytoplasma under electron microscope and its percentage was (96±3)% identified by modified Papanicolaou dying, the same as the result by immunofluorescence for SP-C.ConclusionsHighly Purified and viable AECⅡcould be achieved by our methods and the cell model could be used in further study.Part two Protection of calcitonin gene-related peptide in hyperoxia-induced injury of premature rat typeⅡalveolar epithelial cellsObjectiveTo explore the influence of 60% oxygen and the effects of calcitonin gene-related peptide(CGRP) on typeⅡalveolar epithelial cells(AECⅡ) isolated from premature rat lung in vitro.Methods AECⅡwere isolated from 19d fetal rat lung and cultured for 12h to attach. Then AECⅡwere randomly divided into four groups: air group, hyperoxia group, hyperoxia plus CGRP group, hyperoxia plus CGRP and CGRP8-37 group. AECⅡof air group and hyperoxia group were exposed to 21% or 60% oxygen respectively for 24h while hyperoxia plus CGRP group were added with CGRP and hyperoxia plus CGRP and CGRP8-37 group with CGRP and CGRP8-37(CGRP receptor antagonist) before placed into 60% oxygen. Concentrations of maleic dialdehyde(MDA), superoxide dismutase(SOD) and total antioxidant capacity (TAOC) in culture cells were detected by ultraviolet spectrophotometer. Reactive oxygen species (ROS) and apoptosis rate of AECII were analyzed by flow cytometry and the mRNA level of surfactant associated protein C (SP-C) was measured by RT-PCR.ResultsThe levels of MDA, ROS and apoptosis cell number were increased whereas TAOC, SOD and SP-C mRNA expression declined in hyperoxia group compared with those in air group(P<0.01). Reversely, MDA, ROS and apoptosis rate were significantly lower and levels of TAOC, SOD and SP-C mRNA expression were significantly higher in group hyperoxia plus CGRP group than in hyperoxia group or hyperoxia plus CGRP and CGRP8-37 group.Conclusions Exposure to 60% oxygen for 24h could cause oxidative injury, induce apoptosis and decrease SP-C mRNA level of AECII in vitro in premature rats, while CGRP may play a protective role against hyperoxic lung injury by antioxidant, inhibition of AEC apoptosis and promotion of the SP-C mRNA expression.Part three The effects of calcitonin gene-related peptide on proliferation of premature rat typeⅡalveolar epithelial cells exposed to 60% oxygenObjectiveTo explore the influence of 60% oxygen and the effects of calcitonin gene-related peptide(CGRP) on growth of typeⅡalveolar epithelial cells(AECⅡ) isolated from premature rat lung in vitro.MethodsAECⅡwere isolated from 19d fetal rat lung and cultured for 12h to attach. Then AECⅡwere randomly divided into six groups: air, air/CGRP, air/CGRP8-37, hyperoxia, O2/CGRP, and O2/CGRP8-37. AECⅡwere exposed to FiO2 21%(air) or 60% (hyperoxia) for 24h respectively. Air/CGRP and O2/CGRP were carried out by adding CGRP into medium and then putting the plates into air or 60% oxygen for 24h. Air/CGRP8-37 and O2/CGRP8-37 were done by adding both CGRP and CGRP8-37 into cultural fluid before placing the plate into air or 60% oxygen. MTT assay was taken to determine the optimal concentration of CGRP by evaluating the effects of different concentration of CGRP(10-10～10-7M) on growth of normal AECⅡ. Cell proliferation ability was determined by MTT assay and cell cycles by flow cytometry. The mRNA levels and protein contents of surfactant protein C (SP-C) in six groups were measured by RT-PCR and immunofluorescence respectively.ResultsThe 10-8M of CGRP was choose in the study for that MTT results showed CGRP promoted growth of AECⅡin a dose-dependent manner in a range of 10-10～10-8M. CGRP in a concentration of 10-8M could also increase the rate of cells subjected to S and G2/M phases in cell cycles and raised the expression of SP-C mRNA and protein in normal culture of AECⅡ. When exposed to 60% oxygen for 24h, AECⅡshowed a decreased viability, enhanced proportion of cell in G0/G1 phase with a corresponding decline of rates in S and G2/M phases, and reduced levels of mRNA and proteins of SP-C. But addition with 10-8M of CGRP prior to hyperoxia treatment promoted AECⅡproliferation, enhanced the cell proportions of S and G2/M phases, and raised the expression of SP-C mRNA and protein.Conclusions Exposure to 60% oxygen for 24h could inhibited AECⅡgrowth, proliferation and differentiation whereas CGRP could partially counteract the inhibition effects of hyperoxia on AECⅡand play a protective role against hyperoxia-induced lung injury.Part four The protection of CGRP on hyperoxia-induced typeⅡalveolar epithelial cell injury mediated by PKCalpha pathwayObjectiveTo explore the mechanism of CGRP in protecting AECⅡfrom hyperoxia-induced injury through the determination of the signal transduction molecular -- PKC alpha and NF -κB.MethodsAECⅡwere isolated from 19d fetal rat lung and cultured for 12h to attach. Then AECⅡwere randomly divided into six groups: air, air/CGRP, air/CGRP8-37, hyperoxia, O2/CGRP, and O2/CGRP8-37. AECⅡwere exposed to FiO2 21%(air) or 60% (hyperoxia) for 24h respectively. Air/CGRP and O2/CGRP were carried out by adding CGRP into medium and then putting the plates into air or 60% oxygen for 24h. Air/CGRP8-37 and O2/CGRP8-37 were done by adding both CGRP and CGRP8-37 into cultural fluid before placing the plate into air or 60% oxygen. Western blot was taken to detect the fraction of PKCalpha in membrane and cytosol and translocation of NF-κB was observed under laser confocal microscopy.ResultsWhen CGRP was added into the medium in normal cultural condition, AECⅡshow an increased ratio of membrane to cytoplasm fraction of PKCalpha and strong fluorescence of NF-κB in nucleus compared with those in air and air/CGRP8-37(p < 0.01). After exposure to hyperoxia, the ratio of membrane to cytoplasm fraction of PKC declined and the fluorescence of NF-κB in nucleus enhanced compared with those in air. The ratio of PKCalpha and fluorescence of NF-κB were increased in O2/CGRP compared with those in hyperoxia and O2/CGRP8-37. The differences among groups were significant.ConclusionsThe PKC alpha mediated signal transduction of CGRP and participated in the process that CGRP protected AECⅡfrom hyperoxia-induced injury, while NF-κB is a downstream molecular of PKC alpha, executing in part the function of the PKC alpha signal.更多还原