【作者】 卢红艳；

【导师】 常立文；

【作者基本信息】 华中科技大学 ， 儿科新生儿， 2007， 博士

【摘要】 第一部分高氧对早产大鼠肺泡Ⅱ型上皮细胞增殖和转分化的影响【目的】建立高氧细胞损伤模型,探讨高氧对早产大鼠肺泡Ⅱ型上皮细胞(AECⅡ)增殖和转分化的影响,为高氧肺损伤时肺泡化障碍及肺损伤修复机制的研究提供实验依据。【方法】原代培养早产大鼠(孕19～20 d)AECⅡ,于接种15 h贴壁并更换培养液后,随机分为空气组和高氧组。高氧组通入95% O2-5% CO2 10 min后置于370C、5%CO2培养箱中培养,空气组直接置于370C、5%CO2培养箱中培养。于培养后24、48及72 h,收获各组细胞。高氧对早产大鼠AECII转分化的影响:利用倒置相差显微镜和透射电镜观察细胞的形态变化;免疫细胞化学染色法检测AECⅡ特异性肺泡表面活性蛋白-C(SP-C)及肺泡Ⅰ型上皮细胞(AECⅠ)特异性蛋白水通道蛋白5(AQP5)的表达;RT-PCR和流式细胞术分别检测SP-C、AQP5 mRNA及蛋白的表达。高氧对早产大鼠AECII增殖的影响:利用血球计数板计数法对培养细胞计数;台盼蓝拒染法检测细胞活力;流式细胞术检测细胞周期;免疫荧光法检测Ki67表达。【结果】空气组AECII在培养后其数目不断增加,高氧组给氧后48和72 h,细胞数目减少,细胞活力降低。高氧使G0/G1期细胞比例显著增多,S和G2/M期细胞比例明显减少。高氧组给氧后24、48及72 h,Ki67+细胞的表达率及荧光指数( FI )较相同时间点空气组均明显降低( P<0.05或P<0.01 )。同时,随着给氧时间延长,原代培养的AECⅡ伸展变平,失去其板层体及微绒毛,丧失AECⅡ的特性,获得AECⅠ样外观。伴随其形态学改变,AECⅡ逐渐停止表达其特异性蛋白SP-C,开始表达AECⅠ特异性蛋白AQP5。高氧组给O2后24、48及72 h,SP-C mRNA、SP-C+细胞的表达率及荧光指数较同时间点空气组明显降低( P<0.05或P<0.01 ),AQP5的上述指标在给O2后24及48 h则较空气组明显增加( P<0.05或P<0.01 ),给O2后72 h,AQP5的表达开始减弱,与同时间点空气组相比无明显差异(P﹥0.05)。【结论】(1)原代培养的早产大鼠AECⅡ暴露在高氧环境中发生G1期阻滞,Ki67表达减少,细胞增殖受抑,可能是高氧肺损伤肺泡化障碍发生的原因之一;(2)体外培养的AECⅡ可自然转分化,但暴露在高氧环境中其转分化明显加速,高氧诱导早产大鼠AECⅡ转分化在不成熟肺泡上皮细胞损伤修复中起重要作用。第二部分与成纤维细胞共培养下早产大鼠肺泡Ⅱ型上皮细胞增殖与转分化的研究【目的】建立早产大鼠肺泡Ⅱ型上皮细胞(AECⅡ)与肺成纤维细胞(LF)共培养模型,观察与LF共培养下早产大鼠AECⅡ增殖与转分化的生物学特性,为进一步研究AECⅡ与LF在肺发育及肺损伤中的相互作用提供实验基础。【方法】分离、纯化并签定早产大鼠AECⅡ和LF,利用PCF插入式Millicell培养皿和6孔板构建AEC-LF共培养模型,AECⅡ以5×105/mL的密度接种到6孔板中,LF以1×106/mL的密度接种到PCF插入式Millicell培养皿中,以6孔板中不放入套皿单独培养的AECⅡ为对照。于单独培养和共培养后2 d和4 d,利用倒置相差显微镜观察AECⅡ形态和基本生长情况;血球计数板计数各组不同时间点细胞数;台盼蓝染色检测细胞活力;RT-PCR和流式细胞术分别检测肺泡表面活性蛋白-C(SP-C)、水通道蛋白5(AQP5)mRNA及蛋白质表达;流式细胞术检测细胞周期及Ki67表达。【结果】(1)成功构建出早产大鼠AECⅡ和LF共培养模型,倒置相差显微镜下观察细胞活力可。(2)AECⅡ单独培养4 d,细胞分散生长,胞核伸展体积变大,核仁减少,部分转分化为AECⅠ样细胞;与LF共培养4 d ,细胞呈大的聚集样生长,细胞体积无明显增大,胞核仍呈圆形。与单独培养组相比,AECII与LF共培养2 d及4 d ,其SP-C mRNA及蛋白质表达明显增加(P <0.01),而AQP5 mRNA及蛋白质表达则明显减少(P <0.05或P <0.01)。上述细胞形态学及免疫标记物检测结果提示,与LF共培养时,AECⅡ能较好地保留其细胞形态,向AECⅠ转分化减少。(3)共培养2 d与单独培养2 d相比,AECⅡ的活力和细胞数目无明显差异。单独培养4 d,尽管AECII数目较单独培养2 d仍有增加,但增加幅度不明显,且细胞活力明显下降,而共培养4 d,AECII数目较单独培养4 d有明显增加,且(95.2±4.9)%的AECII台盼蓝仍拒染。与单独培养组相比,共培养2 d及4 d ,G0/G1期细胞均明显减少(P <0.01),而G2/M及S期细胞均明显增加(P <0.05或P <0.01),且表达Ki67+细胞的比率及荧光指数明显增加(P <0.01),说明与LF共培养时可促进AECII增殖。【结论】体外构建的早产大鼠AECⅡ与LF共培养模型,部分模拟了体内微环境。AECⅡ和LF共培养有利于保持AECⅡ的增殖和分化功能,可用于体外研究肺发育和肺损伤时AECⅡ和LF的相互作用。第三部分早产大鼠肺发育过程中SP-C和AQP5的动态表达及高氧的影响【目的】观察早产大鼠生后肺组织肺表面活性蛋白C(SP-C)和水通道蛋白5(AQP5)表达的动态变化及高氧的影响,探讨SP-C及AQP5在肺发育及肺损伤中的作用,为高氧肺损伤发生机制提供理论依据。【方法】剖宫术取出孕21 d (足月为22 d ) SD早产鼠,生后12～24 h内随机分为空气组和高氧组。高氧组持续暴露于85% O2中,空气组置于同一室内常压空气中。两组分别于暴露后1、4、7、10和14 d时,提取肺组织,采用免疫组织化学法对肺组织SP-C和AQP5表达定位,RT-PCR测定SP-C和AQP5 mRNA的表达,Western-blot法检测SP-C和AQP5蛋白表达。【结果】(1)早产大鼠生后不同时间肺组织均有SP-C表达,以生后1 d表达最明显,1 d以后在正常肺发育时下降,其阳性染色信号主要定位于AECⅡ。与同时间点空气组相比,高氧暴露1 d时,肺组织SP-C mRNA及蛋白表达显著降低(P <0.01),以后增加,高氧7 d增加最明显(p<0.01),高氧14 d时SP-C表达较空气组又减弱(p<0.05)。(2)早产大鼠生后肺组织AQP5表达不断增强,其阳性染色主要定位于AECⅠ。与同时间点空气组相比,高氧暴露1 d时,仅肺组织AQP5 mRNA表达显著高于对照组(P <0.05),而高氧暴露4、7、10及14 d时,其mRNA及蛋白表达均明显减少(P <0.05或P <0.01)。【结论】SP-C和AQP5参与了早产大鼠肺发育及高氧肺损伤的生理与病理过程;高氧暴露导致AQP5及SP-C表达下调是促使高氧肺损伤发生发展的重要因素。更多还原

【Abstract】 Part 1 Effects of Hyperoxia on Proliferation and Transdifferentiation of TypeⅡAlveolar Epithelial Cells from Premature Delivery RatsObjective To establish a hyperoxia-exposed type II alveolar epithelial cells ( AECIIs ) injury model to investigate effects in vitro of hyperoxia on the proliferation and transdifferentiation of AECIIs in premature delivery rats.Methods AECIIs were gained by primary culture from 19 d fetal rat lung. After purified and adherence to the culture flask for 15 h, AECIIs were randomly assigned to two groups: air control groups and hyperoxia groups. The hyperoxia groups were filled with 95 % oxygen-5 % CO2 at 3 L/min for 10 min, and then incubated at 37°C in a CO2 incubator, while the air control groups were incubated at 37°C in a CO2 incubator directly. After cultured for 24, 48 and 72 h, cells were harvested. The morphological change of cells was observed under inverted phase contrast microscope and transmission electron microscope. AECII–specific protein-surfactant protein C ( SP-C ) and typeⅠalveolar epithelial cells ( AECⅠ)-specific protein aquaporin5 ( AQP5 ) were detected by immunocytochemical staining. The expression levels of SP-C and AQP5 mRNAs and their proteins were observed by RT-PCR and flow cytometry. Cell number was counted every 24 h with a hemacytometer. Cell viability was determined by Trypan blue dye exclusion. Flow cytometry were used as assays for cell cycle. The expression of Ki67 was dected by immumofluorescence method and flow cytometry.Results Cell number increased in cultures exposed to room air. Compared with air control groups, Cells exposed to hyperoxia for 48 and 72 h had reduced cell number and cell viability. The expression rate of Ki67 in positive cells and fluorescence index(FI) decreased markedly after exposure to hyperoxia for 24、48 and 72 hours. Hyperoxia increased the percentage of cells in G1 phase and decreased the percentage in S and G2/M phases of the cell cycle ( P<0.05 or P<0.01 ). At the same time, after exposure to hyperoxia, primarily cultured AECIIs rapidly lost their characteristics and gained some AECⅠ-like outward appearance. Cuboidal AECIIs spreaded and flattened, lost lamellar bodies and microvilli. With these morphological changes, AECIIs stopped expression of AECII–specific protein SP-C and expressed AECⅠ-associated protein AQP5. Compared with air control groups, the expression rate of SP-C mRNA in positive cells and fluorescence index ( FI ) decreased markedly in 3 groups of exposure to hyperoxia for 24、48 and 72 hours ( P<0.05 or P<0.01 ),but the AQP5 expression increased significantly in 2 groups of exposure to hyperoxia for 24 and 48 hours ( P<0.05 or P<0.01 ). The expression of AQP5 began decreasing and had no notable difference compared with exposure to air for 72 hours ( P﹥0.05 ).Conclusion (1) Hyperoxia could inhibite proliferation of primary cultured AECIIs from fetal rat lung in G1 phase of the cell cycle and decrease the expression rate of Ki67. This proliferation arrest may be associated with the pathogenesis of chronic lung disease of premature infants. (2) Although transdifferentiation of AECIIs soccured spontaneously in vitro, it was accelerated on exposure to hyperoxia. Hyperoxia-induced AECIIs transdifferentiation may play a key role in the repairation of alveolar epithelial cell injury in the premature delivery rat lung. Part 2 Proliferation and Transdifferentiation Characteristics of TypeⅡAlveolar Epithelial Cells Co-cultured with Lung FibroblastsObjective To set up a co-culture model of typeⅡalveolar epithelial cells ( AECIIs ) with lung fibroblasts ( LFs ) from fetal rat lung, observe the proliferation and transdifferentiation characteristics of AECIIs, and to further study the interaction between AECII and LF in lung development and injury in vitro.Methods AECIIs and LFs were isolated and purified. A co-cultured model of AECIIs and LFs was set up by PCF Millicell culture plate inserts and six-well cluster dishes. AECIIs were plated into the wells of six-well cluster dishes at a density of 5×105/mL. LFs were plated into the Millicell culture plate inserts at a density of 1×106/mL. Control groups consisted of AECIIs cultured with Millicell culture plate inserts containing no LF, or AECIIs cultured by themselves. After incubated for 2 and 4 days, the morphological change and growth of cells were observed under inverted phase contrast microscope. Cell number was counted with a hemacytometer, cell viability was determined by Trypan blue dye exclusion. The SP-C and AQP5 expressions in mRNA and protein levels were observed by RT-PCR and flow cytometry. Flow cytometry was also used to detect the cell cycle and Ki67 expression of AECIIs.Results (1) A co-cultured model of AECⅡs and LFs was set up well, and this coculture model constructed in vitro mimiced microenvironment in vivo. (2) AECIIs cultured alone for 4 days, AECIIs grew scatteredly with flattened nuclei, greaten volum, decreased chromatospherite and transdifferentiated partly into AECⅠ-like cells. Co-culture of AECIIs with LFs for 4 days, AECIIs also grew aggregates with rounded nuclei and had no obvious change in cell volum. Compared with the control groups, the SP-C expression in mRNA and protein levels increased markedly(P <0.01)after cocultured with LFs for 2 and 4 days, while the AQP5 expression decreased(P <0.05 or P <0.01). The cell morphology and immun marker results indicated that AECIIs could keep its morphology well, and the transdifferentiation of AECIIs to AECⅠs was decreased when co-cultured with LFs. (3) There was no notable difference between coculture groups and control groups on 2 days in cell number and viability of AECIIs. After co-clutured with LFs for 4 days, AECIIs also kept its morphology, cell viability. Proliferation of AECIIs increased significantly than that of AECIIs cultured by themselves, and (95.2±4.9)% cells retained the ability to exclude dye. Cocultured with LFs for 2 and 4 days, the percentage of cells in G0/G1 phases decreased ( P <0.01 ), while the percentage of cells in S and G2/M phases increased ( P <0.01 or P <0.05 ), and the expression rate of Ki67 in positive cells and fluorescence index(FI) also increased ( P <0.01 or P <0.05 ), these suggested that cocultured with LFs could promote the proliferation of AECIIs.Conclusion The coculture model constructed in vitro mimics microenvironment in vivo. Differentiation and proliferation function of AECⅡcan be maintained in this coculture model, this can be used to study the the interaction between AECⅡand LF in lung development and injury in vitro.Part 3 Effects of Hyperoxia on the Dynamic Expression of SP-C and AQP5 in Premature Rats Lung DevelopmentObjective To observe the dynamic expressions of surfactant protein C ( SP-C ) and Aquaporin5 ( AQP5 ) in premature rats and after inhaling hyperoxia and investigate roles of SP-C and AQP5 in lung development and hyperoxia lung injury.Methods Gestation 21 day ( term=22 day ) Sprague-Dawley ( SD ) fetuses were randomly assigned to air control group and hyperoxia group within 12～24 h after birth. Hypreoxia group was exposed to about 85% oxygen and air group in room air. After 1 to 14 days of exposure, lung tissue was extracted. Immunohistochemistry was used mainly for the location of the SP-C and AQP5 expressions in lung tissue, SP-C and AQP5 mRNA were detected by reverse transcription polymerase chain reaction ( RT-PCR ). SP-C and AQP5 expressions in protein levels were detected by western-blot. Results (1) The expression of SP-C could be found at various time after birth in premature rats, and the positive staining was restricted to the AECⅡ. At day 1 after birth, the expression of SP-C was highest and began to decrease at day 4. Compared with air control group, the SP-C mRNA and protein decreased significantly in hyperoxia group of day 1 ( P <0.01 ). After exposure to hyperoxia for 7 to 10 days, the SP-C expression increased remarkedly ( P <0.01 or P <0.05 ) but decreased again by 14 days of hyperoxia ( P <0.05 ). (2) The expression of lung AQP5 in premature rats increased persistently after birth, and the positive staining was restricted to the AECⅠ. Compared with the air control group, the AQP5 mRNA in hyperoxia group of day 1 increased significantly ( P <0.05 ), but after exposure to hyperoxia for 4 to 14 days, the AQP5 expression decreased remarkedly in mRNA and protein levels ( P <0.05 or P <0.01 ).Conclusions SP-C and AQP5 may have participated the physiological and pathological process in premature rats lung development and hyperoxia lung injury. Hyperoxia exposure lead to a down regulation or functional impairment of the SP-C and AQP5 expression , this may be an important factor for the development of hyperoxia lung injury.更多还原