BMP-7在慢性肺疾病新生鼠的表达及对肺成纤维细胞作用的研究
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摘要
早产儿慢性肺疾病(Chronic Lung Disease,CLD)是早产儿在肺发育未成熟的基础上,因高氧、感染、气压伤等原因引起肺部的远期合并症。近年来,随着产科技术的逐渐提高、机械通气的普遍开展及肺表面活性物质的广泛使用,使得极低或超低出生体重儿存活率在不断提高,也使CLD的发生呈上升趋势,严重影响新生儿的生存质量。因此,探索CLD肺纤维化的发生机制及寻找有效的防治方法是目前新生儿领域的热点研究之一
大量研究结果表明,高浓度氧是诱发早产儿CLD的最主要原因和最危险因素。吸入高浓度氧气后,引起肺部氧化应激反应。大量的细胞因子参与高氧引发的炎症反应,直接引起或加重肺损伤。其中转化生长因子-β1(Transforming growth factor-β1,TGF-β1)在肺损伤中的致肺纤维化作用已被证实。研究发现,高氧致慢性肺疾病新生鼠模型中,TGF-β1蛋白和nRNA的表达增强。TGF-β1不仅自身在肺炎发生和肺纤维化过程中具有重要作用,而且能诱导肺脏高水平表达结缔组织生长因子,加快肺纤维化的发生。但与TGF-β1同属于TGF-β超家族成员的骨形态发生蛋白-7(bone morphogenetic protein,BMP-7)在肺损伤中的作用研究较少,有研究证实：BMP-7与拮抗纤维化作用密切相关,BMP-7可能在拮抗器官(肝脏,肾脏,心脏)纤维化中发挥重要的作用。但其在高氧致早产儿CLD肺纤维化中的作用及机制尚不清楚,因此探讨BMP-7在CLD中的作用,可能为寻求早产儿CLD的有效防治措施奠定基础。
本研究以高氧诱导的新生鼠CLD模型及原代培养的肺成纤维细胞为对象,通过Real Time PCR分子生物学技术、流式细胞术等技术,动态观察肺组织中BMP-7的表达规律及与肺纤维化的关系,并试图通过BMP-7作用原代培养的新生鼠肺成纤维细胞的体外实验,阐明BMP-7对成纤维细胞增殖及细胞周期的影响。
材料与方法
一、动物实验部分
(一)实验动物
足月新生Wistar大鼠80只,体重5-9克,雌雄不限,随机分为实验组(高氧组)和对照组(空气组),每组40只。
(二)动物模型的制备
参照我们已报道的方法制备高氧诱导新生鼠CLD模型。实验组将足月新生的Wistar大鼠生后即置于氧箱中,持续输入氧气,维持氧浓度为0.90,用钠石灰吸收CO2,CO2浓度<0.5%,温度为25-27℃,湿度50%-70%。每天定时开箱0.5小时,添加水、饲料及更换垫料,并每天与对照组交换代母鼠,以避免因氧中毒而致喂养能力下降。对照组吸入空气,具体方法及控制因素同实验组。
(三)标本的采集和检测指标
每组分别于实验后的3,7,14和21天随机选取10只麻醉后处死,分离肺组织。将左肺组织置于4%多聚甲醛中固定,应用HE染色方法进行肺组织病理改变观察,应用免疫组织化学技术进行肺组织BMP-7蛋白表达的检测。其余肺组织于-80℃冰箱中冻存,应用Real Time PCR分子生物学技术进行BMP-7mRNA表达的检测。
二、细胞培养部分
(一)新生鼠肺成纤维细胞的分离和培养
按Kelleher等建立的方法分离与培养新生鼠肺成纤维细胞,将健康足月1日龄内的新生Wistar大鼠经10%水合氯醛麻醉致死后迅速分离出肺组织,用D-Hanks液洗净后剪碎肺组织至1mm3大小；加胰酶于37℃水温下振荡消化；用含10%胎牛血清的DMEM吹打终止胰酶作用；用400目过滤网过滤杂质保留上清；800转／秒离心,弃上清保留沉淀；将沉淀重悬于含10%胎牛血清的DMEM中,调整细胞密度并将细胞接种于25cm3的培养瓶中,将培养瓶置于恒温培养箱中培养；待细胞融合成单层膜生长后按1：2传代。于第三代进行分组实验。
(二)新生鼠肺成纤维细胞的鉴定
通过观察倒置相差显微镜下肺成纤维细胞的形态及应用波形蛋白免疫细胞化学技术对成纤维细胞进行鉴定。
(三)标本的处理和检测指标
收集并固定经过处理的各时点的细胞,应用MTT法测定0ng／ml(加入等量的无血清培养液)、5ng／ml、10ng／ml、50ng／ml的BMP-7作用下肺成纤维细胞的生长曲线；应用FCM法测定肺成纤维细胞的细胞周期,并将其分成两组：Ⅰ、分别用0ng／ml(加入等量培养基),5ng／ml,10ng／ml,50ng／ml的BMP-7作用于新生鼠LF,于24小时收集细胞,测定LF细胞周期的变化,Ⅱ、用50ng／ml的BMP-7(实验组)作用新生鼠LF,分别于12、24、48小时进行细胞收集,同时在这三个时间点设立对照组(加入等量的无血清培养液),测定LF细胞周期的变化。
三、统计学分析
采用SPSS17.0统计学软件进行分析,所有数据均以平均值±标准差(x±s)表示,两组间比较应用t检验,多组间比较采用ANOVA分析,相关性分析采用Spearman分析,以P<0.05表示差异有显著性,P<0.0表示差异极具显著性。
结果
一、肺组织形态学改变
3-21天空气组肺泡间隔变薄,肺泡结构逐渐规整；高氧组3-7天,肺泡间隔及肺泡腔内以炎性细胞渗出和红细胞渗出为主,随着高氧时间的延长,高氧组14-21天肺组织纤维化逐渐形成。
二、肺组织纤维化评分
与对照组相比,14天,21天时实验组肺纤维化评分有统计学差异,p<0.05。
三、肺组织BMP-7蛋白的表达
(一)BMP-7表达部位
空气组BMP-7在血管内皮细胞,支气管和细支气管上皮细胞有表达。高氧组3d可见肺上皮细胞、间质成纤维细胞表达强烈的BMP-7,7d,14d可见肺上皮细胞、成纤维细胞表达BMP-7逐渐减弱,21d时BMP-7在这些细胞中的表达明显减弱。
(二)BMP-7蛋白表达
实验开始后3d、7d实验组肺组织BMP-7蛋白表达明显高于对照组,且有统计学意义；14d、21d实验组BMP-7蛋白表达强度与对照组比较无差异。
(三)BMP-7mRNA表达
实验组3d,7dBMP-7mRNA表达明显高于对照组,且有统计学意义；14d和21d实验组表达逐渐下降,且14d与对照组比较无差异,21d与对照组有统计学意义。
四、相关性分析
高氧暴露下的新生鼠肺组织BMP-7蛋白表达与纤维化评分呈明显负相关：r=—0.692,P<0.01。
五、成纤维细胞鉴定
光镜下观察,可见细胞呈梭形排列,免疫组化可见成纤维细胞细胞浆被染成棕黄色(×100),胞浆中有呈丝状的波形蛋白。
六、BMP-7刺激下新生鼠肺成纤维细胞生长曲线
在0ng／ml、5ng／ml、10ng／ml、50ng／ml的BMP-7作用下,肺成纤维细胞生长速度逐渐减慢。
七、BMP-7刺激下新生鼠肺成纤维细胞细胞周期变化
(一)浓度依赖曲线
用不同浓度BMP-7(0ng／ml、5ng／ml、10ng／ml、50ng／ml)作用于新生鼠LF,作用24小时后,测定细胞周期的变化情况,可见随着BMP-7浓度的增加,LF在细胞周期中处于G1期细胞的百分比逐渐增多。
(二)时间依赖曲线
用50ng／ml的BMP-7作用新生鼠LF,分别于12、24、48h收集细胞,测定细胞周期,可见随着时间的延长,LF在细胞周期中处于G1期细胞的百分比逐渐增多。
结论
1、高氧致新生鼠CLD肺组织中,随着高氧时间的延长,肺组织纤维化逐渐形成,肺组织BMP-7蛋白和基因的表达呈下降水平,且肺组织BMP-7蛋白表达与肺组织纤维化明显负相关。
2、随着BMP-7浓度及作用时间的增加,原代培养新生鼠肺成纤维细胞的细胞增殖减弱,其细胞周期处于G1期的细胞百分比逐渐增多,表明BMP-7可能使得G1／S调控点的作用增强,G2／M调控点的作用减弱,从而使更多的细胞停滞在G1期,进而达到抑制细胞增殖的作用。
Preface
Chronic lung disease of prematurity (CLD) in premature infants is immature lung development, based on the result of high oxygen, infection, pressure causes lung injury, such as the long-term complications. In recent years, with antenatal glucocorticoid application,to carry out general mechanical ventilation and pulmonary surfactant widely used, making a very low or extremely low birth weight infant survival rate has been improving, but also the occurrence of CLD into an upward trend seriously affected the quality of life of newborns. Therefore, to explore the occurrence of pulmonary fibrosis CLD mechanism and how to reduce neonatal pulmonary fibrosis is the most hot spots in the area of the studies.
A large number of cytokines in hyperoxia-induced inflammatory response, directly cause or worsen lung injury. In which TGF-β1 in lung injury-induced pulmonary fibrosis has been confirmed, a large number of studies found that hyperoxia-induced chronic lung disease in neonatal rats with hyperoxia-induced CLD, TGF-βprotein and mRNA expression increased. TGF-βnot only to their own place of pneumonia and pulmonary fibrosis plays an important role, but also to induce high levels of expression of connective tissue growth factor in lung, to accelerate the occurrence of pulmonary fibrosis. But belong to the same TGF-βsuperfamily member BMP-7 with TGF-β1 in lung injury research less, research has shown:BMP-7 is closely related with the antagonist fibrosis:BMP-7 may play an important role in antagonistic organ fibrosis (liver, kidney, heart). However, in hyperoxia-induced pulmonary fibrosis in CLD in premature, the effect and mechanism is not clear, so explore the BMP-7 in the role of CLD, which may be effective for CLD in preterm lay the foundation for prevention and control measures.
In this study, at the basic of neonatal rats with hyperoxia-induced CLD and primary culture of lung fibroblasts, by Real Time PCR molecular biology techniques, flow cytometry (FCM) techniques and so on, dynamic observation the expression of BMP-7 in lung tissue and the relations with pulmonary fibrosis,and by intervene primary culture of neonatal rat lung fibroblasts in vitro experiments with BMP-7,try to clarify the effect of BMP-7 on fibroblast froliferation and the cell cycle.
Materials and Methods
Animal experiment
(一) experimental Animal
Full-term newborn Wistar rats were 80,male and female open
(二) Animal model and subgroup
Reference to the method we have reported preparation neonatal rats with hyperoxia-induced CLD models.The hyperoxia group inspired oxygen (concentration 0.90), and the control group inhaled air.
(三)specimen collection and processing
Pups from each group were killed on days 1,3,7,14,21 after anesthesia with 10% chloral hydrate. the left lungs were placed in 4% paraformaldehyde for hematoxylin-eosin stain and immunohistoehemistry,and the rest lungs were placed in-80℃Refrigerator frozen for Real Time PCR.
二Cell culture experiment
(一)Lung fibroblast culture
Isolate the lung fibroblast of neonatal rats (1days) according to the method of Kellehe. Culture primary and passage cells.
(二) Lung fibroblast identification
During the 3rd passage,By observing the cell morphology under inverted phase contrast microscope and use the vimentin immunohistochemistry to indentify fibroblast.
(三)BMP-7 stimulation test and specimen collection
When the conjugation of third passage cells was 70%,serum-free medium was replaced. The subgroup experiment started 24 hours.In the condition of 21% oxygen concentration,different dose of BMP-7(0ng/ml,5ng/ml, lOng/ml,50ng.ml) was used to stimulate lung fibroblast of neonatal rats. After 24 hours,the cells was ethanol fixed and preserved in-20℃refrigerator.We used the 50 ng/ml BMP-7 to stimulate the fibroblast, ethanol fixed the cells at different times(12,24,48h) and preserved in-20℃refrigerator. The control group (without BMP-7 stimulation at 12,24,48h)was set.
We used MTT to measure the condiction of lung fibroblast growth and draw the growth curve. Collection of lung fibroblasts fixed with ethanol, PI staining, using the FACSAria flow cytometer of U.S. BD company and analysis system for analysis of each sample to measure the expression of cell cycle.
三Statistical analysis
SPSS 17.0 was used to perform statistical analysis, with all datas expressed as x±s, T-test was used in two groups, Spearman was used to analyse the correlation and ANOVA was used to analyse multigroups and it had significance when P<0.05.
Results
一The histmorphology findings of the lung tissue
On day 3 of experiment,the alveolar spetum was thinner and the alveolar structure of the air group was more regular meanwhile in hyperoxia group,there was a few red blood cell which exuded to alveolus.From 7d to 21d,the alveolar septum was more thinner and the alveolar structure in air group became regular gradually.In hyperoxia group on day 7, it was observed that there was inflammatory response, more interstitial cells, lung septum degraded and lung edema; on day 14,the interstitial cells was increased with the alveolar septum thickened obviously and lung fibrosis emerged gradually; on day 21, the interstitial cells increased obviously with alveolar septum much thicker.The normal alveolar structure is disappeared and pulmonary fibrosis is formation.
二lung fibrosis scores
On 14d and 21d,there was statistical difference between control and experiment droup (p<0.05).
三The expression of BMP-7 protein in the lungs
(一) The expression location of BMP-7 protein
In control group, BMP-7 was expressed in vascular endothelial cell, bronchi and bronchiole epithelium.In experiment group, on 3d, BMP-7 protein was expressed strongly in pulmonary epithelial cells,alveolar septum and interstitial fibroblasts. On 7d,14d the expression of BMP-7 protein in pulmonary epithelial cells and fibroblasts gradually weakened.On 21d,the expression of BMP-7 protein in these cells decreased significantly.
(二) The expression strength of BMP-7 protein
On day 3d,7d,the expression of BMP-7 was signigicantly higher in experiment group than in control group.on day 14d,21d,there was no significant deviation in the expression of BMP-7 protein between experiment group and control group.
(三) The expression strength of BMP-7 mRNA
It is similar to the expression strength of BMP-7 protein
四Correlation analysis
Spearman method was used for correlation analysis:the expression of BMP-7 protein in hyperoxia lung tissue was obviously negetive correlation with lung fibrosis scores:r=-0.692, p<0.01.
五Identification of fibroblasts
The cytoplasm of fibroblast was stained buffy in which there was slikness Vimentin under light microscope(×100).
六The growth curve in lung fibroblast of neonatal rat under the stimulation of BMP-7
Under the stimulation of BMP-7 with the concentration of 0ng/ml、5ng/ml、10ng/ml、50ng/ml,the lung fibroblast growth slowed down.
七The change of cell cycle in lung fibroblast of neonatal rats under the stimulation of BMP-7
Under the stimulation of BMP-7 with the concentration of 0ng/ml、5ng/ml、10ng/m、50ng/ml,the cell cycle in experiments groups individually after 24h.Compered with 0ng/ml group,the p<0.05 in both 10ng/ml and 50ng/ml groups but p>0.05 in 5ng/ml group.When compared between groups of 5ng/ml、10ng/ml、50ng/ml,p<0.05 in all.0ng/ml and 50ng/ml BMP-7 was used to stimulate lung fibroblast and the change of cell cycle was tested after 12,24and 48h.After 12h,the cell cycle in 50ng/ml compared between 0ng/ml groups,p<0.05. After 24h,the cell cycle in 50ng/ml compared between 0ng/ml groups,p<0.05. After 48h the cell cycle in 50ng/ml compared between 0ng/ml groups,p<0.05.Under the stimulation of 50ng/ml BMP-7,p<0.05between groups after 12,24,48 hours.
Conclusion
1 neonatal rats with hyperoxia-induced CLD, with the prolong of hyperoxia,the fibroblast appear,the level expression of BMP-7 protein and genes in lung tissue become less and less. and the expression of BMP-7 protein in lung tissue of neonatal rats had negative correlation with lung fibrosis.
2 With the concentration of BMP-7 and time increase,the proliferation of neonatal rats lung fibroblasts become down.the percents of G1 is up.It suggests that BMP-7 may result in G1/S control point of the enhanced,G2/M control point weakened,so that more cells stuck into the G1 phase,and to inhibit cell proliferation.
大量研究结果表明,高浓度氧是诱发早产儿CLD的最主要原因和最危险因素。吸入高浓度氧气后,引起肺部氧化应激反应。大量的细胞因子参与高氧引发的炎症反应,直接引起或加重肺损伤。其中转化生长因子-β1(Transforming growth factor-β1,TGF-β1)在肺损伤中的致肺纤维化作用已被证实。研究发现,高氧致慢性肺疾病新生鼠模型中,TGF-β1蛋白和nRNA的表达增强。TGF-β1不仅自身在肺炎发生和肺纤维化过程中具有重要作用,而且能诱导肺脏高水平表达结缔组织生长因子,加快肺纤维化的发生。但与TGF-β1同属于TGF-β超家族成员的骨形态发生蛋白-7(bone morphogenetic protein,BMP-7)在肺损伤中的作用研究较少,有研究证实：BMP-7与拮抗纤维化作用密切相关,BMP-7可能在拮抗器官(肝脏,肾脏,心脏)纤维化中发挥重要的作用。但其在高氧致早产儿CLD肺纤维化中的作用及机制尚不清楚,因此探讨BMP-7在CLD中的作用,可能为寻求早产儿CLD的有效防治措施奠定基础。
本研究以高氧诱导的新生鼠CLD模型及原代培养的肺成纤维细胞为对象,通过Real Time PCR分子生物学技术、流式细胞术等技术,动态观察肺组织中BMP-7的表达规律及与肺纤维化的关系,并试图通过BMP-7作用原代培养的新生鼠肺成纤维细胞的体外实验,阐明BMP-7对成纤维细胞增殖及细胞周期的影响。
材料与方法
一、动物实验部分
(一)实验动物
足月新生Wistar大鼠80只,体重5-9克,雌雄不限,随机分为实验组(高氧组)和对照组(空气组),每组40只。
(二)动物模型的制备
参照我们已报道的方法制备高氧诱导新生鼠CLD模型。实验组将足月新生的Wistar大鼠生后即置于氧箱中,持续输入氧气,维持氧浓度为0.90,用钠石灰吸收CO2,CO2浓度<0.5%,温度为25-27℃,湿度50%-70%。每天定时开箱0.5小时,添加水、饲料及更换垫料,并每天与对照组交换代母鼠,以避免因氧中毒而致喂养能力下降。对照组吸入空气,具体方法及控制因素同实验组。
(三)标本的采集和检测指标
每组分别于实验后的3,7,14和21天随机选取10只麻醉后处死,分离肺组织。将左肺组织置于4%多聚甲醛中固定,应用HE染色方法进行肺组织病理改变观察,应用免疫组织化学技术进行肺组织BMP-7蛋白表达的检测。其余肺组织于-80℃冰箱中冻存,应用Real Time PCR分子生物学技术进行BMP-7mRNA表达的检测。
二、细胞培养部分
(一)新生鼠肺成纤维细胞的分离和培养
按Kelleher等建立的方法分离与培养新生鼠肺成纤维细胞,将健康足月1日龄内的新生Wistar大鼠经10%水合氯醛麻醉致死后迅速分离出肺组织,用D-Hanks液洗净后剪碎肺组织至1mm3大小；加胰酶于37℃水温下振荡消化；用含10%胎牛血清的DMEM吹打终止胰酶作用；用400目过滤网过滤杂质保留上清；800转／秒离心,弃上清保留沉淀；将沉淀重悬于含10%胎牛血清的DMEM中,调整细胞密度并将细胞接种于25cm3的培养瓶中,将培养瓶置于恒温培养箱中培养；待细胞融合成单层膜生长后按1：2传代。于第三代进行分组实验。
(二)新生鼠肺成纤维细胞的鉴定
通过观察倒置相差显微镜下肺成纤维细胞的形态及应用波形蛋白免疫细胞化学技术对成纤维细胞进行鉴定。
(三)标本的处理和检测指标
收集并固定经过处理的各时点的细胞,应用MTT法测定0ng／ml(加入等量的无血清培养液)、5ng／ml、10ng／ml、50ng／ml的BMP-7作用下肺成纤维细胞的生长曲线；应用FCM法测定肺成纤维细胞的细胞周期,并将其分成两组：Ⅰ、分别用0ng／ml(加入等量培养基),5ng／ml,10ng／ml,50ng／ml的BMP-7作用于新生鼠LF,于24小时收集细胞,测定LF细胞周期的变化,Ⅱ、用50ng／ml的BMP-7(实验组)作用新生鼠LF,分别于12、24、48小时进行细胞收集,同时在这三个时间点设立对照组(加入等量的无血清培养液),测定LF细胞周期的变化。
三、统计学分析
采用SPSS17.0统计学软件进行分析,所有数据均以平均值±标准差(x±s)表示,两组间比较应用t检验,多组间比较采用ANOVA分析,相关性分析采用Spearman分析,以P<0.05表示差异有显著性,P<0.0表示差异极具显著性。
结果
一、肺组织形态学改变
3-21天空气组肺泡间隔变薄,肺泡结构逐渐规整；高氧组3-7天,肺泡间隔及肺泡腔内以炎性细胞渗出和红细胞渗出为主,随着高氧时间的延长,高氧组14-21天肺组织纤维化逐渐形成。
二、肺组织纤维化评分
与对照组相比,14天,21天时实验组肺纤维化评分有统计学差异,p<0.05。
三、肺组织BMP-7蛋白的表达
(一)BMP-7表达部位
空气组BMP-7在血管内皮细胞,支气管和细支气管上皮细胞有表达。高氧组3d可见肺上皮细胞、间质成纤维细胞表达强烈的BMP-7,7d,14d可见肺上皮细胞、成纤维细胞表达BMP-7逐渐减弱,21d时BMP-7在这些细胞中的表达明显减弱。
(二)BMP-7蛋白表达
实验开始后3d、7d实验组肺组织BMP-7蛋白表达明显高于对照组,且有统计学意义；14d、21d实验组BMP-7蛋白表达强度与对照组比较无差异。
(三)BMP-7mRNA表达
实验组3d,7dBMP-7mRNA表达明显高于对照组,且有统计学意义；14d和21d实验组表达逐渐下降,且14d与对照组比较无差异,21d与对照组有统计学意义。
四、相关性分析
高氧暴露下的新生鼠肺组织BMP-7蛋白表达与纤维化评分呈明显负相关：r=—0.692,P<0.01。
五、成纤维细胞鉴定
光镜下观察,可见细胞呈梭形排列,免疫组化可见成纤维细胞细胞浆被染成棕黄色(×100),胞浆中有呈丝状的波形蛋白。
六、BMP-7刺激下新生鼠肺成纤维细胞生长曲线
在0ng／ml、5ng／ml、10ng／ml、50ng／ml的BMP-7作用下,肺成纤维细胞生长速度逐渐减慢。
七、BMP-7刺激下新生鼠肺成纤维细胞细胞周期变化
(一)浓度依赖曲线
用不同浓度BMP-7(0ng／ml、5ng／ml、10ng／ml、50ng／ml)作用于新生鼠LF,作用24小时后,测定细胞周期的变化情况,可见随着BMP-7浓度的增加,LF在细胞周期中处于G1期细胞的百分比逐渐增多。
(二)时间依赖曲线
用50ng／ml的BMP-7作用新生鼠LF,分别于12、24、48h收集细胞,测定细胞周期,可见随着时间的延长,LF在细胞周期中处于G1期细胞的百分比逐渐增多。
结论
1、高氧致新生鼠CLD肺组织中,随着高氧时间的延长,肺组织纤维化逐渐形成,肺组织BMP-7蛋白和基因的表达呈下降水平,且肺组织BMP-7蛋白表达与肺组织纤维化明显负相关。
2、随着BMP-7浓度及作用时间的增加,原代培养新生鼠肺成纤维细胞的细胞增殖减弱,其细胞周期处于G1期的细胞百分比逐渐增多,表明BMP-7可能使得G1／S调控点的作用增强,G2／M调控点的作用减弱,从而使更多的细胞停滞在G1期,进而达到抑制细胞增殖的作用。
Preface
Chronic lung disease of prematurity (CLD) in premature infants is immature lung development, based on the result of high oxygen, infection, pressure causes lung injury, such as the long-term complications. In recent years, with antenatal glucocorticoid application,to carry out general mechanical ventilation and pulmonary surfactant widely used, making a very low or extremely low birth weight infant survival rate has been improving, but also the occurrence of CLD into an upward trend seriously affected the quality of life of newborns. Therefore, to explore the occurrence of pulmonary fibrosis CLD mechanism and how to reduce neonatal pulmonary fibrosis is the most hot spots in the area of the studies.
A large number of cytokines in hyperoxia-induced inflammatory response, directly cause or worsen lung injury. In which TGF-β1 in lung injury-induced pulmonary fibrosis has been confirmed, a large number of studies found that hyperoxia-induced chronic lung disease in neonatal rats with hyperoxia-induced CLD, TGF-βprotein and mRNA expression increased. TGF-βnot only to their own place of pneumonia and pulmonary fibrosis plays an important role, but also to induce high levels of expression of connective tissue growth factor in lung, to accelerate the occurrence of pulmonary fibrosis. But belong to the same TGF-βsuperfamily member BMP-7 with TGF-β1 in lung injury research less, research has shown:BMP-7 is closely related with the antagonist fibrosis:BMP-7 may play an important role in antagonistic organ fibrosis (liver, kidney, heart). However, in hyperoxia-induced pulmonary fibrosis in CLD in premature, the effect and mechanism is not clear, so explore the BMP-7 in the role of CLD, which may be effective for CLD in preterm lay the foundation for prevention and control measures.
In this study, at the basic of neonatal rats with hyperoxia-induced CLD and primary culture of lung fibroblasts, by Real Time PCR molecular biology techniques, flow cytometry (FCM) techniques and so on, dynamic observation the expression of BMP-7 in lung tissue and the relations with pulmonary fibrosis,and by intervene primary culture of neonatal rat lung fibroblasts in vitro experiments with BMP-7,try to clarify the effect of BMP-7 on fibroblast froliferation and the cell cycle.
Materials and Methods
Animal experiment
(一) experimental Animal
Full-term newborn Wistar rats were 80,male and female open
(二) Animal model and subgroup
Reference to the method we have reported preparation neonatal rats with hyperoxia-induced CLD models.The hyperoxia group inspired oxygen (concentration 0.90), and the control group inhaled air.
(三)specimen collection and processing
Pups from each group were killed on days 1,3,7,14,21 after anesthesia with 10% chloral hydrate. the left lungs were placed in 4% paraformaldehyde for hematoxylin-eosin stain and immunohistoehemistry,and the rest lungs were placed in-80℃Refrigerator frozen for Real Time PCR.
二Cell culture experiment
(一)Lung fibroblast culture
Isolate the lung fibroblast of neonatal rats (1days) according to the method of Kellehe. Culture primary and passage cells.
(二) Lung fibroblast identification
During the 3rd passage,By observing the cell morphology under inverted phase contrast microscope and use the vimentin immunohistochemistry to indentify fibroblast.
(三)BMP-7 stimulation test and specimen collection
When the conjugation of third passage cells was 70%,serum-free medium was replaced. The subgroup experiment started 24 hours.In the condition of 21% oxygen concentration,different dose of BMP-7(0ng/ml,5ng/ml, lOng/ml,50ng.ml) was used to stimulate lung fibroblast of neonatal rats. After 24 hours,the cells was ethanol fixed and preserved in-20℃refrigerator.We used the 50 ng/ml BMP-7 to stimulate the fibroblast, ethanol fixed the cells at different times(12,24,48h) and preserved in-20℃refrigerator. The control group (without BMP-7 stimulation at 12,24,48h)was set.
We used MTT to measure the condiction of lung fibroblast growth and draw the growth curve. Collection of lung fibroblasts fixed with ethanol, PI staining, using the FACSAria flow cytometer of U.S. BD company and analysis system for analysis of each sample to measure the expression of cell cycle.
三Statistical analysis
SPSS 17.0 was used to perform statistical analysis, with all datas expressed as x±s, T-test was used in two groups, Spearman was used to analyse the correlation and ANOVA was used to analyse multigroups and it had significance when P<0.05.
Results
一The histmorphology findings of the lung tissue
On day 3 of experiment,the alveolar spetum was thinner and the alveolar structure of the air group was more regular meanwhile in hyperoxia group,there was a few red blood cell which exuded to alveolus.From 7d to 21d,the alveolar septum was more thinner and the alveolar structure in air group became regular gradually.In hyperoxia group on day 7, it was observed that there was inflammatory response, more interstitial cells, lung septum degraded and lung edema; on day 14,the interstitial cells was increased with the alveolar septum thickened obviously and lung fibrosis emerged gradually; on day 21, the interstitial cells increased obviously with alveolar septum much thicker.The normal alveolar structure is disappeared and pulmonary fibrosis is formation.
二lung fibrosis scores
On 14d and 21d,there was statistical difference between control and experiment droup (p<0.05).
三The expression of BMP-7 protein in the lungs
(一) The expression location of BMP-7 protein
In control group, BMP-7 was expressed in vascular endothelial cell, bronchi and bronchiole epithelium.In experiment group, on 3d, BMP-7 protein was expressed strongly in pulmonary epithelial cells,alveolar septum and interstitial fibroblasts. On 7d,14d the expression of BMP-7 protein in pulmonary epithelial cells and fibroblasts gradually weakened.On 21d,the expression of BMP-7 protein in these cells decreased significantly.
(二) The expression strength of BMP-7 protein
On day 3d,7d,the expression of BMP-7 was signigicantly higher in experiment group than in control group.on day 14d,21d,there was no significant deviation in the expression of BMP-7 protein between experiment group and control group.
(三) The expression strength of BMP-7 mRNA
It is similar to the expression strength of BMP-7 protein
四Correlation analysis
Spearman method was used for correlation analysis:the expression of BMP-7 protein in hyperoxia lung tissue was obviously negetive correlation with lung fibrosis scores:r=-0.692, p<0.01.
五Identification of fibroblasts
The cytoplasm of fibroblast was stained buffy in which there was slikness Vimentin under light microscope(×100).
六The growth curve in lung fibroblast of neonatal rat under the stimulation of BMP-7
Under the stimulation of BMP-7 with the concentration of 0ng/ml、5ng/ml、10ng/ml、50ng/ml,the lung fibroblast growth slowed down.
七The change of cell cycle in lung fibroblast of neonatal rats under the stimulation of BMP-7
Under the stimulation of BMP-7 with the concentration of 0ng/ml、5ng/ml、10ng/m、50ng/ml,the cell cycle in experiments groups individually after 24h.Compered with 0ng/ml group,the p<0.05 in both 10ng/ml and 50ng/ml groups but p>0.05 in 5ng/ml group.When compared between groups of 5ng/ml、10ng/ml、50ng/ml,p<0.05 in all.0ng/ml and 50ng/ml BMP-7 was used to stimulate lung fibroblast and the change of cell cycle was tested after 12,24and 48h.After 12h,the cell cycle in 50ng/ml compared between 0ng/ml groups,p<0.05. After 24h,the cell cycle in 50ng/ml compared between 0ng/ml groups,p<0.05. After 48h the cell cycle in 50ng/ml compared between 0ng/ml groups,p<0.05.Under the stimulation of 50ng/ml BMP-7,p<0.05between groups after 12,24,48 hours.
Conclusion
1 neonatal rats with hyperoxia-induced CLD, with the prolong of hyperoxia,the fibroblast appear,the level expression of BMP-7 protein and genes in lung tissue become less and less. and the expression of BMP-7 protein in lung tissue of neonatal rats had negative correlation with lung fibrosis.
2 With the concentration of BMP-7 and time increase,the proliferation of neonatal rats lung fibroblasts become down.the percents of G1 is up.It suggests that BMP-7 may result in G1/S control point of the enhanced,G2/M control point weakened,so that more cells stuck into the G1 phase,and to inhibit cell proliferation.
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