大气混合污染物对大鼠肺表面活性蛋白A变化的影响
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摘要
本研究模拟大气污染现状,制备大气混合污染物的动物模型,研究大气颗粒污染物及多种气体污染物对大鼠所致肺损伤的作用机制。
本实验通过对大鼠进行气管注入PM2.5混悬液进行染尘,动态吸入含有SO2、NO2、CO的空气混合气进行染毒的方式,制备动物模型。采用ELISA法分析大鼠血清及支气管肺泡灌洗液(BALF)中SP-A含量的变化;采用免疫组化法分析大鼠肺组织中SP-A表达的变化;采用实时荧光定量RT-PCR技术分析大鼠肺组织中SP-A的mRNA表达的变化,对大气混合污染物对大鼠呼吸道的损伤状况进行探讨。
实验结果显示,染毒7d组大鼠肺组织中SP-A mRNA表达与对照组比较明显升高,提示SP-A是早期肺损伤的敏感指标; SP-A可缓冲炎症反应,有代偿性保护作用。大鼠染毒30d组,肺泡Ⅱ型上皮细胞受损,肺组织中SP-A表达、肺泡灌洗液中SP-A含量均呈现明显下降,血清中SP-A水平含量升高明显,提示SP-A是反映肺泡Ⅱ型上皮细胞功能受损的特异性指标。大气混合污染物对大鼠呼吸系统产生了损害,使肺泡Ⅱ型上皮细胞受损,引起肺泡毛细血管内皮损伤,肺泡-毛细血管屏障破坏。本研究结果为大气污染所致肺损伤生物学标志物的研究提供了科学依据。
In recent years, air pollution was increasingly serious and could pose threats to human health. Air pollutants were complex components that were formed from a variety of pollutants. The main pollutants in the pollutant particles was the particulate matter(PM10)which diameter was less than 10μm. Recent studies have found that particulate matter which diameter was less than 2.5μm could enter into the lung tissue and blood deeply. PM2.5 was more harmful to the human body. Otherwise, with the development of people’s living standard and the rapid industry, energy consumption and the emission of SO2、NO2、CO were increased. Pulmonary surfactant protein A(SP-A )was found firstly and strongly expressed in the alveolar epithelial cells. SP-A showed lung-specific for its small expression out of the lung. SP-A function, synthesis and secretion were very complex. Many human lung diseases were observed with the abnormal changes of SP-A. Therefore, SP-A could be as a biological marker to determine the extent of lung injury.
Objective:
To analyze the change of SP-A expression in serum, lung tissue and bronchoalveolar lavage fluid (BALF), animal models which was made by simulating the atmospheric pollution were prepared to study the damage conditions of the deep respiratory in rats. It provided scientific basis for further research of lung injury caused by air pollution through studying the common mechanism .
Methods:
78 Wistar rats were randomly divided into three experimental groups(1d、7d、30d group)and three control groups(control 1d group、control 7d group、control 30d group).13 rats every group respectively .Rats were anesthetizeed by ether and injected into trachea one-time non-exposed. 1ml which contain 10mg PM2.5 saline suspension were injected into each experimental group rats. 1ml saline were injected into each rats of control groups. The day after contamination, experimental groups were inhaled with Air mixture of which concentrations of SO2、NO2、CO were 15、12、400mg/m3 respectively for 4 hours . Normal air was absorbed by control groups. Rats were killed by groups in 2d、8d、31d after contamination . Blood from abdominal aorta were collected through ether anesthesia and was centrifugalized by 2500r/min for 10min. Serum samples were stored at -80℃. Tracheal intubation was applied to each group. Bronchoalveolar were lavaged by 3ml saline one time (total 3 times)in 37℃. Supernant was stored at -80℃.Rats were operated and taked out right middle lob which is about 80mg. Three rats in each group were derived from bronchial and lung tissue that the tissue were fixed with 10% neutral buffered formalin for immunohistochemistry experiments.
Measurements and Methods:
The concentrations of SP-A in serum and BALF were examinated by ELISA; The concentrations in lung tissue were measured with immunohistochemistry;The contents of mRNA of SP-A in lung tissue were measured through real time RT-qPCR.
Results:
1. The levels of SP-A in rat serum.
There was no significant difference of SP-A in rat serum between 1d and 7d exposure group and control group(P>0.05). As compared with control group, the SP-A of serum were increased in 30d exposure group(P<0.01).Compared with 1d and 7d exposure group, the increasement in SP-A of serum was observed in 30d exposure group(P<0.01).
2. The levels of SP-A in rat BALF.
There was no significant deviation of SP-A in rat BALF between 1d and 7d exposure group and control group(P>0.05). As compared with control group, the SP-A of serum decreased significantly in 30d exposure group(P<0.01).Compared with 1d and 7d exposure group, the decreaement in SP-A of BALF was observed in 30d exposure group(P<0.01).
3. SP-A protein expression in rat lung tissue.
The results of immunohistochemistry showed that SP-A rich expression in rat lung tissue were observed in 1d exposure group and 7d exposure group compared with control group. There were a huge of round or oval brown granules in alveolar typeⅡcells and some macrophages in 1d exposure group and 7d exposure group. As compared with control group, the brown granules were decreased apparently in 30d exposure group. There was no significant difference of SP-A between1d and 7d exposure group and control group(P>0.05). Compared with control group, the decreasement in SP-A was observed in 30d exposure group(P<0.01). Compared with 30d exposure group, the decreasement in SP-A was observed in 1d exposure group and 7d exposure group(P<0.01).
4. SP-A mRNA expression in rat lung tissue
The results of RT-RCR showed that the SP-A mRNA of 1d and 30d exposure group and control group did not change apparently(P>0.05). As compared with control group, the SP-A of mRNA were increased apparently in 7d exposure group(P<0.01). Compared with 1d exposure group and 30d exposure group, SP-A in 7d exposure group significantly increased(P<0.01).
Conclusions:
SP-A was an important component of lung surfactant which functions were more complex. Lung-specific expression of SP-A had very important clinic significance. Rats were used in this study to detect the mechanism and changes of SP-A when mixture pollutants in atmosphere leaded to lung injury. The results showed:
1.SP-A had the function of innate immune defense and could delay Inflammatory response for elevation of SP-A transcription level. The changes of transcription levels indicated that SP-A was a sensitive indicator in early lung injury.
2.There were no significant differences in the contents of SP-A beween 1d and 7d in early lung injury from lung tissues, alveolus douche, serum,respectively.
3.Alveolar typeⅡcells were damaged severely and SP-A expression in lung tissue and BALF decreased significantly in late lung injury which was positively correlated with the degree of acute lung injury. SP-A in serum increased apparently which was negative correlation with the degree of acute lung injury. These results suggested that SP-A was specific index for pulmonary typeⅡcells function impairment .
In a word, SP-A was closely related with acute lung injury, which suggested that the levels of SP-A could be used as a sensitive biological indicator of lung damage caused by air-pollutants . It provided scientific basis for further research of lung injury caused by air pollution.
本实验通过对大鼠进行气管注入PM2.5混悬液进行染尘,动态吸入含有SO2、NO2、CO的空气混合气进行染毒的方式,制备动物模型。采用ELISA法分析大鼠血清及支气管肺泡灌洗液(BALF)中SP-A含量的变化;采用免疫组化法分析大鼠肺组织中SP-A表达的变化;采用实时荧光定量RT-PCR技术分析大鼠肺组织中SP-A的mRNA表达的变化,对大气混合污染物对大鼠呼吸道的损伤状况进行探讨。
实验结果显示,染毒7d组大鼠肺组织中SP-A mRNA表达与对照组比较明显升高,提示SP-A是早期肺损伤的敏感指标; SP-A可缓冲炎症反应,有代偿性保护作用。大鼠染毒30d组,肺泡Ⅱ型上皮细胞受损,肺组织中SP-A表达、肺泡灌洗液中SP-A含量均呈现明显下降,血清中SP-A水平含量升高明显,提示SP-A是反映肺泡Ⅱ型上皮细胞功能受损的特异性指标。大气混合污染物对大鼠呼吸系统产生了损害,使肺泡Ⅱ型上皮细胞受损,引起肺泡毛细血管内皮损伤,肺泡-毛细血管屏障破坏。本研究结果为大气污染所致肺损伤生物学标志物的研究提供了科学依据。
In recent years, air pollution was increasingly serious and could pose threats to human health. Air pollutants were complex components that were formed from a variety of pollutants. The main pollutants in the pollutant particles was the particulate matter(PM10)which diameter was less than 10μm. Recent studies have found that particulate matter which diameter was less than 2.5μm could enter into the lung tissue and blood deeply. PM2.5 was more harmful to the human body. Otherwise, with the development of people’s living standard and the rapid industry, energy consumption and the emission of SO2、NO2、CO were increased. Pulmonary surfactant protein A(SP-A )was found firstly and strongly expressed in the alveolar epithelial cells. SP-A showed lung-specific for its small expression out of the lung. SP-A function, synthesis and secretion were very complex. Many human lung diseases were observed with the abnormal changes of SP-A. Therefore, SP-A could be as a biological marker to determine the extent of lung injury.
Objective:
To analyze the change of SP-A expression in serum, lung tissue and bronchoalveolar lavage fluid (BALF), animal models which was made by simulating the atmospheric pollution were prepared to study the damage conditions of the deep respiratory in rats. It provided scientific basis for further research of lung injury caused by air pollution through studying the common mechanism .
Methods:
78 Wistar rats were randomly divided into three experimental groups(1d、7d、30d group)and three control groups(control 1d group、control 7d group、control 30d group).13 rats every group respectively .Rats were anesthetizeed by ether and injected into trachea one-time non-exposed. 1ml which contain 10mg PM2.5 saline suspension were injected into each experimental group rats. 1ml saline were injected into each rats of control groups. The day after contamination, experimental groups were inhaled with Air mixture of which concentrations of SO2、NO2、CO were 15、12、400mg/m3 respectively for 4 hours . Normal air was absorbed by control groups. Rats were killed by groups in 2d、8d、31d after contamination . Blood from abdominal aorta were collected through ether anesthesia and was centrifugalized by 2500r/min for 10min. Serum samples were stored at -80℃. Tracheal intubation was applied to each group. Bronchoalveolar were lavaged by 3ml saline one time (total 3 times)in 37℃. Supernant was stored at -80℃.Rats were operated and taked out right middle lob which is about 80mg. Three rats in each group were derived from bronchial and lung tissue that the tissue were fixed with 10% neutral buffered formalin for immunohistochemistry experiments.
Measurements and Methods:
The concentrations of SP-A in serum and BALF were examinated by ELISA; The concentrations in lung tissue were measured with immunohistochemistry;The contents of mRNA of SP-A in lung tissue were measured through real time RT-qPCR.
Results:
1. The levels of SP-A in rat serum.
There was no significant difference of SP-A in rat serum between 1d and 7d exposure group and control group(P>0.05). As compared with control group, the SP-A of serum were increased in 30d exposure group(P<0.01).Compared with 1d and 7d exposure group, the increasement in SP-A of serum was observed in 30d exposure group(P<0.01).
2. The levels of SP-A in rat BALF.
There was no significant deviation of SP-A in rat BALF between 1d and 7d exposure group and control group(P>0.05). As compared with control group, the SP-A of serum decreased significantly in 30d exposure group(P<0.01).Compared with 1d and 7d exposure group, the decreaement in SP-A of BALF was observed in 30d exposure group(P<0.01).
3. SP-A protein expression in rat lung tissue.
The results of immunohistochemistry showed that SP-A rich expression in rat lung tissue were observed in 1d exposure group and 7d exposure group compared with control group. There were a huge of round or oval brown granules in alveolar typeⅡcells and some macrophages in 1d exposure group and 7d exposure group. As compared with control group, the brown granules were decreased apparently in 30d exposure group. There was no significant difference of SP-A between1d and 7d exposure group and control group(P>0.05). Compared with control group, the decreasement in SP-A was observed in 30d exposure group(P<0.01). Compared with 30d exposure group, the decreasement in SP-A was observed in 1d exposure group and 7d exposure group(P<0.01).
4. SP-A mRNA expression in rat lung tissue
The results of RT-RCR showed that the SP-A mRNA of 1d and 30d exposure group and control group did not change apparently(P>0.05). As compared with control group, the SP-A of mRNA were increased apparently in 7d exposure group(P<0.01). Compared with 1d exposure group and 30d exposure group, SP-A in 7d exposure group significantly increased(P<0.01).
Conclusions:
SP-A was an important component of lung surfactant which functions were more complex. Lung-specific expression of SP-A had very important clinic significance. Rats were used in this study to detect the mechanism and changes of SP-A when mixture pollutants in atmosphere leaded to lung injury. The results showed:
1.SP-A had the function of innate immune defense and could delay Inflammatory response for elevation of SP-A transcription level. The changes of transcription levels indicated that SP-A was a sensitive indicator in early lung injury.
2.There were no significant differences in the contents of SP-A beween 1d and 7d in early lung injury from lung tissues, alveolus douche, serum,respectively.
3.Alveolar typeⅡcells were damaged severely and SP-A expression in lung tissue and BALF decreased significantly in late lung injury which was positively correlated with the degree of acute lung injury. SP-A in serum increased apparently which was negative correlation with the degree of acute lung injury. These results suggested that SP-A was specific index for pulmonary typeⅡcells function impairment .
In a word, SP-A was closely related with acute lung injury, which suggested that the levels of SP-A could be used as a sensitive biological indicator of lung damage caused by air-pollutants . It provided scientific basis for further research of lung injury caused by air pollution.
引文
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