Effects Of Mixed Air Pollutants On The Expression Of CC16 In Rats

Objective:Breath system is the target organ of air pollution. Air mixde pollutants (PM10,SO2,NO2 and CO) entering alveolus and bronchia can damnify breath system. Mechanisms of disease of breath system induced by air pollution have not been clear so far. Many experiments about air pollution were only composed of one air pollutant. Experiments simulating true air pollution in order to explore the mechanism of respiratory disease, the change of CC16 in lung and bronchia and expression of CC16 mRNA which was affected by particular matter( PM )and mixture of many kinds of pollutants in air have not been reported yet. CC16 (Clara cell secretary 16-kD protein)is secreted by Clara cell found mainly distribute in bronchiole. CC16 has many biological functions such as anti-inflammation,antioxidation,immunoregulation and suppressing cancer cells. CC16 plays important roles in acute lung injury (ALI).It can protect respiratory system from oxidation and inflammation. CC16 is mainly expressed in lung and bronchia also founed BALF (bronchoalveolar lavage fluid). The level of CC16 in serum has relationship with compound and secretion of CC16 in bronchia. It was generally recognized that CC16 in serum came from bronchia. CC16 as one differential protein of lung epidermis has become a biomarker to detect functions of Clara cell and lung epidermis.In this experiment we established animal model patterns with air mixed pollutants (PM10,SO2,NO2,CO) ,through measured CC16 levels in BALF and serum and mRNA expression in lung ,to investigate the mechanism of lung injury ,search sensitive biomarker and provided theory for diagnosing and curing respiratory diseases.Methods1. Collection of PM10PM10 was collected in winter at ShenYang medical college. Apparatus used to collect PM10 were maid in Japan. Filtrated films were 55mm. Air flux was 20~30L/min. we collected PM10 for 2 monthes,24h/d. Filtrated films were taken in -20oC environment after desiccation and meager.2. Preparation of liquid with PM10Filtrated films were washed by ultrasonic using normal saline for 20min,and computer quantity of PM10. Higher does groups' concentration was 22.5mg/ml. Middle does groups' concentration was 15mg/ml.Lower does groups' concentration was 7.5mg/ml.3. Preparation of air mixture(SO2,NO2,CO)The air mixed pollutants were provided by DaLian special gas Company including higher concentration, middle concentration and lower concentration. Each concentration was 12 times higher than that of experiment. When these air mixtures were used in this experiment, they would be diluted 12 times.4. Establishment of animal model48 Wistar rats with 200~240g weight were randomly divided into 3 experimental groups( 1d dose group , 15d dose group , 30d lower dose group , 30d middle dose group, 30d dose group) and 3 control groups(1d control group ,15d control group,30d control group).Each group had 8 rats. Rats anaesthetized by aether through trachea. Experimental groups were injected liquid with PM10 with 1ml. Each does group' quantity of PM10 were 22.5mg.Control groups were injected normal saline 1ml. Experimental groups inhaled air mixture of concentration, which were diluted 12 times . The concentrations of SO2, NO2 and CO were 22.5, 18, 600mg/m3 in experimental groups. 1d, 15d and 30dgroups rats separately inhaled air mixture for 1d,15,and 30d. Control groups inhaled normal air.5. Taking samplesOn the 1st, 15th and 30th day after inhalation of air mixed gas, we got blood in abdominal aorta of anaesthetized rats by ethylether .Then serum separated from blood was placed in -80℃fridge. we got BALF with 37℃normal saline lavaging bronchi and alveoli of Wistar mice. BALF was placed in -80℃fridge. Lung tissue taken in right about 80mg was placed in the EP canal without RNA enzyme which was used to take hall RNA. Other lung tissue was placed in 10% which was used to dye or be observed by electric microscope. .6 .Mensurated target and Methods(1) Utilization of RT-PCR to measure expression of CC16 mRNA in lung.(2) Utilization immunity histochhemistry to measure CC16'expression in lung.(3)Utilization of ELISA to measure CC16 level in BALF and serumResults1.The mRNA expression of CC16 in lungThe mRNA expression levels of CC16 in 1d experimental group were significantly lower than that of control group (p<0.05). In 15d experimental group, the mRNA expression levels of CC16 had trend to resume and the mRNA expression levels of CC16 in 30d experimental group were significantly higher than that of control group (p<0.05)2.The measurement of CC16 in lungThe CC16 protein expression levels in 1d experimental group were significantly lower than that of control group (p<0.05). However in 30d experimental group, the protein expression levels of CC16 were significantly higher than that of control group (p<0.05).In 15d experimental group, there was no significant change.3.The measurement of CC16 in BALFThe level of CC16 in BALF was significantly lower in 1d experimental group than that of control group (p<0.05).The level of CC16 in 15d experimental group had trend to resume . The level of CC16 in 30d experimental group was no significantly greater than that of control. Howeve significantly greater than that of 1d experimental group (p>0.05) .4.The measurement of CC16 in serumCompared with control group , the level of CC16 in 1d and 15d experimental group had no significant change(p>0.05). The level of CC16 in 30d experimental group were significantly higher than that of both control group and 1d experimental group,respectively.(p<0.05)Conclusion1.In acute inflammation forepart,the expression of CC16mRNA in lung decreased.The CC16 levels both in the lung and its BALF was decreased significantly.2.In the late stage of acute inflammation,the expression of CC16 mRNA and CC16 protein in lung increased while the level of CC16 in BALF and serum also increased.3.The level of CC16 in serum may be used as a peripheral marker in the acute inflammation.