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The Study of Young Rats with Acute Lung Injury on the Alterations of Alveolar Epithelial Type Ⅱ Cell and SP-A and SP-D and the Interference of Dexamethasone

Author: ShuLinHua
Tutor: WeiKeLun
School: China Medical University
Course: Pediatrics
Keywords: Acute lung injury Alveolar epithelial type Ⅱ cell Ultrastructure Lamellar body Pulmonary surfactant Pulmonary surfactant protein A and D Gene expression of Pulmonary surfactant
CLC: R725.6
Type: PhD thesis
Year: 2006
Downloads: 237
Quote: 4
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Abstract


IntroductionAcute lung injury (ALI) and acute respiratory distress syndrome (ARDS) refer to a syndrome with pulmonary inflammation and the increase of the permi-bility caused by various reasons except cardiogenic elements which can lead to a-cute, progress and anoxic respiratory failure. Pathological features of ALI/ ARDS are pulmonary edema and microatelectasis caused by the diffused damage of capillaries. It is thought that the nature of ALI/ARDS comes from the extensive damage of pulmonary endothelial cells and pulmonary epithelial cells due to the over inflammatory reactions. ALI/ARDS is the clinical crisis which threatens the children at different ages. ALI/ARDS is one of the very common diseases with potential danger. The foreign statistics showed that 10 % children in PICU suffered from ALI. It was about 5 % ~ 10 % children with ALI in PICU in China, or even more higher. ALI can be caused by abortion, pulmonary infection, inhalation, drowning, toxication, trauma, ventilation and hypoimmune responsibility. ALI is the early stage of ARDS. The new statistic data indicated that the case fatality rate of ARDS is more than 60 %. Moer and more emphysis has been being focused on ALI/ARDS in the clinical research projects.Pulmonary surfactant (PS) is synthesized and secreted by alveolar epithelial type II cell (AEC - II ). It consists of cholesterol, phospholipids and peo-teins. PS plays an important role in reducing the alveolar surface tension and keeping the gas - fluid balance in trachea.Pulmonary surfactant protein is composed of protein A, protein B, protein C, and protein D ( SP - A.SP - B.SP - C.SP - D). SP - ANSP - D play an important role on pulmonary innate immune response, such as agglutination with micropathogen, chemotaxis, promotion for the phagocytosis of macrophage.Since SP - A was discovered in 1970s, great deal of research has been done on SP, for instance, SP - A. It showed that SP - A and SP - D altered in lung tissue and bronchial alveolar lavage fluid (BALF) in ALI induced by LPS. SP - A and SP - D were elevated by the drop of LPS in trachea. The early alterations of AEC - II and LB were reported by Fehrenbach in 1998. The study on the ultrastructure of AEC - II of rats with ALI/ARDS has been reported since 2003. The alterations of AEC - H of neonatal and adult rats with ALI induced by LPS were reported in 2005. The changes of pulmonary surfactant system are the main pathogenic mechanisms of ALI/ARDS. SP - A and SP - D have been approved to be closely associated with respsiratory diseasesSo far the real - time synchro - systematic study on AEC - E , LB, SP - A mRNA, SP - D mRNA, SP - A, and SP - D in the aspects of genomics, protei-nomics and untrastructure in AO induced by LPS haven t been reported in and out of China, including the interference of Dexamethasone on ALI caused by LPS. The exact alterations of SP - D in lung tissue of ALI havent been reached an agreement internationally. Even though there has been no report on the study of SP - D in China.We focus on the synchrosystemic study of ALI lung tissue induced by LPS on AEC - II ultrastructure, SP - A mRNA, SP - D mRNA, SP - A, and SP - D in the aspects of genomics, proteinomics and micromorphology synchronically, and the effects of Dexamethasone on them, in order to explore the pathogenic mechanism of ALI for the exact and effective therapy of climes.Materials and Methods1. MaterialsThe ALI models of Sprague - Dawley 21 days old rats were established by intraperitoneal injecting of LPS (4mg/kg E. coli O55:B5, Sigma). 0.9 % NSwith same amount was given for the nonnal control group, Dexamethasone (5mg/kg) was administered 1 hour later after LPS had been used in treatment group. The rats adopted for the experiment were executed at 6, 12, 24, 36, 48, and 72 h after anesthetized, with 10% chloral hydrate (4 mL/kg). Samples (8 rats each time point) were taken from the right lungs. One mm3 of samples were obtained from the lower part of right lung and were fixed with 2. 5% glutaralde-hyde. Then they were stored for the transmission electron microscope examination. The remains were kept in RNase free Eppendorf tubes in the 80 Tl refrigerator in order to test SP - A mNRA, SP - D mRNA, SP - A and SP - D.2. MethodsTransmission electron microscope was employed for the study of ultrastruc-ture of AEC - II. The expressions of SP - A mRNA and SP - D mRNA of lung tissue were detected with the method of RT - PCR. Western Blot was employed for the measurement of SP - A and SP - D in lung tissue at different groups. Statistical analysis was performed with SPSS 12.0 statistical package, and the data was expressed as x ± s, the comparison between groups was made by analysis of variance as well as SLD method, student t - test, and corrective t - test ( when variance -equality was not accepted).ResultsPart one;The alterations of the ultrastructure of AEC - II in the lung tissues of Sprague - Dawley21 days old rats in ALI, treatment and normal contral groups The ultrastructures of AEC - H in ALI group have been changed greatly compared with normal control. It had closed relationship with the development of the illness. The microvilli on the surface of AEC - E disappeared at 24 h. LBs increased in number, enlarged in size, LBs were rearranged like a ring around the nucleus. Electron density reduced. Emtying enhanced. Vacuole -like deformity occurred. Giant LBs presented at 48 h. Ruptured LBs and the residue were seen at 48 and 72 h. The number of LBs reduced at 72 h. At the beginning of ALI, the metabolism of AEC - II increased with mitosis. There were two nu-clei in one AEC - H. The morphology of nucleus of AEC - H became irregular with the disappearance of some border. There were two nucleoli in some nuclei of AEC - II. Finally the volume of nuclei shrunk in size with irregular shape, accompanied by the abnormality of chromosome. Karyolysis occured in some of the nuclei.Compared with ALI group, a serial changes of AEC - II in LB, Mi, and nuclei under electron microscope have been taken placed after the administration of Dexamethasone. Exocytosis of AEC - II indicated the enhanced secretion after 24 h of the administration of Dexamethasone. LBs were accumulated on the same side where the exocytosis was performed. The density of LBs with the homogeneous volume reduced. The number of LBs reduced with the weakness of density after 48 h of Dexamethasone administration. LBs emptied with unhomo-geneous volume and irregular in size. The residues of LBs were seen. The prominent phenomenon was about Mi. The number of Mi increased. Some Mi accumulated. Some were swollen. Some were increased in volume. The cristae of Mi were ruptured. At 72 h the number of LBs increased, LBs rearranged for the second time just like a ring around the nucleus with the similarity in ALI group at 24 h. The rearrangement of LBs showed the self - reregulation of AEC - II to the stimuli induced by LPS so as to meet the requirement of the body. At 24 h after Dexamethasone administered, the nuclei of AEC - H shrunk. Its volume decreased. The nuclei concentrated. The granules in the nuclei presented. At 48 h, the shape of nuclei was irregular, some of the border was not clear. At 72 h of Dexamethasone administration, the nuclei increased in size. Karyoplasm of nuclei was homogeneous. Some of the nucleoli were at the centre of the nuclei. The borders of the nuclei were clear. Hyperplasia of AEC - II was more active with mitosis.Part two: The effects of LPS and Dexamethasone on SP - A mRNA and SP -DmRNAin the lung tissues of Sprague - Dawley 21 days old rats 1. The effects of LPS and Dexamethasone on SP - A mRNA The expressions of SP - A mRNA in ALI group were quite different at every time after the injection of LPS. There was no difference in normal control group.ANOVA and t - test were employed in the statistics. Internal comparison in ALI group: F = 6.302, no variance, P < 0.01, there was significance. SP - A mR-NA in ALI group was downregulated at 12 h, reached to the lowest point at 24 h, and kept at the lowest level for 24 h compared normal control group ( all P < 0.01). After 48 h SP - A mRNA began to climb up. There were no significande between ALI and normal control groups at 6, 12, and 72 h, P >0. 05. That meant SP - A mRNA was driven down by LPS after a certain period of time and kept at the lowest level for some time and then upregulated.When treatmemt group was compared with ALI group, SP - A mRNA was significantly upregulated. After the t - test between these two groups at the same time point, P <0.01 at 6, 12, 24, 48, 72 h except at 36 h. That elucidated SP - A mRNA in treatment group was upregulated greatly.There was some similarity on the expression of SP — A mRNA between ALI group and treatment group. They dropped first and then went up. They paralleled in the most part of time. But there was quite difference between them. The SP - A mRNA level in treatment group was much higher than that of ALI group. It was longer for SP - A mRNA to have been kept at the lowest level in ALI group than that of treatment group.The effects of LPS and Dexamethasone on SP - D mRNAThe expression of SP - D mRNA in ALI group caused by LPS was obviously downregulated at each time point ( except 72 h ) compared with nonnal control group, P <0.01, the significance is great. The expression of SP - D mRNA in treatment group at every time point was much stronger than that of ALI group with paralleled manner. ANOVA and t - test have been adopted. In ALI group, F = 343.146, no variance, P <0.01, there was great difference between every two groups at each time point. In the treatment group, F = 19. 300, no variance, P <0.01, there was great difference between groups at each time point. ALI group compared with normal group at the same time point, SP - D mRNA dropped at 6 h and reached to the lowest point at 36 h (0. 27 0. 05 ) , then climbed up. There was great significance at 6, 12, 24, 36, 48 h, P <0. 01, but there was no at 72 h compared with normal contral group. When treatment group was compared with ALI group at each time point, P <0. 01, that meantthere was great significane between these two groups on the expressions of SP -D mRNA. It elucidated that SP - D mNRA was driven upward by Dexametha-sone. As the treatment group was compared with normal control group, the expression of SP - D mRNA at 6, 12 h, P >0. 05. That meant the expression of SP - D mRNA nearly reached to the normal level at 6, 12 h in the early stage of ALI caused by LPS. At 24, 36, 48, 72 h, P <0.01, that meant there was e-normous significance, but the implication was quite different. At 24, 36 h, SP - D mRNA was higher than that of AO group though it was lower than that of normal control group. At 48, 72 h, SP - D mRNA was higher than both ALI and normal control groups. That meant the synthesis of SP - D mRNA was increased.The Effects of LPS and Dexamethasone on SP - A mRNA and SP - D mRNA and their comparisonThere were no difference for SP - A mRNA in ALI group compared with normal control group at6,12h(P>0.05). SP-A mRNA was upregulated at 24, 36, 48 h compared with normal control group ( P < 0. 01). SP - A increased at 24, 36, 48 h compared with normal control group. SP-A mRNA and SP-A both changed synchronally at the opposite directions. Both SP-A mRNA and SP-A were elevated greatly in treatment group compared with ALI group. The expression of SP - A mRNA in treatment group at 6, 12, 24, 48, 72 h was completely higher than that of ALI group (P<0.01). SP-A in treatment group at 24, 36, 48, 72 h was absolutely higher than that of SP - A in All group ( P < 0.01). Both SP-A mRNA and SP - A were lifted by Dexamethasone synchronally in the up - direction.SP - D mRNA in ALI group was downregulated at every time point (except 72 h) compared with normal control group (P <0.01). And it was kept at the lowest level at 24, 36 h for 12 hrs. SP - D in ALI group was downregulated at 36, 48, 72 h compared with normal control group ( P < 0. 01). SP - D was at the lowest point at 48 h (0.92 0.11). There was time difference between SP -D mRNA and SP - D, SP - D mRNA dropped first and followed by SP - D. The effect of SP - D mRNA on SP - D exerted after a certain peroid of time. The death of rats in ALI was serious in ALI group at this time point. SP - D mRNAwas obviously upregulated in treatment group under the interference of Dexam-ethasone compared with ALI group (P<0.01). SP-D was elevated in treatment group at 36, 48, 72 h ( P < 0.01). SP - D in treatment group at 6, 12, 24, 36, 48 h nearly reached to the normal level compare with normal control group ( P > 0.05 ). SP - D in treatment group at 72 h was lower than that of normal control group ( P < 0. 05 ). There was no death in treatment group. The effect of SP - D mNRA on SP - D was exhibited after a certain time. Both SP -D mRNA and SP - D altered in the same direction with time difference.Part three: The effects of LPS and Dexamethasone on SP - A and SP-Din the lung tissues of Sprague - Dawley 21 days old rats1. The effects of LPS and Dexamethasone on SP — AThere was no difference at 6 and 12 h in ALI group compared with normal control group, P >0.05. SP - A in ALI group rose up significantly at 24, 36, and 48 h compared with normal group, P <0.01, there was great difference. It reached to the top at 36 h (6. 94 0. 80). SP - A in ALI group was lower at 72 h than that of normal contral group, P <0.01, there was great difference between them. There was no difference on the expression of SP - A between treatment and ALI groups at 6 and 12 h, P >0.05. SP - A in treatment group elevated at 24, 36, 48 h, P <0.01, great significance existed. And it was on the top at 24 h (9.14 0.77). SP - A fell at 72 h, but still higher than that of the ALI group, P<0.01.2 The effects of LPS and Dexamethasone on SP - DThere was no difference for SP - D at 6, 12, 24 h in lung tissue in ALI group compared with normal control group, P >0. 05. The significance was great between ALI and normal control groups at 36, 48, 72 h, P <0.01, and reached to the nadir at 48 h, 0. 92 0.11.There was no alteration for SP-D in treatment group at 6, 12, and 24 h compared with ALI group, P >0.05. SP-D rose up at 36, 48, 72 h contrasted with ALI group, P <0. 01. So there was great significance between them. The SP-D level in treatment group was nearly the same like normal control group except at 72 h point ( P > 0. 05 ). It showed that there was no significance between these two groups. It implied that SP-D could be recovered to the normallevel, but hard to exceed the normal level.3 The comparison between the effects of LPS and Dexamethasone on SP - A and SP - DSP - A and SP - D didnt changed at 6 and 12 h when they were campared with their own normal control groups respectively (P >0.05). Great alterations broke out for SP - A and SP - D from 24 to 72 h when they contrasted with their own normal control groups. SP - A rose up higher than its normal control group, SP - D fell down lower than its normal control group. The tracks of SP - A and SP - D driven by LPS were just synchronic and in the reverse directions. The differences between SP - A and SP - D were that SP - A started at 24 h (P < 0. 01) and but for SP - D began at 36 h ( P < 0. 01) as they compared with their own normal control groups respectively. SP - A began to go up from 24 h ( P < 0. 01) continuously and SP - D from 36 h (P <0.01) after Dexamethasone was administered.4 The comparison between the effects of LPS and Dexamethasone on SP - A mRNA and SP - A, SP - D mNRA and SP - DThere was no change for SP - A mRNA and SP - A at 6 and 12 h in ALI group (P>0.05). SP-A mRNA dropped at 24, 36, and 48 h (P<0.01);but SP-A went up at 24, 36, 48, and 72 h ( P <0. 01). SP-A mRNA and SP-A altered at the same time but in the opposite directions. SP-A mRNA and SP-A changed greatly in treatment group. The expression of SP - A mRNA was much higher at 6, 12, 24, 48, and 72 h in treatment group compared with ALI group ( P <0.01). SP - A was higher at 24, 36, 48, and 72 h in treatment group compared with ALI group (P<0.01). SP-A mRNA and SP-A developed in the same direction at the same time.SP - D mRNA in ALI grouop fell down greatly compared with normal control group (P <0.01) except at 72 h time point. And it lay on the nadir from 24 to 36 h. SP - D dropped at 36 h and made significant difference at 36, 48, and 72 h (P<0.01) compared with normal control group, and reached to the nadir at 48 h (0.92 0.11). There was a hysteresis for SP - D to follow SP - D mRNA. That meant the influence of SP - D mNRA on SP - D exerted after a certain period of time. It was coincident with the actual fact that the mortality of rats inALI group at 48 h was much higher than that of others when SP - D was at the nadir. The actions of SP - D mRNA and SP - D in ALI group behaviored unsyn-chronously with the same direction. SP - D followed the drop of SP - D mRNA after a certain period of time. SP - D mNRA and SP - D both elevated in treatment group compared with ALI group (P<0.01). SP-D rose up at 36, 48, 72 h, contrasted with ALI group, P <0.01;compared with normal contral group at 72 h, P <0.05. There was no death in the treament group. The influence of SP-D mRNA on SP - D emerged after a certain time. SP-D mRNA and SP -D both in treatment group acted unsynchronically in the same direction.Conclusion1. The damage of AEC - II is the early characteristic of ALI induced by LPS. ALI has close relationship with the injury of organelle and nucleus of AEC- II. The damage to the organelle at different time was quite different.2. Dexamethasone palys an active role on secretion of PS, proliferation of AEC - II , reducing the damage to LB, stabilizing the nuclei, and inhibiting the apoptosis of AEC - II.3. The rearrangement of LBs in AEC - II under the action of Dexamethasone made a foundation for the secretion of AEC - II. The overcompensation of Mi provided plenty of energy for the whole process. All these changes indicated the repairment for the ultrastructure of AEC - H and the recovery of its function.4. SP - A mRNA and SP-D mRNA were driven down in the different manners at different time points by LPS and were maintained at the lowest level for a certain period of time. Both of them can be driven up by Dexamethasone.5. SP - A and SP — D didn t change at the early stage of ALI in duced by LPS. Then they developed in the different ways. SP - A rose up, but SP-D fell down. The separation between SP - A and SP-D showed that the compensation of SP - A was much more powerful than that of SP - D. It maybe relate to the recovery of the structure, function and proliferation of AEC - II , including the rearrangement of LBs in AEC - II ?6. The mortality of SD rats with ALI occurred when SP-D was at the na-dir. This implied the content of SP - D in lung tissue has closed relationship to the severity of ALI, and it’ s maybe more important than SP - A. But the cooperation between SP - A and SP - D was not excluded.7. The levels of SP - A and SP - D in ALI groups can be improved greatly by Dexamethasone after a certain period of time. The recovery of SP - D in treatment group was nearly to the normal group level.

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