| Objective:To find out whether there is the expression of NMDA receptors in the E15 to E21 rat fetal lung, and by E15 rat fetal lung organ culture model,to observe NMDAR's effects on rat fetal lung development and the effect on the expression of TTF-1 which plays an important role in the regulation of fetal lung Developmen.Methods:1 Preparation of pregnant rat:Put a 200-250gSD female rat and a 300-350g male rat in the same cage together, the next morning if there is vaginal plugs, then define it as 0 days'pregnant(EO).2 Measure the expression of NMDAR mRNA of fetal lung tissue: With the method of Fluorescence quantitative PCR measuring the mRNA expression of the E15 to E21 fetal lung's NMDA1, NMDA2A, NMDA2B, NMDA2C, NMDA2D, NMDA3A, NMDA3B receptors' mRNA.3. Fetal lung organ culture and groups:Put the fetal lung on the microporous membrane in transwell plates (diameter:0.8um), it is at the junction of gas and liquid, use serum-free BGJb medium containing penicillin (100U/mL), chloramphenicol (100ug/mL),37℃,5% CO2 culture. Experimental groups:control group:the medium without any treatment. Treatment group:NMDA (sigma company) group, a final concentration of lmmol/L4.General condition of the cultivation fetal lungs:the general area changes of fetal lung per 24 hour, wet weight of fetal lung per 48 hour, branching of fetal lung.5.Pathological examination of fetal lung:epithelial cell differen-tiation, saccular circumference per unit, saccular area per unit, lung parenchyma area per unit, the ratio of saccular area and lung parenchyma area.6. Measure of TTF-1 mRNA:With the method of Fluorescence quantitative PCR measuring the changes of TTF-1 mRNA between the control group and treatment group..7. Statistical analysis:The experimental data with mean±standard deviation, compared among groups using single factor analysis of variance (one-way ANOVA) and SNK-q test,compare the difference between two groups with T test.p<0.05 was considered statistically significant.Results:1 NMDAR expression in fetal lung:All the E15-E21 rat fetal lungs express the NMDAR1, NMDAR2A,NMDAR2B,NMDAR2C, NMDA-R2D,NMDAR3A, NMDAR3B.From E15 to E17,fetal lung expresses NMDAR3C most, from E18 to E20,expresses NMDAR2A most,E21 fetal lung expresses NMD-AR2D most.2:Fetal lung culture conditions:with the incubation time going, fetal lung wet weight increased (p<0.05), areaes increased (p<0.05), bronchial branches a lot.3. HE staining of the cultured fetal lung:With the culture time, the lung epithelial cells differentiation from high columnar epithelial cells to cuboidal cells, saccular circumference per unit increased (p<0.01), sacuular area per unit increased (p<0.05), the ratio of saccular area/the lung parenchyma area increased (p<0.05), lung parenchyma area per unit decreased(p<0.05).4. Cultivate fetal lung TTF-1 mRNA expression factor:With the culture time, the TTF-1 mRNA expression was increased (p<0.05).5.NMDA effects on cultured fetal lung:Compared with the control group, the saccular circumference per unit in the NMDA group decreased (p＜0.05), sacuular area per unit decreased (p＜0.01), the ratio of saccular area/the lung parenchyma area decreased (p＜0.01), lung parenchyma area per unit increased (p＜0.01). But the fetal wet lung area, fetal lung weight, TTF-1 mRNA expression show no significant difference between control group and NMDA group (p＞0.05)Conclusion:1 successfully established the rat fetal lung organ culture model 2 The first observation of the expression of NMDAR in E15-E21 normal fetal rat lung and their changes during the embryonic period.3 The first observation that the activation of NMDAR could inhibit the development of the rat fetal lung,and the mechanism has nothing to do with the TTF-1 mRNA expression... |
PDF Full Text Request