妊娠期糖尿病大鼠的胎鼠肺结构和肺表面活性蛋白B、C及其调控因子表达

Fetal lung structures and expression of pulmonary surfactant proteins of B and C and their modulating factors in rat with gestational diabetes mellitus

目的 通过对GDM大鼠胎鼠的肺组织结构及肺表面活性蛋白（surfactant proteins,SP）-B、SP-C、甲状腺转录因子（thyroid transcription factor,TTF）-1、多形性腺瘤样因子（pleiomorphic adenoma gene like,PLAGL）-2蛋白表达水平进行研究,探讨GDM大鼠胎鼠肺发育障碍的机制. 方法 清洁级健康成年Sprague-Dawley大鼠构建孕鼠GDM模型,共20只造模成功,对照组为20只正常孕鼠.于妊娠第21天检测孕鼠随机血糖,并行剖宫取胎术,胎鼠计数并称重.透射电子显微镜下观察肺泡Ⅱ型上皮细胞的超微结构.每组随机取60只胎鼠,取肺组织制作360张石蜡切片,随机取100张不连续切片,光镜下观察肺组织形态结构;随机取另外100张不连续切片,免疫组织化学法检测SP-B、SP-C、TTF-1和PLAGL-2的蛋白定位及表达.每组取9只胎鼠,蛋白质印迹技术检测胎鼠肺组织中SP-B、SP-C、TTF-l和PLAGL-2蛋白水平.每组取27只胎鼠,荧光实时定量聚合酶链反应检测胎鼠肺组织中SP-B、SP-C、TTF-1和PLAGL-2的mRNA水平.采用独立样本t检验进行统计学分析. 结果 GDM组孕鼠平均随机血糖值高于对照组[（26.8±2.8）与（4.9±0.5）mmol/L,t=-34.05,P=0.00].GDM组胎鼠平均体重高于对照组[（5.6±0.6）与（5.2±0.5）g,t=-1.97,P=o.03].GDM组与对照组肺泡个数分别为（10.1±1.6）与（12.1±1.3）个,肺泡面积分别为（986.9±5.5）与（1 257.3±5.0） μm2,GDM组均低于对照组（t值分别为9.84和27.53）;肺泡间隔分别为（11.5±6.2）与（9.9±4.3）μm,GDM组高于对照组（t=-2.17）,差异均有统计学意义（P值均＜ 0.05）.透射电镜下,可见GDM组肺泡Ⅱ型上皮细胞表面微绒毛粗短、减少,板层小体数量减少.胎肺组织中SP-B、SP-C、TTF-1和PLAGL-2蛋白均沿胞浆呈颗粒状分布,GDM组4种蛋白表达的平均吸光度分别为1.15±0.12、1.23±0.06、0.87±0.21与1.21±0.18,对照组分别为1.22±0.05、1.31±0.14、1.12±0.09与1.33±0.07,GDM组均低于对照组,差异有统计学意义（t值分别为2.40、2.35、4.89和2.77,P值均＜0.01）.GDM组胎肺组织中SP-B、SP-C、TTF-1和PLAGL-2的蛋白水平分别为0.57±0.09、0.45±0.03、1.50±0.04和1.11±0.04,对照组分别为0.81±0.03、0.66±0.04、1.69±0.05和1.46±0.07,GDM组均低于对照组,差异有统计学意义（t值分别为11.77、11.09、8.80和13.37,P值均＜0.01）.GDM组胎肺组织中SP-B、SP-C、TTF-1和PLAGL-2mRNA水平分别为0.60±0.04、0.79±0.04、0.81±0.03和0.79±0.05,对照组分别为1.06±0.19、1.03±0.24、1.03±0.18和1.02±0.19,GDM组均低于对照组,差异有统计学意义（t值分别为6.80、2.98、3.54和3.54,P值均＜0.01）. 结论 GDM大鼠的胎鼠肺组织中SP-B和SP-C蛋白水平均明显下降,可能由于TTF-1和/或PLAGL-2基因表达下调所致;SP-B和SP-C蛋白减少与胎肺结构的病理变化有密切关联.

Objective To explore the lung developmental disorder of rats with gestational diabetes mellitus （GDM） via investigating the GDM rat fetal lung structures and expression of pulmonary surfactant proteins （SP）-B,SP-C,thyroid transcription factor （TTF）-1 and pleiomorphic adenoma gene like （PLAGL）-2.Methods Sprague-Dawley rats were used to construct the GDM model.Twenty GDM rats were used as GDM group and 20 normal pregnant rats as control group.Cesarean section was performed on day 21 of gestation and random blood sugar was detected,and fetal rats were counted and weighed.Ultrastructure of the fetal lungs was studied by transmission electron microscopy.Sixty fetal rats were selected randomly in each group,and 360 paraffin sections were made from fetal lungs.One hundred discontinuous paraffin sections were picked up in each group to observe morphological and structural changes under optical microscope.The other one hundred discontinuous paraffin sections were picked up in each group to detect the location and expression of SP-B,SP-C,TTF-1 and PLAGL-2 protein by immunohistochemistry.Nine fetal rats were selected randomly to detect the expression level of SP-B,SP-C,TTF-1 and PLAGL-2 proteins in fetal lung tissues by Western blotting.Twenty seven fetal rats were selected randomly to detect the mRNAs level of SP-B,SP-C,TTF-1 and PLAGL-2 by real-time quantitative polymerase chain reaction.Independent sample t-test was used for statistical analysis.Results The average random blood glucose level in GDM group was significantly higher than that in control group [（26.8± 2.8） vs （4.9± 0.5） mmol/L,t=-34.05,P=0.00].The average weight of fetal rats in GDM group was higher than that in control group [（5.6±0.6） vs （5.2±0.5） g,t=-1.97,P=0.03].Alveolar number （10.1 ± 1.6 vs 12.1 ± 1.3） and alveolar area [（986.9 ± 5.5） vs （1 257.3± 5.0） μ m2] in GDM group was less than that in control group （t=9.84 and 27.53,both P 〈 0.05）.Alveolar septum [（11.5±6.2） vs （9.9±4.3） μm] in GDM group was higher than that in control group （t=-2.17,P 〈 0.05）.Microvillus in type] cells were short and the number of lamellar bodies was significantly decreased in GDM group.SP-B,SP-C,TTF-1 and PLAGL-2 proteins were distributed in the cytoplasm in granular form.The average value of absorbance of SP-B,SP-C,TTF-1 and PLAGL-2 proteins in GDM group was 1.15±0.12,1.23±0.06,0.87±0.21 and 1.21 ±0.18 respectively;and that in control group was 1.22±0.05,1.31 ±0.14,1.12±0.09 and 1.33 ±0.07 respectively.The value in GDM group was lower than that in control group （t=2.40,2.35,4.89,and 2.77 respectively,all P 〈 0.01）.The expression level of SP-B,SP-C,TTF-1 and PLAGL-2 proteins in GDM group was 0.57± 0.09,0.45±0.03,1.50±0.04 and 1.11 ±0.04 respectively;and that in control group was 0.81 ±0.03,0.66±0.04,1.69±0.05 and 1.46±0.07 respectively.The value in GDM group was lower than that in control group （t=1 1.77,11.09,8.80 and 13.37,respectively,all P 〈 0.01）.The mRNA level of SP-B,SP-C,TTF-1 and PLAGL-2 in GDM group was 0.60±0.04,0.79±0.04,0.81 ±0.03 and 0.79±0.05 respectively;and that in control group was 1.06±0.19,1.03±0.24,1.03±0.18 and 1.02±0.19 respectively.The value in GDM group was lower than that in control group （t=6.80,2.98,3.54 and 3.54 respectively,all P 〈 0.01）.Conclusions The protein expression level of SP-B and SP-C in fetal lungs of GDM rats decreases obviously,possibly because of the down-regulation of the gene expression of TTF-1 and/or PLAGL-2.The pathological changes in fetal lungs of GDM rats might be associated with the descending level of SP-B and SP-C protein.