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地塞米松对大鼠肺形态结构及基质金属蛋白酶2、9及其抑制剂表达影响的实验研究

Effects of Postnatal Dexamethasone on Lung Morphogenesis and Matrix Metalloproteimase -2 and -9, and Their Tissue Inhibitors in the Deceloping Rat Lung

作者: 专业:儿科学 导师:赵德育 年度:2010 学位:硕士  院校: 南京医科大学

Keywords

Dexamethasone(DEX), lung, morphogenesis, MMP-2 gene, MMP-9 gene, TIMP-1 gene, TIMP-2 gene

        目的地塞米松(DEX)能显著改善早产儿肺功能、促进肺部早期发育,但其对肺发育的远期影响及其分子机制目前尚不清楚。肺发育过程离不开细胞外基质(ECM)的重塑,这其中由于基质金属蛋白酶(MMPs)及其组织抑制剂(TIMPs)参与了维持ECM合成和降解的平衡,因此在生后肺发育中起着重要作用。本研究拟以新生大鼠为研究对象,通过生后给予不同剂量地塞米松,观察大鼠在肺发育不同时期肺组织形态结构的差异和基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、金属蛋白酶组织抑制剂-1(TIMP-1)、金属蛋白酶组织抑制剂-2(TIMP-2)表达水平的变化,从而探讨生后糖皮质激素的应用对肺发育的影响及可能的作用机理。方法120只新生大鼠随机平均分为三组。小剂量DEX组:生后第5天起连续5天腹腔注射DEX 0.2mg/(kg·d)。大剂量DEX组:生后第5天起连续5天腹腔注射DEX0.5 mg/(kg·d)。生理盐水组:生后第5天起连续5天腹腔注射等量生理盐水。各组大鼠分别在生后第10天(撤药后第1天)、14天、21天(断乳期)、45天(青春期)随机选取10只处死,分离肺组织。通过光镜观察,分析比较各组大鼠肺形态发育的差异,RT-PCR方法检测MMP-2、MMP-9、TIMP-1、TIMP-2基因mRNA的表达,Western-Blot方法检测MMP-9、TIMP-1蛋白表达量。结果1.HE染色光镜观察结果:生后10天和14天,DEX用药组与同龄生理盐水对照组相比,肺泡数目减少、肺泡腔结构扩大,肺泡间隔厚度较薄、周围结缔组织减少;生后21天,DEX用药组与同龄对照组相比肺泡数目减少,肺泡腔结构扩大,且外观呈大小不一,肺泡间隔厚度增加;生后45天DEX用药组与同龄对照组相比肺泡数目减少,肺泡腔结构扩大。其中两DEX用药组在生后同一时间点每视野肺泡计数均低于对照组(P<0.01),平均肺泡表面积均高于对照组(P<0.05)。两DEX用药组平均肺泡间隔厚度在生后第10天、14天均低于对照组(P<0.01),在生后第21天均高于对照组(P<0.05)。2.RT-PCR结果:在生后各时间点,两DEX用药组大鼠肺组织MMP-2基因mRNA的表达与生理盐水对照组相比均未见统计学差异(P均>0.05);两DEX用药组TIMP-2基因mRNA的表达均低于对照组(P均<0.05),且生后14天及21天,两DEX组之间差异有统计学意义(P<0.05);MMP-2/TIMP-2比值在生后14天和21天两DEX用药组均高于对照组(P均<0.05),其中生后21天两DEX组之间差异有统计学意义(P<0.05)。两DEX用药组MMP-9基因mRNA的表达在生后14天与对照组相比均未见统计学差异(P均>0.05),在生后21天和45天均高于对照组(P均<0.05),且生后21天两DEX组之间差异有统计学意义(P<0.05);两DEX用药组TIMP-1基因mRNA的表达在生后14天和45天与对照组相比均未见统计学差异(P均>0.05),在生后21天均高于对照组(P均<0.05);MMP-9/TIMP-1比值在生后21天和45天两DEX用药组均高于对照组(P均<0.05),其中生后21天两DEX组之间差异有统计学意义(P<0.05)。3.Western-blot结果:两DEX用药组MMP-9基因蛋白的表达在生后14天与对照组相比均未见统计学差异(P均>0.05),在生后21天和45天均高于对照组,但此差异仅在生后21天时有统计学意义(P均<0.05);两DEX用药组TIMP-1基因蛋白的表达在生后14天和45天与对照组相比均未见统计学差异(P均>0.05),在生后21天均高于对照组(P均<0.05)。结论1.生后用药DEX可促进肺部早期发育,小剂量DEX即可促进肺部成熟,增加用药剂量虽然肺部发育成熟度有所提高,但肺泡化受阻的现象也愈加明显——肺泡直径变大,肺泡次级隔发育受阻,总肺泡数减少。肺泡化不完全最终导致肺泡结构简单化,其影响可持续直至成年。2.生后用药DEX可影响大鼠肺组织中MMP-9、TIMP-1、TIMP-2基因的mRNA及蛋白的表达。3.生后用药DEX可能是通过在肺发育不同时间点下调TIMP-2以及上调MMP-9和TIMP-1的表达水平来引起MMPs/TIMPs失衡,导致ECM重塑异常,最终影响大鼠肺形态发育。其中MMP-9/TIMP-1失衡持续至成年,有可能在气道重塑的发生中起到了重要作用,可能与后期哮喘的发生有关。
    Objective:Postnatal glucocorticoids such as dexamethasone (DEX) can improvepulmonary function in premature newborn infants, but its long-term effects on lungdevelopment and its molecular mechanism is still unclear.MMPs and TIMPs havebeen shown to play important roles in the development of lung by maintaining thebalance between the synthesis and degradation of ECM components. To investigatethe effects of DEX on lung morphogenesis and the expression of MMP-2、MMP-9and their tissue inhibitors, newborn rat models were used to compare the effects ofvarious dosages of DEX therapy on postnatal development of rat lung.Methods:120 newborn rats were divided into three groups randomly: For the smalldosage and large dosage DEX groups, i.p with 0.2mg/(kg·d), 0.5mg/(kg·d)respectively on the 5th、6th、7th、8th and 9th day after birth. For the control group, i.pwith 0.5ml saline on the5th、6th、7th、8th and 9th day after birth. 10 rats from eachgroup were randomly chosen and sacrificed on the 10th(1 day post-Dexadministration), 14th, 21st(weaning), 45th(adolescence) day after birth. Thehistological structures of rat lungs were observed with light microscope. TheRT-PCR、Western-Blot methods were used to detect the mRNA and protein level ofMMP-2、MMP-9、TIMP-1 and TIMP-2 genes. Results:1. Under the light microscope, during the first 2 weeks, rat lung in the twoDEX groups had fewer alveolar numbers, larger alveolar space, and thinneralveolar septum compared with control group (P<0.01).On the 21st and 45th days,the airspaces of rat lungs in the two treatment groups were still enlarged, lessnumerous(P<0.01), an "emphysematous" condition of the lungs could be observedespecially in the large dosage DEX group.2. RT-PCR results: The mRNA level of MMP-9 gene was significantly enhancedin study groups compared with control on the 21st and 45th days(all P<0.05). ThemRNA level of TIMP-1 gene was significantly enhanced in study groups comparedwith control on the 21st day(all P<0.05). MMP-9/TIMP-1 ratio was significantlyenhanced on the 21st and 45th days(all P<0.05). There was no statistic difference inthe expression of MMP-2 between the DEX groups and control group at all timeintervals. The expressions of TIMP-2 was downregulated in study groups comparedwith control at all time intervals(all P<0.05). MMP-2/TIMP-2 ratio wassignificantly enhanced on the 14th and 21st days(all P<0.05).3. Western-blot results:The expressions of MMP-9 protein was significantlyenhanced in study groups compared with control on the 21st days(all P<0.05). Theexpressions of TIMP-1 protein was significantly enhanced in study groups comparedwith control on the 21st day(all P<0.05).Conclusions:1. Using DEX postnatal could promote lung development obviously, even thesmall dosage of DEX may induce the lung mature. Increasing the doses of DEX notonly improves the level of the lung development, but also has negative effect on ratlung morphogenesis. With the dose increased, the normal alveolization was inhibited,resulted in reduction of alveolar numbers, alteration of alveolar space and septum,and the impact continued well into adulthood. 2. Postnatal DEX therapy changed the expressions of the mRNA and the proteinof MMP-9、TIMP-1 and TIMP-2 genes in the developing rat lung.3. The changes of the expressions of MMP-9、TIMP-1 and TIMP-2 genes mayinvolved in the effects of DEX therapy on rat lung morphogenesis, and the imbalanceof MMP-9/TIMP-1, which may be related to the occurrence of asthma, continued toadult.
        

地塞米松对大鼠肺形态结构及基质金属蛋白酶2、9及其抑制剂表达影响的实验研究

缩略词表4-5
中文摘要5-8
英文摘要8-10
前言11-14
参考文献14-16
材料与方法16-29
结果29-38
讨论38-45
参考文献45-48
综述48-59
附录59-60
致谢60-61
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