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同型半胱氨酸所致大鼠仔鼠先天性心脏病与PAX-8基因表达的研究
Study on the Relationship between Homocysteinemia, Congential Heart Disease and PAX-8 Gene
【作者】 康昭海；
【导师】 王新；
【作者基本信息】 中南大学 ， 妇产科学， 2009， 硕士
【摘要】 目的:通过同型半胱氨酸（Homocysteine,Hcy）干预孕鼠,建立大鼠仔鼠先天性心脏畸形模型,探讨Hcy与先天性心脏病（congenitalheart defect,CHD）PAX-8基因表达的关系及叶酸（Folic Acid,FA）、VitB12对Hcy的干预作用,从心脏发育的分子学角度探讨CHD发生的病因。方法:40只S-D孕鼠随机分为四组:高剂量Hcy建模组（A组）、低剂量Hcy建模组（B组）、高剂量Hcy+(FA+VitB12)干预组（C组）及对照组（D组）,每组各10只孕鼠。大鼠自妊娠第7天开始腹腔内注射不同剂量的Hcy:A组2%Hcy 2mg/10g/d、B组1%Hcy1mg/10g/d、C组2%Hcy 2mg/10g/d、D组注射生理盐水0.1ml/10g/d,C组自确定妊娠起,按FA 0.05mg/10g/d+VitB12 1mg/10g╱d给药,至孕鼠自然分娩时停药。每日观察孕鼠,待其自然分娩当日,体视显微镜下解剖仔鼠,观察仔鼠心脏畸形情况。选取A、B、C三组内VSD仔鼠心脏各5例,非CHD仔鼠心脏各15例,D组非CHD仔鼠心脏15例,提取RNA,应用real time PCR检测PAX-8 mRNA在仔鼠心脏的表达情况;同时选取A、B、C三组VSD仔鼠心脏各2例、非CHD仔鼠心脏各5例、D组非CHD仔鼠心脏5例,5%甲醛固定,石蜡包埋,应用免疫组化检测PAX-8蛋白在仔鼠心脏的表达情况。结果:1、A、B、C、D四组平均产仔数分别为:9.04只、12.1只、11.56只及13.56只,A、B、C三组平均产仔数分别与D组比较,p值均＜0.01;A、B、C三组平均产仔数之间两两比较,p值均＜0.01,提示外源性Hcy可导致孕鼠产仔数减少,通过对孕鼠用FA+Vit B12干预,可增加孕鼠产仔数。2、A、B、C、D四组仔鼠心脏畸形率分别为:13.5%、9.17%、8.77%及0.74%,A、B、C三组之间两两比较,p值均＞0.05;A、B、C三组分别与D组比较,p值均＜0.01,提示外源性Hcy可引起仔鼠心脏畸形。3、PAX-8 mRNA在A、B、C、D四组组内及组间仔鼠心脏表达的比较:PAX-8 mRNA在VSD仔鼠心脏细胞中的表达均低于非CHD仔鼠,p值均＜0.01。A、B、C三组VSD仔鼠心脏细胞中的PAX-8mRNA表达比较:B组＞A组,A组＞C组,p值均＜0.01,B组与C组比较p＞0.05。A、B、C、D四组非CHD仔鼠心脏组织中的PAX-8mRNA表达比较,p值均＞0.05。VSD仔鼠心肌组织PAX-8 mRNA表达水平与孕鼠Hcy注射剂量呈负相关,r=-0.949,p＜0.01。4、PAX-8蛋白在A、B、C、D四组组内及组间VSD仔鼠心脏细胞中表达均低于非CHD仔鼠,p值均＜0.01。PAX-8蛋白在A、B、C三组VSD仔鼠之间心脏中的表达比较无差异,p值均＞0.05。四组非CHD仔鼠心脏细胞中的表达比较无差异,p值均＞0.05。结论:1、PAX-8mRNA在VSD胎鼠心肌中的呈低表达,且与Hcy剂量呈负相关,PAX-8基因的低表达可能与VSD的形成有关。2、高水平的Hcy具有胚胎毒性,孕期FA与VitB12干预可增加平均胎产数,降低Hcy的胚胎毒性。
【Abstract】 Objective To create a mode of congenital abnormal heart of fetal rats by intervening pregnant S-D rats with homocysteine（Hcy）,and to investigate the relationship between congenital heart disease（CHD） and homocysteine（Hcy）,PAX-8 gene,Folic Acid（FA）,VitB12.And to explore the etiology of CHD from molecular genetics of heart development.Methods The 40 pregnant rats were randomly divided into four groups:high dose group of 2%Hcy（Group-A）,low dose group of 1%Hcy（Group-B）,intervention group of FA(FA、VitB12+2%Hcy,Group-C) and control group（Group-D）.Each group had 10 pregnant rats and the rats were injected intraperitoneally with Hcy on the seventh day of pregnancy.The dosage in each group was as follows:A-2%Hcy 2mg/10g/d,B-1%Hcy 1mg/10g/d,C-2%Hcy 2mg/10g/d,D- the same amount of saline.Rats in Group-C were administrated with FA 0.05mg/10g/d and VitB12 1mg/10g/d from the first pregnant day to the day of fetal rats borne naturally.The heart of fetal rats of each group were observed by stereoscope on the day when they are borne naturally.RNA extraction was performed from one part of heart in fetal rats of each group,mRNA expression of PAX-8 in hearts of these fetal rats of each group were detected by Real-time quantitative PCR.Expression of PAX-8 protein in heart of these fetal rats of each group were assessed by immunohistochemistryResults1、The difference of the average number of births among the 4 groups is significant（p＜0.01）,and the average number of births of each group was 9.04,12.1,11.56 and 13.56 respectively.The average number of births of Group-D was significantly higher than that of Group-A, Group-B and Group-C respectively,p＜0.01.The differences of average number of births between A,B and C are significant（P＜0.01）.2、The difference of the abnormal heart incidence of fetal rats among the 4 groups is significant（p＜0.01）,and the abnormal heart incidence of each group was 13.5%、9.17%、8.77%and 0.74%respectively.The differences of abnormal heart incidence of fetal rats between A,B and C are not significant（P＞0.05）.The abnormal heart rate of fetal rats of Group-D was significantly lower than that of Group-A,Group-B and Group-C respectively,p＜0.013、The difference of mRNA expression of PAX-8 in heart of fetal rats among the 4 groups is significant（p＜0.01）.The mRNA expression of PAX-8 in heart of fetal rats with VSD was significantly lower than that of fetal rats without CHD.The difference of mRNA expression of PAX-8 in heart of fetal rats between A and B,A and C are significant（p＜0.01）. There was no deference between B and C（p＞0.05）.The difference of mRNA expression of PAX-8 in heart of fetal rats without CHD among the 4 groups is not significant（P＞0.05）.The mRNA expression of PAX-8 in hearts of fetal rats was correlated with the levels of Hcy injected negatively.The low expression of PAX-8 mRNA may cause cardiovascular abnormality of fetal rats.4、The difference of expression of PAX-8 protein in heart of fetal rats among the 4 groups is significant（p＜0.01）.The expression of PAX-8 protein in heart of fetal rats with VSD was significantly lower than that of fetal rats without CHD.The difference of expression of PAX-8 protein in heart of fetal rats between A,B,and C are not significant（p＞0.05）.The difference of expression of PAX-8 protein in heart of fetal rats without CHD among the 4 groups is not significant（P＞0.05）.Conclusion1、The mRNA expression of PAX-8 in fetal rats with VSD is lower than that of normal heart,and correlates with the levels of Hcy injected negatively.The low expression of PAX-8 mRNA may be related to the formation of VSD.2、FA and VitB 12 intervention during pregnancy may increase the average number of births,and reduce the embryo toxicity of Hcy.