高同型半胱氨酸血症对大鼠血脑屏障影响机制的实验研究

华中科技大学

硕士学位论文

高同型半胱氨酸血症对大鼠血脑屏障影响机制的实验研究

姓名：程鹤

申请学位级别：硕士

专业：神经病学

指导教师：彭海

20090501

华 中 科 技 大 学 硕 士 学 位 论 文中文摘要

第一部分 高同型半胱氨酸血症对大鼠血脑屏障的影响

目的 建立高同型半胱氨酸血症大鼠模型，观察高同型半胱氨酸血症(HHcy)对大鼠血脑屏障的影响

方法 36只Sprague-Dawley(SD)大鼠随机分为两组(1)对照组(n=18)，(2) 模型组(n=18)。根据观测的不同时间点，每组大鼠再分为三个小组(1天组，4周组，8周组)，每小组各6只。 采用按体重皮下注射蛋氨酸法建立高同型半胱氨酸血症大鼠模型，在不同的时间点(1天，4周，8周)处死各组大鼠，采用高效液相色谱法检测大鼠血浆同型半胱氨酸（Hcy）含量，用比色法检测大鼠大脑伊文思蓝(EB)的含量来比较血脑屏障通透性的改变，以透射电镜方法观察大鼠大脑额叶微小血管基底膜形态学改变。 结果 对照组大鼠在不同时间点血浆Hcy水平和脑组织中EB含量均无明显变化；模型组血浆Hcy水平随着时间的延长显著增高，与同组第一天和同周不同组比较均有显著性差异（P<0.05）。模型组大脑中EB含量在第4周时先上升然后在第8周时又有所下降，与同组第一天和同周不同组比较均有显著性差异（P<0.05）。对照组在不同时间点电镜下血管基底膜结构正常，模型组第4周和第8周电镜下观察基底膜出现不同程度的厚薄不均、周围水肿、破裂等改变。

结论 高同型半胱氨酸血症可以引起血管基底膜破坏，导致血脑屏障结构和功能的破坏。

关键词: 高同型半胱氨酸血症；同型半胱氨酸；大鼠；血脑屏障

华 中 科 技 大 学 硕 士 学 位 论 文第二部分 MMP-2在HHcy致血脑屏障损伤中的作用及联合维生素

的干预效果

目的 观察MMP-2在HHcy致血脑屏障损伤中的作用及联合维生素的干预效果，为预防和治疗脑血管病寻找新的方法和途径。

方法 54只Sprague-Dawley(SD)大鼠随机分为三组(1)对照组(n=18)， (2)模型组(n=18)，(3)维生素组(n=18)，根据观测的不同时间点，每组大鼠再分为三个小组(1天组，4周组，8周组)，每小组各6只。 采用按体重皮下注射蛋氨酸法建立高同型半胱氨酸血症大鼠模型，在不同的时间点(1天，4周，8周)处死各组大鼠。采用高效液相色谱法检测大鼠血浆Hcy含量；经逆转录聚合酶链反应(RT-PCR)方法观察脑组织MMP-2 mRNA表达水平；用比色法检测大鼠大脑伊文思蓝(EB)的含量来比较血脑屏障通透性的改变。

结果 对照组和维生素组大鼠在不同时间点血浆Hcy水平无明显变化，而模型组血浆Hcy水平随着时间的延长显著增高（P<0.05）。对照组和维生素组在不同时间点脑组织MMP-2 mRNA表达水平极低，模型组第4周和第8周脑组织MMP-2 mRNA表达水平随着时间的延长显著增高（P<0.05）。模型组大脑中EB含量在第4周时先上升然后在第8周时又有所下降，与同组第一天和同周不同组比较均有显著性差异（P<0.05）。

结论 高同型半胱氨酸血症可能通过诱导MMP-2 mRNA表达上调而导致血管基底膜破坏，引起血脑屏障通透性破坏，通过补充叶酸和维生素B可以降低血浆同型半胱氨酸水平从而保护血脑屏障，预防脑血管病。

关键词: 高同型半胱氨酸血症； MMP-2；血脑屏障；叶酸

华 中 科 技 大 学 硕 士 学 位 论 文Abstract

Part one: Effect of Hyperhomocysteinemia on Blood-Brain Barrier

of Rats

Objective The aim are to establish hyperhomocysteinemia(HHcy) and to observe the effect of HHcy on Boold-Brain Barrie of rats.

Methods 36 normal male SD rats were distributed randomly to two groups：

(1)control group (n=18)，(2)model group (n=18). Each group was divided into three teams and each team had six rats. Rats given dl-methionine subcutaneous every day for eight weeks were to erect up HHcy. At the different time，rats were killed. The plasma homocysteine(Hcy) level was detected by high performance liquid chromatography(HPLC). The permeability of blood-brain barrier was observed with colorimetry and by detecting the content of Evans Blue leaked out of the brain issue.The micro-artery histological changes of the frontal lobe were examined by electron microscope.

Result The mean plasma homocysteine(Hcy) level and the contents of Evans Blue of model group was significantly higher than those of control group at four week and at eight week.At the same time(P<0.05)，The breakdown of blood-brain barrier appeared on electron microscope in model group.

Conclusions These results indicate HHcy induce the breakdown of the construction and the function of blood-brain barrier.

key words hyperhomocysteinemia；homocysteine；rat；blood-brain barrier

华 中 科 技 大 学 硕 士 学 位 论 文Part two Experimental studies of the relationship between the transcription of MMP-2 mRNA and the change of Boold-Brain Barrier and the interference of the compounded vitamin

Objective To investigate the relationship between the transcription of MMP-2 mNNA and the change of Boold-Brain Barrier，and the interference of the compounded vitamin.

Methods 54 normal male SD rats were randomly distributed into three groups：

(1)control group (n=18)；(2)model group (n=18)；(3)vitamin group (n=18). Each group was divided into three teams and each team had six rats. Rats given dl-methionine subcutaneous every day for eight weeks were to erecte up Hyperhomocysteinemia(HHcy).At the different time，rats were killed. The plasma homocysteine(Hcy) level was detected by high performance liquid chromatography(HPLC). The transcription of MMP-2 mRNA in different groups was detected by RT-PCR.The permeability of blood-brain barrier was observed with colorimetry and by detecting the content of Evans Blue leaked out of the brain issue.

Result The mean plasma homocysteine(Hcy) level of group 2 was significantly higher than those of group 1 and group 3 in four week and in eight week. The transcription of MMP-2 mRNA and the content of Evans Blue in group 2 leaked out of the brain tissue were increased obviously，but they were not changed in group 1 and group 3 at the different time.

Conclusions These results indicate HHcy stimulates transcriptions of MMP-2 mRNA which may play a role in the breakdown of blood-brain barrier. Folate and Vitamin

B could reduce the plasma homocysteine level to prevent cerebrovascular diseases.

key words hyperhomocysteinemia; MMP-2; blood-brain barrier; folate

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